Patent application title: ENZYMATIC REPLACEMENT THERAPY AND ANTISENSE THERAPY FOR POMPE DISEASE
Inventors:
Erik Van Der Wal (Rotterdam, NL)
Atze Jacobus Bergsma (Rotterdam, NL)
Wilhelmus Wenceslaus Matthias Pijnappel (Rotterdam, NL)
Antje Tjitske Van Der Ploeg (Rotterdam, NL)
Arnoldus Reuser (Rotterdam, NL)
IPC8 Class: AA61K3847FI
USPC Class:
1 1
Class name:
Publication date: 2021-10-21
Patent application number: 20210322526
Abstract:
The present invention is direct to the treatment of Pompe disease by
administration of an enzyme or nucleic acid encoding for said enzyme
suitable for Enzyme Replacement Therapy for Pompe disease in combination
with the administration of an antisense oligomeric compound that
modulates the splicing of acid alpha-glucosidase (GAA) gene.Claims:
1-35. (canceled)
36. Antisense oligomeric compound comprising a sequence selected from the group consisting of SEQ ID NO: 1590-1594 and sequences having at least 80% identity thereof.
37-49. (canceled)
50. Antisense oligomeric compound as claimed in claim 36, wherein at least one of the nucleotides is modified.
51. Antisense oligomeric compound as claimed in claim 50, wherein the oligomeric compound is uniformly modified.
52. Antisense oligomeric compound as claimed in claim 36, wherein the sugar of one or more nucleotides is modified.
53. Antisense oligomeric compound as claimed in claim 52, wherein the sugar modification is 2'-O-methyl or 2'-O-methoxyethyl.
54. Antisense oligomeric compound as claimed in claim 36, wherein the base of one or more nucleotides is modified.
55. Antisense oligomeric compound as claimed in claim 36, wherein the backbone of the oligomeric compound is modified.
56. Antisense oligomeric compound as claimed in claim 36, wherein the oligomeric compound is morpholino phosphorothioate, or morpholino phosphorodiamidate, or tricyclo DNA.
57. Method of modulating splicing of GAA pre-mRNA in a cell comprising contacting the cell with an antisense oligomeric compound as claimed in claim 36.
58. Method for treating Pompe disease in a patient comprising administering to the patient an effective amount of an antisense oligomeric compound according to claim 36.
59. Method to restore the function of GAA in a cell comprising the administration of the antisense oligomeric compound according to claim 36.
60. Method of correcting abnormal gene expression in a cell of a subject, the method comprising administering to the subject an antisense oligomeric compound according to claim 36.
61. Method of claim 60, wherein the cell is a muscular cell.
62. Method according to claim 57 wherein the cell comprises at least one mutation selected from the group c.-32-13T>G, c.-32-3C>G, c.547-6, c.1071, c.1254, and c.1552-30.
63. Method according to claim 58 wherein the patient comprises at least one mutation selected from the group c.-32-13T>G, c.-32-3C>G, c.547-6, c.1071, c.1254, and c.1552-30.
64. Method according to claim 59 wherein the cell comprises at least one mutation selected from the group c.-32-13T>G, c.-32-3C>G, c.547-6, c.1071, c.1254, and c.1552-30.
65. Method according to claim 60 wherein the cell comprises at least one mutation selected from the group c.-32-13T>G, c.-32-3C>G, c.547-6, c.1071, c.1254, and c.1552-30.
66. Method according to claim 58 wherein exon inclusion is accomplished.
67. Method according to claim 66, wherein inclusion of exon 2 is accomplished.
68. A pharmaceutical composition comprising at least one antisense oligomeric compound according to claim 1.
69. A pharmaceutical composition according to claim 68 wherein said composition further comprises a pharmaceutical acceptable excipient and/or a cell delivery agent.
Description:
[0001] The invention is related to a combination of enzymatic replacement
therapy (ERT) or gene therapy and antisense oligonucleotides for the
treatment of Pompe disease and to pharmaceutical compositions comprising
the antisense oligonucleotides and enzymes. The invention is also related
to a method to modulate the splicing of pre-mRNA of the GAA gene and to
treatment of Pompe disease.
BACKGROUND
[0002] Pompe disease also known as acid maltase deficiency or Glycogen storage disease type II is an autosomal recessive metabolic disorder which damages many cells throughout the body and in particular muscle cells and nerve cells, but also other cells throughout the body. The damage is caused by an accumulation of glycogen in the lysosome due to a deficiency of the lysosomal enzyme acid alpha-glucosidase. The build-up of glycogen and the consequences thereof affect various body tissues, particularly, skeletal muscles, heart muscles and smooth muscles of various organs and body parts such as blood vessels, gastrointestinal tract, uterus, bladder, the liver; and the central and perferal nervous system. As a consequence the clinical symptoms are broad. Progressive skeletal muscle weakness (myopathy) is a hallmark of the disease.
[0003] In Pompe disease, a protein, acid alpha-glucosidase (EC 3.2.1.20), also known as acid maltase, which is a lysosomal hydrolase, is defective. The protein is an enzyme that normally degrades the alpha-1,4 and alpha-1,6 linkages in glycogen, maltose and isomaltose and is required for the degradation of 1-3% of cellular glycogen. The deficiency of this enzyme results in the accumulation of structurally normal glycogen in lysosomes. The defective metabolism of glycogen in the lysosomes may also lead to (secondary) storage of glycogen in the cytoplasm and autophagic build-up. Excessive glycogen storage within lysosomes may interrupt normal functioning of other organelles and lead to cellular injury.
[0004] The defective alpha-glucosidase protein or reduced amount of alpha-glucosidase protein and activity is the result of mutations (or variations) with in the GAA gene. Some of these GAA mutations may lead to alternative splicing and thereby to absent or a reduced amount of alpha-glucosidase protein or activity. The GAA gene is located on long arm of chromosome 17 at 17q25.2-q25.3 (base pair 75,689,876 to 75,708,272). The gene spans approximately 20 kb and contains 20 exons with the first exon being noncoding.
[0005] Although over 460 GAA mutations have been described (http://cluster15.erasmusmc.nl/klgn/pompe/mutations.html), only a few splicing mutations have been characterized. Severe mutations that completely abrogate GAA enzyme activity cause the classic infantile form of Pompe disease with onset of symptoms shortly after birth, hypertrophic cardiomyopathy, general skeletal muscle weakness, and respiratory failure and death within the first 1.5 years of life if left untreated. Milder mutations leave partial GAA enzyme activity and result in a milder phenotype with onset of symptoms varying from childhood to adulthood. In general, a higher residual alpha-glucosidase activity in primary fibroblasts is associated with later onset of Pompe disease.
[0006] Enzyme replacement therapy (ERT) has been developed for Pompe disease, in which recombinant human GAA protein is administered intravenously every two weeks. This treatment is aimed to increase the intracellular level of alpha-glucosidase activity in affected cells and tissues and thereby reduce or prevent glycogen storage and eventually symptoms of the disease. The treatment can rescue the lives of classic infantile patients and delay disease progression of later onset patients, but the effects are heterogeneous.
[0007] Pompe disease is an autosomal recessive inheritable disorder. One of the most common mutation in Pompe disease is the IVS1 mutation, c.-32-13T>G, a transversion (T to G) mutation and occurs among infants, children, juveniles and adults with this disorder. This mutation interrupts a site of RNA splicing.
[0008] Antisense oligonucleotides (antisense oligomeric compounds) are currently being tested in clinical trials for their ability to modulate splicing. A classical example is Duchenne muscular dystrophy. In this disease, mutation hotspots are present in certain exons. Using antisense oligomeric compounds, the mutated exon is skipped and the mutation is bypassed. This results in a slightly shorter protein that is still partial functional. It is straightforward to induce exon skipping using antisense oligomeric compounds, because it is evident that the antisense oligomeric compound must be targeted to the relevant splice site. Also in Epidermolysis bullosa (WO2013053819) and in Leber congenital amaurosis symptoms (WO2012168435) antisense oligonucleotides are used for exon skipping.
[0009] For the IVS1 mutation in Pompe, such a strategy does not work. The IVS mutation causes a skipping of exon 2 resulting in the deletion of the canonical translation start side and leads to non-sense mediated decay and thus no protein is transcribed. For antisense therapy to work for the IVS1 mutation in Pompe disease, it needs to induce exon inclusion. However, it is very difficult to induce exon inclusion, because it relies on targeting a splicing repressor sequence, which cannot be reliably predicted. For the IVS1 mutation, an antisense oligomeric compound that blocks a splicing repressor sequence may promote exon 2 inclusion in the presence of the IVS1 mutation. It is known that such repressor sequences may be present anywhere in the gene, either in an exon (termed exonic splicing silencer or ESS) or in an intron (termed intronic splicing silencer or ISS) and maybe close to the mutation or far away or maybe close to the affected splice site or far away from it.
[0010] Although a number of antisense compounds that are capable of modulating splicing of a target gene in vitro have been reported, there remains a need to identify compounds that may modulate the splicing of the GAA gene.
[0011] Enzyme replacement therapy (ERT) with acid alpha-glucosidase (GAA), has been used for infantile, childhood and adult Pompe patients also called classic infantile or infantile onset and late onset forms. The ERT modifies the natural course of the disease, however targeting of the main target tissues and cells is a challenge. For example 15-40% of the body is composed of skeletal muscle and to be corrective each individual cell in the body needs to reached. The enzyme needs to be taken up by cells via endocytosis, which seems most efficient when it is targeted to receptors on the cell surface such as the mannose 6-phosphate/IGF II receptor. This mannose 6-phosphate/IGF II receptor recognizes various ligands such as mannose 6-phosphate, IGF II and Gluc-NAC. Thus ERT with these ligands show a better uptake. The current registered ERT is targeted at the M6P part of the M6P/IGF II receptor, but there is also ERT underdevelopment with an increased amount of M6P ligands or with IGF II linked to it. Another problem with ERT is that some patients develop antibodies to the administered GAA enzyme reducing the effect of ERT and these patients respond poorly to the treatment. In addition, ERT requires purified recombinant human GAA which is difficult to make and is expensive. Furthermore, recombinant human GAA is has a relative short half life ranging and therefore must be administered intravenously every 2 weeks (or every week), which is cumbersome for patients.
[0012] It is therefore an object of the invention to provide an improved treatment for Pompe Disease. Another object of the invention is to provide an improved ERT treatment of Pompe Disease. Another object of the invention is to provide an antisense compound that is capable of targeting exonic splicing silencer (ESS) or in an intronic splicing silencer (ISS). Yet another object of the invention is to provide a antisense compound that is capable of targeting the IVS-1 mutation. It is further an object of the invention to improve the enzyme replacement therapy of GAA enzyme in patients. The present invention meets one or more of the objects.
[0013] The present invention combines two strategies which are different. ERT or gene therapy enhances the activity of glycogen breakdown administration of administration of a foreign GAA enzyme, whereas antisense therapy improves or enhances the intracellular production of the patients own GAA enzyme.
SUMMARY OF THE INVENTION
[0014] The present invention is directed to a composition for use for the treatment of Pompe disease, said composition comprising an enzyme or nucleic acid encoding for said enzyme suitable for Enzyme Replacement Therapy for Pompe disease, wherein said treatment is a combination of the administration of said enzyme or said nucleic acid encoding for said enzyme and the administration of an antisense oligomeric that modulates the splicing of acid alpha-glucosidase (GAA) enzyme.
[0015] The present invention is directed to a treatment of Pompe disease by administration of an enzyme or nucleic acid encoding for said enzyme suitable for Enzyme Replacement Therapy for Pompe disease in combination with the administration of an antisense oligomeric compound that modulates the splicing of acid alpha-glucosidase (GAA) enzyme gene.
[0016] Optionally the enzyme suitable for Enzyme Replacement Therapy for Pompe disease is an enzyme that breaks down glycogen such as acid alpha glycosidase (GAA). The nucleic acid encoding for said enzyme suitable for Enzyme Replacement Therapy for Pompe disease may be used in gene therapy. Optionally the nucleic acid is in a vector or other means that enables the translation of the enzyme. Optionally the modulation of the splicing is to increase the activity of glycogen break-down. Optionally the modulation of the splicing is to increase the activity of acid alpha-glucosidase (GAA) enzyme gene. Optionally the modulation of the splicing is to increase the activity of GAA to at least 120% of the activity of GAA enzyme without the modulation of the splicing of the GAA gene. Optionally the modulation of the splicing is to increase the activity of GAA to at least 25%% of the activity of a wild type GAA enzyme.
[0017] Optionally the antisense oligomeric compound modulates adherent splicing of acid alpha-glucosidase (GAA) enzyme gene.
[0018] Optionally the antisense oligomeric compound modulates splicing by an activity selected from the group consisting of promotion of exon inclusion, inhibition of a cryptic splicing site, inhibition of intron inclusion, recovering of reading frame, inhibition of splicing silencer sequence, activation of spicing enhancer sequence or any combination thereof.
[0019] Optionally the antisense oligomeric compound modulates splicing by promotion of exon inclusion, optionally exon 2, or exon 6.
[0020] Optionally the antisense oligomeric compound modulates splicing by inhibition of a cryptic splicing site.
[0021] Optionally the antisense oligomeric compound modulates splicing by inhibition of intron inclusion.
[0022] Optionally the antisense oligomeric compound modulates splicing by recovering of the reading frame.
[0023] Optionally the antisense oligomeric compound modulates splicing by inhibition of splicing silencer sequence.
[0024] Optionally the antisense oligomeric compound modulates splicing by activation of spicing enhancer sequence.
[0025] Optionally the antisense oligomeric compound targets a nucleic acid sequence of the GAA gene selected from the group consisting of SEQ ID NO: 1, 37-40, 1584-1589 and nucleotide polymorphism of SEQ ID NO: 1, 37-40, 1584-1589.
[0026] Optionally said enzyme or said nucleic acid encoding for said enzyme and the antisense oligomeric compound is administered simultaneously or separately. Optionally said nucleic acid encoding for said enzyme and said antisense oligomeric compound are administered simultaneously or in one treatment composition. Optionally said enzyme and said antisense oligomeric compound are administered simultaneously or in one treatment composition. Optionally said nucleic acid encoding for said enzyme and said antisense oligomeric compound are administered on separate occasions or in separate treatment compositions. Optionally said enzyme and said antisense oligomeric compound are administered on separate occasions or in separate treatment compositions. Optionally the treatment uses said enzyme and said nucleic acid encoding for said enzyme. Optionally said enzyme and said nucleic acid encoding for said enzyme are administered simultaneously or in one treatment composition. Optionally the treatment uses said enzyme and said nucleic acid encoding for said enzyme. Optionally said enzyme and said nucleic acid encoding for said enzyme are administered on separate occasions or in separate treatment compositions. Optionally said enzyme and said nucleic acid encoding for said enzyme and said antisense oligomeric compound are administered simultaneously or in one treatment composition.
[0027] Optionally the administration route is selected from the group consisting of oral, parenteral, intravenous, intra-arterial, subcutaneous, intraperitoneal, ophthalmic, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intradermal, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal, transdermal, or via inhalation, or combinations thereof. Optionally the administration route for the enzyme or the nucleic acid encoding for said enzyme is intravenous. Optionally the administration route of said enzyme or said nucleic acid encoding for said enzyme and the administration route of said antisense oligomeric compound are the same or different. Optionally the administration route for said antisense oligomeric compound is intravenous. Optionally the administration route for said antisense oligomeric compound is orally. Optionally the administration route for the enzyme or the nucleic acid encoding for said enzyme is orally. It is explicitly envisioned to combine various administration routes in the present invention.
[0028] Optionally said enzyme or said nucleic acid encoding for said enzyme is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days. Optionally said enzyme or said nucleic acid encoding for said enzyme is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weeks. Optionally said enzyme or said nucleic acid encoding for said enzyme is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 months. Optionally said enzyme or said nucleic acid encoding for said enzyme is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, years. It is explicitly envisioned that various frequencies of administration as indicated here are combined. For example 8 weeks of administration once every week and thereafter 24 weeks of administration of once every 2 weeks.
[0029] Optionally said antisense oligomeric compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days. Optionally said antisense oligomeric compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weeks. Optionally said antisense oligomeric compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 months. Optionally said antisense oligomeric compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, years. It is explicitly envisioned that various frequencies of administration as indicated here are combined. For example 8 weeks of administration once every week and thereafter 24 weeks of administration of once every 2 weeks. 10. Also various combinations of the frequencies of administration of the antisense oligomeric compound in combination with various combination of the frequencies of administration of the said enzyme or said nucleic acid encoding for said enzyme are explicitly envisioned in the present invention. For example said enzyme is administered once every two weeks and the antisense oligomeric compound is administered once every 4 weeks.
[0030] Optionally said enzyme or said nucleic acid encoding for said enzyme is administered in a dose of about 1-100 mg/kg, optionally 2-90 mg/kg, 3-80 mg/kg, 5-75 mg/kg, 7-70 mg/kg, 10-60 mg/kg, 12-55 mg/kg, 15-50 mg/kg, 17-45 mg/kg, 20-40 mg/kg, 22-35 mg/kg, 25-30 mg/kg.
[0031] Optionally said antisense oligomeric compound is administered in a dose of about 0.05 to 1000 mg/kg, optionally about 0.1 to 900 mg/kg, 1-800 mg/kg, 2-750 mg/kg, 3-700 mg/kg, 4-600 mg/kg, 5-500 mg/kg, 7 to 450 mg/kg, 10 to 400 mg/kg, 12 to 350 mg/kg, 15 to 300 mg/kg, 17 to 250 mg/kg, 20 to 220 mg/kg, 22 to 200 mg/kg, 25 to 180 mg/kg, 30 to 150 mg/kg, 35 to 125 mg/kg, 40 to 100 mg/kg, 45 to 75 mg/kg, 50-70 mg/kg.
[0032] Optionally said enzyme or said nucleic acid encoding for said enzyme or said antisense oligomeric compound is administered in combination with a chaperone such as an Active Site-Specific Chaperone (ASSC). Optionally said enzyme is administered in combination with a chaperone Optionally said nucleic acid encoding for said enzyme is administered in combination with a chaperone Optionally said antisense oligomeric compound is administered in combination with a chaperone Suitable chaperones are 1-deoxynojirimycin and derivatives thereof. Suitable examples of chaperones are 1-deoxynojirimycin N-(n-nonyl) deoxynojirimycin (NN-DNJ), N-(n-butyl) deoxynojirimycin (NB-DNJ), N-octyl-4-epi-B-valienamine, N-acetylglucosamine-thiazoline, N-(7-oxadecyl)deoxynojirimycin (NO-DNJ) and N-(n-dodecyl)deoxynojirimycin (ND-DNJ), 1-deoxygalactonojirimycin, N-alkylderivative of 1-deoxynojirimycin. 1-deoxynojirimycin and derivatives thereof are also suitable for substrate reduction.
[0033] Optionally the administration is in combination with genistein. Optionally in a dose of genistein of 1-100 mg/kg per day, optionally of 5-90 mg/kg per day, optionally 10-80-mg/kg per clay, optionally 15-75 mg/kg per day, optionally 20-70 mg/kg per day, optionally 25-60 mg/kg per day, 30-55 mg/kg per day, 35-50 mg/kg per day, 40-45 mg/kg per day. Optionally said enzyme or said nucleic acid encoding for said enzyme or said antisense oligomeric compound is administered in combination with a genistein. Optionally said enzyme is administered in combination with a genistein Optionally said nucleic acid encoding for said enzyme is administered in combination with a genistein Optionally said antisense oligomeric compound is administered in combination with a genistein.
[0034] Optionally the administration is in combination with cell penetrating peptides. Optionally the administration is in combination with a targeting ligand. Optionally said cell penetrating peptide and/or targeting ligand is present on the antisense oligomeric compound. Optionally said cell penetrating peptide and/or targeting ligand is present on said nucleic acid encoding for said enzyme. Optionally said cell penetrating peptide and/or targeting ligand is present on said enzyme.
[0035] Optionally the enzyme is an acid alpha-glucosidase (GAA) enzyme. Optionally said enzyme is a modification, variant, analogue, fragment, portion, or functional derivative, thereof. Optionally said enzyme is a modification, variant, analogue, fragment, portion, or functional derivative of GAA enzyme. The present invention explicitly encompasses all forms of recombinant human acid alpha-glucosidase which may be based on all natural or genetically modified forms of either human GAA cDNA, or human GAA gene, or combinations thereof, including those forms that are created by codon optimization. Suitable GAA enzyme include an enzyme selected from the group consisting of Myozyme and lysozyme, neo-GAA (carbohydrate modified forms of alglucosidase-alpha), BMN-701 (BioMarin: Gilt GAA for Pompe disease, in which rhGAA is fused with an IGF-II peptide) rhGGAA (Oxyrane: recombinant human acid alpha-glucosidase produced in genetically modified yeast cells and enriched in mannose 6-phosphate content), rhGAA modified by conjugation, for example to mannose-6-phosphate groups or to IGF-II peptides. said enzyme is selected from the group consisting of a recombinant human GAA, Myozyme, Lumizyme, neoGAA, Gilt GAA (BMN-701), or rhGGAA.
[0036] Optionally the composition or treatment comprises more than one antisense oligomeric compound. Optionally, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more different antisense oligomeric compounds are used for the composition and/or treatment.
[0037] Optionally the antisense oligomeric compound is selected from the group comprising SEQ ID NO: 2-33, 541-1583, 1590-1594, and sequences having at least 80% identity thereof. Optionally the antisense oligomeric compound is selected from the group comprising SEQ ID NO: 2-33 and sequences having at least 80% identity thereof. Optionally the antisense oligomeric compound is SEQ ID NO: 12 or SEQ ID NO: 33 and sequences having at least 80% identity thereof.
[0038] Optionally the antisense oligomeric compound is complementary to a sequence selected from the group comprising SEQ ID NO: 1, 37-40, 1584-1589, and sequences having at least 80% identity thereof.
[0039] Optionally at least one of the nucleotides of the is antisense oligomeric compound is modified. Optionally all of the nucleotides in the antisense oligomeric compound are modified. Optionally the modifications in the antisense oligomeric compound is the same for each nucleotide or different. Various combinations of modification of the nucleotides is explicitly envisioned in the present invention.
[0040] Optionally the sugar of one or more nucleotides of the is antisense oligomeric compound is modified. Optionally the sugar modification is 2'-O-methyl. Optionally the sugar modification2'-O-methoxyethyl.
[0041] Optionally the base of one or more nucleotides of the antisense oligomeric compound is modified.
[0042] Optionally the backbone of the antisense oligomeric compound is modified, Optionally the backbone of the antisense oligomeric compound is a morpholino phosphorothioate. Optionally the backbone of the antisense oligomeric compound is a morpholino phosphorodiamidate. Optionally the backbone of the antisense oligomeric compound is a tricyclo-DNA.
[0043] Optionally the antisense oligomeric compound and/or the enzyme or nucleic acid coding for said enzyme is present in a carrier selected from the group of exosomes, nanoparticles, micelles, liposomes, or microparticles. The carrier may enhance the uptake of the antisense oligomeric compound and/or the enzyme or nucleic acid coding for said enzyme into the cells. Optionally the composition or the treatment comprises compounds that enhance the uptake of the antisense oligomeric compound and/or the enzyme or nucleic acid coding for said enzyme into the cells. Suitable compounds that enhance the uptake into the cells are Polyethylimine, conjugated pluronic copolymers, lipids, e.g. patisiran, ICAM-targeted nanocarriers, peptide Pip6a or cationic nanoemulsions. A skilled person is well suited to find compounds that enhance the uptake of the antisense oligomeric compound and/or the enzyme or nucleic acid coding for said enzyme into the cells.
[0044] The present invention is also directed to a pharmaceutical composition comprising at least one antisense oligomeric compound as defined in aspects of the present invention and/or embodiments thereof and a enzyme as defined in aspects of the present invention and/or embodiments thereof.
[0045] Optionally the pharmaceutical composition further comprises a pharmaceutical acceptable excipient and/or a cell delivery agent. Suitable cell delivery agents are carriers selected from the group of exosomes, nanoparticles, micelles, liposomes, or microparticles. Optionally the pharmaceutical composition comprises compounds that enhance the uptake of the antisense oligomeric compound and/or the enzyme or nucleic acid coding for said enzyme into the cells. Suitable compounds that enhance the uptake into the cells are Polyethylimine, conjugated pluronic copolymers, lipids, e.g. patisiran, ICAM-targeted nanocarriers. peptide Pip6a or cationic nanoemulsions. A skilled person is well suited to find compounds that enhance the uptake of the antisense oligomeric compound and/or the enzyme or nucleic acid coding for said enzyme into the cells.
[0046] Optionally the pharmaceutical composition further comprises a chaperone such as a Active Site-Specific Chaperone (ASSC). Suitable chaperones are 1-deoxynojirimycin and derivatives thereof. Suitable examples of chaperones are 1-deoxynojirimycin N-(n-nonyl)deoxynojirimycin (NN-DNJ), N-(n-butyl)deoxynojirimycin (NB-DNJ), N-octyl-4-epi-.beta.-valienamine, N-acetylglucosamine-thiazoline, N-(7-oxadecyl)deoxynojirimycin (NO-DNJ) and N-(n-dodecyl)deoxynojirimycin (ND-DNJ), 1-deoxygalactonojirimycin, N-alkylderivative of 1-deoxynojirimycin. 1-deoxynojirimycin and derivatives thereof are also suitable for substrate reduction.
[0047] Optionally the pharmaceutical composition further comprises genistein. Optionally the pharmaceutical composition further comprises genistein in a dose of 1-100 mg/kg per clay, optionally of 5-90 mg/kg per day, optionally 10-80-mg/kg per day, optionally 15-75 mg/kg per day, optionally 20-70 mg/kg per clay, optionally 25-60 mg/kg per clay, 30-55 mg/kg per clay, 35-50 mg/kg per day, 40-45 mg/kg per day. Optionally the pharmaceutical composition comprises said enzyme or said nucleic acid encoding for said enzyme or said antisense oligomeric compound in combination with a genistein. Optionally the pharmaceutical composition comprises said enzyme in combination with a genistein Optionally the pharmaceutical composition comprises said nucleic acid encoding for said enzyme in combination with a genistein Optionally the pharmaceutical composition comprises said antisense oligomeric compound in combination with a genistein.
[0048] Optionally the pharmaceutical composition further comprises cell penetrating peptides. Optionally the pharmaceutical composition further comprises a targeting ligand. Optionally said cell penetrating peptide and/or targeting ligand is present on the antisense oligomeric compound. Optionally said cell penetrating peptide and/or targeting ligand is present on said nucleic acid encoding for said enzyme. Optionally said cell penetrating peptide and/or targeting ligand is present on said enzyme.
[0049] Optionally in the pharmaceutical composition the enzyme is an acid alpha-glucosidase (GAA) enzyme. Optionally said enzyme is a modification, variant, analogue, fragment, portion, or functional derivative, thereof. Optionally said enzyme is a modification, variant, analogue, fragment, portion, or functional derivative of GAA enzyme. The present invention explicitly encompasses all forms of recombinant human acid alpha-glucosidase which may be based on all natural or genetically modified forms of either human GAA cDNA, or human GAA gene, or combinations thereof, including those forms that are created by codon optimization. Suitable GAA enzyme include an enzyme selected from the group consisting of Myozyme and lysozyme, neo-GAA (carbohydrate modified forms of alglucosidase-alpha), BMN-701 (BioMarin: Gilt GAA for Pompe disease, in which rhGAA is fused with an IGF-II peptide) rhGGAA (Oxyrane: recombinant human acid alpha-glucosidase produced in genetically modified yeast cells and enriched in mannose 6-phosphate content), rhGAA modified by conjugation, for example to mannose-6-phosphate groups or to IGF-II peptides. said enzyme is selected from the group consisting of a recombinant human GAA, Myozyme, Lumizyme, neoGAA, Gilt GAA (BMN-701), or rhGGAA.
[0050] Optionally the pharmaceutical composition comprises the enzyme in an amount of about 1-50 mg/mL enzyme. Optionally the enzyme is present in the pharmaceutical composition in an amount of 2-45 mg/mL, 3-40 mg/mL, 5-35 mg/mL, 7-30 mg/mL, 10-25 mg/mL, 12-22 mg/mL, or 15-20, mg/mL.
[0051] Optionally the pharmaceutical composition comprises more than one antisense oligomeric compound. Optionally, the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more different antisense oligomeric compounds.
[0052] Optionally the pharmaceutical composition comprises the antisense oligomeric compound in an amount of about 1-50 mg/mL enzyme. Optionally the antisense oligomeric compound is present in the pharmaceutical composition in an amount of 2-45 mg/mL, 3-40 mg/mL, 5-35 mg/mL, 7-30 mg/mL, 10-25 mg/mL, 12-22 mg/mL, or 15-20, mg/mL.
[0053] Optionally the pharmaceutical composition comprises a carrier selected from the group consisting of exosomes, nanoparticles, micelles, liposomes, and microparticles.
[0054] The present invention is also directed to a sequences selected from the group comprising SEQ ID NO: 1590-1594 and sequences having at least 80% identity thereof.
[0055] The present invention is also directed to a sequences selected from the group comprising SEQ ID NO: 1590-1594 and sequences having at least 80% identity thereof for use in the treatment Pompe disease.
[0056] The present invention is also directed to a method of modulating splicing of GAA pre-mRNA in a cell comprising:
[0057] contacting the cell with an antisense oligomeric compound selected from the group comprising SEQ ID NO: 1590-1594 and sequences having at least 80% identity thereof.
[0058] The present invention is also directed to a method for treating Pompe disease in a patient comprising administering said patient with an effective amount of an antisense oligomeric compound selected from the group comprising SEQ ID NO: 1590-1594 and sequences having at least 80% identity thereof.
[0059] The present invention is also directed to a method to restore the function of GAA in a cell wherein said method comprises the administration of an antisense oligomeric compound selected from the group comprising SEQ ID NO: 1590-1594 and sequences having at least 80% identity thereof. The present invention is also directed to a method of correcting abnormal gene expression in a cell, Optionally a muscular cell, of a subject, the method comprising administering to the subject an antisense oligomeric compound selected from the group comprising SEQ ID NO: 1590-1594 and sequences having at least 80% identity thereof. Optionally in said methods the cell or the patient comprises at least one mutation selected from the group c.-32-13T>G, c.-32-3C>G, c.547-6, c.1071, c.1254, and c.1552-30, Optionally the cell or patient comprises mutation c.-32-3C>G or c.-32-13T>G. Optionally in said methods exon inclusion is accomplished, optionally inclusion of exon 2.
[0060] The present invention is also directed to a pharmaceutical composition comprising at least one antisense oligomeric compound selected from the group comprising SEQ ID NO: 1590-1594 and sequences having at least 80% identity thereof. Optionally the pharmaceutical composition further comprises a pharmaceutical acceptable excipient and/or a cell delivery agent.
DETAILED DESCRIPTION
[0061] The principle behind antisense technology is that an antisense compound, which hybridizes to a target nucleic acid, modulates gene expression activities such as transcription, splicing or translation. This sequence specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes or gene products involved in disease.
[0062] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence, resulting in exon-exon junctions at the site where exons are joined. Targeting exon-exon junctions can be useful in situations where aberrant levels of a normal splice product are implicated in disease, or where aberrant levels of an aberrant splice product are implicated in disease. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions can also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also suitable targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts" and are also suitable targets. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA. Single-stranded antisense compounds such as oligonucleotide compounds that work via an RNase H mechanism are effective for targeting pre-mRNA. Antisense compounds that function via an occupancy-based mechanism are effective for redirecting splicing as they do not, for example, elicit RNase H cleavage of the mRNA, but rather leave the mRNA intact and promote the yield of desired splice product(s).
[0063] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants." More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants." Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants." If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.
[0064] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.
[0065] As used herein, "antisense mechanisms" are all those involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
[0066] As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of" meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
[0067] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
[0068] The terms "individual", "patient", and "subject" are used interchangeably herein and refer to mammals, in particular primates and Optionally humans.
[0069] The term "exon" refers to a portion of a gene that is present in the mature form of mRNA. Exons include the ORF (open reading frame), i.e., the sequence which encodes protein, as well as the 5' and 3' UTRs (untranslated regions). The UTRs are important for translation of the protein. Algorithms and computer programs are available for predicting exons in DNA sequences (Grail, Grail 2 and Genscan and US 20040219522 for determining an exon-intron junctions).
[0070] As used herein, the term "protein coding exon" refers to an exon which codes (or at least partially codes) for a protein (or part of a protein). The first protein coding exon in an mRNA is the exon which contains the start codon. The last protein encoding exon in an mRNA is the exon which contains the stop codon. The start and stop codons can be predicted using any number of well-known programs in the art.
[0071] As used herein, the term "internal exon" refers to an exon that is flanked on both its 5' and 3' end by another exon. For an mRNA comprising n exons, exon 2 to exon (n-1) are the internal exons. The first and last exons of an mRNA are referred to herein as "external exons".
[0072] The term "intron" refers to a portion of a gene that is not translated into protein and while present in genomic DNA and pre-mRNA, it is removed in the formation of mature mRNA.
[0073] The term "messenger RNA" or "mRNA" refers to RNA that is transcribed from genomic DNA and that carries the coding sequence for protein synthesis. Pre-mRNA (precursor mRNA) is transcribed from genomic DNA. In eukaryotes, pre-mRNA is processed into mRNA, which includes removal of the introns, i.e., "splicing", and modifications to the 5' and 3' end (e.g., polyadenylation). mRNA typically comprises from 5' to 3'; a 5'cap (modified guanine nucleotide), 5' UTR (untranslated region), the coding sequence (beginning with a start codon and ending with a stop codon), the 3' UTR, and the poly(A) tail.
[0074] The term "nucleic acid sequence" or "nucleic acid molecule" or polynucleotide are used interchangeably and refer to a DNA or RNA molecule in single or double stranded form. An "isolated nucleic acid sequence" refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a cell.
[0075] A "mutation" in a nucleic acid molecule is a change of one or more nucleotides compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more nucleotides. A "point mutation" is the replacement of a single nucleotide, or the insertion or deletion of a single nucleotide.
[0076] Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as "substantially identical" or "essentially similar" when they are optimally aligned by for example the programs GAP or BESTFIT or the Emboss program "Needle" (using default parameters, see below) share at least a certain minimal percentage of sequence identity (as defined further below). These programs use the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximising the number of matches and minimises the number of gaps. Generally, the default parameters are used, with a gap creation penalty=10 and gap extension penalty=0.5 (both for nucleotide and protein alignments). For nucleotides the default scoring matrix used is DNAFULL and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignments and scores for percentage sequence identity may for example be determined using computer programs, such as EMBOSS (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). Alternatively sequence similarity or identity may be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences have "substantial sequence identity" if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, Optionally 90%, 95%, 98%, 99% or more (as determined by Emboss "needle" using default parameters, i.e. gap creation penalty=10, gap extension penalty=0.5, using scoring matrix DNAFULL for nucleic acids an Blosum62 for proteins). Such sequences are also referred to as `variants` herein, e.g. other variants of antisense oligomeric compounds. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3'- and/or 5'-side are 100% identical.
[0077] The term "hybridisation" as used herein is generally used to mean hybridisation of nucleic acids at appropriate conditions of stringency as would be readily evident to those skilled in the art depending upon the nature of the probe sequence and target sequences. Conditions of hybridisation and washing are well known in the art, and the adjustment of conditions depending upon the desired stringency by varying incubation time, temperature anchor ionic strength of the solution are readily accomplished. See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989. The choice of conditions is dictated by the length of the sequences being hybridised, in particular, the length of the probe sequence, the relative G-C content of the nucleic acids and the amount of mismatches to be permitted. Low stringency conditions are preferred when partial hybridisation between strands that have lesser degrees of complementarity is desired. When perfect or near perfect complementarity is desired, high stringency conditions are preferred. For typical high stringency conditions, the hybridisation solution contains 6.times.S.S.C., 0.01 M EDTA, 1.times.Denhardt's solution and 0.5% SOS. hybridisation is carried out at about 68.degree. C. for about 3 to 4 hours for fragments of cloned DNA and for about 12 to about 16 hours for total eukaryotic DNA. For lower stringencies the temperature of hybridisation is reduced to about 42.degree. C. below the melting temperature (TM) of the duplex. The TM is known to be a function of the G-C content and duplex length as well as the ionic strength of the solution.
[0078] The term "allele(s)" means any of one or more alternative forms of a gene at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. One allele is present on each chromosome of the pair of homologous chromosomes. These may be identical alleles of the gene (homozygous) or two different alleles (heterozygous).
[0079] Mutant allele" refers herein to an allele comprising one or more mutations in the coding sequence (mRNA, cDNA or genomic sequence) compared to the wild type allele. Such mutation(s) (e.g. insertion, inversion, deletion and/or replacement of one or more nucleotide(s)) may lead to the encoded protein having reduced in vitro and/or in vivo functionality (reduced function) or no in vitro and/or in vivo functionality (loss-of-function), e.g. due to the protein e.g. being truncated or having an amino acid sequence wherein one or more amino acids are deleted, inserted or replaced. Such changes may lead to the protein having a different conformation, being targeted to a different sub-cellular compartment, having a modified catalytic domain, having a modified binding activity to nucleic acids or proteins, etc, it may also lead to a different splicing event.
[0080] A "fragment" of the gene or nucleotide sequence or antisense oligomeric compound refers to any subset of the molecule, e.g., a shorter polynucleotide or oligonucleotide.
[0081] A "variant" refers to a molecule substantially similar to the antisense oligomeric compound or a fragment thereof, such as a nucleotide substitution variant having one or more substituted nucleotides, but which maintains the ability to hybridize with the particular gene. Optionally the variant comprises the mutations as identified by the invention. Variants also include longer sequences.
[0082] An "analogue" refers to a non-natural molecule substantially similar to or functioning in relation to either the entire molecule, a variant or a fragment thereof.
[0083] As used herein, the terms "precursor mRNA" or "pre-mRNA" refer to an immature single strand of messenger ribonucleic acid (mRNA) that contains one or more intervening sequence(s) (introns). Pre-mRNA is transcribed by an RNA polymerase from a DNA template in the cell nucleus and is comprised of alternating sequences of introns and coding regions (exons). Once a pre-mRNA has been completely processed by the splicing out of introns and joining of exons, it is referred to as "messenger RNA" or "mRNA," which is an RNA that is comprised exclusively of exons. Eukaryotic pre-mRNAs exist only transiently before being fully processed into mRNA. When a pre-mRNA has been properly processed to an mRNA sequence, it is exported out of the nucleus and eventually translated into a protein by ribosomes in the cytoplasm.
[0084] As used herein, the terms "splicing" and "processing" refers to the modification of a pre-mRNA following transcription, in which introns are removed and exons are joined. Pre-mRNA splicing involves two sequential biochemical reactions. Both reactions involve the spliceosomal transesterification between RNA nucleotides. In a first reaction, the 2'-OH of a specific branch-point nucleotide within an intron, which is defined during spliceosome assembly, performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site forming a lariat intermediate. In a second reaction, the 3'-OH of the released 5' exon performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat. Pre-mRNA splicing is regulated by intronic silencer sequence (ISS), exonic silencer sequences (ESS) and terminal stem loop (TSL) sequences.
[0085] As used herein, the terms "intronic silencer sequences (ISS)" and "exonic silencer sequences (TSL)" refer to sequence elements within introns and exons, respectively, that control alternative splicing by the binding of trans-acting protein factors within a pre-mRNA thereby resulting in differential use of splice sites. Typically, intronic silencer sequences are less conserved than the splice sites at exon-intron junctions.
[0086] As used herein, "modulation of splicing" refers to altering the processing of a pre-mRNA transcript such that there is an increase or decrease of one or more splice products, or a change in the ratio of two or more splice products. Modulation of splicing can also refer to altering the processing of a pre-mRNA transcript such that a spliced mRNA molecule contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion in one or more exons, or additional sequence not normally found in the spliced mRNA (e.g., intron sequence).
[0087] As used herein, "splice site" refers to the junction between an exon and an intron in a pre-mRNA (unspliced RNA) molecule (also known as a "splice junction"). A "cryptic splice site" is a splice site that is not typically used but may be used when the usual splice site is blocked or unavailable or when a mutation causes a normally dormant site to become an active splice site. An "aberrant splice site" is a splice site that results from a mutation in the native DNA and pre-mRNA.
[0088] As used herein, "splice products" or "splicing products" are the mature mRNA molecules generated from the process of splicing a pre-mRNA. Alternatively spliced pre-mRNAs have at least two different splice products. For example, a first splicing product may contain an additional exon, or portion of an exon, relative to a second splicing product. Splice products of a selected pre-mRNA can be identified by a variety of different techniques well known to those of skill in the art.
[0089] As used herein "splice donor site" refers to a splice site found at the 5' end of an intron, or alternatively, the 3' end of an exon. Splice donor site is used interchangeably with "5' splice site." As used herein "splice acceptor site" refers to a splice site found at the 3' end of an intron, or alternatively, the 5' end of an exon. Splice acceptor site is used interchangeably with "3' splice site."
[0090] As used herein, "targeting" or "targeted to" refer to the process of designing an oligomeric compound such that the compound hybridizes with a selected nucleic acid molecule or region of a nucleic acid molecule. Targeting an oligomeric compound to a particular target nucleic acid molecule can be a multistep process. The process usually begins with the identification of a target nucleic acid whose expression is to be modulated. As used herein, the terms "target nucleic acid" and "nucleic acid encoding GAA" encompass DNA encoding GAA, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. As disclosed herein, the target nucleic acid encodes GAA.
[0091] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result.
[0092] As used herein, "target mRNA" refers to the nucleic acid molecule to which the oligomeric compounds provided herein are designed to hybridize. In the context of the present disclosure, target mRNA is usually unspliced mRNA, or pre-mRNA. In the context of the present invention, the target mRNA is GAA mRNA or GAA pre-mRNA.
[0093] "Region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Target regions may include, for example, a particular exon or intron, or may include only selected nucleotides within an exon or intron which are identified as appropriate target regions. Target regions may also be splicing repressor sites. Within regions of target nucleic acids are segments. "Segments" are defined as smaller or sub-portions of regions within a target nucleic acid. "Sites," as used in the present invention, are defined as unique nucleobase positions within a target nucleic acid. As used herein, the "target site" of an oligomeric compound is the 5'-most nucleotide of the target nucleic acid to which the compound binds.
[0094] Target degradation can include an RNase H, which is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit cleavage by RNAse H. Occupancy-based antisense mechanisms, whereby antisense compounds hybridize yet do not elicit cleavage of the target, include inhibition of translation, modulation of splicing, modulation of poly(A) site selection and disruption of regulatory RNA structure. For the present invention "RNA-like" antisense compounds for use in occupancy-based antisense mechanisms are preferred.
[0095] In the context of the present disclosure, an oligomeric compound "targeted to a splice site" refers to a compound that hybridizes with at least a portion of a region of nucleic acid encoding a splice site or a compound that hybridizes with an intron or exon in proximity to a splice site, such that splicing of the mRNA is modulated.
[0096] The term "oligomeric compound" refers to a polymeric structure capable of hybridizing to a region of a nucleic acid molecule. This term includes oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and chimeric combinations of these. Oligomeric compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular. Moreover, branched structures are known in the art. Oligomeric compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.
[0097] The term "antisense oligonucleotide, AON, or antisense oligomeric compound" refers to an oligonucleotide that is capable of interacting with and/or hybridizing to a pre-mRNA or an mRNA having a complementary nucleotide sequence thereby modifying gene expression and/or splicing. Enzyme-dependent antisense oligonucleotides include forms that are dependent on RNase H activity to degrade target mRNA, and include single-stranded DNA, RNA, and phosphorothioate antisense. Steric blocking antisense oligonucleotides (RNase-H independent antisense) interfere with gene expression or other mRNA-dependent cellular processes by binding to a target sequence of mRNA. Steric blocking antisense includes 2'-0 alkyl antisense oligonucleotides, Morpholino antisense oligonucleotides, and tricyclo-DNA antisense oligonucleotides. Steric blocking antisense oligonucleotides are preferred in the present invention.
[0098] As used herein, antisense oligonucleotides that are "RNase H-independent" are those compounds which do not elicit cleavage by RNase H when hybridized to a target nucleic acid. RNase H-independent oligomeric compounds modulate gene expression, such as splicing, by a target occupancy-based mechanism. Rnase H-independent antisense oligonucleotides are preferred in the present invention.
[0099] As used herein, "hybridization" means the pairing of complementary strands of oligomeric compounds. In the context of the present disclosure, an oligomeric compound is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target nucleic acid sequences. One of skill in the art will be able to determine when an oligomeric compound is specifically hybridizable.
[0100] As used herein, "complementary" refers to a nucleic acid molecule that can form hydrogen bond(s) with another nucleic acid molecule by either traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides. In reference to the antisense oligomeric compound of the present disclosure, the binding free energy for a antisense oligomeric compound with its complementary sequence is sufficient to allow the relevant function of the antisense oligomeric compound to proceed and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of ex vivo or in vivo therapeutic treatment. Determination of binding free energies for nucleic acid molecules is well known in the art (see e.g., Turner et ah, CSH Symp. Quant. Biol. 1/7:123-133 (1987); Frier et al, Proc. Nat. Acad. Sci. USA 83:9373-77 (1986); and Turner et al, J. Am. Chem. Soc. 109:3783-3785 (1987)). Thus, "complementary" (or "specifically hybridizable") are terms that indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between a antisense oligomeric compound and a pre-mRNA or mRNA target. It is understood in the art that a nucleic acid molecule need not be 100% complementary to a target nucleic acid sequence to be specifically hybridizable. That is, two or more nucleic acid molecules may be less than fully complementary. Complementarity is indicated by a percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid molecule. For example, if a first nucleic acid molecule has 10 nucleotides and a second nucleic acid molecule has 10 nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides between the first and second nucleic acid molecules represents 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively. Percent complementarity of an oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). "Perfectly" or "fully" complementary nucleic acid molecules means those in which all the contiguous residues of a first nucleic acid molecule will hydrogen bond with the same number of contiguous residues in a second nucleic acid molecule, wherein the nucleic acid molecules either both have the same number of nucleotides (i.e., have the same length) or the two molecules have different lengths.
[0101] As used herein, "uniformly modified" or "fully modified" refers to an oligomeric compound, an antisense oligonucleotide, or a region of nucleotides wherein essentially each nucleoside is a sugar modified nucleoside having uniform modification.
[0102] As used herein, a "chimeric oligomeric compound", "chimeric antisense compound" or "chimeric antisense oligonucleotide compound" is a compound containing two or more chemically distinct regions, each comprising at least one monomer unit (i.e., a nucleotide in the case of an oligonucleotide compound). The term "chimeric antisense compound" specifically refers to an antisense compound, having at least one sugar, nucleobase and/or internucleoside linkage that is differentially modified as compared to the other sugars, nucleotides and internucleoside linkages within the same oligomeric compound. The remainder of the sugars, nucleotides and internucleoside linkages can be independently modified or unmodified. In general a chimeric oligomeric compound will have modified nucleosides that can be in isolated positions or grouped together in regions that will define a particular motif. Chimeric oligomeric compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. In the context of the present disclosure, a "chimeric RNase H-independent antisense compound" is an antisense compound with at least two chemically distinct regions, but which is not susceptible to cleavage by RNase H when hybridized to a target nucleic acid.
[0103] As used herein, a "nucleoside" is a base-sugar combination and "nucleotides" are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
[0104] As used herein, a nucleoside with a modified sugar residue is any nucleoside wherein the ribose sugar of the nucleoside has been substituted with a chemically modified sugar moiety. In the context of the present disclosure, the chemically modified sugar moieties include, but are not limited to, 2'-O-methoxyethyl, 2'-fluoro, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, 2'-carbamate, 2'-aminooxy, 2'-acetamido and locked nucleic acid.
[0105] As used herein, compounds "resistant to RNase H degradation" are antisense compounds having a least one chemical modification that increases resistance of the compound to RNase H cleavage. Such modifications include, but are not limited to, nucleotides with sugar modifications. As used herein, a nucleotide with a modified sugar includes, but is not limited to, any nucleotide wherein the 2'-deoxyribose sugar has been substituted with a chemically modified sugar moiety. In the context of the present invention, chemically modified sugar moieties include, but are not limited to, 2'-O-(2-methoxyethyl), 2'-fluoro, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, 2'-carbamate, 2'-aminooxy, 2'-acetamido, locked nucleic acid (LNA) and ethylene bridged nucleic acid (ENA). Modified compounds resistant to RNase H cleavage are thoroughly described herein and are well know to those of skill in the art.
[0106] In the context of the present disclosure, "cellular uptake" refers to delivery and internalization of oligomeric compounds into cells. The oligomeric compounds can be internalized, for example, by cells grown in culture (in vitro), cells harvested from an animal (ex vivo) or by tissues following administration to an animal (in vivo).
[0107] By "subject" is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. "Subject" also refers to an organism to which the nucleic acid molecules of this disclosure can be administered. In one embodiment of the invention and/or embodiments thereof, a subject is a mammal or mammalian cell. In another embodiment, a subject is a human or human cell.
[0108] As used herein, the term "therapeutically effective amount" means an amount of antisense oligomeric compound that is sufficient, in the subject (e.g., human) to which it is administered, to treat or prevent the stated disease, disorder, or condition. The antisense oligomeric compound of the instant disclosure, individually, or in combination or in conjunction with other drugs, can be used to treat diseases or conditions discussed herein. For example, to treat a particular disease, disorder, or condition, the antisense oligomeric compound can be administered to a patient or can be administered to other appropriate cells evident to those skilled in the art, individually or in combination with one or more drugs, under conditions suitable for treatment. In the present invention the disease is Optionally Pompe disease.
[0109] As used herein, the phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Optionally, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
[0110] As used herein, the term "isolated" means that the referenced material is removed from its native environment, e.g., a cell. Thus, an isolated biological material can be free of some or all cellular components, i.e. components of the cells in which the native material occurs naturally (e.g., cytoplasmic or membrane component).
[0111] The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e. contaminants, inducing native materials from which the material is obtained. For example, a purified tc-DNA antisense oligomeric compound is Optionally substantially free of cell or culture components, including tissue culture components, contaminants, and the like. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Optionally, purified material substantially free of contaminants is at least 50% pure; more Optionally, at least 90% pure, and more Optionally still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
[0112] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, "about" or "consisting essentially of mean+-20% of the indicated range, value, or structure, unless otherwise indicated.
[0113] As used herein, the terms "include" and "comprise" are used synonymously. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.
[0114] The term "about" or "approximately" means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, Optionally within 50%, more Optionally within 20%, more Optionally still within 10%, and even more Optionally within 5% of a given value or range. The allowable variation encompassed by the term "about" or "approximately" depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. According to the invention, a "subject" or "patient" is a human or non-human animal. Although the animal subject is Optionally a human, the compounds and compositions of the invention have application in veterinary medicine as well, e.g., for the treatment of domesticated species such as canine, feline, and various other pets; farm animal species such as bovine, equine, ovine, caprine, porcine, etc.; wild animals, e.g., in the wild or in a zoological garden; and avian species, such as chickens, turkeys, quail, songbirds, etc.
[0115] The term "enzyme replacement therapy" or "ERT" refers to the introduction of a non-native, purified enzyme into an individual having a deficiency in such enzyme. The administered enzyme can be obtained from natural sources or by recombinant expression. The term also refers to the introduction of a purified enzyme in an individual otherwise requiring or benefiting from administration of a purified enzyme, e.g., suffering from protein insufficiency. The introduced enzyme may be a purified, recombinant enzyme produced in vitro, or enzyme purified from isolated tissue or fluid, such as, e.g., placenta or animal milk, or from plants.
[0116] As used herein, the term "active site-specific chaperone" or ASSC refers to any molecule including a protein, peptide, nucleic acid, carbohydrate, etc. that specifically interacts reversibly with an active site of a protein and enhances formation of a stable molecular conformation. As used herein, "active site-specific chaperone" does not include endogenous general chaperones present in the ER of cells such as Bip, calnexin or calreticulin, or general, non-specific chemical chaperones such as deuterated water, DMSO, or TMAO.
[0117] The term "purified" as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained. For example, a purified protein is Optionally substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is Optionally substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material Optionally, purified material substantially free of contaminants is at least 95% pure; more Optionally, at least 97% pure, and more Optionally still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. In a specific embodiment, purified means that the level of contaminants is below a level acceptable to regulatory authorities for safe administration to a human or non-human animal.
[0118] As used herein, the terms "mutant" and "mutation" mean any detectable change in genetic material, e.g., DNA, or any process, mechanism or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., RNA, protein or enzyme) expressed by a modified gene or DNA sequence.
[0119] As used herein the term "mutant protein" refers to proteins translated from genes containing genetic mutations that result in altered protein sequences. In a specific embodiment, such mutations result in the inability of the protein to achieve its native conformation under the conditions normally present in the ER. The failure to achieve this conformation results in these proteins being degraded, rather than being transported through their normal pathway in the protein transport system to their proper location within the cell. Other mutations can result in decreased activity or more rapid turnover.
[0120] As used herein the term "wild-type gene" refers to a nucleic acid sequences which encodes a protein capable of having normal biological functional activity in vivo. The wild-type nucleic acid sequence may contain nucleotide changes that differ from the known, published sequence, as long as the changes result in amino acid substitutions having little or no effect on the biological activity. The term wild-type may also include nucleic acid sequences engineered to encode a protein capable of increased or enhanced activity relative to the endogenous or native protein.
[0121] As used herein, the term "wild-type protein" refers to any protein encoded by a wild-type gene that is capable of having functional biological activity when expressed or introduced in vivo. The term "normal wild-type activity" refers to the normal physiological function of a protein in a cell. Such functionality can be tested by any means known to establish functionality of a protein. The term "genetically modified" refers to cells that express a particular gene product following introduction of a nucleic acid comprising a coding sequence which encodes the gene product, along with regulatory elements that control expression of the coding sequence. Introduction of the nucleic acid may be accomplished by any method known in the art including gene targeting and homologous recombination. As used herein, the term also includes cells that have been engineered to express or overexpress an endogenous gene or gene product not normally expressed by such cell, e.g., by gene activation technology.
[0122] The phrase "pharmaceutically acceptable", whether used in connection with the pharmaceutical compositions of the invention, refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Optionally, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are Optionally employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition.
[0123] The terms "therapeutically effective dose" and "effective amount" refer to the amount of the compound that is sufficient to result in a therapeutic response. In embodiments where an ASSC and enzyme such as GAA are administered in a complex, the terms "therapeutically effective dose" and "effective amount" may refer to the amount of the complex that is sufficient to result in a therapeutic response. A therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy. Thus, a therapeutic response will generally be an amelioration of one or more symptoms or sign of a disease or disorder.
[0124] It should be noted that a concentration of the chaperone that is inhibitory during in vitro production, transportation, or storage of the purified therapeutic protein may still constitute an "effective amount" for purposes of this invention because of dilution (and consequent shift in binding due to the change in equilibrium), bioavailability and metabolism of the chaperone upon administration in vivo.
[0125] The term `alkyl` refers to a straight or branched hydrocarbon group consisting solely of carbon and hydrogen atoms, containing no unsaturation, and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl).
[0126] The term "alkenyl" refers to a C2-C20 aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be a straight or branched chain, e.g., ethenyl, 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl.
[0127] The term "cycloalkyl" denotes an unsaturated, non-aromatic mono- or multicyclic hydrocarbon ring system such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. Examples of multicyclic cycloalkyl groups include perhydronapththyl, adamantyl and norbornyl groups bridged cyclic group or sprirobicyclic groups, e.g., Spiro (4,4) non-2-yl.
[0128] The term "aryl" refers to aromatic radicals having in the range of about 6 to about 14 carbon atoms such as phenyl, naphthyl, tetrahydronapthyl, indanyl, biphenyl.
[0129] The term "heterocyclic" refers to a stable 3- to 15-membered ring radical which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this invention, the heterocyclic ring radical may be a monocyclic or bicyclic ring system, which may include fused or bridged ring systems, and the nitrogen, carbon, oxygen or sulfur atoms in the heterocyclic ring radical may be optionally oxidized to various oxidation states. In addition, a nitrogen atom, where present, may be optionally quaternized; and the ring radical may be partially or fully saturated (e.g. heteroaromatic or heteroaryl aromatic). The heterocyclic ring radical may be attached to the main structure at any heteroatom or carbon atom that results in the creation of a stable structure.
[0130] The term "heteroaryl" refers to a heterocyclic ring wherein the ring is aromatic.
[0131] The substituents in the `substituted alkyl`, `substituted alkenyl`, `substituted cycloalkyl`, `substituted aryl` and `substituted heteroaryl` may be the same or different, with one or more selected from the groups hydrogen, halogen, acetyl, nitro, carboxyl, oxo (.dbd.O), CF.sub.3, --OCF.sub.3, NH.sub.2, --C(.dbd.O)-alkyl.sub.2, OCH.sub.3, or optionally substituted groups selected from alkyl, alkoxy and aryl. The term "halogen" refers to radicals of fluorine, chlorine, bromine and iodine.
[0132] GAA Enzyme
[0133] Human GAA is synthesized as a 110 kDal precursor (Wisselaar et al. (1993) J. Biol. Chem. 268(3): 2223-31). The mature form of the enzyme is a mixture of monomers of 70 and 76 kD (Wisselaar et al. (1993) J. Biol. Chem. 268(3): 2223-31). The precursor enzyme has seven potential glycosylation sites and four of these are retained in the mature enzyme (Wisselaar et al. (1993) J. Biol. Chem. 268(3): 2223-31). The proteolytic cleavage events which produce the mature enzyme occur in late endosomes or in the lysosome (Wisselaar et al. (1993) J. Biol. Chem. 268(3): 2223-31). The C-terminal 160 amino acids are absent from the mature 70 and 76 kD species. It has been reported that the C-terminal portion of the protein, although cleaved from the rest of the protein during processing, remains associated with the major species Moreland et al. (Nov. 1, 2004) J. Biol. Chem. Manuscript 404008200).
[0134] The enzyme of GAA may be obtained from a cell endogenously expressing the enzyme or GAA, or the enzyme or GAA may be a recombinant human enzyme or GAA (rhGAA), as described herein. Optionally the recombinant human enzyme or rhGAA is a full length wild-type enzyme. Optionally the recombinant human enzyme or rhGAA comprises a subset of the amino acid residues present in a wild-type enzyme or GAA, wherein the subset includes the amino acid residues of the wild-type enzyme or GAA that form the active site for substrate binding and/or substrate reduction. As such, the present invention contemplates an recombinant human enzyme or rhGAA that is a fusion protein comprising the wild-type enzyme or GAA active site for substrate binding and/or substrate reduction, as well as other amino acid residues that may or may not be present in the wild type enzyme or GAA.
[0135] The enzyme or GAA may be obtained from commercial sources or may be obtained by synthesis techniques known to a person of ordinary skill in the art. The wild-type enzyme can be purified from a recombinant cellular expression system (e.g., mammalian cells or insect cells-see generally U.S. Pat. No. 5,580,757.; U.S. Pat. Nos. 6,395,884 and 6,458,574, 6,461,609, 6,210,666; 6,083,725.; U.S. Pat. No. 6,451,600.; U.S. Pat. No. 5,236,838.; and U.S. Pat. No. 5,879,680.), human placenta, or animal milk (see e.g. U.S. Pat. No. 6,188,045.). After the infusion, the exogenous enzyme is expected to be taken up by tissues through non-specific or receptor-specific mechanism. In general, the uptake efficiency (without use of an chaperone) is not high, and the circulation time of the exogenous protein is short (Ioannu et al, Am. J. Hum. Genet. 2001; 68: 14-25). In addition, the exogenous protein is unstable and subject to rapid intracellular degradation in vitro. Other synthesis techniques for obtaining GAA suitable for pharmaceutical may be found, for example, in U.S. Pat. Nos. 7,560,424 and 7,396,811, U.S. Published Application Nos. 2009/0203575, 2009/0029467, 2008/0299640, 2008/0241118, 2006/0121018, and 2005/0244400, U.S. Pat. Nos. 7,423,135, 6,534,300, and 6,537,785; International Published Application No. 2005/077093 and U.S. Published Application Nos. 2007/0280925, and 2004/0029779. These references are hereby incorporated by reference in their entirety.
[0136] Optionally the GAA is alglucosidase alfa, which consists of the human enzyme acid alpha-glucosidase (GAA), encoded by any of nine observed haplotypes of this gene.
The GAA or enzyme suitable for ERT may be a modification, variant, analogue, fragment, portion, or functional derivative, thereof.
[0137] The uptake of the enzyme or GAA may be enhanced by functionalizing the enzyme or GAA by targets for receptors selected from the group consisting of mannose 6-phosphate receptor, insulin like growth factor II receptor, mannose receptor, galactose receptor, fucose receptor, N-Acetylglucosamine (GlcNAc) receptor, plasminogen activator receptor, IGF 1 receptor, insulin receptor; transferrin receptor, cation-dependent mannose-6-phosphate receptor (CD-MPR).
[0138] Functional derivatives" of the enzyme or GAA as described herein are fragments, variants, analogs, or chemical derivatives of the enzyme which retain at least a portion of the enzyme activity or immunological cross reactivity with an antibody specific for the enzyme.
[0139] A fragment or portion of enzyme refers to any subset of the molecule.
[0140] The enzyme or GAA may be modified with a compound selected from the group consisting of mannose 6-phosphate, peptide insulin-like growth factor-2.
[0141] Peptide insulin-like growth factor-2 is used in glycosylation-independent lysosomal targeting (GILT).
[0142] Optionally the enzyme or GAA is produced by recombinant DNA technology in a Chinese hamster ovary cell line.
[0143] Optionally the enzyme or GAA is produced by a glycoengineered yeast platform (e.g. based on the yeast Yarrowia lipolytica).
[0144] Optionally the enzyme or GAA is produced by transgene rabbits and collected via the milk of these transgene rabbits.
[0145] GAA enzyme is available as Myozyme (Sanofi) Lumizyme (Sanofi) OXY2810 (Oxyrane), IGF2-GAA (Biomarin) BMN-701 (Biomarin), Reveglucosidase alfa (Biomarin).
[0146] Chaperone or ASSC (active site-specific chaperone) may be obtained using synthesis techniques known to one of ordinary skill in the art. For example, ASSC that may be used in the present application, such as 1-DNJ may be prepared as described in U.S. Pat. Nos. 6,274,597 and 6,583,158, and U.S. Published Application No. 2006/0264467, each of which is hereby incorporated by reference in its entirety.
[0147] Optionally, the ASSC is a-homonojirimycin and the GAA is hrGAA (e.g., Myozyme.RTM. or Lumizyme.RTM.). Optionally the ASSC is castanospermine and the GAA is hrGAA (e.g., Myozyme.RTM. or Lumizyme.RTM.). The ASSC (e.g. -homonojirimycin and castanospermine) may be obtained from synthetic libraries (see, e.g., Needels et al., Proc. Natl. Acad. Sci. USA 1993; 90: 10700-4; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 1993; 90: 10922-10926; Lam et al., PCT Publication No. WO 92/00252; Kocis et al., PCT Publication No. WO 94/28028) which provide a source of potential ASSC's. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through Res. 1986; 155:1 19-29. Optionally ASSC's useful for the present invention are inhibitors of lysosomal enzymes and include glucose and galactose imino-sugar derivatives as described in Asano et al., J. Med. Chem. 1994; 37:3701-06; Dale et al, Biochemistry 1985; 24:3530-39; Goldman et al., J. Nat. Prod. 1996; 59:1137-42; Legler et al, Carbohydrate Res. 1986; 155; 1 19-29. Such derivatives include those that can be purchased from commercial sources such as Toronto Research Chemicals, Inc. (North York, On. Canada) and Sigma.
[0148] Optionally, the route of administration is subcutaneous. Other routes of administration may be oral or parenteral, including intravenous, intraarterial, intraperitoneal, ophthalmic, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intradermal, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, intrapulmonary, intranasal, transmucosal, transdermal, or via inhalation. Intrapulmonary delivery methods, apparatus and drug preparation are described, for example, in U.S. Pat. Nos. 5,785,049, 5,780,019, and 5,775,320, each incorporated herein by reference. In some embodiments, the method of intradermal delivery is by iontophoretic delivery via patches; one example of such delivery is taught in U.S. Pat. No. 5,843,015, which is incorporated herein by reference. Administration may be by periodic injections of a bolus of the preparation, or as a sustained release dosage form over long periods of time, or by intravenous or intraperitoneal administration, for example, from a reservoir which is external (e.g., an IV bag) or internal (e.g., a bioerodable implant, a bioartificial organ, or a population of implanted GAA production cells). See, e.g., U.S. Pat. Nos. 4,407,957 and 5,798,113, each incorporated herein by reference. Intrapulmonary delivery methods and apparatus are described, for example, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, each incorporated herein by reference. Other useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposomal delivery, needle-delivered injection, needle-less injection, nebulizer, aeorosolizer, electroporation, and transdermal patch. Needle-less injector devices are described in U.S. Pat. Nos. 5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications of which are herein incorporated by reference. Any of the GAA preparation described herein can administered in these methods.
[0149] Optionally the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound may be administered in combination with an Active Site-Specific Chaperone (ASSC) for the GAA enzyme (e.g., 1-deoxynojirimycin (DNJ, 1-DNJ)). The ASSC enables higher concentrations of the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound in a pharmaceutical composition. In combination with an ASSC the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound may be administered at a concentration between about 5 and about 250 mg/mL. Optionally, the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound is combined with an ASSC at a high concentration, for example, at a concentration selected from the group consisting of about 25-240 mg/mL, about 80-200 mg/mL, about 115-160 mg/mL. Optionally, the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound is combined with an ASSC, wherein the ASSC is present at a concentration between about 5 mg/mL and about 200 mg/mL, optionally between about 32 mg/mL and about 160 mg/mL. Optionally, t the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound is combined with an ASSC, wherein the ASSC is present at a concentration between about 0.5 mM and about 20 mM. GAA enzyme combined with an ASSC can remain soluble at a high concentration (e.g., 25 mg/mL) and remain non-aggregated while maintaining a viscosity suitable for injection (e.g., subcutaneous administration). Optionally the compositions of the present invention comprise more than about 5 mg/mL of the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound.
[0150] Optionally, the compositions of the invention comprise about 5-25 mg/mL the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound and about 1-10 mM DNJ.
[0151] Optionally, 1-deoxynojirimycin-HCl, or a pharmaceutically acceptable salt thereof, may be administered to a subject in a dose of between about 10 mg/kg to 1000 mg/kg, Optionally administered orally, either prior to, concurrent with, or after administration of the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound.
[0152] Optionally the method of treating Pompe Disease comprises administering the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound biweekly, weekly or once per two weeks for up to about 10 weeks in combination with from about 1 to about 5000 mg/kg of an ASSC (e.g., 1-DNJ-HCl) prior to, and in regular intervals after, the infusion of the enzyme or GAA enzyme or nucleic acid encoding the enzyme or GAA enzyme and/or the antisense oligomeric compound. For example, the ASSC could be administered within two hours of the infusion, and then administered at regular intervals once, twice, three-times, four-times, five-times or six-times within 24 hours post-infusion. Optionally, the GAA is Myozyme.RTM. and is administered via infusion once per week and the ASSC (e.g., 1-DNJ-HCl) is administered at 10 mg/kg, 100 mg/kg or 1000 mg/kg 30 minutes prior to infusion, and then 8, 16, and 24 hours after each Myozyme.RTM. infusion.
[0153] Optionally, the GAA is Lumizyme.RTM. and is administered via infusion once per week and the ASSC (e.g., 1-DNJ-HCl) is administered at
##STR00001##
10 mg/kg, 100 mg/kg or 1000 mg/kg 30 minutes prior to infusion, and then 8, 16, and 24 hours after each Lumizyme.RTM. infusion.
[0154] It is believed that acid alpha-glucosidase (GAA) functions to remove terminal glucose residues from lysosomal glycogen. Some genetic mutations reduce GAA trafficking and maturation. The pharmacological chaperone 1-DNJ increases GAA levels by selectively binding and stabilizing the enzyme in a proper conformation which restores proper protein trafficking to the lysosome. Optionally, the ASSC is administered as described in International Publication No. 2008/134628, which is hereby incorporated by reference in its entirety.
[0155] The ASSC is a small molecule inhibitor of the GAA enzyme, including reversible competitive inhibitors of the GAA enzyme. Optionally the ASSC may be represented by the formula:
##STR00002##
[0156] where R.sub.1 is H or a straight or branched alkyl, cycloalkyl, alkoxyalkyl or aminoalkyl containing 1-12 carbon atoms optionally substituted with an --OH, --COOH, --Cl, --F, --CF.sub.3, --OCF.sub.3, --O--C(O)N-(alkyl).sub.2; and R.sub.2 is H or a straight or branched alkyl, cycloalkyl, or alkoxylalkyl containing 1-9 carbon atoms; including pharmaceutically acceptable salts, esters and prodrugs thereof. Optionally the ASSC is 1-deoxynojirimycin (1-DNJ), which is represented by the following formula:
[0157] or a pharmaceutically acceptable salts, esters or prodrug of 1-deoxynojirimycin. Optionally, the salt is hydrochloride salt (i.e. 1-deoxynojirimycin-HCl). Optionally, the ASSC is N-butyl-deoxynojirimycin (NB-DNJ; Zavesca.RTM., Actelion Pharmaceuticals Ltd, Switzerland), which is represented by the following formula:
##STR00003##
[0158] or a pharmaceutically acceptable salt, ester or prodrug of NB-DNJ.
[0159] Optionally the ASSC is C.sub.10H.sub.19NO.sub.4, which is represented by the following formula:
##STR00004##
[0160] or a pharmaceutically acceptable salt, ester or prodrug of C.sub.10H.sub.19NO.sub.4. Optionally the salt is hydrochloride salt.
[0161] Optionally, the ASSC is C.sub.12H.sub.23NO.sub.4, which is represented by the following formula:
##STR00005##
[0162] or a pharmaceutically acceptable salt, ester or prodrug of C.sub.12H.sub.23NO.sub.4. Optionally, the salt is hydrochloride salt.
[0163] Patients with complete absence of GAA enzyme are cross-reactive immunololgical material (CRIM) negative, and develop high titer antibody to rhGAA. Patients with GAA protein detectable by western blot are classified as CRIM-positive. Whereas the majority of CRIM-positive patients have sustained therapeutic responses to ERT, or gene therapy CRIM-negative patients almost uniformly do poorly, experiencing rapid clinical decline because of the development of sustained, high-titer antibodies to rhGAA.
[0164] A combination of rituximab with methotrexate with or without intravenous gammaglobulins (WIG) may be used to induce tolerance induction of CRIM negative patients. The treatment may be prophylactically to avoid antibody to rhGAA or may be given to patients that have already developed anti-rhGAA. Rituximab may be given in a dose of 100-1000 mg/kg, or in a dose of 150-900 mg/kg, or in a dose of 200-800 mg/kg, or in a dose of 250-750 mg/kg, or in a dose of 300-600 mg/kg, or in a dose of 350-500 mg/kg, or in a dose of 400-450 mg/kg.
[0165] Rituximab may be given once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days. Optionally Rituximab is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weeks. Optionally Rituximab is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 months. Optionally Rituximab is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, years.
[0166] Methotrexate may be given in a dose of 0.1-10 mg/kg, or in a dose of 0.2-5 mg/kg, or in a dose of 0.3-2 mg/kg, or in a dose of 0.4-1 mg/kg or in a dose of 0.5-0.7 mg/kg. Methotrexate may be given once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days. Optionally Methotrexate is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weeks. Optionally Methotrexate is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 months. Optionally Methotrexate is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, years. Administration of Methotrexate may be based on hematologic tolerance. IVIG may be given in a dose of 0.1-10 mg/kg, or in a dose of 0.2-5 mg/kg, or in a dose of 0.3-2 mg/kg, or in a dose of 0.4-1 mg/kg or in a dose of 0.5-0.7 mg/kg. IVIG may be given once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days. Optionally IVIG is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 weeks. Optionally IVIG is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 months. Optionally IVIG is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, years. Treatment may be given until rhGAA antibody titer is down to zero. Various combinations of administration of the enzyme or GAA and Rituximab, and/or Methotrexate and/or IVIG is explicitly envisioned in the present invention.
[0167] In one aspect, the invention is directed to an antisense oligomeric compound. Previous work by others has resulted in the design of antisense oligomeric compounds that promote exon exclusion in several human disorders including Duchenne Muscular Dystrophy (DMD). The strategy is simple and straightforward and relies on blocking a well-defined splice site. This results in exon skipping, thereby removing the exon containing the pathogenic gene variant. The resulting mRNA is a little bit shorter resulting in expression of a truncated protein with considerable residual activity, sufficient to at least partially alleviate the disease. The strategy is simple because canonical splice sites are known for virtually all genes. The only requirement is to design an antisense oligomeric compound that binds to the canonical splice site in the pre-mRNA, which will result in blocking of that site and skipping of the exon involved.
[0168] A much more difficult task is the reverse process: to promote inclusion rather than exclusion of an exon. To promote exon inclusion, a splice repressor may be blocked using an antisense oligomeric compound. It is however unknown where splice repressors are located. These can be present in introns or in exons and are named intronic or exonic splice silencers (ISSs or ESSs, respectively). There is software available to predict the presence of such silences but these are very unreliable. This is further illustrated by our own experience using the minigene system containing GAA exon 1-3, which failed to confirm activity of predicted splice silencer motifs. The idea to promote exon 2 inclusion of GAA with an antisense oligomeric compound to treat Pompe disease is entirely novel.
[0169] sequences targeting SEQ ID NO: 1 are able to enhance inclusion of GAA exon 2. Also sequences targeting SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, were found to be able to enhance inclusion of GAA exon 2. It is to be noted that targeting means that at least part of the sequence SEQ ID NO: 1 is targeted, e.g. by a sequence that hybridizes with at least a part or by the sequence SEQ ID NO: 1, or that binds to at least a part of SEQ ID NO: 1. Sequences that target may be shorter or longer than the target sequence.
[0170] Sequence in cDNA to which AON anneals* sequence of AON (5'-SEQ ID NO:
TABLE-US-00001 c-32-156_-210 1 GCTCTGCACTCCCCTGCTGGAGCTTTTCTCGCCCTTCCTTCTGGC CCTCTCCCCA c-32-156_-200 37 GCTCTGCACTCCCCTGCTGGAGCTTTTCTCGCCCTTCCTTCTGGC c-32-160_-190 38 TGCACTCCCCTGCTGGAGCTTTTCTCGCCCT c-32-160_195 39 TGCACTCCCCTGCTGGAGCTTTTCTCGCCCTTCCTT c-32-165_-195 40 TCCCCTGCTGGAGCTTTTCTCGCCCTTCCTT
[0171] Optionally the sequences targeting SEQ ID NO: 1 hybridize with at least a part of SEQ ID NO: 1. Sequences that hybridize may be shorter or longer than the target sequence. Nucleotide sequences SEQ ID NO: 2-33 are oligomers that are able to enhance GAA exon 2 inclusion.
[0172] Two variant antisense oligomeric compounds, one of 21 nucleotides (SEQ ID NO: 33) and one of 25 nucleotides (SEQ ID NO: 12), were tested and both were found to enhance exon 2 inclusion. This was accompanied by enhanced GAA enzyme activity of at least 2 fold. It is known that patients with the IVS1 variant have .about.15% leaky wild type splicing. The enhancement of 2 fold results in enzyme activities of -30%, which are known to be above the disease threshold of 20% and thus are anticipated to restore at least a part, or even fully the lysosomal glycogen degradation.
[0173] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound selected from the group comprising SEQ ID NO: 2-33 and variants and fragments having at least 80% identity thereof. The antisense oligomeric compound may also target single nucleotide polymorphism of SEQ ID NO: 1, 37, 38, 39, 40. It should be noted that it may not necessary to have the full length of SEQ ID NO: 2-33, fragments having a shorter or longer sequence are also envisioned. The inventors have found the target genomic sequence which enables the inclusion of exon 2 of GAA and a skilled person is capable of finding suitable sequences that target this target genomic sequence, such as SEQ ID NO: 1, 37, 38, 39, 40 and single nucleotide polymorphisms thereof. Exemplary sequences that target this target genomic sequence, such as SEQ ID NO: 1, 37, 38, 39, or 41 may be SEQ ID NO: 2-33, but also variants and fragments having at least 80% identity thereof. In particular shorter fragments such as fragments with 18, 19, 20, 21, 22, 23, or 24 nucleotides of SEQ ID NO: 2-33 are envisioned.
[0174] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound complementary to a polynucleotide having a sequence selected from the group comprising SEQ ID NO: 1, 37-40 and single nucleotide polymorphisms thereof. Also sequences having at least 80% identity to antisense oligomeric compound complementary to a polynucleotide having a sequence selected from the group comprising SEQ ID NO: 1, 37-40 are envisioned. Antisense oligomeric compound that target one or more than one single nucleotide polymorphisms may be designed.
[0175] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound targeting a sequence selected from the group comprising the genomic sequence c-32-156_-210.
[0176] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound comprising sequences selected from the group comprising SEQ ID NO: 2-33, 41-1583, 1590-1594 and sequences having at least 80% identity thereof.
[0177] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to antisense oligomeric compound comprising a sequences selected from the group comprising SEQ ID NO: 2-33, and 41-540, 1590-1594.
[0178] In one aspect or embodiment of aspects and/or embodiments thereof the invention is directed to an antisense oligomeric compound complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of the following mutation c.-32-13T>G, c.-32-3C>G c.-32-102T>C, c.-32-56C>T, c.-32-46G>A, c.-32-28C>A, c.-32-28C>T, c.-32-21G>A, c.7G>A, c.11G>A, c.15_17AAA, c.17C>T, c.19_21AAA, c.26_28AAA, c.33_35AAA, c.39G>A, c.42C>T, c.90C>T, c.112G>A, c.137C>T, c.164C>T, c.348G>A, c.373C>T, c.413T>A, c.469C>T, c.476T>C, c.476T>G, c.478T>G, c.482C>T, c.510C>T, c.515T>A, c.520G>A, c.546+11C>T, c.546+14G>A, c.546+19G>A, c.546+23C>A, c.547-6, c.1071, c.1254, c.1552-30, c.1256A>T, c.1551+1G>T, c.546G>T, 0.17C>T, c.469C>T, c.546+23C>A, c.-32-102T>C, c.-32-56C>T, c.11G>A, c.112G>A, c.137C>T.
[0179] The above identified mutations have been found to modulate splicing. Targeting the location of the mutation may also modulate the splicing. It is therefore understood that the antisense oligomeric compound targets the location the mutation. The nomenclature of the mutation identifies the location and the mutation. It is understood that the antisense oligomeric compound targets the location of the mutation, and the mutation does not need to be present in the genomic sequence or in the pre-mRNA. The location of the mutation is thus the location of the mutated nucleotide, or the location of the wild type nucleotide of the mutation. The antisense oligomeric compound may be targeted to a sequence comprising nucleotides upstream and nucleotides downstream of the location of the mutation. Optionally the antisense oligomeric compound target a sequence comprising 2-50 nucleotides upstream, and/or 2-50 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 3-45 nucleotides upstream, and/or 3-45 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 5-40 nucleotides upstream, and/or 5-40 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 6-35 nucleotides upstream, and/or 6-35 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 7-33 nucleotides upstream, and/or 7-33 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 8-30 nucleotides upstream, and/or 8-30 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 9-28 nucleotides upstream, and/or 9-28 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 10-25 nucleotides upstream, and/or 10-25 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 11-22 nucleotides upstream, and/or 11-22 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 12-20 nucleotides upstream, and/or 12-20 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 13-18 nucleotides upstream, and/or 13-18 nucleotides downstream of the location of the mutation, more Optionally the antisense oligomeric compound target a sequence comprising 14-16 nucleotides upstream, and/or 14-16 nucleotides downstream of the location of the mutation.
[0180] The nomenclature is well known to a skilled person and can be found in Dunnen and Antonarakis Human mutation 15:7-12(2000) and Antonarakis SE, the Nomenclature Working Group. 1998. Recommendations for a nomenclature system for human gene mutations. Hum Mutat 11:1-3 and on the website (http://www.dmd.nl/mutnomen.html. Genomic positions may also be found on www.pompecenter.nl. All of these are incorporated by reference.
[0181] Optionally the genomic nucleic acid sequence is pre-mRNA.
[0182] These antisense oligomeric compound are useful in the treatment of glycogen storage disease type II/Pompe disease.
[0183] In one aspect or the target sequence is an intronic splicing silencer or ISS. Optionally of the invention and/or embodiments thereof of an aspect and/or embodiments of the invention the target sequence is the GCTCTGCACTCCCCTGCTGGAGCTTTTCTCGCCCTTCCTTCTGGCCCTC TCCCCA (SEQ ID NO: 1). It should be noted that also naturally occurring single nucleotide polymorphism are included. Antisense oligomeric compounds targeting SEQ ID NO: 1 are a very suitable to treat Pompe patients. Exemplary antisense oligomeric compounds targeting SEQ ID NO: 1 are SEQ ID NO: 2-33 and in particular SEQ ID NO: 12 and SEQ ID NO 33. However the invention is not limited to these two sequences. A skilled person is capable of designing antisense oligomeric compounds against target sequence SEQ ID NO: 1, 37, 38, 39, or 40. The antisense oligomeric compounds against target sequenced SEQ ID NO: 1 may have length of 10 to 100 nucleotides, Optionally 11 to 75 nucleotides, Optionally 12 to 73 nucleotides, Optionally 13 to 70 nucleotides, Optionally 14 to 65 nucleotides, Optionally 15 to 60 nucleotides, Optionally 16 to 55 nucleotides, Optionally 17 to 50 nucleotides, Optionally 18 to 45 nucleotides, Optionally 19 to 40 nucleotides, Optionally 20 to 38 nucleotides, Optionally 21 to 35 nucleotides, Optionally 22 to 33 nucleotides, Optionally 23 to 30 nucleotides, Optionally 24 to 29 nucleotides, Optionally 25 to 28 nucleotides, Optionally 26 to 27 nucleotides.
Hereunder exemplary antisense oligomeric compounds targeting SEQ ID NO: 1 are given
TABLE-US-00002 Sequence in cDNA to which Seq AON anneals* sequence of AON (5'.fwdarw.3'): ID c.-32-180_-156 TGGGGAGAGGGCCAGAAGGAAGGGC 2 c.-32-181_-157 GGGGAGAGGGCCAGAAGGAAGGGCG 3 c.-32-182_-158 GGGAGAGGGCCAGAAGGAAGGGCGA 4 c.-32-183_-159 GGAGAGGGCCAGAAGGAAGGGCGAG 5 c.-32-184_-160 GAGAGGGCCAGAAGGAAGGGCGAGA 6 c.-32-185_-161 AGAGGGCCAGAAGGAAGGGCGAGAA 7 c.-32-186_-162 GAGGGCCAGAAGGAAGGGCGAGAAA 8 c.-32-187_-163 AGGGCCAGAAGGAAGGGCGAGAAAA 9 c.-32-188_-164 GGGCCAGAAGGAAGGGCGAGAAAAG 10 c.-32-189_-165 GGCCAGAAGGAAGGGCGAGAAAAGC 11 c.-32-190_-166 GCCAGAAGGAAGGGCGAGAAAAGCT 12 c.-32-191_-167 CCAGAAGGAAGGGCGAGAAAAGCTC 13 c.-32-192_-168 CAGAAGGAAGGGCGAGAAAAGCTCC 14 c.-32-193_-169 AGAAGGAAGGGCGAGAAAAGCTCCA 15 c.-32-194_-170 GAAGGAAGGGCGAGAAAAGCTCCAG 16 c.-32-195_-171 AAGGAAGGGCGAGAAAAGCTCCAGC 17 c.-32-196_-172 AGGAAGGGCGAGAAAAGCTCCAGCA 18 c.-32-197_-173 GGAAGGGCGAGAAAAGCTCCAGCAG 19 c.-32-198_-174 GAAGGGCGAGAAAAGCTCCAGCAGG 20 c.-32-199_-175 AAGGGCGAGAAAAGCTCCAGCAGGG 21 c.-32-200_-176 AGGGCGAGAAAAGCTCCAGCAGGGG 22 c.-32-201_-177 GGGCGAGAAAAGCTCCAGCAGGGGA 23 c.-32-202_-178 GGCGAGAAAAGCTCCAGCAGGGGAG 24 c.-32-203_-179 GCGAGAAAAGCTCCAGCAGGGGAGT 25 c.-32-204_-180 CGAGAAAAGCTCCAGCAGGGGAGTG 26 c.-32-205_-181 GAGAAAAGCTCCAGCAGGGGAGTGC 27 c.-32-206_-182 AGAAAAGCTCCAGCAGGGGAGTGCA 28 c.-32-207_-183 GAAAAGCTCCAGCAGGGGAGTGCAG 29 c.-32-208_-184 AAAAGCTCCAGCAGGGGAGTGCAGA 30 c.-32-209_-185 AAAGCTCCAGCAGGGGAGTGCAGAG 31 c.-32-210_-186 AAGCTCCAGCAGGGGAGTGCAGAGC 32 c.-32-187_-167 CCAGAAGGAAGGGCGAGAAAA 33
[0184] In the above examples the sequences are 25 nucleotides long however longer variants or shorter fragment are also envisioned. Exemplary is SEQ ID NO: 33 which is only 21 nucleotides long and comprises the same nucleotides as SEQ ID NO: 12 but is shorter. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 2-33 and fragments and variants thereof having at least 80% sequence identity. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 2-33 and fragments and variants thereof having at least 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% sequence identity to SEQ ID NO: 2-33.
[0185] The present invention is also directed to sequences that are at least 80% identical to SEQ ID NO: 2-33. Optionally at least 85% identical to SEQ ID NO: 2-33, more Optionally at least 88% identical to SEQ ID NO: 2-33, more Optionally at least 90% identical to SEQ ID NO: 2-33. more Optionally at least 91% identical to SEQ ID NO: 2-33, more Optionally at least 92% identical to SEQ ID NO: 2-33, more Optionally at least 93% identical to SEQ ID NO: 2-33, more Optionally at least 94% identical to SEQ ID NO: 2-33, more Optionally at least 95% identical to SEQ ID NO: 2-33, more Optionally at least 96% identical to SEQ ID NO: 2-33, more Optionally at least 97% identical to SEQ ID NO: 2-33, more Optionally at least 98% identical to SEQ ID NO: 2-33, more Optionally at least 99% identical to SEQ ID NO: 2-33.
[0186] Preferred antisense sequences are SEQ ID NO: 12, and SEQ ID NO:33 or sequences that are at least 80% identical thereto, Optionally at least 85% identical, more Optionally at least 88% identical, more Optionally at least 90% identical, more Optionally at least 91% identical, more Optionally at least 92% identical, more Optionally at least 93% identical, more Optionally at least 94% identical, more Optionally at least 95% identical, more Optionally at least 96% identical, more Optionally at least 97% identical, more Optionally at least 98% identical, more Optionally at least 99% identical to SEQ ID NO: 12, and/or 33.
[0187] Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 2-33, wherein the fragment is 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long.
[0188] Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 2-33, wherein the fragment is 17, 18, 19, 20, 21, or 22 nucleotides long. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 2-33, wherein the fragment is 19, 20, or 21 nucleotides long.
[0189] The antisense oligomeric compounds may be selected from the group of SEQ ID NO: 41-540:
TABLE-US-00003 Sequence in GAA cDNA to which AON Seq anneals AON sequence 5'.fwdarw.3' ID c.-32-319_-300 CCAAACAGCTGTCGCCTGGG 41 c.-32-299_-280 AGGTAGACACTTGAAACAGG 42 c.-32-279_-260 CCCAGGAAGACCAGCAAGGC 43 c.-32-259_-240 TCAAACACGCTTAGAATGTC 44 c.-32-239_-220 GTCTGCTAAAATGTTACAAA 45 c.-32-219_-200 GAGTGCAGAGCACTTGCACA 46 c.-32-199_-180 CGAGAAAAGCTCCAGCAGGG 47 c.-32-179_-160 GAGAGGGCCAGAAGGAAGGG 48 c.-32-159_-140 GCCCTGCTGTCTAGACTGGG 49 c.-32-139_-120 AGGTGGCCAGGGTGGGTGTT 50 c.-32-119_-100 GCACCCAGGCAGGTGGGGTA 51 c.-32-99_-80 CAACCGCGGCTGGCACTGCA 52 c.-32-79_-60 TCAAAGCAGCTCTGAGACAT 53 c.-32-59_-40 GGGCGGCACTCACGGGGCTC 54 c.-32-39_-20 GCTCAGCAGGGAGGCGGGAG 55 c.-32-19_-0 CCTGCGGGAGAAGAAAGCGG 56 c.-30_-12 GCCTGGACAGCTCCTACAGG 57 c.-10_+9 CACTCCCATGGTTGGAGATG 58 c.10_+29 TGGGAGCAGGGCGGGTGCCT 59 c.30_+49 CGCAGACGGCCAGGAGCCGG 60 c.50_+69 GGTTGCCAAGGACACGAGGG 61 c.70_+89 ATGTGCCCCAGGAGTGCAGC 62 c.90_+109 GCAGGAAATCATGGAGTAGG 63 c.110_+129 ACTCAGCTCTCGGGGAACCA 64 c.130_+149 TCCAGGACTGGGGAGGAGCC 65 c.150_+169 GGTGAGCTGGGTGAGTCTCC 66 c.170_+189 TGGTCTGCTGGCTCCCTGCT 67 c.190_+209 GCCTGGGCATCCCGGGGCCC 68 c.210_+229 CTCTGGGACGGCCGGGGTGT 69 c.230_+249 GTCGCACTGTGTGGGCACTG 70 c.250_+269 AAGCGGCTGTTGGGGGGGAC 71 c.270_+289 CCTTGTCAGCCCCGCAATCG 72 c.290_+309 GCACTGTTCCTGGGTGATGG 73 c.310_+329 TAGCAACAGCCGCGGGCCTC 74 c.330_+349 GCCCCTGCTTTGCAGGGATG 75 c.350_+369 CCCCATCTGGGCTCCCTGCA 76 c.370_+389 GGGAAGAAGCACCAGGGCTG 77 c.390_+409 TGTAGCTGGGGTAGCTGGGT 78 c.410_+429 GGAGCTCAGGTTCTCCAGCT 79 c.430_+449 GCCGTGTAGCCCATTTCAGA 80 c.450_+469 GGGTGGTACGGGTCAGGGTG 81 c.470_+489 GTCCTTGGGGAAGAAGGTGG 82 c.490_+509 TCCAGCCGCAGGGTCAGGAT 83 c.510_+529 TCTCAGTCTCCATCATCACG 84 c.530_+546 GTGAAGTGGAGGCGGT 85 c.-32-225_-206 AGAGCACTTGCACAGTCTGC 86 c.-32-223_-204 GCAGAGCACTTGCACAGTCT 87 c.-32-221_-202 GTGCAGAGCACTTGCACAGT 88 c.-32-217_-198 GGGAGTGCAGAGCACTTGCA 89 c.-32-215_-196 AGGGGAGTGCAGAGCACTTG 90 c.-32-213_-194 GCAGGGGAGTGCAGAGCACT 91 c.-32-185_-166 GCCAGAAGGAAGGGCGAGAA 92 c.-32-183_-164 GGGCCAGAAGGAAGGGCGAG 93 c.-32-181_-162 GAGGGCCAGAAGGAAGGGCG 94 c.-32-177_-158 GGGAGAGGGCCAGAAGGAAG 95 c.-32-175_-156 TGGGGAGAGGGCCAGAAGGA 96 c.-32-173_-154 ACTGGGGAGAGGGCCAGAAG 97 variants that affect aberrant splicing of exon AON sequence designed 2 caused by IVS1 in GAA to block the region exon 1-3 minigene surrounding the identified Seq system splice element (5'.fwdarw.3') ID c.-32-102C > T CACCCAGGCAGGTGGGGTAAGGTGG 98 AGCACCCAGGCAGGTGGGGTAAGGT 99 GCAGCACCCAGGCAGGTGGGGTAAG 100 CTGCAGCACCCAGGCAGGTGGGGTA 101 CACTGCAGCACCCAGGCAGGTGGGG 102 GGCACTGCAGCACCCAGGCAGGTGG 103 CTGGCACTGCAGCACCCAGGCAGGT 104 GGCTGGCACTGCAGCACCCAGGCAG 105 GCGGCTGGCACTGCAGCACCCAGGC 106 CCGCGGCTGGCACTGCAGCACCCAG 107 TCAACCGCGGCTGGCACTGCAGCAC 108 ACCCAGGCAGGTGGGGTAAGGTGGC 109 GCACCCAGGCAGGTGGGGTAAGGTG 110 CAGCACCCAGGCAGGTGGGGTAAGG 111 TGCAGCACCCAGGCAGGTGGGGTAA 112 ACTGCAGCACCCAGGCAGGTGGGGT 113 GCACTGCAGCACCCAGGCAGGTGGG 114 TGGCACTGCAGCACCCAGGCAGGTG 115 GCTGGCACTGCAGCACCCAGGCAGG 116 CGGCTGGCACTGCAGCACCCAGGCA 117 CGCGGCTGGCACTGCAGCACCCAGG 118 ACCGCGGCTGGCACTGCAGCACCCA 119 CAACCGCGGCTGGCACTGCAGCACC 120 ATCAACCGCGGCTGGCACTGCAGCA 121 c.-32-56C > T, GGCTCTCAAAGCAGCTCTGAGACAT 122 c-32-46G > A, GGGGCTCTCAAAGCAGCTCTGAGAC 123 c.-32-28C > A, ACGGGGCTCTCAAAGCAGCTCTGAG 124 c.-32-28C > T, TCACGGGGCTCTCAAAGCAGCTCTG 125 c.-32-21G > A ACTCACGGGGCTCTCAAAGCAGCTC 126 GCACTCACGGGGCTCTCAAAGCAGC 127 CGGCACTCACGGGGCTCTCAAAGCA 128 GGCGGCACTCACGGGGCTCTCAAAG 129 GGGGCGGCACTCACGGGGCTCTCAA 130 GAGGGGCGGCACTCACGGGGCTCTC 131 GGGAGGGGCGGCACTCACGGGGCTC 132 GCGGGAGGGGCGGCACTCACGGGGC 133 AGGCGGGAGGGGCGGCACTCACGGG 134 GGAGGCGGGAGGGGCGGCACTCACG 135 AGGGAGGCGGGAGGGGCGGCACTCA 136 GCAGGGAGGCGGGAGGGGCGGCACT 137 CAGCAGGGAGGCGGGAGGGGCGGCA 138 CTCAGCAGGGAGGCGGGAGGGGCGG 139 GGCTCAGCAGGGAGGCGGGAGGGGC 140 CGGGCTCAGCAGGGAGGCGGGAGGG 141 AGCGGGCTCAGCAGGGAGGCGGGAG 142 AAAGCGGGCTCAGCAGGGAGGCGGG 143 AGAAAGCGGGCTCAGCAGGGAGGCG 144 GAAGAAAGCGGGCTCAGCAGGGAGG 145 GAGAAGAAAGCGGGCTCAGCAGGGA 146 GGGAGAAGAAAGCGGGCTCAGCAGG 147 GCGGGAGAAGAAAGCGGGCTCAGCA 148 CTGCGGGAGAAGAAAGCGGGCTCAG 149 GCCTGCGGGAGAAGAAAGCGGGCTC 150 AGGCCTGCGGGAGAAGAAAGCGGGC 151 ACTCCCATGGTTGGAGATGGCCTGG 152 TCACTCCCATGGTTGGAGATGGCCT 153 CCTCACTCCCATGGTTGGAGATGGC 154 TGCCTCACTCCCATGGTTGGAGATG 155 GGTGCCTCACTCCCATGGTTGGAGA 156 CGGGTGCCTCACTCCCATGGTTGGA 157 GGCGGGTGCCTCACTCCCATGGTTG 158 AGGGCGGGTGCCTCACTCCCATGGT 159 GCAGGGCGGGTGCCTCACTCCCATG 160 GAGCAGGGCGGGTGCCTCACTCCCA 161 GGGAGCAGGGCGGGTGCCTCACTCC 162 GTGGGAGCAGGGCGGGTGCCTCACT 163 CGGTGGGAGCAGGGCGGGTGCCTCA 164 GCCGGTGGGAGCAGGGCGGGTGCCT 165 GAGCCGGTGGGAGCAGGGCGGGTGC 166 AGGAGCCGGTGGGAGCAGGGCGGGT 167 CCAGGAGCCGGTGGGAGCAGGGCGG 168 GGCCAGGAGCCGGTGGGAGCAGGGC 169 ACGGCCAGGAGCCGGTGGGAGCAGG 170 AGACGGCCAGGAGCCGGTGGGAGCA 171 GCAGACGGCCAGGAGCCGGTGGGAG 172 GCGCAGACGGCCAGGAGCCGGTGGG 173 GGGCGCAGACGGCCAGGAGCCGGTG 174 GAGGGCGCAGACGGCCAGGAGCCGG 175 ACGAGGGCGCAGACGGCCAGGAGCC 176 ACACGAGGGCGCAGACGGCCAGGAG 177 GGACACGAGGGCGCAGACGGCCAGG 178 AAGGACACGAGGGCGCAGACGGCCA 179 CCAAGGACACGAGGGCGCAGACGGC 180 TGCCAAGGACACGAGGGCGCAGACG 181 GCTCTCAAAGCAGCTCTGAGACATC 182 GGGCTCTCAAAGCAGCTCTGAGACA 183 CTCACGGGGCTCTCAAAGCAGCTCT 184 CACTCACGGGGCTCTCAAAGCAGCT 185 GGCACTCACGGGGCTCTCAAAGCAG 186 GCGGCACTCACGGGGCTCTCAAAGC 187 GGGCGGCACTCACGGGGCTCTCAAA 188 AGGGGCGGCACTCACGGGGCTCTCA 189 GGAGGGGCGGCACTCACGGGGCTCT 190 CGGGAGGGGCGGCACTCACGGGGCT 191 GGCGGGAGGGGCGGCACTCACGGGG 192 GAGGCGGGAGGGGCGGCACTCACGG 193 GGGAGGCGGGAGGGGCGGCACTCAC 194 CAGGGAGGCGGGAGGGGCGGCACTC 195 AGCAGGGAGGCGGGAGGGGCGGCAC 196 TCAGCAGGGAGGCGGGAGGGGCGGC 197 GCTCAGCAGGGAGGCGGGAGGGGCG 198 GGGCTCAGCAGGGAGGCGGGAGGGG 199 GCGGGCTCAGCAGGGAGGCGGGAGG 200 AAGCGGGCTCAGCAGGGAGGCGGGA 201 GAAAGCGGGCTCAGCAGGGAGGCGG 202 AAGAAAGCGGGCTCAGCAGGGAGGC 203 AGAAGAAAGCGGGCTCAGCAGGGAG 204 GGAGAAGAAAGCGGGCTCAGCAGGG 205 CGGGAGAAGAAAGCGGGCTCAGCAG 206 TGCGGGAGAAGAAAGCGGGCTCAGC 207 CCTGCGGGAGAAGAAAGCGGGCTCA 208 GGCCTGCGGGAGAAGAAAGCGGGCT 209 CAGGCCTGCGGGAGAAGAAAGCGGG 210 CGGGGCTCTCAAAGCAGCTCTGAGA 211 CACGGGGCTCTCAAAGCAGCTCTGA 212 c.7G > A, c.11G > A, CTCCCATGGTTGGAGATGGCCTGGA 213 c.15_17AAA, c.17C > T, CACTCCCATGGTTGGAGATGGCCTG 214 c.19_21AAA, CTCACTCCCATGGTTGGAGATGGCC 215 c.26_28AAA, GCCTCACTCCCATGGTTGGAGATGG 216 c.33_35AAA, c.39G > A, GTGCCTCACTCCCATGGTTGGAGAT 217 c.42C > T GGGTGCCTCACTCCCATGGTTGGAG 218 GCGGGTGCCTCACTCCCATGGTTGG 219 GGGCGGGTGCCTCACTCCCATGGTT 220
CAGGGCGGGTGCCTCACTCCCATGG 221 AGCAGGGCGGGTGCCTCACTCCCAT 222 GGAGCAGGGCGGGTGCCTCACTCCC 223 TGGGAGCAGGGCGGGTGCCTCACTC 224 GGTGGGAGCAGGGCGGGTGCCTCAC 225 CCGGTGGGAGCAGGGCGGGTGCCTC 226 AGCCGGTGGGAGCAGGGCGGGTGCC 227 GGAGCCGGTGGGAGCAGGGCGGGTG 228 CAGGAGCCGGTGGGAGCAGGGCGGG 229 GCCAGGAGCCGGTGGGAGCAGGGCG 230 CGGCCAGGAGCCGGTGGGAGCAGGG 231 GACGGCCAGGAGCCGGTGGGAGCAG 232 CAGACGGCCAGGAGCCGGTGGGAGC 233 CGCAGACGGCCAGGAGCCGGTGGGA 234 GGCGCAGACGGCCAGGAGCCGGTGG 235 AGGGCGCAGACGGCCAGGAGCCGGT 236 CGAGGGCGCAGACGGCCAGGAGCCG 237 CACGAGGGCGCAGACGGCCAGGAGC 238 GACACGAGGGCGCAGACGGCCAGGA 239 AGGACACGAGGGCGCAGACGGCCAG 240 CAAGGACACGAGGGCGCAGACGGCC 241 GCCAAGGACACGAGGGCGCAGACGG 242 TTGCCAAGGACACGAGGGCGCAGAC 243 c.90C > T, c.112G > A, GGATGTGCCCCAGGAGTGCAGCGGT 244 c.137C > T, c.164C > T TAGGATGTGCCCCAGGAGTGCAGCG 245 AGTAGGATGTGCCCCAGGAGTGCAG 246 GGAGTAGGATGTGCCCCAGGAGTGC 247 ATGGAGTAGGATGTGCCCCAGGAGT 248 TCATGGAGTAGGATGTGCCCCAGGA 249 AATCATGGAGTAGGATGTGCCCCAG 250 GAAATCATGGAGTAGGATGTGCCCC 251 AGGAAATCATGGAGTAGGATGTGCC 252 GCAGGAAATCATGGAGTAGGATGTG 253 CAGCAGGAAATCATGGAGTAGGATG 254 ACCAGCAGGAAATCATGGAGTAGGA 255 GAACCAGCAGGAAATCATGGAGTAG 256 GGGAACCAGCAGGAAATCATGGAGT 257 CGGGGAACCAGCAGGAAATCATGGA 258 CTCGGGGAACCAGCAGGAAATCATG 259 CTCTCGGGGAACCAGCAGGAAATCA 260 AGCTCTCGGGGAACCAGCAGGAAAT 261 TCAGCTCTCGGGGAACCAGCAGGAA 262 ACTCAGCTCTCGGGGAACCAGCAGG 263 CCACTCAGCTCTCGGGGAACCAGCA 264 AGCCACTCAGCTCTCGGGGAACCAG 265 GGAGCCACTCAGCTCTCGGGGAACC 266 GAGGAGCCACTCAGCTCTCGGGGAA 267 GGGAGGAGCCACTCAGCTCTCGGGG 268 TGGGGAGGAGCCACTCAGCTCTCGG 269 ACTGGGGAGGAGCCACTCAGCTCTC 270 GGACTGGGGAGGAGCCACTCAGCTC 271 CAGGACTGGGGAGGAGGCACTCAGC 272 TCCAGGACTGGGGAGGAGCCACTCA 273 CCTCCAGGACTGGGGAGGAGCCACT 274 CTCCTCCAGGACTGGGGAGGAGCCA 275 GTCTCCTCCAGGACTGGGGAGGAGC 276 GAGTCTCCTCCAGGACTGGGGAGGA 277 GTGAGTCTCCTCCAGGACTGGGGAG 278 GGGTGAGTCTCCTCCAGGACTGGGG 279 CTGGGTGAGTCTCCTCCAGGACTGG 280 AGCTGGGTGAGTCTCCTCCAGGACT 281 TGAGCTGGGTGAGTCTCCTCCAGGA 282 GGTGAGCTGGGTGAGTCTCCTCCAG 283 CTGGTGAGCTGGGTGAGTCTCCTCC 284 TGCTGGTGAGCTGGGTGAGTCTCCT 285 CCTGCTGGTGAGCTGGGTGAGTCTC 286 TCCCTGCTGGTGAGCTGGGTGAGTC 287 GCTCCCTGCTGGTGAGCTGGGTGAG 288 TGGCTCCCTGCTGGTGAGCTGGGTG 289 GCTGGCTCCCTGCTGGTGAGCTGGG 290 CTGCTGGCTCCCTGCTGGTGAGCTG 291 GTCTGCTGGCTCCCTGCTGGTGAGC 292 GATGTGCCCCAGGAGTGCAGCGGTT 293 AGGATGTGCCCCAGGAGTGCAGCGG 294 GTAGGATGTGCCCCAGGAGTGCAGC 295 GAGTAGGATGTGCCCCAGGAGTGCA 296 TGGAGTAGGATGTGCCCCAGGAGTG 297 CATGGAGTAGGATGTGCCCCAGGAG 298 ATCATGGAGTAGGATGTGCCCCAGG 299 AAATCATGGAGTAGGATGTGCCCCA 300 GGAAATCATGGAGTAGGATGTGCCC 301 CAGGAAATCATGGAGTAGGATGTGC 302 AGCAGGAAATCATGGAGTAGGATGT 303 CCAGCAGGAAATCATGGAGTAGGAT 304 AACCAGCAGGAAATCATGGAGTAGG 305 GGAACCAGCAGGAAATCATGGAGTA 306 GGGGAACCAGCAGGAAATCATGGAG 307 TCGGGGAACCAGCAGGAAATCATGG 308 TCTCGGGGAACCAGCAGGAAATCAT 309 GCTCTCGGGGAACCAGCAGGAAATC 310 CAGCTCTCGGGGAACCAGCAGGAAA 311 CTCAGCTCTCGGGGAACCAGCAGGA 312 CACTCAGCTCTCGGGGAACCAGCAG 313 GCCACTCAGCTCTCGGGGAACCAGC 314 GAGCCACTCAGCTCTCGGGGAACCA 315 AGGAGCCACTCAGCTCTCGGGGAAC 316 GGAGGAGCCACTCAGCTCTCGGGGA 317 GGGGAGGAGCCACTCAGCTCTCGGG 318 CTGGGGAGGAGCCACTCAGCTCTCG 319 GACTGGGGAGGAGCCACTCAGCTCT 320 AGGACTGGGGAGGAGCCACTCAGCT 321 CCAGGACTGGGGAGGAGCCACTCAG 322 CTCCAGGACTGGGGAGGAGCCACTC 323 TCCTCCAGGACTGGGGAGGAGCCAC 324 TCTCCTCCAGGACTGGGGAGGAGCC 325 AGTCTCCTCCAGGACTGGGGAGGAG 326 TGAGTCTCCTCCAGGACTGGGGAGG 327 GGTGAGTCTCCTCCAGGACTGGGGA 328 TGGGTGAGTCTCCTCCAGGACTGGG 329 GCTGGGTGAGTCTCCTCCAGGACTG 330 GAGCTGGGTGAGTCTCCTCCAGGAC 331 GTGAGCTGGGTGAGTCTCCTCCAGG 332 TGGTGAGCTGGGTGAGTCTCCTCCA 333 GCTGGTGAGCTGGGTGAGTCTCCTC 334 CTGCTGGTGAGCTGGGTGAGTCTCC 335 CCCTGCTGGTGAGCTGGGTGAGTCT 336 CTCCCTGCTGGTGAGCTGGGTGAGT 337 GGCTCCCTGCTGGTGAGCTGGGTGA 338 CTGGCTCCCTGCTGGTGAGCTGGGT 339 TGCTGGCTCCCTGCTGGTGAGCTGG 340 TCTGCTGGCTCCCTGCTGGTGAGCT 341 GGTCTGCTGGCTCCCTGCTGGTGAG 342 c.348G > A, c.373C > T AGCCCCTGCTTTGCAGGGATGTAGC 343 GCAGCCCCTGCTTTGCAGGGATGTA 344 CTGCAGCCCCTGCTTTGCAGGGATG 345 CCCTGCAGCCCCTGCTTTGCAGGGA 346 CTCCCTGCAGCCCCTGCTTTGCAGG 347 GGCTCCCTGCAGCCCCTGCTTTGCA 348 TGGGCTCCCTGCAGCCCCTGCTTTG 349 TCTGGGCTCCCTGCAGCCCCTGCTT 350 CATCTGGGCTCCCTGCAGCCCCTGC 351 CCCATCTGGGCTCCCTGCAGCCCCT 352 GCCCCATCTGGGCTCCCTGCAGCCC 353 CTGCCCCATCTGGGCTCCCTGCAGC 354 GGCTGCCCCATCTGGGCTCCCTGCA 355 AGGGCTGCCCCATCTGGGCTCCCTG 356 CCAGGGCTGCCCCATCTGGGCTCCC 357 CACCAGGGCTGCCCCATCTGGGCTC 358 AGCACCAGGGCTGCCCCATCTGGGC 359 GAAGCACCAGGGCTGCCCCATCTGG 360 AAGAAGCACCAGGGCTGCCCCATCT 361 GGAAGAAGCACCAGGGCTGCCCCAT 362 TGGGAAGAAGCACCAGGGCTGCCCC 363 GGTGGGAAGAAGCACCAGGGCTGCC 364 TGGGTGGGAAGAAGCACCAGGGCTG 365 GCTGGGTGGGAAGAAGCACCAGGGC 366 GCCCCTGCTTTGCAGGGATGTAGCA 367 CAGCCCCTGCTTTGCAGGGATGTAG 368 TGCAGCCCCTGCTTTGCAGGGATGT 369 CCTGCAGCCCCTGCTTTGCAGGGAT 370 TCCCTGCAGCCCCTGCTTTGCAGGG 371 GCTCCCTGCAGCCCCTGCTTTGCAG 372 GGGCTCCCTGCAGCCCCTGCTTTGC 373 CTGGGCTCCCTGCAGCCCCTGCTTT 374 ATCTGGGCTCCCTGCAGCCCCTGCT 375 CCATCTGGGCTCCCTGCAGCCCCTG 376 CCCCATCTGGGCTCCCTGCAGCCCC 377 TGCCCCATCTGGGCTCCCTGCAGCC 378 GCTGCCCCATCTGGGCTCCCTGCAG 379 GGGCTGCCCCATCTGGGCTCCCTGC 380 CAGGGCTGCCCCATCTGGGCTCCCT 381 ACCAGGGCTGCCCCATCTGGGCTCC 382 GCACCAGGGCTGCCCCATCTGGGCT 383 AAGCACCAGGGCTGCCCCATCTGGG 384 AGAAGCACCAGGGCTGCCCCATCTG 385 GAAGAAGCACCAGGGCTGCCCCATC 386 GGGAAGAAGCACCAGGGCTGCCCCA 387 GTGGGAAGAAGCACCAGGGCTGCCC 388 GGGTGGGAAGAAGCACCAGGGCTGC 389 CTGGGTGGGAAGAAGCACCAGGGCT 390 AGCTGGGTGGGAAGAAGCACCAGGG 391 c.413T > A CAGCTTGTAGCTGGGGTAGCTGGGT 392 TCCAGCTTGTAGCTGGGGTAGCTGG 393 TCTCCAGCTTGTAGCTGGGGTAGCT 394 GTTCTCCAGCTTGTAGCTGGGGTAG 395 AGGTTCTCCAGCTTGTAGCTGGGGT 396 TCAGGTTCTCCAGCTTGTAGCTGGG 397 GCTCAGGTTCTCCAGCTTGTAGCTG 398 GAGCTCAGGTTCTCCAGCTTGTAGC 399 AGGAGCTCAGGTTCTCCAGCTTGTA 400 AGAGGAGCTCAGGTTCTCCAGCTTG 401 TCAGAGGAGCTCAGGTTCTCCAGCT 402 TTTCAGAGGAGCTCAGGTTCTCCAG 403 AGCTTGTAGCTGGGGTAGCTGGGTG 404 CCAGCTTGTAGCTGGGGTAGCTGGG 405 CTCCAGCTTGTAGCTGGGGTAGCTG 406 TTCTCCAGCTTGTAGCTGGGGTAGC 407 GGTTCTCCAGCTTGTAGCTGGGGTA 408 CAGGTTCTCCAGCTTGTAGCTGGGG 409 CTCAGGTTCTCCAGCTTGTAGCTGG 410 AGCTCAGGTTCTCCAGCTTGTAGCT 411 GGAGCTCAGGTTCTCCAGCTTGTAG 412 GAGGAGCTCAGGTTCTCCAGCTTGT 413 CAGAGGAGCTCAGGTTCTCCAGCTT 414 TTCAGAGGAGCTCAGGTTCTCCAGC 415 ATTTCAGAGGAGCTCAGGTTCTCCA 416 c.469C > T, c.476T > C, GGGGTGGTACGGGTCAGGGTGGCCG 417 c.476T > G, c.478T > G, TGGGGGTGGTACGGGTCAGGGTGGC 418 c.482C > T GGTGGGGGTGGTACGGGTCAGGGTG 419 AAGGTGGGGGTGGTACGGGTCAGGG 420 AGAAGGTGGGGGTGGTACGGGTCAG 421 GAAGAAGGTGGGGGTGGTACGGGTC 422 GGGAAGAAGGTGGGGGTGGTACGGG 423 TGGGGAAGAAGGTGGGGGTGGTACG 424 CTTGGGGAAGAAGGTGGGGGTGGTA 425 TCCTTGGGGAAGAAGGTGGGGGTGG 426 TGTCCTTGGGGAAGAAGGTGGGGGT 427 GATGTCCTTGGGGAAGAAGGTGGGG 428 AGGATGTCCTTGGGGAAGAAGGTGG 429 TCAGGATGTCCTTGGGGAAGAAGGT 430 GGTCAGGATGTCCTTGGGGAAGAAG 431 AGGGTCAGGATGTCCTTGGGGAAGA 432 GCAGGGTCAGGATGTCCTTGGGGAA 433 CCGCAGGGTCAGGATGTCCTTGGGG 434 AGCCGCAGGGTCAGGATGTCCTTGG 435 GGGTGGTACGGGTCAGGGTGGCCGT 436 GGGGGTGGTACGGGTCAGGGTGGCC 437 GTGGGGGTGGTACGGGTCAGGGTGG 438 AGGTGGGGGTGGTACGGGTCAGGGT 439 GAAGGTGGGGGTGGTACGGGTCAGG 440 AAGAAGGTGGGGGTGGTACGGGTCA 441 GGAAGAAGGTGGGGGTGGTACGGGT 442 GGGGAAGAAGGTGGGGGTGGTACGG 443 TTGGGGAAGAAGGTGGGGGTGGTAC 444 CCTTGGGGAAGAAGGTGGGGGTGGT 445 GTCCTTGGGGAAGAAGGTGGGGGTG 446 ATGTCCTTGGGGAAGAAGGTGGGGG 447 GGATGTCCTTGGGGAAGAAGGTGGG 448 CAGGATGTCCTTGGGGAAGAAGGTG 449 GTCAGGATGTCCTTGGGGAAGAAGG 450 GGGTCAGGATGTCCTTGGGGAAGAA 451 CAGGGTCAGGATGTCCTTGGGGAAG 452 CGCAGGGTCAGGATGTCCTTGGGGA 453 GCCGCAGGGTCAGGATGTCCTTGGG 454 CAGCCGCAGGGTCAGGATGTCCTTG 455 c.510C > T, c.515T > A, CGTCCAGCCGCAGGGTCAGGATGTC 456 c.520G > A CACGTCCAGCCGCAGGGTCAGGATG 457 ATCACGTCCAGCCGCAGGGTCAGGA 458 TCATCACGTCCAGCCGCAGGGTCAG 459 CATCATCACGTCCAGCCGCAGGGTC 460 TCCATCATCACGTCCAGCCGCAGGG 461 TCTCCATCATCACGTCCAGCCGCAG 462 AGTCTCCATCATCACGTCCAGCCGC 463 TCAGTCTCCATCATCACGTCCAGCC 464 TCTCAGTCTCCATCATCACGTCCAG 465 GTTCTCAGTCTCCATCATCACGTCC 466
CGGTTCTCAGTCTCCATCATCACGT 467 GGCGGTTCTCAGTCTCCATCATCAC 468 GAGGCGGTTCTCAGTCTCCATCATC 469 TGGAGGCGGTTCTCAGTCTCCATCA 470 AGTGGAGGCGGTTCTCAGTCTCCAT 471 GAAGTGGAGGCGGTTCTCAGTCTCC 472 GTCCAGCCGCAGGGTCAGGATGTCC 473 ACGTCCAGCCGCAGGGTCAGGATGT 474 TCACGTCCAGCCGCAGGGTCAGGAT 475 CATCACGTCCAGCCGCAGGGTCAGG 476 ATCATCACGTCCAGCCGCAGGGTCA 477 CCATCATCACGTCCAGCCGCAGGGT 478 CTCCATCATCACGTCCAGCCGCAGG 479 GTCTCCATCATCACGTCCAGCCGCA 480 CAGTCTCCATCATCACGTCCAGCCG 481 CTCAGTCTCCATCATCACGTCCAGC 482 TTCTCAGTCTCCATCATCACGTCCA 483 GGTTCTCAGTCTCCATCATCACGTC 484 GCGGTTCTCAGTCTCCATCATCACG 485 AGGCGGTTCTCAGTCTCCATCATCA 486 GGAGGCGGTTCTCAGTCTCCATCAT 487 GTGGAGGCGGTTCTCAGTCTCCATC 488 AAGTGGAGGCGGTTCTCAGTCTCCA 489 TGAAGTGGAGGCGGTTCTCAGTCTC 490 c.546+11C > T, TGCCCTGCCCACCGTGAAGTGGAGG 491 c.546+14G > A, CCTGCCCTGCCCACCGTGAAGTGGA 492 c.546+19G > A, CCCCTGCCCTGCCCACCGTGAAGTG 493 c.546+23C > A CGCCCCTGCCCTGCCCACCGTGAAG 494 CCCGCCCCTGCCCTGCCCACCGTGA 495 GCCCTGCCCACCGTGAAGTGGAGGC 496 CTGCCCTGCCCACCGTGAAGTGGAG 497 CCCTGCCCTGCCCACCGTGAAGTGG 498 GCCCCTGCCCTGCCCACCGTGAAGT 499 CCGCCCCTGCCCTGCCCACCGTGAA 500 CCCCGCCCCTGCCCTGCCCACCGTG 501 GCCCCCGCCCCTGCCCTGCCCACCG 502 CCGCCCCCGCCCCTGCCCTGCCCAC 503 CGCCGCCCCCGCCCCTGCCCTGCCC 504 GCCGCCGCCCCCGCCCCTGCCCTGC 505 TGGCCGCCGCCCCCGCCCCTGCCCT 506 CCTGGCCGCCGCCCCCGCCCCTGCC 507 GCCCTGGCCGCCGCCCCCGCCCCTG 508 CTGCCCTGGCCGCCGCCCCCGCCCC 509 CTCTGCCCTGGCCGCCGCCCCCGCC 510 CCCTCTGCCCTGGCCGCCGCCCCCG 511 CACCCTCTGCCCTGGCCGCCGCCCC 512 CGCACCCTCTGCCCTGGCCGCCGCC 513 CGCGCACCCTCTGCCCTGGCCGCCG 514 CCCCCGCCCCTGCCCTGCCCACCGT 515 CGCCCCCGCCCCTGCCCTGCCCACC 516 GCCGCCCCCGCCCCTGCCCTGCCCA 517 CCGCCGCCCCCGCCCCTGCCCTGCC 518 GGCCGCCGCCCCCGCCCCTGCCCTG 519 CTGGCCGCCGCCCCCGCCCCTGCCC 520 CCCTGGCCGCCGCCCCCGCCCCTGC 521 TGCCCTGGCCGCCGCCCCCGCCCCT 522 TCTGCCCTGGCCGCCGCCCCCGCCC 523 CCTCTGCCCTGGCCGCCGCCCCCGC 524 ACCCTCTGCCCTGGCCGCCGCCCCC 525 GCACCCTCTGCCCTGGCCGCCGCCC 526 GCGCACCCTCTGCCCTGGCCGCCGC 527 c.547-6 AGAGATGGGGGTTTATTGATGTTCC 528 GAAGAGATGGGGGTTTATTGATGTT 529 TAGAAGAGATGGGGGTTTATTGATG 530 TCTAGAAGAGATGGGGGTTTATTGA 531 GATCTAGAAGAGATGGGGGTTTATT 532 TTGATCTAGAAGAGATGGGGGTTTA 533 CTTTGATCTAGAAGAGATGGGGGTT 534 ATCTTTGATCTAGAAGAGATGGGGG 535 GGATCTTTGATCTAGAAGAGATGGG 536 CTGGATCTTTGATCTAGAAGAGATG 537 AGCTGGATCTTTGATCTAGAAGAGA 538 TTAGCTGGATCTTTGATCTAGAAGA 539 TGTTAGCTGGATCTTTGATCTAGAA 540
[0190] In the above examples the sequences are 25 nucleotides long however longer variants or shorter fragment are also envisioned. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 41-540 and fragments and variants thereof having at least 80% sequence identity. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 41-540 and fragments and variants thereof having at least 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% sequence identity to SEQ ID NO: 41-540.
[0191] The present invention is also directed to sequences that are at least 80% identical to SEQ ID NO: 41-540. Optionally at least 85% identical to SEQ ID NO: 41-540, more Optionally at least 88% identical to SEQ ID NO: 41-540, more Optionally at least 90% identical to SEQ ID NO: 41-540. more Optionally at least 91% identical to SEQ ID NO: 41-540, more Optionally at least 92% identical to SEQ ID NO: 41-540, more Optionally at least 93% identical to SEQ ID NO: 41-540, more Optionally at least 94% identical to SEQ ID NO: 41-540, more Optionally at least 95% identical to SEQ ID NO: 41-540, more Optionally at least 96% identical to SEQ ID NO: 41-540, more Optionally at least 97% identical to SEQ ID NO: 41-540, more Optionally at least 98% identical to SEQ ID NO: 41-540, more Optionally at least 99% identical to SEQ ID NO: 41-540.
[0192] Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 41-540, wherein the fragment is 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 41-540, wherein the fragment is 17, 18, 19, 20, 21, or 22 nucleotides long. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 41-540, wherein the fragment is 19, 20, or 21 nucleotides long.
[0193] Optionally of the invention and/or embodiments thereof the target sequence provides exclusion of intron 6. It was found that SEQ ID NO: 1584 provides the target sequence for exclusion of intron 6.
[0194] Optionally of the invention and/or embodiments thereof of an aspect and/or embodiments of the invention the target sequence is the AACCCCAGAGCTGCTTCCCTTCCAGATGTGGTCCTGCAGCCGAGCCCT GCCCTTAGCTGGAGGTCGACAGGTGGGATCCTGGATGTCTACATCTTC CTGGGCCCAGAGCCCAAGAGCGTGGTGCAGCAGTACCTGGACGTTGTG GGTAGGGCCTGCTCCCTGGCCGCGGCCCCCGCCCCAAGGCTCCCTCCT CCCTCCCTCATGAAGTCGGCGTTGGCCTGCAGGATACCCGTTCATGCC GCCATACTGGGGCCTGGGCTTCCACCTGTGCCGCTGGGGCTACTCCTC CACCGCTATCACCCGCCAGGTGGTGGAGAACATGACCAGGGCCCACTT CCCCCTGGTGAGTTGGGGTGGTGGCAGGGGAG (SEQ ID NO: 1584). It should be noted that also naturally occurring single nucleotide polymorphism are included.
[0195] Also the following genomic sequences are target sequences for exclusion of intron 6 of GAA:
TABLE-US-00004 Sequence in cDNA to which antisense oligomeric compound Seq anneals* sequence of region (5'.fwdarw.3'): ID c.956- AACCCCAGAGCTGCTTCCCTTCCAGAT 1584 25_1194+25 GTGGTCCTGCAGCCGAGCCCTGCCCTT AGCTGGAGGTCGACAGGTGGGATCCTG GATGTCTACATCTTCCTGGGCCCAGAG CCCAAGAGCGTGGTGCAGCAGTACCTG GACGTTGTGGGTAGGGCCTGCTCCCTG GCCGCGGCCCCCGCCCCAAGGCTCCCT CCTCCCTCCCTCATGAAGTCGGCGTTG GCCTGCAGGATACCCGTTCATGCCGCC ATACTGGGGCCTGGGCTTCCACCTGTG CCGCTGGGGCTACTCCTCCACCGCTAT CACCCGCCAGGTGGTGGAGAACATGAC CAGGGCCCACTTCCCCCTGGTGAGTTG GGGTGGTGGCAGGGGAG c.956-25_1004 AACCCCAGAGCTGCTTCCCTTCCAGAT 1585 GTGGTCCTGCAGCCGAGCCCTGCCCTT AGCTGGAGGTCGACAGGTGG c.1005_1075+3 GATCCTGGATGTCTACATCTTCCTGGG 1586 CCCAGAGCCCAAGAGCGTGGTGCAGCA GTACCTGGACGTTGTGGGTA c.1075+4_1076-2 GGGCCTGCTCCCTGGCCGCGGCCCCCG 1587 CCCCAAGGCTCCCTCCTCCCTCCCTCA TGAAGTCGGCGTTGGCCTGC c.1076-2_1147 AGGATACCCGTTCATGCCGCCATACTG 1588 GGGCCTGGGCTTCCACCTGTGCCGCTG GGGCTACTCCTCCACCGCTA c.1148_1194+25 TCACCCGCCAGGTGGTGGAGAACATGA 1589 CCAGGGCCCACTTCCCCCTGGTGAGTT GGGGTGGTGGCAGGGGAG
[0196] It is to be noted that targeting means that at least part of the sequence SEQ ID NO: 1584-1589 is targeted, e.g. by a sequence that hybridizes with at least a part or by the sequence SEQ ID NO: 1584-1589, or that binds to at least a part of SEQ ID NO: 1584-1589. Sequences that target may be shorter or longer than the target sequence.
[0197] Optionally the sequences targeting SEQ ID NO: 1584-1589 hybridize with at least a part of SEQ ID NO: 1584-1589. Sequences that hybridize may be shorter or longer than the target sequence. Nucleotide sequences SEQ ID NO: 541-1583 are oligomers that are able to enhance GAA intron 6 exclusion.
[0198] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound selected from the group comprising SEQ ID NO: 541-1583 and variants and fragments having at least 80% identity thereof. The antisense oligomeric compound may also target single nucleotide polymorphism of SEQ ID NO: 1584-1589. It should be noted that it may not necessary to have the full length of SEQ ID NO: 541-1583, fragments having a shorter or longer sequence are also envisioned. The inventors have found the target genomic sequence which enables the exclusion of intron 6 and a skilled person is capable of finding suitable sequences that target this target genomic sequence, such as SEQ ID NO: 1584-1589 and single nucleotide polymorphisms thereof. Exemplary sequences that target this target genomic sequence, such as SEQ ID NO: 1584-1589 may be SEQ ID NO: 541-1583, but also variants and fragments having at least 80% identity thereof. In particular shorter fragments such as fragments with 18, 19, 20, 21, 22, 23, or 24 nucleotides of SEQ ID NO: 541-1583 are envisioned.
[0199] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound complementary to a polynucleotide having a sequence selected from the group comprising SEQ ID NO: 1584-1589 and single nucleotide polymorphisms thereof. Also sequences having at least 80% identity to antisense oligomeric compound complementary to a polynucleotide having a sequence selected from the group comprising SEQ ID NO: 1584-1589 are envisioned. Antisense oligomeric compound that target one or more than one single nucleotide polymorphisms may be designed.
[0200] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound targeting a sequence selected from the group comprising the genomic sequence c.956-25_1194+25.
[0201] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to an antisense oligomeric compound comprising sequences selected from the group comprising SEQ ID NO: 41-1583, 1590-1594 and sequences having at least 80% identity thereof.
[0202] In one aspect or embodiment of aspects and/or embodiments thereof, the invention is directed to antisense oligomeric compound comprising a sequences selected from the group comprising SEQ ID NO: 541-1583, 1590-1594.
[0203] Antisense oligomeric compounds targeting SEQ ID NO: 1584 are a very suitable to treat Pompe patients. Exemplary antisense oligomeric compounds targeting SEQ ID NO: 1584 are SEQ ID NO: 541-1853. However the invention is not limited to these sequences. A skilled person is capable of designing antisense oligomeric compounds against target sequence SEQ ID NO: 1584, 1885, 1586, 1587, 1588, 1589. The antisense oligomeric compounds against target sequenced SEQ ID NO: 1584, 1885, 1586, 1587, 1588, or 1589 may have length of 10 to 100 nucleotides, Optionally 11 to 75 nucleotides, Optionally 12 to 73 nucleotides, Optionally 13 to 70 nucleotides, Optionally 14 to 65 nucleotides, Optionally 15 to 60 nucleotides, Optionally 16 to 55 nucleotides, Optionally 17 to 50 nucleotides, Optionally 18 to 45 nucleotides, Optionally 19 to 40 nucleotides, Optionally 20 to 38 nucleotides, Optionally 21 to 35 nucleotides, Optionally 22 to 33 nucleotides, Optionally 23 to 30 nucleotides, Optionally 24 to 29 nucleotides, Optionally 25 to 28 nucleotides, Optionally 26 to 27 nucleotides.
[0204] The antisense oligomeric compounds may be selected from the group of SEQ ID NO541-1583:
TABLE-US-00005 Sequence in cDNA to which AON anneals for intron 6 Seq exclusion AON sequence 5'.fwdarw.3' ID c.956-25_-1 CTGGAAGGGAAGCAGCTCTGGGGTT 541 c.956-24_956 TCTGGAAGGGAAGCAGCTCTGGGGT 542 c.956-23_957 ATCTGGAAGGGAAGCAGCTCTGGGG 543 c.956-22_958 CATCTGGAAGGGAAGCAGCTCTGGG 544 c.956-21_959 ACATCTGGAAGGGAAGCAGCTCTGG 545 c.956-20_960 CACATCTGGAAGGGAAGCAGCTCTG 546 c.956-19_961 CCACATCTGGAAGGGAAGCAGCTCT 547 c.956-18_962 ACCACATCTGGAAGGGAAGCAGCTC 548 c.956-17_963 GACCACATCTGGAAGGGAAGCAGCT 549 c.956-16_964 GGACCACATCTGGAAGGGAAGCAGC 550 c.956-15_965 AGGACCACATCTGGAAGGGAAGCAG 551 c.956-14_966 CAGGACCACATCTGGAAGGGAAGCA 552 c.956-13_967 GCAGGACCACATCTGGAAGGGAAGC 553 c.956-12_968 TGCAGGACCACATCTGGAAGGGAAG 554 c.956-11_969 CTGCAGGACCACATCTGGAAGGGAA 555 c.956-10_970 GCTGCAGGACCACATCTGGAAGGGA 556 c.956-9_971 GGCTGCAGGACCACATCTGGAAGGG 557 c.956-8_972 CGGCTGCAGGACCACATCTGGAAGG 558 c.956-7_973 TCGGCTGCAGGACCACATCTGGAAG 559 c.956-6_974 CTCGGCTGCAGGACCACATCTGGAA 560 c.956-5_975 GCTCGGCTGCAGGACCACATCTGGA 561 c.956-4_976 GGCTCGGCTGCAGGACCACATCTGG 562 c.956-3_977 GGGCTCGGCTGCAGGACCACATCTG 563 c.956-2_978 AGGGCTCGGCTGCAGGACCACATCT 564 c.956-1_979 CAGGGCTCGGCTGCAGGACCACATC 565 c.956_980 GCAGGGCTCGGCTGCAGGACCACAT 566 c.957_981 GGCAGGGCTCGGCTGCAGGACCACA 567 c.958_982 GGGCAGGGCTCGGCTGCAGGACCAC 568 c.959_983 AGGGCAGGGCTCGGCTGCAGGACCA 569 c.960_984 AAGGGCAGGGCTCGGCTGCAGGACC 570 c.961_985 TAAGGGCAGGGCTCGGCTGCAGGAC 571 c.962_986 CTAAGGGCAGGGCTCGGCTGCAGGA 572 c.963_987 GCTAAGGGCAGGGCTCGGCTGCAGG 573 c.964_988 AGCTAAGGGCAGGGCTCGGCTGCAG 574 c.965_989 CAGCTAAGGGCAGGGCTCGGCTGCA 575 c.966_990 CCAGCTAAGGGCAGGGCTCGGCTGC 576 c.967_991 TCCAGCTAAGGGCAGGGCTCGGCTG 577 c.968_992 CTCCAGCTAAGGGCAGGGCTCGGCT 578 c.969_993 CCTCCAGCTAAGGGCAGGGCTCGGC 579 c.970_994 ACCTCCAGCTAAGGGCAGGGCTCGG 580 c.971_995 GACCTCCAGCTAAGGGCAGGGCTCG 581 c.972_996 CGACCTCCAGCTAAGGGCAGGGCTC 582 c.973_997 TCGACCTCCAGCTAAGGGCAGGGCT 583 c.974_998 GTCGACCTCCAGCTAAGGGCAGGGC 584 c.975_999 TGTCGACCTCCAGCTAAGGGCAGGG 585 c.976_1000 CTGTCGACCTCCAGCTAAGGGCAGG 586 c.977_1001 CCTGTCGACCTCCAGCTAAGGGCAG 587 c.978_1002 ACCTGTCGACCTCCAGCTAAGGGCA 588 c.979_1003 CACCTGTCGACCTCCAGCTAAGGGC 589 c.980_1004 CCACCTGTCGACCTCCAGCTAAGGG 590 c.981_1005 CCCACCTGTCGACCTCCAGCTAAGG 591 c.982_1006 TCCCACCTGTCGACCTCCAGCTAAG 592 c.983_1007 ATCCCACCTGTCGACCTCCAGCTAA 593 c.984_1008 GATCCCACCTGTCGACCTCCAGCTA 594 c.985_1009 GGATCCCACCTGTCGACCTCCAGCT 595 c.986_1010 AGGATCCCACCTGTCGACCTCCAGC 596 c.987_1011 CAGGATCCCACCTGTCGACCTCCAG 597 c.988_1012 CCAGGATCCCACCTGTCGACCTCCA 598 c.989_1013 TCCAGGATCCCACCTGTCGACCTCC 599 c.990_1014 ATCCAGGATCCCACCTGTCGACCTC 600 c.991_1015 CATCCAGGATCCCACCTGTCGACCT 601 c.992_1016 ACATCCAGGATCCCACCTGTCGACC 602 c.993_1017 GACATCCAGGATCCCACCTGTCGAC 603 c.994_1018 AGACATCCAGGATCCCACCTGTCGA 604 c.995_1019 TAGACATCCAGGATCCCACCTGTCG 605 c.996_1020 GTAGACATCCAGGATCCCACCTGTC 606 c.997_1021 TGTAGACATCCAGGATCCCACCTGT 607 c.998_1022 ATGTAGACATCCAGGATCCCACCTG 608 c.999_1023 GATGTAGACATCCAGGATCCCACCT 609 c.1000_1024 AGATGTAGACATCCAGGATCCCACC 610 c.1001_1025 AAGATGTAGACATCCAGGATCCCAC 611 c.1002_1026 GAAGATGTAGACATCCAGGATCCCA 612 c.1003_1027 GGAAGATGTAGACATCCAGGATCCC 613 c.1004_1028 AGGAAGATGTAGACATCCAGGATCC 614 c.1005_1029 CAGGAAGATGTAGACATCCAGGATC 615 c.1006_1030 CCAGGAAGATGTAGACATCCAGGAT 616 c.1007_1031 CCCAGGAAGATGTAGACATCCAGGA 617 c.1008_1032 GCCCAGGAAGATGTAGACATCCAGG 618 c.1009_1033 GGCCCAGGAAGATGTAGACATCCAG 619 c.1010_1034 GGGCCCAGGAAGATGTAGACATCCA 620 c.1011_1035 TGGGCCCAGGAAGATGTAGACATCC 621 c.1012_1036 CTGGGCCCAGGAAGATGTAGACATC 622 c.1013_1037 TCTGGGCCCAGGAAGATGTAGACAT 623 c.1014_1038 CTCTGGGCCCAGGAAGATGTAGACA 624 c.1015_1039 GCTCTGGGCCCAGGAAGATGTAGAC 625 c.1016_1040 GGCTCTGGGCCCAGGAAGATGTAGA 626 c.1017_1041 GGGCTCTGGGCCCAGGAAGATGTAG 627 c.1018_1042 TGGGCTCTGGGCCCAGGAAGATGTA 628 c.1019_1043 TTGGGCTCTGGGCCCAGGAAGATGT 629 c.1020_1044 CTTGGGCTCTGGGCCCAGGAAGATG 630 c.1021_1045 TCTPGGGCTCTGGGCCCAGGAAGAT 631 c.1022_1046 CTCTTGGGCTCTGGGCCCAGGAAGA 632 c.1023_1047 GCTCTTGGGCTCTGGGCCCAGGAAG 633 c.1024_1048 CGCTCTTGGGCTCTGGGCCCAGGAA 634 c.1025_1049 ACGCTCTTGGGCTCTGGGCCCAGGA 635 c.1026_1050 CACGCTCTTGGGCTCTGGGCCCAGG 636 c.1027_1051 CCACGCTCTTGGGCTCTGGGCCCAG 637 c.1028_1052 ACCACGCTCTTGGGCTCTGGGCCCA 638 c.1029_1053 CACCACGCTCTTGGGCTCTGGGCCC 639 c.1030_1054 GCACCACGCTCTTGGGCTCTGGGCC 640 c.1031_1055 TGCACCACGCTCTTGGGCTCTGGGC 641 c.1032_1056 CTGCACCACGCTCTTGGGCTCTGGG 642 c.1033_1057 GCTGCACCACGCTCTTGGGCTCTGG 643 c.1034_1058 TGCTGCACCACGCTCTTGGGCTCTG 644 c.1035_1059 CTGCTGCACCACGCTCTTGGGCTCT 645 c.1036_1060 ACTGCTGCACCACGCTCTTGGGCTC 646 c.1037_1061 TACTGCTGCACCACGCTCTTGGGCT 647 c.1038_1062 GTACTGCTGCACCACGCTCTTGGGC 648 c.1039_1063 GGTACTGCTGCACCACGCTCTTGGG 649 c.1040_1064 AGGTACTGCTGCACCACGCTCTTGG 650 c.1041_1065 CAGGTACTGCTGCACCACGCTCTTG 651 c.1042_1066 CCAGGTACTGCTGCACCACGCTCTT 652 c.1043_1067 TCCAGGTACTGCTGCACCACGCTCT 653 c.1044_1068 GTCCAGGTACTGCTGCACCACGCTC 654 c.1045_1069 CGTCCAGGTACTGCTGCACCACGCT 655 c.1046_1070 ACGTCCAGGTACTGCTGCACCACGC 656 c.1047_1071 AACGTCCAGGTACTGCTGCACCACG 657 c.1048_1072 CAACGTCCAGGTACTGCTGCACCAC 658 c.1049_1073 ACAACGTCCAGGTACTGCTGCACCA 659 c.1050_1074 CACAACGTCCAGGTACTGCTGCACC 660 c.1051_1075 CCACAACGTCCAGGTACTGCTGCAC 661 c.1052_1075+1 CCCACAACGTCCAGGTACTGCTGCA 662
c.1053_1075+2 ACCCACAACGTCCAGGTACTGCTGC 663 c.1054_1075+3 TACCCACAACGTCCAGGTACTGCTG 664 c.1055_1075+4 CTACCCACAACGTCCAGGTACTGCT 665 c.1056_1075+5 CCTACCCACAACGTCCAGGTACTGC 666 c.1057_1075+6 CCCTACCCACAACGTCCAGGTACTG 667 c.1058_1075+7 GCCCTACCCACAACGTCCAGGTACT 668 c.1059_1075+8 GGCCCTACCCACAACGTCCAGGTAC 669 c.1060_1075+9 AGGCCCTACCCACAACGTCCAGGTA 670 c.1061_1075+10 CAGGCCCTACCCACAACGTCCAGGT 671 c.1062_1075+11 GCAGGCCCTACCCACAACGTCCAGG 672 c.1063_1075+12 AGCAGGCCCTACCCACAACGTCCAG 673 c.1064_1075+13 GAGCAGGCCCTACCCACAACGTCCA 674 c.1065_1075+14 GGAGCAGGCCCTACCCACAACGTCC 675 c.1066_1075+15 GGGAGCAGGCCCTACCCACAACGTC 676 c.1067_1075+16 AGGGAGCAGGCCCTACCCACAACGT 677 c.1068_1075+17 CAGGGAGCAGGCCCTACCCACAACG 678 c.1069_1075+18 CCAGGGAGCAGGCCCTACCCACAAC 679 c.1070_1075+19 GCCAGGGAGCAGGCCCTACCCACAA 680 c.1071_1075+20 GGCCAGGGAGCAGGCCCTACCCACA 681 c.1072_1075+21 CGGCCAGGGAGCAGGCCCTACCCAC 682 c.1073_1075+22 GCGGCCAGGGAGCAGGCCCTACCCA 683 c.1074_1075+23 CGCGGCCAGGGAGCAGGCCCTACCC 684 c.1075_1075+24 CCGCGGCCAGGGAGCAGGCCCTACC 685 C.1075+1_+25 GCCGCGGCCAGGGAGCAGGCCCTAC 686 C.1075+2_+26 GGCCGCGGCCAGGGAGCAGGCCCTA 687 C.1075+3_+27 GGGCCGCGGCCAGGGAGCAGGCCCT 688 C.1075+4_+28 GGGGCCGCGGCCAGGGAGCAGGCCC 689 C.1075+5_+29 GGGGGCCGCGGCCAGGGAGCAGGCC 690 C.1075+6_+30 CGGGGGCCGCGGCCAGGGAGCAGGC 691 C.1075+7_+31 GCGGGGGCCGCGGCCAGGGAGCAGG 692 C.1075+8_+32 GGCGGGGGCCGCGGCCAGGGAGCAG 693 C.1075+9_+33 GGGCGGGGGCCGCGGCCAGGGAGCA 694 C.1075+10_+34 GGGGCGGGGGCCGCGGCCAGGGAGC 695 C.1075+11_+35 TGGGGCGGGGGCCGCGGCCAGGGAG 696 C.1075+12_+36 TTGGGGCGGGGGCCGCGGCCAGGGA 697 C.1075+13_+37 CTTGGGGCGGGGGCCGCGGCCAGGG 698 C.1075+14_+38 CCTTGGGGCGGGGGCCGCGGCCAGG 699 C.1075+15_+39 GCCTTGGGGCGGGGGCCGCGGCCAG 700 C.1075+16_+40 AGCCTTGGGGCGGGGGCCGCGGCCA 701 C.1075+17_1076-39 GAGCCTTGGGGCGGGGGCCGCGGCC 702 C.1075+18_1076-38 GGAGCCTTGGGGCGGGGGCCGCGGC 703 C.1075+19_1076-37 GGGAGCCTTGGGGCGGGGGCCGCGG 704 C.1075+20_1076-36 AGGGAGCCTTGGGGCGGGGGCCGCG 705 C.1075+21_1076-35 GAGGGAGCCTTGGGGCGGGGGCCGC 706 C.1075+22_1076-34 GGAGGGAGCCTTGGGGCGGGGGCCG 707 C.1075+23_1076-33 AGGAGGGAGCCTTGGGGCGGGGGCC 708 C.1075+24_1076-32 GAGGAGGGAGCCTTGGGGCGGGGGC 709 C.1075+25_1076-31 GGAGGAGGGAGCCTTGGGGCGGGGG 710 C.1075+26_1076-30 GGGAGGAGGGAGCCTTGGGGCGGGG 711 C.1075+27_1076-29 AGGGAGGAGGGAGCCTTGGGGCGGG 712 C.1075+28_1076-28 GAGGGAGGAGGGAGCCTTGGGGCGG 713 C.1075+29_1076-27 GGAGGGAGGAGGGAGCCTTGGGGCG 714 C.1075+30_1076-26 GGGAGGGAGGAGGGAGCCTTGGGGC 715 C.1075+31_1076-25 AGGGAGGGAGGAGGGAGCCTTGGGG 716 C.1075+32_1076-24 GAGGGAGGGAGGAGGGAGCCTTGGG 717 C.1075+33_1076-23 TGAGGGAGGGAGGAGGGAGCCTTGG 718 C.1075+34_1076-22 ATGAGGGAGGGAGGAGGGAGCCTTG 719 C.1075+35_1076-21 CATGAGGGAGGGAGGAGGGAGCCTT 720 C.1075+36_1076-20 TCATGAGGGAGGGAGGAGGGAGCCT 721 C.1075+37_1076-19 TTCATGAGGGAGGGAGGAGGGAGCC 722 C.1075+38_1076-18 CTTCATGAGGGAGGGAGGAGGGAGC 723 C.1075+39_1076-17 ACTTCATGAGGGAGGGAGGAGGGAG 724 C.1075+40_1076-16 GACTTCATGAGGGAGGGAGGAGGGA 725 c.1076-39_-15 CGACTTCATGAGGGAGGGAGGAGGG 726 c.1076-38_-14 CCGACTTCATGAGGGAGGGAGGAGG 727 c.1076-37_-13 GCCGACTTCATGAGGGAGGGAGGAG 728 c.1076-36_-12 CGCCGACTTCATGAGGGAGGGAGGA 729 c.1076-35_-11 ACGCCGACTTCATGAGGGAGGGAGG 730 c.1076-34_-10 AACGCCGACTTCATGAGGGAGGGAG 731 c.1076-33_-9 CAACGCCGACTTCATGAGGGAGGGA 732 c.1076-32_-8 CCAACGCCGACTTCATGAGGGAGGG 733 c.1076-31_-7 GCCAACGCCGACTTCATGAGGGAGG 734 c.1076-30_-6 GGCCAACGCCGACTTCATGAGGGAG 735 c.1076-29_-5 AGGCCAACGCCGACTTCATGAGGGA 736 c.1076-28_-4 CAGGCCAACGCCGACTTCATGAGGG 737 c.1076-27_-3 GCAGGCCAACGCCGACTTCATGAGG 738 c.1076-26_-2 TGCAGGCCAACGCCGACTTCATGAG 739 c.1076-25_-1 CTGCAGGCCAACGCCGACTTCATGA 740 c.1076-24_1076 CCTGCAGGCCAACGCCGACTTCATG 741 c.1076-23_1077 TCCTGCAGGCCAACGCCGACTTCAT 742 c.1076-22_1078 ATCCTGCAGGCCAACGCCGACTTCA 743 c.1076-21_1079 TATCCTGCAGGCCAACGCCGACTTC 744 c.1076-20_1080 GTATCCTGCAGGCCAACGCCGACTT 745 c.1076-19_1081 GGTATCCTGCAGGCCAACGCCGACT 746 c.1076-18_1082 GGGTATCCTGCAGGCCAACGCCGAC 747 c.1076-17_1083 CGGGTATCCTGCAGGCCAACGCCGA 748 c.1076-16_1084 ACGGGTATCCTGCAGGCCAACGCCG 749 c.1076-15_1085 AACGGGTATCCTGCAGGCCAACGCC 750 c.1076-14_1086 GAACGGGTATCCTGCAGGCCAACGC 751 c.1076-13_1087 TGAACGGGTATCCTGCAGGCCAACG 752 c.1076-12_1088 ATGAACGGGTATCCTGCAGGCCAAC 753 c.1076-11_1089 CATGAACGGGTATCCTGCAGGCCAA 754 c.1076-10_1090 GCATGAACGGGTATCCTGCAGGCCA 755 c.1076-9_1091 GGCATGAACGGGTATCCTGCAGGCC 756 c.1076-8_1092 CGGCATGAACGGGTATCCTGCAGGC 757 c.1076-7_1093 GCGGCATGAACGGGTATCCTGCAGG 758 c.1076-6_1094 GGCGGCATGAACGGGTATCCTGCAG 759 c.1076-5_1095 TGGCGGCATGAACGGGTATCCTGCA 760 c.1076-4_1096 ATGGCGGCATGAACGGGTATCCTGC 761 c.1076-3_1097 TATGGCGGCATGAACGGGTATCCTG 762 c.1076-2_1098 GTATGGCGGCATGAACGGGTATCCT 763 c.1076-1_1099 AGTATGGCGGCATGAACGGGTATCC 764 c.1076_1100 CAGTATGGCGGCATGAACGGGTATC 765 c.1077_1101 CCAGTATGGCGGCATGAACGGGTAT 766 c.1078_1102 CCCAGTATGGCGGCATGAACGGGTA 767 c.1079_1103 CCCCAGTATGGCGGCATGAACGGGT 768 c.1080_1104 GCCCCAGTATGGCGGCATGAACGGG 769 c.1081_1105 GGCCCCAGTATGGCGGCATGAACGG 770 c.1082_1106 AGGCCCCAGTATGGCGGCATGAACG 771 c.1083_1107 CAGGCCCCAGTATGGCGGCATGAAC 772 c.1084_1108 CCAGGCCCCAGTATGGCGGCATGAA 773 c.1085_1109 CCCAGGCCCCAGTATGGCGGCATGA 774 c.1086_1110 GCCCAGGCCCCAGTATGGCGGCATG 775 c.1087_1111 AGCCCAGGCCCCAGTATGGCGGCAT 776 c.1088_1112 AAGCCCAGGCCCCAGTATGGCGGCA 777 c.1089_1113 GAAGCCCAGGCCCCAGTATGGCGGC 778 c.1090_1114 GGAAGCCCAGGCCCCAGTATGGCGG 779 c.1091_1115 TGGAAGCCCAGGCCCCAGTATGGCG 780 c.1092_1116 GTGGAAGCCCAGGCCCCAGTATGGC 781 c.1093_1117 GGTGGAAGCCCAGGCCCCAGTATGG 782 c.1094_1118 AGGTGGAAGCCCAGGCCCCAGTATG 783 c.1095_1119 CAGGTGGAAGCCCAGGCCCCAGTAT 784 c.1096_1120 ACAGGTGGAAGCCCAGGCCCCAGTA 785 c.1097_1121 CACAGGTGGAAGCCCAGGCCCCAGT 786 c.1098_1122 GCACAGGTGGAAGCCCAGGCCCCAG 787
c.1099_1123 GGCACAGGTGGAAGCCCAGGCCCCA 788 c.1100_1124 CGGCACAGGTGGAAGCCCAGGCCCC 789 c.1101_1125 GCGGCACAGGTGGAAGCCCAGGCCC 790 c.1102_1126 AGCGGCACAGGTGGAAGCCCAGGCC 791 c.1103_1127 CAGCGGCACAGGTGGAAGCCCAGGC 792 c.1104_1128 CCAGCGGCACAGGTGGAAGCCCAGG 793 c.1105_1129 CCCAGCGGCACAGGTGGAAGCCCAG 794 c.1106_1130 CCCCAGCGGCACAGGTGGAAGCCCA 795 c.1107_1131 GCCCCAGCGGCACAGGTGGAAGCCC 796 c.1108_1132 AGCCCCAGCGGCACAGGTGGAAGCC 797 c.1109_1133 TAGCCCCAGCGGCACAGGTGGAAGC 798 c.1110_1134 GTAGCCCCAGCGGCACAGGTGGAAG 799 c.1111_1135 AGTAGCCCCAGCGGCACAGGTGGAA 800 c.1112_1136 GAGTAGCCCCAGCGGCACAGGTGGA 801 c.1113_1137 GGAGTAGCCCCAGCGGCACAGGTGG 802 c.1114_1138 AGGAGTAGCCCCAGCGGCACAGGTG 803 c.1115_1139 GAGGAGTAGCCCCAGCGGCACAGGT 804 c.1116_1140 GGAGGAGTAGCCCCAGCGGCACAGG 805 c.1117_1141 TGGAGGAGTAGCCCCAGCGGCACAG 806 c.1118_1142 GTGGAGGAGTAGCCCCAGCGGCACA 807 c.1119_1143 GGTGGAGGAGTAGCCCCAGCGGCAC 808 c.1120_1144 CGGTGGAGGAGTAGCCCCAGCGGCA 809 c.1121_1145 GCGGTGGAGGAGTAGCCCCAGCGGC 810 c.1122_1146 AGCGGTGGAGGAGTAGCCCCAGCGG 811 c.1123_1147 TAGCGGTGGAGGAGTAGCCCCAGCG 812 c.1124_1148 ATAGCGGTGGAGGAGTAGCCCCAGC 813 c.1125_1149 GATAGCGGTGGAGGAGTAGCCCCAG 814 c.1126_1150 TGATAGCGGTGGAGGAGTAGCCCCA 815 c.1127_1151 GTGATAGCGGTGGAGGAGTAGCCCC 816 c.1128_1152 GGTGATAGCGGTGGAGGAGTAGCCC 817 c.1129_1153 GGGTGATAGCGGTGGAGGAGTAGCC 818 c.1130_1154 CGGGTGATAGCGGTGGAGGAGTAGC 819 c.1131_1155 GCGGGTGATAGCGGTGGAGGAGTAG 820 c.1132_1156 GGCGGGTGATAGCGGTGGAGGAGTA 821 c.1133_1157 TGGCGGGTGATAGCGGTGGAGGAGT 822 c.1134_1158 CTGGCGGGTGATAGCGGTGGAGGAG 823 c.1135_1159 CCTGGCGGGTGATAGCGGTGGAGGA 824 c.1136_1160 ACCTGGCGGGTGATAGCGGTGGAGG 825 c.1137_1161 CACCTGGCGGGTGATAGCGGTGGAG 826 c.1138_1162 CCACCTGGCGGGTGATAGCGGTGGA 827 c.1139_1163 ACCACCTGGCGGGTGATAGCGGTGG 828 c.1140_1164 CACCACCTGGCGGGTGATAGCGGTG 829 c.1141_1165 CCACCACCTGGCGGGTGATAGCGGT 830 c.1142_1166 TCCACCACCTGGCGGGTGATAGCGG 831 c.1143_1167 CTCCACCACCTGGCGGGTGATAGCG 832 c.1144_1168 TCTCCACCACCTGGCGGGTGATAGC 833 c.1145_1169 TTCTCCACCACCTGGCGGGTGATAG 834 c.1146_1170 GTTCTCCACCACCTGGCGGGTGATA 835 c.1147_1171 TGTTCTCCACCACCTGGCGGGTGAT 836 c.1148_1172 ATGTTCTCCACCACCTGGCGGGTGA 837 c.1149_1173 CATGTTCTCCACCACCTGGCGGGTG 838 c.1150_1174 TCATGTTCTCCACCACCTGGCGGGT 839 c.1151_1175 GTCATGTTCTCCACCACCTGGCGGG 840 c.1152_1176 GGTCATGTTCTCCACCACCTGGCGG 841 c.1153_1177 TGGTCATGTTCTCCACCACCTGGCG 842 c.1154_1178 CTGGTCATGTTCTCCACCACCTGGC 843 c.1155_1179 CCTGGTCATGTTCTCCACCACCTGG 844 c.1156_1180 CCCTGGTCATGTTCTCCACCACCTG 845 c.1157_1181 GCCCTGGTCATGTTCTCCACCACCT 846 c.1158_1182 GGCCCTGGTCATGTTCTCCACCACC 847 c.1159_1183 GGGCCCTGGTCATGTTCTCCACCAC 848 c.1160_1184 TGGGCCCTGGTCATGTTCTCCACCA 849 c.1161_1185 GTGGGCCCTGGTCATGTTCTCCACC 850 c.1162_1186 AGTGGGCCCTGGTCATGTTCTCCAC 851 c.1163_1187 AAGTGGGCCCTGGTCATGTTCTCCA 852 c.1164_1188 GAAGTGGGCCCTGGTCATGTTCTCC 853 c.1165_1189 GGAAGTGGGCCCTGGTCATGTTCTC 854 c.1166_1190 GGGAAGTGGGCCCTGGTCATGTTCT 855 c.1167_1191 GGGGAAGTGGGCCCTGGTCATGTTC 856 c.1168_1192 GGGGGAAGTGGGCCCTGGTCATGTT 857 c.1169_1193 AGGGGGAAGTGGGCCCTGGTCATGT 858 c.1170_1194 CAGGGGGAAGTGGGCCCTGGTCATG 859 c.1171_1194+1 CCAGGGGGAAGTGGGCCCTGGTCAT 860 c.1172_1194+2 ACCAGGGGGAAGTGGGCCCTGGTCA 861 c.1173_1194+3 CACCAGGGGGAAGTGGGCCCTGGTC 862 c.1174_1194+4 TCACCAGGGGGAAGTGGGCCCTGGT 863 c.1175_1194+5 CTCACCAGGGGGAAGTGGGCCCTGG 864 c.1176_1194+6 ACTCACCAGGGGGAAGTGGGCCCTG 865 c.1177_1194+7 AACTCACCAGGGGGAAGTGGGCCCT 866 c.1178_1194+8 CAACTCACCAGGCCGAAGTGGGCCC 867 c.1179_1194+9 CCAACTCACCAGGGGGAAGTGGGCC 868 c.1180_1194+10 CCCAACTCACCAGGGGGAAGTGGGC 869 c.1181_1194+11 CCCCAACTCACCAGGGGGAAGTGGG 870 c.1182_1194+12 ACCCCAACTCACCAGGGGGAAGTGG 871 c.1183_1194+13 CACCCCAACTCACCAGGGGGAAGTG 872 c.1184_1194+14 CCACCCCAACTCACCAGGGGGAAGT 873 c.1185_1194+15 ACCACCCCAACTCACCAGGGGGAAG 874 c.1186_1194+16 CACCACCCCAACTCACCAGGGGGAA 875 c.1187_1194+17 CCACCACCCCAACTCACCAGGGGGA 876 c.1188_1194+18 GCCACCACCCCAACTCACCAGGGGG 877 c.1189_1194+19 TGCCACCACCCCAACTCACCAGGGG 878 c.1190_1194+20 CTGCCACCACCCCAACTCACCAGGG 879 c.1191_1194+21 CCTGCCACCACCCCAACTCACCAGG 880 c.1192_1194+22 CCCTGCCACCACCCCAACTCACCAG 881 c.1193_1194+23 CCCCTGCCACCACCCCAACTCACCA 882 c.1194_1194+24 TCCCCTGCCACCACCCCAACTCACC 883 c.1194+1_+25 CTCCCCTGCCACCACCCCAACTCAC 884 c.956-25_-5 AAGGGAAGCAGCTCTGGGGTT 885 c.956-24_-4 GAAGGGAAGCAGCTCTGGGGT 886 c.956-23_-3 GGAAGGGAAGCAGCTCTGGGG 887 c.956-22_-2 TGGAAGGGAAGCAGCTCTGGG 888 c.956-21_-1 CTGGAAGGGAAGCAGCTCTGG 889 c.956-20_956 TCTGGAAGGGAAGCAGCTCTG 890 c.956-19_957 ATCTGGAAGGGAAGCAGCTCT 891 c.956-18_958 CATCTGGAAGGGAAGCAGCTC 892 c.956-17_959 ACATCTGGAAGGGAAGCAGCT 893 c.956-16_960 CACATCTGGAAGGGAAGCAGC 894 c.956-15_961 CCACATCTGGAAGGGAAGCAG 895 c.956-14_962 ACCACATCTGGAAGGGAAGCA 896 c.956-13_963 GACCACATCTGGAAGGGAAGC 897 c.956-12_964 GGACCACATCTGGAAGGGAAG 898 c.956-11_965 AGGACCACATCTGGAAGGGAA 899 c.956-10_966 CAGGACCACATCTGGAAGGGA 900 c.956-9_967 GCAGGACCACATCTGGAAGGG 901 c.956-8_968 TGCAGGACCACATCTGGAAGG 902 c.956-7_969 CTGCAGGACCACATCTGGAAG 903 c.956-6_970 GCTGCAGGACCACATCTGGAA 904 c.956-5_971 GGCTGCAGGACCACATCTGGA 905 c.956-4_972 CGGCTGCAGGACCACATCTGG 906 c.956-3_973 TCGGCTGCAGGACCACATCTG 907 c.956-2_974 CTCGGCTGCAGGACCACATCT 908 c.956-1_975 GCTCGGCTGCAGGACCACATC 909 c.956_976 GGCTCGGCTGCAGGACCACAT 910 c.957_977 GGGCTCGGCTGCAGGACCACA 911 c.958_978 AGGGCTCGGCTGCAGGACCAC 912 c.959_979 CAGGGCTCGGCTGCAGGACCA 913
c.960_980 GCAGGGCTCGGCTGCAGGACC 914 c.961_981 GGCAGGGCTCGGCTGCAGGAC 915 c.962_982 GGGCAGGGCTCGGCTGCAGGA 916 c.963_983 AGGGCAGGGCTCGGCTGCAGG 917 c.964_984 AAGGGCAGGGCTCGGCTGCAG 918 c.965_985 TAAGGGCAGGGCTCGGCTGCA 919 c.966_986 CTAAGGGCAGGGCTCGGCTGC 920 c.967_987 GCTAAGGGCAGGGCTCGGCTG 921 c.968_988 AGCTAAGGGCAGGGCTCGGCT 922 c.969_989 CAGCTAAGGGCAGGGCTCGGC 923 c.970_990 CCAGCTAAGGGCAGGGCTCGG 924 c.971_991 TCCAGCTAAGGGCAGGGCTCG 925 c.972_992 CTCCAGCTAAGGGCAGGGCTC 926 c.973_993 CCTCCAGCTAAGGGCAGGGCT 927 c.974_994 ACCTCCAGCTAAGGGCAGGGC 928 c.975_995 GACCTCCAGCTAAGGGCAGGG 929 c.976_996 CGACCTCCAGCTAAGGGCAGG 930 c.977_997 TCGACCTCCAGCTAAGGGCAG 931 c.978_998 GTCGACCTCCAGCTAAGGGCA 932 c.979_999 TGTCGACCTCCAGCTAAGGGC 933 c.980_1000 CTGTCGACCTGCAGCTAAGGG 934 c.981_1001 CCTGTCGACCTCCAGCTAAGG 935 c.982_1002 ACCTGTCGACCTCCAGCTAAG 936 c.983_1003 CACCTGTCGACCTCCAGCTAA 937 c.984_1004 CCACCTGTCGACCTCCAGCTA 938 c.985_1005 CCCACCTGTCGACCTCCAGCT 939 c.986_1006 TCCCACCTGTCGACCTCCAGC 940 c.987_1007 ATCCCACCTGTCGACCTCCAG 941 c.988_1008 GATCCCACCTGTCGACCTCCA 942 c.989_1009 GGATCCCACCTGTCGACCTCC 943 c.990_1010 AGGATCCCACCTGTCGACCTC 944 c.991_1011 CAGGATCCCACCTGTCGACCT 945 c.992_1012 CCAGGATCCCACCTGTCGACC 946 c.993_1013 TCCAGGATCCCACCTGTCGAC 947 c.994_1014 ATCCAGGATCCCACCTGTCGA 948 c.995_1015 CATCCAGGATCCCACCTGTCG 949 c.996_1016 ACATCCAGGATCCCACCTGTC 950 c.997_1017 GACATCCAGGATCCCACCTGT 951 c.998_1018 AGACATCCAGGATCCCACCTG 952 c.999_1019 TAGACATCCAGGATCCCACCT 953 c.1000_1020 GTAGACATCCAGGATCCCACC 954 c.1001_1021 TGTAGACATCCAGGATCCCAC 955 c.1002_1022 ATGTAGACATCCAGGATCCCA 956 c.1003_1023 GATGTAGACATCCAGGATCCC 957 c.1004_1024 AGATGTAGACATCCAGGATCC 958 c.1005_1025 AAGATGTAGACATCCAGGATC 959 c.1006_1026 GAAGATGTAGACATCCAGGAT 960 c.1007_1027 GGAAGATGTAGACATCCAGGA 961 c.1008_1028 AGGAAGATGTAGACATCCAGG 962 c.1009_1029 CAGGAAGATGTAGACATCCAG 963 c.1010_1030 CCAGGAAGATGTAGACATCCA 964 c.1011_1031 CCCAGGAAGATGTAGACATCC 965 c.1012_1032 GCCCAGGAAGATGTAGACATC 966 c.1013_1033 GGCCCAGGAAGATGTAGACAT 967 c.1014_1034 GGGCCCAGGAAGATGTAGACA 968 c.1015_1035 TGGGCCCAGGAAGATGTAGAC 969 c.1016_1036 CTGGGCCCAGGAAGATGTAGA 970 c.1017_1037 TCTGGGCCCAGGAAGATGTAG 971 c.1018_1038 CTCTGGGCCCAGGAAGATGTA 972 c.1019_1039 GCTCTGGGCCCAGGAAGATGT 973 c.1020_1040 GGCTCTGGGCCCAGGAAGATG 974 c.1021_1041 GGGCTCTGGGCCCAGGAAGAT 975 c.1022_1042 TGGGCTCTGGGCCCAGGAAGA 976 c.1023_1043 TTGGGCTCTGGGCCCAGGAAG 977 c.1024_1044 CTTGGGCTCTGGGCCCAGGAA 978 c.1025_1045 TCTTGGGCTCTGGGCCCAGGA 979 c.1026_1046 CTCTTGGGCTCTGGGCCCAGG 980 c.1027_1047 GCTCTTGGGCTCTGGGCCCAG 981 c.1028_1048 CGCTCTTGGGCTCTGGGCCCA 982 c.1029_1049 ACGCTCTTGGGCTCTGGGCCC 983 c.1030_1050 CACGCTCTTGGGCTCTGGGCC 984 c.1031_1051 CCACGCTCTTGGGCTCTGGGC 985 c.1032_1052 ACCACGCTCTTGGGCTCTGGG 986 c.1033_1053 CACCACGCTCTTGGGCTCTGG 987 c.1034_1054 GCACCACGCTCTTGGGCTCTG 988 c.1035_1055 TGCACCACGCTCTTGGGCTCT 989 c.1036_1056 CTGCACCACGCTCTTGGGCTC 990 c.1037_1057 GCTGCACCACGCTCTTGGGCT 991 c.1038_1058 TGCTGCACCACGCTCTTGGGC 992 c.1039_1059 CTGCTGCACCACGCTCTTGGG 993 c.1040_1060 ACTGCTGCACCACGCTCTTGG 994 c.1041_1061 TACTGCTGCACCACGCTCTTG 995 c.1042_1062 GTACTGCTGCACCACGCTCTT 996 c.1043_1063 GGTACTGCTGCACCACGCTCT 997 c.1044_1064 AGGTACTGCTGCACCACGCTC 998 c.1045_1065 CAGGTACTGCTGCACCACGCT 999 c.1046_1066 CCAGGTACTGCTGCACCACGC 1000 c.1047_1067 TCCAGGTACTGCTGCACCACG 1001 c.1048_1068 GTCCAGGTACTGCTGCACCAC 1002 c.1049_1069 CGTCCAGGTACTGCTGCACCA 1003 c.1050_1070 ACGTCCAGGTACTGCTGCACC 1004 c.1051_1071 AACGTCCAGGTACTGCTGCAC 1005 c.1052_1072 CAACGTCCAGGTACTGCTGCA 1006 c.1053_1073 ACAACGTCCAGGTACTGCTGC 1007 c.1054_1074 CACAACGTCCAGGTACTGCTG 1008 c.1055_1075 CCACAACGTCCAGGTACTGCT 1009 c.1056_1075+1 CCCACAACGTCCAGGTACTGC 1010 c.1057_1075+2 ACCCACAACGTCCAGGTACTG 1011 c.1058_1075+3 TACCCACAACGTCCAGGTACT 1012 c.1059_1075+4 CTACCCACAACGTCCAGGTAC 1013 c.1060_1075+5 CCTACCCACAACGTCCAGGTA 1014 c.1061_1075+6 CCCTACCCACAACGTCCAGGT 1015 c.1062_1075+7 GCCCTACCCACAACGTCCAGG 1016 c.1063_1075+8 GGCCCTACCCACAACGTCCAG 1017 c.1064_1075+9 AGGCCCTACCCACAACGTCCA 1018 c.1065_1075+10 CAGGCCCTACCCACAACGTCC 1019 c.1066_1075+11 GCAGGCCCTACCCACAACGTC 1020 c.1067_1075+12 AGCAGGCCCTACCCACAACGT 1021 c.1068_1075+13 GAGCAGGCCCTACCCACAACG 1022 c.1069_1075+14 GGAGCAGGCCCTACCCACAAC 1023 c.1070_1075+15 GGGAGCAGGCCCTACCCACAA 1024 c.1071_1075+16 AGGGAGCAGGCCCTACCCACA 1025 c.1072_1075+17 CAGGGAGCAGGCCCTACCCAC 1026 c.1073_1075+18 CCAGGGAGCAGGCCCTACCCA 1027 c.1074_1075+19 GCCAGGGAGCAGGCCCTACCC 1028 c.1075_1075+20 GGCCAGGGAGCAGGCCCTACC 1029 c.1075+1_+21 CGGCCAGGGAGCAGGCCCTAC 1030 c.1075+2_+22 GCGGCCAGGGAGCAGGCCCTA 1031 c.1075+3_+23 CGCGGCCAGGGAGCAGGCCCT 1032 c.1075+4_+24 CCGCGGCCAGGGAGCAGGCCC 1033 c.1075+5_+25 GCCGCGGCCAGGGAGCAGGCC 1034 c.1075+6_+26 GGCCGCGGCCAGGGAGCAGGC 1035 c.1075+7_+27 GGGCCGCGGCCAGGGAGCAGG 1036 c.1075+8_+28 GGGGCCGCGGCCAGGGAGCAG 1037 c.1075+9_+29 GGGGGCCGCGGCCAGGGAGCA 1038
c.1075+10_+30 CGGGGGCCGCGGCCAGGGAGC 1039 c.1075+11_+31 GCGGGGGCCGCGGCCAGGGAG 1040 c.1075+12_+32 GGCGGGGGCCGCGGCCAGGGA 1041 c.1075+13_+33 GGGCGGGGGCCGCGGCCAGGG 1042 c.1075+14_+34 GGGGCGGGGGCCGCGGCCAGG 1043 c.1075+15_+35 TGGGGCGGGGGCCGCGGCCAG 1044 c.1075+16_+33 TTGGGGCGGGGGCCGCGGCCA 1045 c.1075+17_+37 CTTGGGGCGGGGGCCGCGGCC 1046 c.1075+18_+38 CCTTGGGGCGGGGGCCGCGGC 1047 c.1075+19_+39 GCCTTGGGGCGGGGGCCGCGG 1048 c.1075+20_+40 AGCCTTGGGGCGGGGGCCGCG 1049 c.1075+21_1076-39 GAGCCTTGGGGCGGGGGCCGC 1050 c.1075+22_1076-38 GGAGCCTTGGGGCGGGGGCCG 1051 c.1075+23_1076-37 GGGAGCCTTGGGGCGGGGGCC 1052 c.1075+24_1076-36 AGGGAGCCTTGGGGCGGGGGC 1053 c.1075+25_1076-35 GAGGGAGCCTTGGGGCGGGGG 1054 c.1075+26_1076-34 GGAGGGAGCCTTGGGGCGGGG 1055 c.1075+27_1076-33 AGGAGGGAGCCTTGGGGCGGG 1056 c.1075+28_1076-32 GAGGAGGGAGCCTTGGGGCGG 1057 c.1075+29_1076-31 GGAGGAGGGAGCCTTGGGGCG 1058 c.1075+30_1076-30 GGGAGGAGGGAGCCTTGGGGC 1059 c.1075+31_1076-29 AGGGAGGAGGGAGCCTTGGGG 1060 c.1075+32_1076-28 GAGGGAGGAGGGAGCCTTGGG 1061 c.1075+33_1076-27 GGAGGGAGGAGGGAGCCTTGG 1062 c.1075+34_1076-26 GGGAGGGAGGAGGGAGCCTTG 1063 c.1075+35_1076-25 AGGGAGGGAGGAGGGAGCCTT 1064 c.1075+36_1076-24 GAGGGAGGGAGGAGGGAGCCT 1065 c.1075+37_1076-23 TGAGGGAGGGAGGAGGGAGCC 1066 c.1075+38_1076-22 ATGAGGGAGGGAGGAGGGAGC 1067 c.1075+39_1076-21 CATGAGGGAGGGAGGAGGGAG 1068 c.1075+40_1076-20 TCATGAGGGAGGGAGGAGGGA 1069 c.1076-39_-19 TTCATGAGGGAGGGAGGAGGG 1070 c.1076-38_-18 CTTCATGAGGGAGGGAGGAGG 1071 c.1076-37_-17 ACTTCATGAGGGAGGGAGGAG 1072 c.1076-36_-16 GACTTCATGAGGGAGGGAGGA 1073 c.1076-35_-15 CGACTTCATGAGGGAGGGAGG 1074 c.1076-34_-14 CCGACTTCATGAGGGAGGGAG 1075 c.1076-33_-13 GCCGACTTCATGAGGGAGGGA 1076 c.1076-32_-12 CGCCGACTTCATGAGGGAGGG 1077 c.1076-31_-11 ACGCCGACTTCATGAGGGAGG 1078 c.1076-30_-10 AACGCCGACTTCATGAGGGAG 1079 c.1076-29_-9 CAACGCCGACTTCATGAGGGA 1080 c.1076-28_-8 CCAACGCCGACTTCATGAGGG 1081 c.1076-27_-7 GCCAACGCCGACTTCATGAGG 1082 c.1076-26_-6 GGCCAACGCCGACTTCATGAG 1083 c.1076-25_-5 AGGCCAACGCCGACTTCATGA 1084 c.1076-24_-4 CAGGCCAACGCCGACTTCATG 1085 c.1076-23_-3 GCAGGCCAACGCCGACTTCAT 1086 c.1076-22_-2 TGCAGGCCAACGCCGACTTCA 1087 c.1076-21_-1 CTGCAGGCCAACGCCGACTTC 1088 c.1076-20_1076 CCTGCAGGCCAACGCCGACTT 1089 c.1076-19_1077 TCCTGCAGGCCAACGCCGACT 1090 c.1076-18_1078 ATCCTGCAGGCCAACGCCGAC 1091 c.1076-17_1079 TATCCTGCAGGCCAACGCCGA 1092 c.1076-16_1080 GTATCCTGCAGGCCAACGCCG 1093 c.1076-15_1081 GGTATCCTGCAGGCCAACGCC 1094 c.1076-14_1082 GGGTATCCTGCAGGCCAACGC 1095 c.1076-13_1083 CGGGTATCCTGCAGGCCAACG 1096 c.1076-12_1084 ACGGGTATCCTGCAGGCCAAC 1097 c.1076-11_1085 AACGGGTATCCTGCAGGCCAA 1098 c.1076-10_1086 GAACGGGTATCCTGCAGGCCA 1099 c.1076-9_1087 TGAACGGGTATCCTGCAGGCC 1100 c.1076-8_1088 ATGAACGGGTATCCTGCAGGC 1101 c.1076-7_1089 CATGAACGGGTATCCTGCAGG 1102 c.1076-6_1090 GCATGAACGGGTATCCTGCAG 1103 c.1076-5_1091 GGCATGAACGGGTATCCTGCA 1104 c.1076-4_1092 CGGCATGAACGGGTATCCTGC 1105 c.1076-3_1093 GCGGCATGAACGGGTATCCTG 1106 c.1076-2_1094 GGCGGCATGAACGGGTATCCT 1107 c.1076-1_1095 TGGCGGCATGAACGGGTATCC 1108 c.1076_1096 ATGGCGGCATGAACGGGTATC 1109 c.1077_1097 TATGGCGGCATGAACGGGTAT 1110 c.1078_1098 GTATGGCGGCATGAACGGGTA 1111 c.1079_1099 AGTATGGCGGCATGAACGGGT 1112 c.1080_1100 CAGTATGGCGGCATGAACGGG 1113 c.1081_1101 CCAGTATGGCGGCATGAACGG 1114 c.1082_1102 CCCAGTATGGCGGCATGAACG 1115 c.1083_1103 CCCCAGTATGGCGGCATGAAC 1116 c.1084_1104 GCCCCAGTATGGCGGCATGAA 1117 c.1085_1105 GGCCCCAGTATGGCGGCATGA 1118 c.1086_1106 AGGCCCCAGTATGGCGGCATG 1119 c.1087_1107 CAGGCCCCAGTATGGCGGCAT 1120 c.1088_1108 CCAGGCCCCAGTATGGCGGCA 1121 c.1089_1109 CCCAGGCCCCAGTATGGCGGC 1122 c.1090_1110 GCCCAGGCCCCAGTATGGCGG 1123 c.1091_1111 AGCCCAGGCCCCAGTATGGCG 1124 c.1092_1112 AAGCCCAGGCCCCAGTATGGC 1125 c.1093_1113 GAAGCCCAGGCCCCAGTATGG 1126 c.1094_1114 GGAAGCCCAGGCCCCAGTATG 1127 c.1095_1115 TGGAAGCCCAGGCCCCAGTAT 1128 c.1096_1116 GTGGAAGCCCAGGCCCCAGTA 1129 c.1097_1117 GGTGGAAGCCCAGGCCCCAGT 1130 c.1098_1118 AGGTGGAAGCCCAGGCCCCAG 1131 c.1099_1119 CAGGTGGAAGCCCAGGCCCCA 1132 c.1100_1120 ACAGGTGGAAGCCCAGGCCCC 1133 c.1101_1121 CACAGGTGGAAGCCCAGGCCC 1134 c.1102_1122 GCACAGGTGGAAGCCCAGGCC 1135 c.1103_1123 GGCACAGGTGGAAGCCCAGGC 1136 c.1104_1124 CGGCACAGGTGGAAGCCCAGG 1137 c.1105_1125 GCGGCACAGGTGGAAGCCCAG 1138 c.1106_1126 AGCGGCACAGGTGGAAGCCCA 1139 c.1107_1127 CAGCGGCACAGGTGGAAGCCC 1140 c.1108_1128 CCAGCGGCACAGGTGGAAGCC 1141 c.1109_1129 CCCAGCGGCACAGGTGGAAGC 1142 c.1110_1130 CCCCAGCGGCACAGGTGGAAG 1143 c.1111_1131 GCCCCAGCGGCACAGGTGGAA 1144 c.1112_1132 AGCCCCAGCGGCACAGGTGGA 1145 c.1113_1133 TAGCCCCAGCGGCACAGGTGG 1146 c.1114_1134 GTAGCCCCAGCGGCACAGGTG 1147 c.1115_1135 AGTAGCCCCAGCGGCACAGGT 1148 c.1116_1136 GAGTAGCCCCAGCGGCACAGG 1149 c.1117_1137 GGAGTAGCCCCAGCGGCACAG 1150 c.1118_1138 AGGAGTAGCCCCAGCGGCACA 1151 c.1119_1139 GAGGAGTAGCCCCAGCGGCAC 1152 c.1120_1140 GGAGGAGTAGCCCCAGCGGCA 1153 c.1121_1141 TGGAGGAGTAGCCCCAGCGGC 1154 c.1122_1142 GTGGAGGAGTAGCCCCAGCGG 1155 c.1123_1143 GGTGGAGGAGTAGCCCCAGCG 1156 c.1124_1144 CGGTGGAGGAGTAGCCCCAGC 1157 c.1125_1145 GCGGTGGAGGAGTAGCCCCAG 1158 c.1126_1146 AGCGGTGGAGGAGTAGCCCCA 1159 c.1127_1147 TAGCGGTGGAGGAGTAGCCCC 1160 c.1128_1148 ATAGCGGTGGAGGAGTAGCCC 1161 c.1129_1149 GATAGCGGTGGAGGAGTAGCC 1162 c.1130_1150 TGATAGCGGTGGAGGAGTAGC 1163 c.1131_1151 GTGATAGCGGTGGAGGAGTAG 1164
c.1132_1152 GGTGATAGCGGTGGAGGAGTA 1165 c.1133_1153 GGGTGATAGCGGTGGAGGAGT 1166 c.1134_1154 CGGGTGATAGCGGTGGAGGAG 1167 c.1135_1155 GCGGGTGATAGCGGTGGAGGA 1168 c.1136_1156 GGCGGGTGATAGCGGTGGAGG 1169 c.1137_1157 TGGCGGGTGATAGCGGTGGAG 1170 c.1138_1158 CTGGCGGGTGATAGCGGTGGA 1171 c.1139_1159 CCTGGCGGGTGATAGCGGTGG 1172 c.1140_1160 ACCTGGCGGGTGATAGCGGTG 1173 c.1141_1161 CACCTGGCGGGTGATAGCGGT 1174 c.1142_1162 CCACCTGGCGGGTGATAGCGG 1175 c.1143_1163 ACCACCTGGCGGGTGATAGCG 1176 c.1144_1164 CACCACCTGGCGGGTGATAGC 1177 c.1145_1165 CCACCACCTGGCGGGTGATAG 1178 c.1146_1166 TCCACCACCTGGCGGGTGATA 1179 c.1147_1167 CTCCACCACCTGGCGGGTGAT 1180 c.1148_1168 TCTCCACCACCTGGCGGGTGA 1181 c.1149_1169 TTCTCCACCACCTGGCGGGTG 1182 c.1150_1170 GTTCTCCACCACCTGGCGGGT 1183 c.1151_1171 TGTTCTCCACCACCTGGCGGG 1184 c.1152_1172 ATGTTCTCCACCACCTGGCGG 1185 c.1153_1173 CATGTTCTCCACCACCTGGCG 1186 c.1154_1174 TCATGTTCTCCACCACCTGGC 1187 c.1155_1175 GTCATGTTCTCCACCACCTGG 1188 c.1156_1176 GGTCATGTTCTCCACCACCTG 1189 c.1157_1177 TGGTCATGTTCTCCACCACCT 1190 c.1158_1178 CTGGTCATGTTCTCCACCACC 1191 c.1159_1179 CCTGGTCATGTTCTCCACCAC 1192 c.1160_1180 CCCTGGTCATGTTCTCCACCA 1193 c.1161_1181 GCCCTGGTCATGTTCTCCACC 1194 c.1162_1182 GGCCCTGGTCATGTTCTCCAC 1195 c.1163_1183 GGGCCCTGGTCATGTTCTCCA 1196 c.1164_1184 TGGGCCCTGGTCATGTTCTCC 1197 c.1165_1185 GTGGGCCCTGGTCATGTTCTC 1198 c.1166_1186 AGTGGGCCCTGGTCATGTTCT 1199 c.1167_1187 AAGTGGGCCCTGGTCATGTTC 1200 c.1168_1188 GAAGTGGGCCCTGGTCATGTT 1201 c.1169_1189 GGAAGTGGGCCCTGGTCATGT 1202 c.1170_1190 GGGAAGTGGGCCCTGGTCATG 1203 c.1171_1191 GGGGAAGTGGGCCCTGGTCAT 1204 c.1172_1192 GGGGGAAGTGGGCCCTGGTCA 1205 c.1173_1193 AGGGGGAAGTGGGCCCTGGTC 1206 c.1174_1194 CAGGGGGAAGTGGGCCCTGGT 1207 c.1175_1194+1 CCAGGGGGAAGTGGGCCCTGG 1208 c.1176_1194+2 ACCAGGGGGAAGTGGGCCCTG 1209 c.1177_1194+3 CACCAGGGGGAAGTGGGCCCT 1210 c.1178_1194+4 TCACCAGGGGGAAGTGGGCCC 1211 c.1179_1194+5 CTCACCAGGGGGAAGTGGGCC 1212 c.1180_1194+6 ACTCACCAGGGGGAAGTGGGC 1213 c.1181_1194+7 AACTCACCAGGGGGAAGTGGG 1214 c.1182_1194+8 CAACTCACCAGGGGGAAGTGG 1215 c.1183_1194+9 CCAACTCACCAGGGGGAAGTG 1216 c.1184_1194+10 CCCAACTCACCAGGGGGAAGT 1217 c.1185_1194+11 CCCCAACTCACCAGGGGGAAG 1218 c.1186_1194+12 ACCCCAACTCACCAGGGGGAA 1219 c.1187_1194+13 CACCCCAACTCACCAGGGGGA 1220 c.1188_1194+14 CCACCCCAACTCACCAGGGGG 1221 c.1189_1194+15 ACCACCCCAACTCACCAGGGG 1222 c.1190_1194+16 CACCACCCCAACTCACCAGGG 1223 c.1191_1194+17 CCACCACCCCAACTCACCAGG 1224 c.1192_1194+18 GCCACCACCCCAACTCACCAG 1225 c.1193_1194+19 TGCCACCACCCCAACTCACCA 1226 c.1194_1194+20 CTGCCACCACCCCAACTCACC 1227 c.1194+1_+21 CCTGCCACCACCCCAACTCAC 1228 c.1194+2_+22 CCCTGCCACCACCCCAACTCA 1229 c.1194+3_+23 CCCCTGCCACCACCCCAACTC 1230 c.1194+4_+24 TCCCCTGCCACCACCCCAACT 1231 c.1194+5_+25 CTCCCCTGCCACCACCCCAAC 1232 c.956-25_-8 GGAAGCAGCTCTGGGGTT 1233 c.956-24_-7 GGGAAGCAGCTCTGGGGT 1234 c.956-23_-6 AGGGAAGCAGCTCTGGGG 1235 c.956-22_-5 AAGGGAAGCAGCTCTGGG 1236 c.956-21_-4 GAAGGGAAGCAGCTCTGG 1237 c.956-20_-3 GGAAGGGAAGCAGCTCTG 1238 c.956-19_-2 TGGAAGGGAAGCAGCTCT 1239 c.956-18_-1 CTGGAAGGGAAGCAGCTC 1240 c.956-17_956 TCTGGAAGGGAAGCAGCT 1241 c.956-16_957 ATCTGGAAGGGAAGCAGC 1242 c.956-15_958 CATCTGGAAGGGAAGCAG 1243 c.956-14_959 ACATCTGGAAGGGAAGCA 1244 c.956-13_960 CACATCTGGAAGGGAAGC 1245 c.956-12_961 CCACATCTGGAAGGGAAG 1246 c.956-11_962 ACCACATCTGGAAGGGAA 1247 c.956-10_963 GACCACATCTGGAAGGGA 1248 c.956-9_964 GGACCACATCTGGAAGGG 1249 c.956-8_965 AGGACCACATCTGGAAGG 1250 c.956-7_966 CAGGACCACATCTGGAAG 1251 c.956-6_967 GCAGGACCACATCTGGAA 1252 c.956-5_968 TGCAGGACCACATCTGGA 1253 c.956-4_969 CTGCAGGACCACATCTGG 1254 c.956-3_970 GCTGCAGGACCACATCTG 1255 c.956-2_971 GGCTGCAGGACCACATCT 1256 c.956-1_972 CGGCTGCAGGACCACATC 1257 c.956_973 TCGGCTGCAGGACCACAT 1258 c.957_974 CTCGGCTGCAGGACCACA 1259 c.958_975 GCTCGGCTGCAGGACCAC 1260 c.959_976 GGCTCGGCTGCAGGACCA 1261 c.960_977 GGGCTCGGCTGCAGGACC 1262 c.961_978 AGGGCTCGGCTGCAGGAC 1263 c.962_979 CAGGGCTCGGCTGCAGGA 1264 c.963_980 GCAGGGCTCGGCTGCAGG 1265 c.964_981 GGCAGGGCTCGGCTGCAG 1266 c.965_982 GGGCAGGGCTCGGCTGCA 1267 c.966_983 AGGGCAGGGCTCGGCTGC 1268 c.967_984 AAGGGCAGGGCTCGGCTG 1269 c.968_985 TAAGGGCAGGGCTCGGCT 1270 c.969_986 CTAAGGGCAGGGCTCGGC 1271 c.970_987 GCTAAGGGCAGGGCTCGG 1272 c.971_988 AGCTAAGGGCAGGGCTCG 1273 c.972_989 CAGCTAAGGGCAGGGCTC 1274 c.973_990 CCAGCTAAGGGCAGGGCT 1275 c.974_991 TCCAGCTAAGGGCAGGGC 1276 c.975_992 CTCCAGCTAAGGGCAGGG 1277 c.976_993 CCTCCAGCTAAGGGCAGG 1278 c.977_994 ACCTCCAGCTAAGGGCAG 1279 c.978_995 GACCTCCAGCTAAGGGCA 1280 c.979_996 CGACCTCCAGCTAAGGGC 1281 c.980_997 TCGACCTCCAGCTAAGGG 1282 c.981_998 GTCGACCTCCAGCTAAGG 1283 c.982_999 TGTCGACCTCCAGCTAAG 1284 c.983_1000 CTGTCGACCTCCAGCTAA 1285 c.984_1001 CCTGTCGACCTCCAGCTA 1286 c.985_1002 ACCTGTCGACCTCCAGCT 1287 c.986_1003 CACCTGTCGAGCTGCAGC 1288 c.987_1004 CCACCTGTCGACCTCCAG 1289
c.988_1005 CCCACCTGTCGACCTCCA 1290 c.989_1006 TCCCACCTGTCGACCTCC 1291 c.990_1007 ATCCCACCTGTCGACCTC 1292 c.991_1008 GATCCCACCTGTCGACCT 1293 c.992_1009 GGATCCCACCTGTCGACC 1294 c.993_1010 AGGATCCCACCTGTCGAC 1295 c.994_1011 CAGGATCCCACCTGTCGA 1296 c.995_1012 CCAGGATCCCACCTGTCG 1297 c.996_1013 TCCAGGATCCCACCTGTC 1298 c.997_1014 ATCCAGGATCCCACCTGT 1299 c.998_1015 CATCCAGGATCCCACCTG 1300 c.999_1016 ACATCCAGGATCCCACCT 1301 c.1000_1017 GACATCCAGGATCCCACC 1302 c.1001_1018 AGACATCCAGGATCCCAC 1303 c.1002_1019 TAGACATCCAGGATCCCA 1304 c.1003_1020 GTAGACATCCAGGATCCC 1305 c.1004_1021 TGTAGACATCCAGGATCC 1306 c.1005_1022 ATGTAGACATCCAGGATC 1307 c.1006_1023 GATGTAGACATCCAGGAT 1308 c.1007_1024 AGATGTAGACATCCAGGA 1309 c.1008_1025 AAGATGTAGACATCCAGG 1310 c.1009_1026 GAAGATGTAGACATCCAG 1311 c.1010_1027 GGAAGATGTAGACATCCA 1312 c.1011_1028 AGGAAGATGTAGACATCC 1313 c.1012_1029 CAGGAAGATGTAGACATC 1314 c.1013_1030 CCAGGAAGATGTAGACAT 1315 c.1014_1031 CCCAGGAAGATGTAGACA 1316 c.1015_1032 GCCCAGGAAGATGTAGAC 1317 c.1016_1033 GGCCCAGGAAGATGTAGA 1318 c.1017_1034 GGGCCCAGGAAGATGTAG 1319 c.1018_1035 TGGGCCCAGGAAGATGTA 1320 c.1019_1036 CTGGGCCCAGGAAGATGT 1321 c.1020_1037 TCTGGGCCCAGGAAGATG 1322 c.1021_1038 CTCTGGGCCCAGGAAGAT 1323 c.1022_1039 GCTCTGGGCCCAGGAAGA 1324 c.1023_1040 GGCTCTGGGCCCAGGAAG 1325 c.1024_1041 GGGCTCTGGGCCCAGGAA 1326 c.1025_1042 TGGGCTCTGGGCCCAGGA 1327 c.1026_1043 TTGGGCTCTGGGCCCAGG 1328 c.1027_1044 CTTGGGCTCTGGGCCCAG 1329 c.1028_1045 TCTTGGGCTCTGGGCCCA 1330 c.1029_1046 CTCTTGGGCTCTGGGCCC 1331 c.1030_1047 GCTCTTGGGCTCTGGGCC 1332 c.1031_1048 CGCTCTTGGGCTCTGGGC 1333 c.1032_1049 ACGCTCTTGGGCTCTGGG 1334 c.1033_1050 CACGCTCTTGGGCTCTGG 1335 c.1034_1051 CCACGCTCTTGGGCTCTG 1336 c.1035_1052 ACCACGCTCTTGGGCTCT 1337 c.1036_1053 CACCACGCTCTTGGGCTC 1338 c.1037_1054 GCACCACGCTCTTGGGCT 1339 c.1038_1055 TGCACCACGCTCTTGGGC 1340 c.1039_1056 CTGCACCACGCTCTTGGG 1341 c.1040_1057 GCTGCACCACGCTCTTGG 1342 c.1041_1058 TGCTGCACCACGCTCTTG 1343 c.1042_1059 CTGCTGCACCACGCTCTT 1344 c.1043_1060 ACTGCTGCACCACGCTCT 1345 c.1044_1061 TACTGCTGCACCACGCTC 1346 c.1045_1062 GTACTGCTGCACCACGCT 1347 c.1046_1063 GGTACTGCTGCACCACGC 1348 c.1047_1064 AGGTACTGCTGCACCACG 1349 c.1048_1065 CAGGTACTGCTGCACCAC 1350 c.1049_1066 CCAGGTACTGCTGCACCA 1351 c.1050_1067 TCCAGGTACTGCTGCACC 1352 c.1051_1068 GTCCAGGTACTGCTGCAC 1353 c.1052_1069 CGTCCAGGTACTGCTGCA 1354 c.1053_1070 ACGTCCAGGTACTGCTGC 1355 c.1054_1071 AACGTCCAGGTACTGCTG 1356 c.1055_1072 CAACGTCCAGGTACTGCT 1357 c.1056_1073 ACAACGTCCAGGTACTGC 1358 c.1057_1074 CACAACGTCCAGGTACTG 1359 c.1058_1075 CCACAACGTCCAGGTACT 1360 c.1059_1075+1 CCCACAACGTCCAGGTAC 1361 c.1060_1075+2 ACCCACAACGTCCAGGTA 1362 c.1061_1075+3 TACCCACAACGTCCAGGT 1363 c.1062_1075+4 CTACCCACAACGTCCAGG 1364 c.1063_1075+5 CCTACCCACAACGTCCAG 1365 c.1064_1075+6 CCCTACCCACAACGTCCA 1366 c.1065_1075+7 GCCCTACCCACAACGTCC 1367 c.1066_1075+8 GGCCCTACCCACAACGTC 1368 c.1067_1075+9 AGGCCCTACCCACAACGT 1369 c.1068_1075+10 CAGGCCCTACCCACAACG 1370 c.1069_1075+11 GCAGGCCCTACCCACAAC 1371 c.1070_1075+12 AGCAGGCCCTACCCACAA 1372 c.1071_1075+13 GAGCAGGCCCTACCCACA 1373 c.1072_1075+14 GGAGCAGGCCCTACCCAC 1374 c.1073_1075+15 GGGAGCAGGCCCTACCCA 1375 c.1074_1075+16 AGGGAGCAGGCCCTACCC 1376 c.1075_1075+17 CAGGGAGCAGGCCCTACC 1377 c.1075+1_+18 CCAGGGAGCAGGCCCTAC 1378 c.1075+2_+19 GCCAGGGAGCAGGCCCTA 1379 c.1075+3_+20 GGCCAGGGAGCAGGCCCT 1380 c.1075+4_+21 CGGCCAGGGAGCAGGCCC 1381 c.1075+5_+22 GCGGCCAGGGAGCAGGCC 1382 c.1075+6_+23 CGCGGCCAGGGAGCAGGC 1383 c.1075+7_+24 CCGCGGCCAGGGAGCAGG 1384 c.1075+8_+25 GCCGCGGCCAGGGAGCAG 1385 c.1075+9_+26 GGCCGCGGCCAGGGAGCA 1386 c.1075+10_+27 GGGCCGCGGCCAGGGAGC 1387 c.1075+11_+28 GGGGCCGCGGCCAGGGAG 1388 c.1075+12_+29 GGGGGCCGCGGCCAGGGA 1389 c.1075+13_+30 CGGGGGCCGCGGCCAGGG 1390 c.1075+14_+31 GCGGGGGCCGCGGCCAGG 1391 c.1075+15_+32 GGCGGGGGCCGCGGCCAG 1392 c.1075+16_+33 GGGCGGGGGCCGCGGCCA 1393 c.1075+17_+34 GGGGCGGGGGCCGCGGCC 1394 c.1075+18_+35 TGGGGCGGGGGCCGCGGC 1395 c.1075+19_+36 TTGGGGCGGGGGCCGCGG 1396 c.1075+20_+37 CTTGGGGCGGGGGCCGCG 1397 c.1075+21_+38 CCTTGGGGCGGGGGCCGC 1398 c.1075+22_+39 GCCTTGGGGCGGGGGCCG 1399 c.1075+23_+40 AGCCTTGGGGCGGGGGCC 1400 c.1075+24_1076-39 GAGCCTTGGGGCGGGGGC 1401 c.1075+25_1076-38 GGAGCCTTGGGGCGGGGG 1402 c.1075+26_1076-37 GGGAGCCTTGGGGCGGGG 1403 c.1075+27_1076-36 AGGGAGCCTTGGGGCGGG 1404 c.1075+28_1076-35 GAGGGAGCCTTGGGGCGG 1405 c.1075+29_1076-34 GGAGGGAGCCTTGGGGCG 1406 c.1075+30_1076-33 AGGAGGGAGCCTTGGGGC 1407 c.1075+31_1076-32 GAGGAGGGAGCCTTGGGG 1408 c.1075+32_1076-31 GGAGGAGGGAGCCTTGGG 1409 c.1075+33_1076-30 GGGAGGAGGGAGCCTTGG 1410 c.1075+34_1076-29 AGGGAGGAGGGAGCCTTG 1411 c.1075+35_1076-28 GAGGGAGGAGGGAGCCTT 1412 c.1075+36_1076-27 GGAGGGAGGAGGGAGCCT 1413 c.1075+37_1076-26 GGGAGGGAGGAGGGAGCC 1414 c.1075+38_1076-25 AGGGAGGGAGGAGGGAGC 1415
c.1075+39_1076-24 GAGGGAGGGAGGAGGGAG 1416 c.1075+40_1076-23 TGAGGGAGGGAGGAGGGA 1417 c.1076-39_-22 ATGAGGGAGGGAGGAGGG 1418 c.1076-38_-21 CATGAGGGAGGGAGGAGG 1419 c.1076-37_-20 TCATGAGGGAGGGAGGAG 1420 c.1076-36_-19 TTCATGAGGGAGGGAGGA 1421 c.1076-35_-18 CTTCATGAGGGAGGGAGG 1422 c.1076-34_-17 ACTTCATGAGGGAGGGAG 1423 c.1076-33_-16 GACTTCATGAGGGAGGGA 1424 c.1076-32_-15 CGACTTCATGAGGGAGGG 1425 c.1076-31_-14 CCGACTTCATGAGGGAGG 1426 c.1076-30_-13 GCCGACTTCATGAGGGAG 1427 c.1076-29_-12 CGCCGACTTCATGAGGGA 1428 c.1076-28_-11 ACGCCGACTTCATGAGGG 1429 c.1076-27_-10 AACGCCGACTTCATGAGG 1430 c.1076-26_-9 CAACGCCGACTTCATGAG 1431 c.1076-25_-8 CCAACGCCGACTTCATGA 1432 c.1076-24_-7 GCCAACGCCGACTTCATG 1433 c.1076-23_-6 GGCCAACGCCGACTTCAT 1434 c.1076-22_-5 AGGCCAACGCCGACTTCA 1435 c.1076-21_-4 CAGGCCAACGCCGACTTC 1436 c.1076-20_-3 GCAGGCCAACGCCGACTT 1437 c.1076-19_-2 TGCAGGCCAACGCCGACT 1438 c.1076-18_-1 CTGCAGGCCAACGCCGAC 1439 c.1076-17_1076 CCTGCAGGCCAACGCCGA 1440 c.1076-16_1077 TCCTGCAGGCCAACGCCG 1441 c.1076-15_1078 ATCCTGCAGGCCAACGCC 1442 c.1076-14_1079 TATCCTGCAGGCCAACGC 1443 c.1076-13_1080 GTATCCTGCAGGCCAACG 1444 c.1076-12_1081 GGTATCCTGCAGGCCAAC 1445 c.1076-11_1082 GGGTATCCTGCAGGCCAA 1446 c.1076-10_1083 CGGGTATCCTGCAGGCCA 1447 c.1076-9_1084 ACGGGTATCCTGCAGGCC 1448 c.1076-8_1085 AACGGGTATCCTGCAGGC 1449 c.1076-7_1086 GAACGGGTATCCTGCAGG 1450 c.1076-6_1087 TGAACGGGTATCCTGCAG 1451 c.1076-5_1088 ATGAACGGGTATCCTGCA 1452 c.1076-4_1089 CATGAACGGGTATCCTGC 1453 c.1076-3_1090 GCATGAACGGGTATCCTG 1454 c.1076-2_1091 GGCATGAACGGGTATCCT 1455 c.1076-1_1092 CGGCATGAACGGGTATCC 1456 c.1076_1093 GCGGCATGAACGGGTATC 1457 c.1077_1094 GGCGGCATGAACGGGTAT 1458 c.1078_1095 TGGCGGCATGAACGGGTA 1459 c.1079_1096 ATGGCGGCATGAACGGGT 1460 c.1080_1097 TATGGCGGCATGAACGGG 1461 c.1081_1098 GTATGGCCGCATGAACGG 1462 c.1082_1099 AGTATGGCGGCATGAACG 1463 c.1083_1100 CAGTATGGCGGCATGAAC 1464 c.1084_1101 CCAGTATGGCGGCATGAA 1465 c.1085_1102 CCCAGTATGGCGGCATGA 1466 c.1086_1103 CCCCAGTATGGCGGCATG 1467 c.1087_1104 GCCCCAGTATGGCGGCAT 1468 c.1088_1105 GGCCCCAGTATGGCGGCA 1469 c.1089_1106 AGGCCCCAGTATGGCGGC 1470 c.1090_1107 CAGGCCCCAGTATGGCGG 1471 c.1091_1108 CCAGGCCCCAGTATGGCG 1472 c.1092_1109 CCCAGGCCCCAGTATGGC 1473 c.1093_1110 GCCCAGGCCCCAGTATGG 1474 c.1094_1111 AGCCCAGGCCCCAGTATG 1475 c.1095_1112 AAGCCCAGGCCCCAGTAT 1476 c.1096_1113 GAAGCCCAGGCCCCAGTA 1477 c.1097_1114 GGAAGCCCAGGCCCCAGT 1478 c.1098_1115 TGGAAGCCCAGGCCCCAG 1479 c.1099_1116 GTGGAAGCCCAGGCCCCA 1480 c.1100_1117 GGTGGAAGCCCAGGCCCC 1481 c.1101_1118 AGGTGGAAGCCCAGGCCC 1482 c.1102_1119 CAGGTGGAAGCCCAGGCC 1483 c.1103_1120 ACAGGTGGAAGCCCAGGC 1484 c.1104_1121 CACAGGTGGAAGCCCAGG 1485 c.1105_1122 GCACAGGTGGAAGCCCAG 1486 c.1106_1123 GGCACAGGTGGAAGCCCA 1487 c.1107_1124 CGGCACAGGTGGAAGCCC 1488 c.1108_1125 GCGGCACAGGTGGAAGCC 1489 c.1109_1126 AGCGGCACAGGTGGAAGC 1490 c.1110_1127 CAGCGGCACAGGTGGAAG 1491 c.1111_1128 CCAGCGGCACAGGTGGAA 1492 c.1112_1129 CCCAGCGGCACAGGTGGA 1493 c.1113_1130 CCCCAGCGGCACAGGTGG 1494 c.1114_1131 GCCCCAGCGGCACAGGTG 1495 c.1115_1132 AGCCCCAGCGGCACAGGT 1496 c.1116_1133 TAGCCCCAGCGGCACAGG 1497 c.1117_1134 GTAGCCCCAGCGGCACAG 1498 c.1118_1135 AGTAGCCCCAGCGGCACA 1499 c.1119_1136 GAGTAGCCCCAGCGGCAC 1500 c.1120_1137 GGAGTAGCCCCAGCGGCA 1501 c.1121_1138 AGGAGTAGCCCCAGCGGC 1502 c.1122_1139 GAGGAGTAGCCCCAGCGG 1503 c.1123_1140 GGAGGAGTAGCCCCAGCG 1504 c.1124_1141 TGGAGGAGTAGCCCCAGC 1505 c.1125_1142 GTGGAGGAGTAGCCCCAG 1506 c.1126_1143 GGTGGAGGAGTAGCCCCA 1507 c.1127_1144 CGGTGGAGGAGTAGCCCC 1508 c.1128_1145 GCGGTGGAGGAGTAGCCC 1509 c.1129_1146 AGCGGTGGAGGAGTAGCC 1510 c.1130_1147 TAGCGGTGGAGGAGTAGC 1511 c.1131_1148 ATAGCGGTGGAGGAGTAG 1512 c.1132_1149 GATAGCGGTGGAGGAGTA 1513 c.1133_1150 TGATAGCGGTGGAGGAGT 1514 c.1134_1151 GTGATAGCGGTGGAGGAG 1515 c.1135_1152 GGTGATAGCGGTGGAGGA 1516 c.1136_1153 GGGTGATAGCGGTGGAGG 1517 c.1137_1154 CGGGTGATAGCGGTGGAG 1518 c.1138_1155 GCGGGTGATAGCGGTGGA 1519 c.1139_1156 GGCGGGTGATAGCGGTGG 1520 c.1140_1157 TGGCGGGTGATAGCGGTG 1521 c.1141_1158 CTGGCGGGTGATAGCGGT 1522 c.1142_1159 CCTGGCGGGTGATAGCGG 1523 c.1143_1160 ACCTGGCGGGTGATAGCG 1524 c.1144_1161 CACCTGGCGGGTGATAGC 1525 c.1145_1162 CCACCTGGCGGGTGATAG 1526 c.1146_1163 ACCACCTGGCGGGTGATA 1527 c.1147_1164 CACCACCTGGCGGGTGAT 1528 c.1148_1165 CCACCACCTGGCGGGTGA 1529 c.1149_1166 TCCACCACCTGGCGGGTG 1530 c.1150_1167 CTCCACCACCTGGCGGGT 1531 c.1151_1168 TCTCCACCACCTGGCGGG 1532 c.1152_1169 TTCTCCACCACCTGGCGG 1533 c.1153_1170 GTTCTCCACCACCTGGCG 1534 c.1154_1171 TGTTCTCCACCACCTGGC 1535 c.1155_1172 ATGTTCTCCACCACCTGG 1536 c.1156_1173 CATGTTCTCCACCACCTG 1537 c.1157_1174 TCATGTTCTCCACCACCT 1538 c.1158_1175 GTCATGTTCTCCACCACC 1539 c.1159_1176 GGTCATGTTCTCCACCAC 1540
c.1160_1177 TGGTCATGTTCTCCACCA 1541 c.1161_1178 CTGGTCATGTTCTCCACC 1542 c.1162_1179 CCTGGTCATGTTCTCCAC 1543 c.1163_1180 CCCTGGTCATGTTCTCCA 1544 c.1164_1181 GCCCTGGTCATGTTCTCC 1545 c.1165_1182 GGCCCTGGTCATGTTCTC 1546 c.1166_1183 GGGCCCTGGTCATGTTCT 1547 c.1167_1184 TGGGCCCTGGTCATGTTC 1548 c.1168_1185 GTGGGCCCTGGTCATGTT 1549 c.1169_1186 AGTGGGCCCTGGTCATGT 1550 c.1170_1187 AAGTGGGCCCTGGTCATG 1551 c.1171_1188 GAAGTGGGCCCTGGTCAT 1552 c.1172_1189 GGAAGTGGGCCCTGGTCA 1553 c.1173_1190 GGGAAGTGGGCCCTGGTC 1554 c.1174_1191 GGGGAAGTGGGCCCTGGT 1555 c.1175_1192 GGGGGAAGTGGGCCCTGG 1556 c.1176_1193 AGGGGGAAGTGGGCCCTG 1557 c.1177_1194 CAGGGGGAAGTGGGCCCT 1558 c.1178_1194+1 CCAGGGGGAAGTGGGCCC 1559 c.1179_1194+2 ACCAGGGGGAAGTGGGCC 1560 c.1180_1194+3 CACCAGGGGGAAGTGGGC 1561 c.1181_1194+4 TCACCAGGGGGAAGTGGG 1562 c.1182_1194+5 CTCACCAGGGGGAAGTGG 1563 c.1183_1194+6 ACTCACCAGGGGGAAGTG 1564 c.1184_1194+7 AACTCACCAGGGGGAAGT 1565 c.1185_1194+8 CAACTCACCAGGGGGAAG 1566 c.1186_1194+9 CCAACTCACCAGGGGGAA 1567 c.1187_1194+10 CCCAACTCACCAGGGGGA 1568 c.1188_1194+11 CCCCAACTCACCAGGGGG 1569 c.1189_1194+12 ACCCCAACTCACCAGGGG 1570 c.1190_1194+13 CACCCCAACTCACCAGGG 1571 c.1191_1194+14 CCACCCCAACTCACCAGG 1572 c.1192_1194+15 ACCACCCCAACTCACCAG 1573 c.1193_1194+16 CACCACCCCAACTCACCA 1574 c.1194_1194+17 CCACCACCCCAACTCACC 1575 c.1194+1_+18 GCCACCACCCCAACTCAC 1576 c.1194+2_+19 TGCCACCACCCCAACTCA 1577 c.1194+3_+20 CTGCCACCACCCCAACTC 1578 c.1194+4_+21 CCTGCCACCACCCCAACT 1579 c.1194+5_+22 CCCTGCCACCACCCCAAC 1580 c.1194+6_+23 CCCCTGCCACCACCCCAA 1581 c.1194+7_+24 TCCCCTGCCACCACCCCA 1582 c.1194+8_+25 CTCCCCTGCCACCACCCC 1583 GAA_c.2190-357_-333 TCAGTCAAGTATCTGGAAAGTACGA 1590 GAA_c.2190-355_-335 AGTCAAGTATCTGGAAAGTAC 1591 GAA_c.1249_1273 GGAAGTCCCGGAAGCCAACCTTGTT 1592 GAA_c.1552-46_-26 TGACTCTGCCCAGAGTGAGGA 1593 GAA_c.1755-112_-88 AGCTTTCTGGGATGAGGCAGAGGCT 1594
[0205] In the above examples the sequences are 18, 21 and 25 nucleotides long however longer variants or shorter fragment are also envisioned. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 541-1583, 1590-1594 and fragments and variants thereof having at least 80% sequence identity. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of SEQ ID NO: 541-1583, 1590-1594 and fragments and variants thereof having at least 80%,83%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% sequence identity to SEQ ID NO: 541-1583, 1590-1594.
[0206] Or sequences that are at least 80% identical to SEQ ID NO: 541-1583, 1590-1594. Optionally at least 85% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 88% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 90% identical to SEQ ID NO: 541-1583, 1590-1594. more Optionally at least 91% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 92% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 93% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 94% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 95% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 96% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 97% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 98% identical to SEQ ID NO: 541-1583, 1590-1594, more Optionally at least 99% identical to SEQ ID NO: 541-1583, 1590-1594.
[0207] Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 541-1583, 1590-1594, wherein the fragment is 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 541-1583, 1590-1594, wherein the fragment is 17, 18, 19, 20, 21, or 22 nucleotides long. Optionally of the invention and/or embodiments thereof of the present invention and/or embodiments thereof the antisense oligomeric compounds are selected from the group of fragments SEQ ID NO: 541-1583, 1590-1594, wherein the fragment is 19, 20, or 21 nucleotides long.
[0208] The antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group c.-32-13T>G (IVS1), c.1636+5G>T, c.525delT, c.-32-3C>G, c. 1551+1G>A, c.1075G>A, c.1552-3C>G, c.1437G>A, c.1256A>T, c.1551+1G>T.
[0209] Optionally the genomic nucleic acid sequence is pre-mRNA.
[0210] Optionally of the invention and/or embodiments thereof, the antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group comprising c.-32-3C>G, c.-32-13T>G, c.-32-102T>C, c.-32-56C>T, c.-32-46G>A, c.-32-28C>A, c.-32-28C>T, c.-32-21G>A, c.7G>A, c.11G>A, c.15_17AAA, c.17C>T, c.19_21AAA, c.26_28AAA, c.33_35AAA, c.39G>A, c.42C>T, c.90C>T, c.112G>A, c.137C>T, c.164C>T, c.348G>A, c.373C>T, c.413T>A, c.469C>T, c.476T>C, c.476T>G, c.478T>G, c.482C>T, c.510C>T, c.515T>A, c.520G>A, c.546+11C>T, c.546+14G>A, c.546+19G>A, c.546+23C>A, c.547-6, c.1071, c.1254, and c.1552-30.
[0211] Optionally the genomic nucleic acid sequence is pre-mRNA
[0212] Optionally of the invention and/or embodiments thereof, the antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group comprising c.17C>T c.469C>T c.546+23C>A, c.-32-102T>C c.-32-56C>T c.11G>A c.112G>A c.137C>T.
[0213] Optionally of the invention and/or embodiments thereof, the antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group comprising c.17C>T c.469C>T c.546+23C>A.
[0214] Optionally of the invention and/or embodiments thereof, the antisense oligomeric compound may be also be complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation selected from the group comprising c.-32-102T>C c.-32-56C>T c.11G>A c.112G>A c.137C>T.
[0215] Most preferred are antisense oligomeric compounds that are complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation c.-32-13T>G (IVS1).
[0216] Most preferred are antisense oligomeric compounds that are complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation c.-32-3C>G, c.1256A>T, c.1551+1G>T, c.546G>T.
[0217] Most preferred are antisense oligomeric compounds that are complementary to a genomic nucleic acid sequence of GAA gene targeting the location that comprises the position of a mutation c.-32-3C>G.
[0218] Most preferred are antisense oligomeric compounds that are complementary to a genomic nucleic acid sequence of GAA gene targeting SEQ ID NO: 1.
TABLE-US-00006 (SEQ ID NO: 1) GCTCTGCACTCCCCTGCTGGAGCTTTTCTCGCCCTTCCTTCTGGCCCTC TCCCCA.
[0219] Optionally of the invention and/or embodiments thereof, the antisense oligomeric compound are 8 to 80 nucleotides in length, 9 to 50 nucleotides in length, 10 to 30 nucleotides in length, 12 to 30 nucleotides in length, 15 to 25 nucleotides in length or about 20 nucleotides in length. One of ordinary skill in the art will appreciate that this comprehends antisense compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.
[0220] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 13 to 80 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.
[0221] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 13 to 50 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
[0222] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 13 to 30 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
[0223] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 20 to 30 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.
[0224] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 15 to 25 nucleotides. One having ordinary skill in the art will appreciate that this embodies antisense compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.
[0225] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 20 nucleotides.
[0226] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 19 nucleotides.
[0227] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 18 nucleotides.
[0228] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 17 nucleotides.
[0229] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 16 nucleotides.
[0230] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 15 nucleotides.
[0231] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 14 nucleotides.
[0232] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise 13 nucleotides.
[0233] In one embodiment of the invention and/or embodiments thereof, compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleotides from one of the antisense compounds as claimed. Optionally at least 9 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 10 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 11 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 12 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 13 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 14 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 15 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 16 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 17 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 18 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 19 consecutive nucleotides from one of the antisense compounds as claimed, more Optionally at least 20 consecutive nucleotides from one of the antisense compounds as claimed.
[0234] Any remaining nucleotides from the oligonuclotides may be oligonucleotides that improve resistance to Rnase H, cell-targeting sequences, cell penetrating sequences, marker sequences or any other sequences.
[0235] One having skill in the art armed with the antisense compounds disclosed herein will be able, without undue experimentation, to identify further antisense compounds.
[0236] In order for an antisense oligonucleotide to achieve therapeutic success, oligonucleotide chemistry must allow for adequate cellular uptake (Kurreck, J. (2003) Eur. J. Biochem. 270:1628-1644). Splicing oligonucleotides have traditionally been comprised of uniform modifications that render the oligonucleotide RNA-like, and thus resistant to cleavage by RNase H, which is critical to achieve modulation of splicing. Provided herein are antisense compounds for modulation of splicing.
[0237] Optionally of the invention and/or embodiments thereof, the antisense compounds are chimeric, with regions of RNA-like and DNA-like chemistry. Despite regions of DNA-like chemistry, the chimeric compounds are Optionally RNase H-resistant and effectively modulate splicing of target mRNA in vitro and in vivo. In another preferred embodiment the disclosed antisense oligomeric compounds show enhanced cellular uptake and greater pharmacologic activity compared with uniformly modified oligonucleotides.
[0238] Contemplated herein are antisense oligomeric compound which are targeted to a splice site of a target mRNA or to splicing repressor sequences, or to splicing enhancer sequences, Optionally to splicing repressor sequences. Splice sites include aberrant and cryptic splice sites.
[0239] One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the activity of the antisense compound. Compounds provided herein are therefore directed to those antisense compounds that may contain up to about 20% nucleotides that disrupt base pairing of the antisense compound to the target. Optionally the compounds contain no more than about 15%, more Optionally not more than about 10%, most Optionally not more than 5% or no mismatches. The remaining nucleotides do not disrupt hybridization (e.g., universal bases).
[0240] It is understood in the art that incorporation of nucleotide affinity modifications may allow for a greater number of mismatches compared to an unmodified compound. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One of the skill in the art is capable of determining an appropriate number of mismatches between oligonucleotides, or between an oligonucleotide and a target nucleic acid, such as by determining melting temperature.
[0241] It is known by a skilled person that hybridization to a target mRNA depends on the conditions. "Stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and "stringent conditions" under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.
[0242] Antisense compounds, or a portion thereof, may have a defined percent identity to a SEQ ID NO, or a compound having a specific Isis number. As used herein, a sequence is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in the disclosed sequences would be considered identical as they both pair with adenine. This identity may be over the entire length of the oligomeric compound, or in a portion of the antisense compound (e.g., nucleotides 1-20 of a 27-mer may be compared to a 20-mer to determine percent identity of the oligomeric compound to the SEQ ID NO.) It is understood by those skilled in the art that an antisense compound need not have an identical sequence to those described herein to function similarly to the antisense compound described herein. Shortened versions of antisense compound taught herein, or non-identical versions of the antisense compound taught herein are also contemplated. Non-identical versions are those wherein each base does not have the same pairing activity as the antisense compounds disclosed herein. Bases do not have the same pairing activity by being shorter or having at least one abasic site. Alternatively, a non-identical version can include at least one base replaced with a different base with different pairing activity (e.g., G can be replaced by C, A, or T). Percent identity is calculated according to the number of bases that have identical base pairing corresponding to the SEQ ID NO or antisense compound to which it is being compared. The non-identical bases may be adjacent to each other, dispersed through out the oligonucleotide, or both.
[0243] For example, a 16-mer having the same sequence as nucleotides 2-17 of a 20-mer is 80% identical to the 20-mer. Alternatively, a 20-mer containing four nucleotides not identical to the 20-mer is also 80% identical to the 20-mer. A 14-mer having the same sequence as nucleotides 1-14 of an 18-mer is 78% identical to the 18-mer. Such calculations are well within the ability of those skilled in the art.
[0244] The percent identity is based on the percent of nucleotides in the original sequence present in a portion of the modified sequence. Therefore, a 30 nucleobase antisense compound comprising the full sequence of the complement of a 20 nucleobase active target segment would have a portion of 100% identity with the complement of the 20 nucleobase active target segment, while further comprising an additional 10 nucleobase portion. The complement of an active target segment may constitute a single portion. Optionally of the invention and/or embodiments thereof, the oligonucleotides are at least about 80%, more Optionally at least about 85%, even more Optionally at least about 90%, most Optionally at least 95% identical to at least a portion of the complement of the active target segments presented herein.
[0245] It is well known by those skilled in the art that it is possible to increase or decrease the length of an antisense compound and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7310, 1992, incorporated herein by reference), a series of antisense oligomeric compounds of 13-25 nucleotides in length were tested for their ability to induce cleavage of a target RNA. Antisense oligomeric compounds of 25 nucleotides in length with 8 or 11 mismatch bases near the ends of the antisense oligomeric compounds were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligomeric compounds that contained no mismatches. Similarly, target specific cleavage was achieved using a 13 nucleobase antisense oligomeric compounds, including those with 1 or 3 mismatches. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988, incorporated herein by reference) tested a series of tandem 14 nucleobase antisense oligomeric compounds, and a 28 and 42 nucleobase antisense oligomeric compounds comprised of the sequence of two or three of the tandem antisense oligomeric compounds, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligomeric compounds alone were able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligomeric compounds. It is understood that antisense compounds can vary in length and percent complementarity to the target provided that they maintain the desired activity. Methods to determine desired activity are disclosed herein and well known to those skilled in the art. Optionally of the invention and/or embodiments thereof, the antisense oligomeric compounds have at least 80% complementarity to the target mRNA, more Optionally at least 85% complementarity to the target mRNA, more Optionally at least 90% complementarity to the target mRNA, more Optionally at least 95% complementarity to the target mRNA, more Optionally at least 96% complementarity to the target mRNA, more Optionally at least 97% complementarity to the target mRNA, more Optionally at least 98% complementarity to the target mRNA, more Optionally at least 99% complementarity to the target mRNA, more Optionally at least 100% complementarity to the target mRNA.
[0246] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base (sometimes referred to as a "nucleobase" or simply a "base"). The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage. It is often preferable to include chemical modifications in oligonucleotides to alter their activity. Chemical modifications can alter oligonucleotide activity by, for example: increasing affinity of an antisense oligonucleotide for its target RNA, increasing nuclease resistance, and/or altering the pharmacokinetics of the oligonucleotide. The use of chemistries that increase the affinity of an oligonucleotide for its target can allow for the use of shorter oligonucleotide compounds.
[0247] Antisense compounds provided herein may also contain one or more nucleosides having modified sugar moieties. The furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA) and substitution of an atom or group such as --S--, --N(R)-- or --C(R1)(R2) for the ring oxygen at the 4'-position. Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance. A representative list of preferred modified sugars includes but is not limited to bicyclic modified sugars (BNA's), including LNA and ENA (4'-(CH2)2-O-2' bridge); and substituted sugars, especially 2'-substituted sugars having a 2'-F, 2'-OCH2 or a 2'-O(CH2)2-OCH3 substituent group. Sugars can also be replaced with sugar mimetic groups among others. Methods for the preparations of modified sugars are well known to those skilled in the art. Suitable compounds can comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Also suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and in are from 1 to about 10. Other oligonucleotides comprise one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, C1, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. One modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxyalkoxy group. A further modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O--(CH2)2-O--(CH2)2-N(CH3)2. Other modifications include 2'-methoxy (2'-O--CH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH--CH2), 2'-O-allyl (2'-O-CH2-CH--CH2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. One 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Antisense compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and, 6,147,200.
[0248] In one aspect of the present invention oligomeric compounds include nucleosides modified to induce a 3'-endo sugar conformation. A nucleoside can incorporate modifications of the heterocyclic base, the sugar moiety or both to induce a desired 3'-endo sugar conformation. These modified nucleosides are used to mimic RNA-like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3'-endo conformational geometry.
[0249] In the present invention there is a preference for an RNA type duplex (A form helix, predominantly 3'-endo) as they are RnasH resistant. Properties that are enhanced by using more stable 3'-endo nucleosides include but are not limited to: modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage.
[0250] Nucleoside conformation is influenced by various factors including substitution at the 2', 3' or 4'-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2' position to favor the 3'-endo conformation can be achieved while maintaining the 2'-OH as a recognition element (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3'-endo conformation can be achieved by deletion of the 2'-OH as exemplified by 2' deoxy-2'F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3'-endo conformation positioning the electronegative fluorine atom in the axial position. Representative 2'-substituent groups amenable to the present invention that give A-form conformational properties (3'-endo) to the resultant duplexes include 2'-O-alkyl, 2'-O-substituted alkyl and 2'-fluoro substituent groups. Other suitable substituent groups are various alkyl and aryl ethers and thioethers, amines and monoalkyl and dialkyl substituted amines.
[0251] Other modifications of the ribose ring, for example substitution at the 4'-position to give 4'-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3'-endo conformation. Along similar lines, one or more nucleosides may be modified in such a way that conformation is locked into a C3'-endo type conformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA(TM), Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)
[0252] Preferred modification of the sugar are selected from the group consisting of 2'-O-methyl 2'-O-methoxyethyl, 2'-fluoro, 2'-dimethylaminooxyethoxy, 2'-dimethylaminoethoxyethoxy, 2'-guanidinium, 2'-O-guanidinium ethyl, 2'-carbamate, 2'-aminooxy, 2'-acetamido and locked nucleic acid. In one preferred embodiment, the sugar modification is 2'-O-methyl or 2'-O-methoxyethyl.
[0253] Oligomeric compounds can also include nucleobase (often referred to in the art as heterocyclic base or simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). A "substitution" is the replacement of an unmodified or natural base with another unmodified or natural base. "Modified" nucleotides mean other synthetic and natural nucleotides such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--C[identical to]C--CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleotides include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleotides may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleotides include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleotides are known to those skilled in the art as suitable for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. and are presently suitable base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. It is understood in the art that modification of the base does not entail such chemical modifications as to produce substitutions in a nucleic acid sequence.
[0254] Representative United States patents that teach the preparation of certain of the above noted modified nucleotides as well as other modified nucleotides include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941; and 5,750,692.
[0255] Oligomeric compounds of the present invention may also include polycyclic heterocyclic compounds in place of one or more of the naturally-occurring heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand. The most studied modifications are targeted to guanosines hence they have been termed G-clamps or cytidine analogs. Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one, (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and 6,7,8,9-tetrafluoro-1,3-cliazaphenoxazine-2-one (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388). Incorporated into oligonucleotides these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. Pre-Grant Publications 20030207804 and 20030175906).
[0256] Further helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). Binding studies demonstrated that a single incorporation could enhance the binding affinity of a model oligonucleotide to its complementary target DNA or RNA with a .DELTA.Tm of up to 18.degree. C. relative to 5-methyl cytosine, which is a high affinity enhancement for a single modification. On the other hand, the gain in helical stability does not compromise the specificity of the oligonucleotides.
[0257] Further tricyclic heterocyclic compounds and methods of using them that are amenable to use in the present invention are disclosed in U.S. Pat. Nos. 6,028,183, and 6,007,992.
[0258] The enhanced binding affinity of the phenoxazine derivatives together with their uncompromised sequence specificity makes them valuable nucleobase analogs for the development of more potent antisense-based drugs. In fact, promising data have been derived from in vitro experiments demonstrating that heptanucleotides containing phenoxazine substitutions are capable to activate RNase H, enhance cellular uptake and exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was even more pronounced in case of G-clamp, as a single substitution was shown to significantly improve the in vitro potency of a 20 mer 2'-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).
[0259] Further modified polycyclic heterocyclic compounds useful as heterocyclic bases are disclosed in but not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. Pre-Grant Publication 20030158403.
[0260] The compounds described herein may include internucleoside linking groups that link the nucleosides or otherwise modified monomer units together thereby forming an antisense compound. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (--CH2-N(CH3)-O--CH2-), thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane (--O--Si(H)2-O--); and N,N'-dimethylhydrazine (--CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the antisense compound. Internucleoside linkages having a chiral atom may be prepared racemic, chiral, or as a mixture. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known to those skilled in the art.
[0261] Suitable modified internucleoside linking groups are for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, phosphonoacetate and thiophosphonoacetate (see Sheehan et al., Nucleic Acids Research, 2003, 31(14), 4109-4118 and Dellinger et al., J. Am. Chem. Soc., 2003, 125, 940-950), selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage, i.e., a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
[0262] N3'-P5'-phosphoramidates have been reported to exhibit both a high affinity towards a complementary RNA strand and nuclease resistance (Gryaznov et al., J. Am. Chem. Soc., 1994, 116, 3143-3144). N3'-P5'-phosphoramidates have been studied with some success in vivo to specifically down regulate the expression of the c-myc gene (Skorski et al., Proc. Natl. Acad. Sci., 1997, 94, 3966-3971; and Faira et al., Nat. Biotechnol., 2001, 19, 40-44).
[0263] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050.
[0264] In some embodiments of the invention, oligomeric compounds may have one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular --CH2-NH--O--CH2-, --CH2-N(CH3)-O--CH2- (known as a methylene (methylimino) or MMI backbone), --CH2-O--N(CH3)-CH2-, --CH2-N(CH3)-N(CH3)-CH2- and --O--N(CH3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as --O--P(--O)(OH)--O--CH2-). The MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Amide internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,602,240.
[0265] Some oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
[0266] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439.
[0267] In some embodiments of the invention, the backbone of the antisense oligomeric compound is selected from the group consisting of phosphodiester ODN, phosphoramidate, phosphorothioate, Locked nucleic acids, 2'O methoxyethyl, 2'-deoxy-2'-fluoro-D-arabinose, S-constrained-ethyl, Tricyclo DNA, Morpholino, Vivo-morpholino, Peptide nucleic acids, Unlocked Nucleic Acid, 2'-0,4'-C-ethylene-bridged nucleic acids, 2',4'-bridged nucleic acid, multi-targeting oligonucleotides, 2'-Deoxy-2',4'-difluorouridine, 2'-deoxy-2'-fluoroarabinonucleic acid, 2'-0,4'-C-spirocyclopropylene bridged nucleic acid, amido-bridged nucleic acid, gamma-CF2-aminopropylglycine PNA, constrained altritol nucleic acids, Mixed backbone antisense glucosylceramide synthase oligonucleotide, 3'-fluoro hexitol nucleic acid, positively charged phosphorodiamidate morpholino oligomers, trans-4-hydroxy-L-proline phosphono peptide nucleic acid, constrained methoxyethyl.
[0268] Optionally of the invention and/or embodiments thereof the internucleoside linkage is phosphorothioate, or phosphorodiamidate
[0269] It is further intended that multiple modifications can be made to one or more of the oligomeric compounds of the invention at multiple sites of one or more monomeric subunits (nucleosides are suitable) and/or internucleoside linkages to enhance properties such as but not limited to activity in a selected application.
[0270] The synthesis of numerous of the modified nucleosides amenable to the present invention are known in the art (see for example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenum press). The conformation of modified nucleosides and their oligomers can be estimated by various methods routine to those skilled in the art such as molecular dynamics calculations, nuclear magnetic resonance spectroscopy and CD measurements.
[0271] Optionally of the invention and/or embodiments thereof, the oligomeric compounds of the present invention are morpholino phosphorothioates, or phosphorodiamidate morpholino.
[0272] Another group of oligomeric compounds includes oligonucleotide mimetics. As used herein the term "mimetic" refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target. Representative examples of a sugar mimetic include, but are not limited to, cyclohexenyl or morpholino. Representative examples of a mimetic for a sugar-internucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNA) and morpholino groups linked by uncharged achiral linkages. In some instances a mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art. The heterocyclic base moiety or a modified heterocyclic base moiety is Optionally maintained for hybridization with an appropriate target nucleic acid.
[0273] The compounds described herein may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), [alpha] or [beta], or as (D) or (L) such as for amino acids et al. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms.
[0274] One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA) (Nielsen et al., Science, 1991, 254, 1497-1500). PNAs have favorable hybridization properties, high biological stability and are electrostatically neutral molecules. PNA compounds have been used to correct aberrant splicing in a transgenic mouse model (Sazani et al., Nat. Biotechnol., 2002, 20, 1228-1233). In PNA oligomeric compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA oligomeric compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. PNA compounds can be obtained commercially from Applied Biosystems (Foster City, Calif., USA). Numerous modifications to the basic PNA backbone are known in the art; particularly useful are PNA compounds with one or more amino acids conjugated to one or both termini. For example, 1-8 lysine or arginine residues are useful when conjugated to the end of a PNA molecule. A polyarginine tail may be a suitable for enhancing cell penetration.
[0275] Another class of oligonucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups have been selected to give a non-ionic oligomeric compound. Morpholino-based oligomeric compounds are non-ionic mimetics of oligo-nucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomeric compounds have been studied in zebrafish embryos (see: Genesis, volume 30, issue 3, 2001 and Heasman, J., Dev. Biol., 2002, 243, 209-214). Further studies of morpholino-based oligomeric compounds have also been reported (Nasevicius et at, Nat. Genet., 2000, 26, 216-220; and Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596). Morpholino-based oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506. The morpholino class of oligomeric compounds have been prepared having a variety of different linking groups joining the monomeric subunits. Linking groups can be varied from chiral to achiral, and from charged to neutral. U.S. Pat. No. 5,166,315 discloses linkages including --O--P(--O)(N(CH3)2)-O--; U.S. Pat. No. 5,034,506 discloses achiral intermorpholino linkages; and U.S. Pat. No. 5,185,444 discloses phosphorus containing chiral intermorpholino linkages.
[0276] A further class of oligonucleotide mimetic is referred to as cyclohexene nucleic acids (CeNA). In CeNA oligonucleotides, the furanose ring normally present in a DNA or RNA molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation. Furthermore the incorporation of CeNA into a sequence targeting RNA was stable to serum and able to activate E. coli RNase H resulting in cleavage of the target RNA strand.
[0277] A further modification includes bicyclic sugar moieties such as "Locked Nucleic Acids" (LNAs) in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos. 6,268,490 and 6,670,461). The linkage can be a methylene (--CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, for which the term LNA is used for the bicyclic moiety; in the case of an ethylene group in this position, the term ENA(TM) is used (Singh et at, Chem. Commun., 1998, 4, 455-456; ENA(TM): Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). LNA and other bicyclic sugar analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10[deg.] C.), stability towards 3'-exonucleolytic degradation and good solubility properties. LNAs are commercially available from ProLigo (Paris, France and Boulder, Colo., USA).
[0278] An isomer of LNA that has also been studied is alpha-L-LNA which has been shown to have superior stability against a 3'-exonuclease. The alpha-L-LNAs were incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
[0279] Another similar bicyclic sugar moiety that has been prepared and studied has the bridge going from the 3'-hydroxyl group via a single methylene group to the 4' carbon atom of the sugar ring thereby forming a 3'-C,4'-C-oxymethylene linkage (see U.S. Pat. No. 6,043,060).
[0280] LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNA hybridization was shown to be the most thermally stable nucleic acid type duplex system, and the RNA-mimicking character of LNA was established at the duplex level. Introduction of 3 LNA monomers (T or A) significantly increased melting points (Tm=+15/+11[deg.] C.) toward DNA complements. The universality of LNA-mediated hybridization has been stressed by the formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking of LNA was reflected with regard to the N-type conformational restriction of the monomers and to the secondary structure of the LNA:RNA duplex.
[0281] LNAs also form duplexes with complementary DNA, RNA or LNA with high thermal affinities. Circular dichroism (CD) spectra show that duplexes involving fully modified LNA (esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination of an LNA:DNA duplex confirmed the 3'-endo conformation of an LNA monomer. Recognition of double-stranded DNA has also been demonstrated suggesting strand invasion by LNA. Studies of mismatched sequences show that LNAs obey the Watson-Crick base pairing rules with generally improved selectivity compared to the corresponding unmodified reference strands. DNA-LNA chimeras have been shown to efficiently inhibit gene expression when targeted to a variety of regions (5'-untranslated region, region of the start codon or coding region) within the luciferase mRNA (Braasch et al., Nucleic Acids Research, 2002, 30, 5160-5167).
[0282] Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sc U.S.A., 2000, 97, 5633-5638). The authors have demonstrated that LNAs confer several desired properties. LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. LNA/DNA copolymers exhibited potent antisense activity in assay systems as disparate as G-protein-coupled receptor signaling in living rat brain and detection of reporter genes in Escherichia coli. Lipofectin-mediated efficient delivery of LNA into living human breast cancer cells has also been accomplished. Further successful in vivo studies involving LNA's have shown knock-down of the rat delta opioid receptor without toxicity (Wahlestedt et al., Proc. Natl. Acad. Sci., 2000, 97, 5633-5638) and in another study showed a blockage of the translation of the large subunit of RNA polymerase II (Fluiter et al., Nucleic Acids Res., 2003, 31, 953-962).
[0283] The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
[0284] Analogs of LNA, phosphorothioate-LNA and 2'-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2'-amino-LNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-Amino- and 2'-methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
[0285] Another oligonucleotide mimetic that has been prepared and studied is threose nucleic acid. This oligonucleotide mimetic is based on threose nucleosides instead of ribose nucleosides. Initial interest in (3',2')-alpha-L-threose nucleic acid (TNA) was directed to the question of whether a DNA polymerase existed that would copy the TNA. It was found that certain DNA polymerases are able to copy limited stretches of a TNA template (reported in Chemical and Engineering News, 2003, 81, 9). In another study it was determined that TNA is capable of antiparallel Watson-Crick base pairing with complementary DNA, RNA and TNA oligonucleotides (Chaput et al., J. Am. Chem. Soc., 2003, 125, 856-857).
[0286] In one study (3',2')-alpha-L-threose nucleic acid was prepared and compared to the 2' and 3' amidate analogs (Wu et al., Organic Letters, 2002, 4(8), 1279-1282). The amidate analogs were shown to bind to RNA and DNA with comparable strength to that of RNA/DNA.
[0287] Further oligonucleotide mimetics have been prepared to include bicyclic and tricyclic nucleoside analogs (see Steffens et al., Helv. Chim Acta, 1997, 80, 2426-2439; Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; Renneberg et at, J. Am. Chem. Soc., 2002, 124, 5993-6002; and Renneberg et al., Nucleic acids res., 2002, 30, 2751-2757). These modified nucleoside analogs have been oligomerized using the phosphoramidite approach and the resulting oligomeric compounds containing tricyclic nucleoside analogs have shown increased thermal stabilities (Tm's) when hybridized to DNA, RNA and itself. Oligomeric compounds containing bicyclic nucleoside analogs have shown thermal stabilities approaching that of DNA duplexes.
[0288] Another class of oligonucleotide mimetic is referred to as phosphonomonoester nucleic acids which incorporate a phosphorus group in the backbone. This class of oligonucleotide mimetic is reported to have useful physical and biological and pharmacological properties in the areas of inhibiting gene expression (antisense oligonucleotides, sense oligonucleotides and triplex-forming oligonucleotides), as probes for the detection of nucleic acids and as auxiliaries for use in molecular biology. Further oligonucleotide mimetics amenable to the present invention have been prepared wherein a cyclobutyl ring replaces the naturally occurring furanosyl ring.
[0289] Another modification of the oligomeric compounds of the invention involves chemically linking to the oligomeric compound one or more moieties or conjugates which enhance the properties of the oligomeric compound, such as to enhance the activity, cellular distribution or cellular uptake of the oligomeric compound. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. Nos. 6,287,860 and 6,762,169.
[0290] Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligomeric compounds of the invention may also be conjugated to drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. Pat. No. 6,656,730.
[0291] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.
[0292] Oligomeric compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of an oligomeric compound to enhance properties such as for example nuclease stability. Included in stabilizing groups are cap structures. By "cap structure or terminal cap moiety" is meant chemical modifications, which have been incorporated at either terminus of oligonucleotides (see for example Wincott et al., WO 97/26270). These terminal modifications protect the oligomeric compounds having terminal nucleic acid molecules from exonuclease degradation, and can improve delivery and/or localization within a cell. The cap can be present at either the 5'-terminus (5'-cap) or at the 3'-terminus (3'-cap) or can be present on both termini of a single strand, or one or more termini of both strands of a double-stranded compound. This cap structure is not to be confused with the inverted methylguanosine "5' cap" present at the 5' end of native mRNA molecules. In non-limiting examples, the 5'-cap includes inverted abasic residue (moiety), 4%5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging methylphosphonate moiety (for more details see Wincott et al., International PCT publication No. WO 97/26270).
[0293] Particularly suitable 3'-cap structures include, for example 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and Tyer, 1993, Tetrahedron 49, 1925).
[0294] Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an oligomeric compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.
[0295] In certain embodiments, oligomeric compounds, may be conjugated with a wide variety of different positively charged polymers. Examples of positively charged polymers include peptides, such as argine rich peptides (Examples of positively charged peptides that may be used in the practice of the invention include R9F2C; (RXR)4 XB (where X can be any amino acid); R5F2R4c; (RFF)3; Tat proteins, such as TAT sequence CYGRKKRRQRRR; and (RFF)3R), cationic polymers, such as dendrimeric octaguanindine polymer, and other positively charged molecules as known in the art for conjugation to antisense oligonucleotide compounds. In one embodiment of the invention and/or embodiments thereof, the antisense oligonucleotides are conjugated with positively charged polymer comprising a polymer having a molecular weight that is from about 1,000 to 20,000 Daltons, and Optionally from about 5,000 to 10,000 Daltons. Another example of positively charged polymers is polyethylenimine (PEI) with multiple positively charged amine groups in its branched or unbranched chains PEI has else been widely used as gene and oligomer delivery vesicle.
[0296] Optionally of the invention and/or embodiments thereof the oligomeric compounds are modified with cell penetrating sequences. Suitable cell penetrating sequences include cell penetrating peptides, such as TAT peptide, MPG, Pep-1, MAP, fusogenic, antimicrobial peptides (AMPS), bacteriocidal peptides, fungicidal peptides, virucidal peptides,
[0297] Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of the particles of the invention. The particle of the invention is associated with the CPP peptides either through chemical linkage via covalent bonds or through non-covalent interactions. The function of the CPPs are to deliver the particles into cells, a process that commonly occurs through endocytosis with the cargo delivered to the endosomes of living mammalian cells. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake.
[0298] An exemplary cell penetrating peptide is the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) could be efficiently taken up from the surrounding media by numerous cell types in culture. Other cell penetrating peptides are MPG, Pep-1, transportan, penetratin, CADY, TP, TP10, arginine octamer. polyarginine sequences, Arg8, VP22 HSV-1 structural protein, SAP Proline-rich motifs, Vectocell.RTM. peptides, hCT (9-32), SynB, Pvec, and PPTG1. Cell penetrating peptides may be cationic, essentially containing clusters of polyarginine in their primary sequence or amphipathic. CPPs are generally peptides of less than 30 amino acids, derived from natural or unnatural protein or chimeric sequences.
[0299] Optionally the oligomeric compounds are derivatised with conjugates selected from the group consisting of Pip6a, PEG12, (R/W)9, H5WYG, GalNAc or GN3, M12, PEI-LA, RGD, endosomolytic peptide, peptide, Pep-3, CADY, C6, MPEG-PCL-CH2R4H.sub.2C, (RXR)4XB, R9F2, TAT, (KFF)3K, (RFF)3RXB, (RFF)3R, F-3, Pip2a, B-peptide, B-MSP, Pip5e, PKKKRKV, Penetratin, Lys4, SPACE, Tat-DRBD, (RXR)4, (RxR)3RXB, (KFF)3K, T-cell-derived CPP, PEGPep-3, MPG-8, MPG-8-Chol, PepFect6, P5RHH, R15, Chol-R9.
[0300] Optionally the oligomeric compounds, enzyme, and/or nucleic acid encoding for the enzyme are incorporated or otherwise associated with nanoparticles. Nanoparticles may optionally be modified for targeting specific cells and optimised for penetrating cells. A skilled person is aware of methods to employ nanoparticles for oligomeric compounds delivery to cells.
[0301] Suitable particle are gold particles, silver particle.
[0302] Optionally the nanoparticles are made from material selected from the group consisting of gelatine, hydrophilic gelatine, Arg-Gly-Asp-Polyethylenglycol-stearic acid-chitosan, mesoporous silica.
[0303] Optionally the nanoparticles are made from protein selected from the group consisting of Hematoporphyrin--Bovine serum albumin, Heat-liable enterotoxin subunit B-- Bovine serum albumin, Apotransferin-Bovine serum albumin, Apotransferrin-Lactoferrin, Chitosan-retinoic acid-Albumin, 30Kc19-human-serum-albumin.
[0304] Optionally the nanoparticles are made from a polymer selected from the group consisting of Poly(lactic-co-glyoclic acid), Poly(lactic-co-glyoclic acid)-Chitosan, Poly(lactic-co-glyoclic acid)-eudragit, Poly (lactic acid)-F127-Poly (lactic acid), Polycaprolactone-eudragit RS, Polyacrylic acid, Thiolated Polyacrylic acid, Chitosan, Chitosan-Hydroxy propyl Methyl cellulose Phthalate, Chitosan-PGA-DTPA, Trimethyl chitosan-cysteine conjugate, Lauryl-succinyl-Chitosan, Dextran-poloxamer-Chitosan-albumin, Dextran sulfate-Chitosan, Cholic acid modified dextran sulfate, Alginate-dextran sulfate-Chitosan-albumin, Alginate, Thiolated-Eudragit, Poly-N-isopropylacrylamide, Poly(lactic-co-glyoclic acid), Polyethylenglycol-dithiodipropionate-hyaluronic acid, polycaprolactone, Galactose-Chitosan, O-carboxymethyl-chitosan-Galactose, hyaluronic acid-Galactose, Galactosylated-chitosan-polycaprolactone, Galactosylated-chitosan, poly(alkylene oxide)-poly(propylacrylic acid), Poly (lactic acid), (poly(ethylene imine)), Poly(lactic-co-glyoclic acid).
[0305] Optionally the oligomeric compounds, enzyme, and/or nucleic acid encoding for the enzyme are incorporated or otherwise associated with extracellular vesicles (EV). Extracellular vesicles (EVs) are small vesicles, which are secreted by prokaryotic and eukaryotic cells One may distinguish between three classes of EVs, namely apoptotic bodies (ABs), microvesicles (MVs) and exosomes. Exosomes or extracellular vesicles are derived from cells. The cells may any kind of cell that is capable of producing exosomes. The cells may be patient derived or from donors, cells in culture or heterologous systems from animals or plants. Preferably the exosomes or extracellular vesicles are derived from the human cells. Several approaches may be used for the loading of exosomal or extracellular vesical carriers with therapeutic cargo
[0306] (A) loading naive exosomes or extracellular vesicles isolated from parental cells ex vitro;
[0307] (B) loading parental cells with enzyme, nucleic acid encoding the enzyme and/or antisense oligomeric compound, which is then released in exosomes or extracellular vesicles; and finally,
[0308] (C) transfecting/infecting parental cells with DNA encoding enzyme, and/or antisense oligomeric compound, which are then released in exosomes or extracellular vesicles. Exosomes possess an intrinsic ability to cross biological barriers, including the most difficult to penetrate: the blood brain barrier (BBB).
[0309] Optionally the exosomes or extracellular vesicles comprise the enzyme or GAA. Optionally the exosomes or extracellular vesicles comprise the mRNA for the enzyme or GAA. Optionally the exosomes or extracellular vesicles comprise the antisense oligomeric compound. Optionally the exosomes or extracellular vesicles comprise a DNA construct encoding for the antisense oligomeric compound.
[0310] Optionally the oligomeric compounds, enzyme, and/or nucleic acid encoding for the enzyme are incorporated or otherwise associated with micelles.
[0311] Optionally the oligomeric compounds, enzyme, and/or nucleic acid encoding for the enzyme are incorporated or otherwise associated with liposomes.
[0312] Optionally the oligomeric compounds, enzyme, and/or nucleic acid encoding for the enzyme are incorporated or otherwise associated with microparticles.
[0313] Optionally, the oligomeric compounds are modified with an endosomal escape agent moiety. The endocytic pathway is a major uptake mechanism of cells. Compounds taken up by the endocytic pathway become entrapped in endosomes and may be degraded by specific enzymes in the lysosome. This may be desired or not desired depending on the purpose. If taken up by the endosomes is not desired, endosomal escape agent may be used. Suitable endosomal escape agents may be chloroquine, TAT peptide.
[0314] It is not necessary for all positions in a given oligomeric compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even within a single nucleoside within an oligomeric compound.
[0315] The present invention also includes oligomeric compounds which are chimeric compounds. "Chimeric" oligomeric compounds or "chimeras," in the context of this invention, are single- or double-stranded oligomeric compounds, such as oligonucleotides, which contain two or more chemically distinct regions, each comprising at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. Chimeric antisense oligonucleotides are one form of oligomeric compound. These oligonucleotides typically contain at least one region which is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, alteration of charge, increased stability and/or increased binding affinity for the target nucleic acid.
[0316] Chimeric oligomeric compounds of the invention can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922.
[0317] Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
[0318] Oligomeric compounds of the present invention can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
[0319] The following precursor compounds, including amidites and their intermediates can be prepared by methods routine to those skilled in the art; 5'-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC 5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5'-O-Dimethoxytityl-2'-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, (5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N4-benzoyl-5-methylcytidin- -3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2'-Fluorodeoxyadenosine, 2'-Fluorodeoxyguanosine, 2'-Fluorouridine, 2'-Fluorodeoxycytidine, 2'-O-(2-Methoxyethyl) modified amidites, 2'-O-(2-methoxyethyl)-5-methyluridine intermediate, 5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, (5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi- n-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5'-O-Dimethoxytrityl-2'-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N<4>-benzoyl-5-methyl-cy- tidine penultimate intermediate, (5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N<4>-be- nzoyl-5-methylcytidin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), (5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N<6>-be- nzoyladenosin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), (5'-0-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N<- ;4>-isobutyrylguanosin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphorami- dite (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and 2'-O-(dimethylaminooxyethyl) nucleoside amidites, 2'-(Dimethylaminooxyethoxy) nucleoside amidites, 5'-O-tert-Butyldiphenylsilyl-0<2>-2'-anhydro-5-methyluridine, 5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine, 2'-O-((2-phthalimidoxy)ethyl)-5'-t-butyldiphenylsilyl-5-methyluridine, 5'-O-tert-butyldiphenylsilyl-2'-O-((2-formadoximinooxy)ethyl)-5-methyluri- dine, 5'-O-tert-Butyldiphenylsilyl-2'-O--(N,N dimethylaminooxyethyl)-5-methyluridine, 2'-O-(dimethylaminooxyethyl)-5-methyluridine, 5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine, 5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-((2-cyanoe- thyl)-N,N-diisopropylphosphoramidite), 2'-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(- 4,4'-dimethoxytrityl)guanosine-3'4(2-cyanoethyl)-N,N-diisopropylphosphoram- idite), 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites, 2'-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl 5'-O-dimethoxytrityl-2'-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine and 5'-O-Dimethoxytrityl-2'-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
[0320] The preparation of such precursor compounds for oligonucleotide synthesis are routine in the art and disclosed in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743.
[0321] 2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites can be purchased from commercial sources (e.g. Chemgenes, Needham, Mass. or Glen Research, Inc. Sterling, Va.). Other 2'-O-alkoxy substituted nucleoside amidites can be prepared as described in U.S. Pat. No. 5,506,351.
[0322] Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C) nucleotides can be synthesized routinely according to published methods (Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham, Mass.).
[0323] 2'-fluoro oligonucleotides can be synthesized routinely as described (Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841) and U.S. Pat. No. 5,670,633.
[0324] 2'-O-Methoxyethyl-substituted nucleoside amidites can be prepared routinely as per the methods of Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.
[0325] Aminooxyethyl and dimethylaminooxyethyl amidites can be prepared routinely as per the methods of U.S. Pat. No. 6,127,533.
[0326] Phosphorothioate-containing oligonucleotides (P-S) can be synthesized by methods routine to those skilled in the art (see, for example, Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press). Phosphinate oligonucleotides can be prepared as described in U.S. Pat. No. 5,508,270.
[0327] Alkyl phosphonate oligonucleotides can be prepared as described in U.S. Pat. No. 4,469,863.
[0328] 3'-Deoxy-3'-methylene phosphonate oligonucleotides can be prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.
[0329] Phosphoramidite oligonucleotides can be prepared as described in U.S. Pat. No. 5,256,775 or 5,366,878.
[0330] Alkylphosphonothioate oligonucleotides can be prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).
[0331] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides can be prepared as described in U.S. Pat. No. 5,476,925.
[0332] Phosphotriester oligonucleotides can be prepared as described in U.S. Pat. No. 5,023,243.
[0333] Borano phosphate oligonucleotides can be prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0334] 4'-thio-containing oligonucleotides can be synthesized as described in U.S. Pat. No. 5,639,873.
[0335] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P--O or P--S linkages can be prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.
[0336] Formacetal and thioformacetal linked oligonucleosides can be prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564.
[0337] Ethylene oxide linked oligonucleosides can be prepared as described in U.S. Pat. No. 5,223,618.
[0338] Peptide nucleic acids (PNAs) can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, 5,719,262, 6,559,279 and 6,762,281.
[0339] Oligomeric compounds incorporating at least one 2'-O-protected nucleoside by methods routine in the art. After incorporation and appropriate deprotection the 2'-O-protected nucleoside will be converted to a ribonucleoside at the position of incorporation. The number and position of the 2-ribonucleoside units in the final oligomeric compound may vary from one at any site or the strategy can be used to prepare up to a full 2'-OH modified oligomeric compound.
[0340] The main RNA synthesis strategies that are presently being used commercially include 5'-[beta]-DMT-2'-O-t-butyldimethylsilyl (TBDMS), 5'-O-DMT-2'-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP), 2'-O-[(triisopropylsilyl)oxy]methyl (2'-O-CH2-O--Si(iPr)3 (TOM), and the 5'-O-silyl ether-2'-ACE (5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether (DOD)-2'-O-bis(2-acetoxyethoxy)methyl (ACE). Some companies currently offering RNA products include Pierce Nucleic Acid Technologies (Milwaukee, Wis.), Dharmacon Research Inc. (a subsidiary of Fisher Scientific, Lafayette, Colo.), and Integrated DNA Technologies, Inc. (Coralville, Iowa). One company, Princeton Separations, markets an RNA synthesis activator advertised to reduce coupling times especially with TOM and TBDMS chemistries. Such an activator would also be amenable to the oligomeric compounds of the present invention.
[0341] All of the aforementioned RNA synthesis strategies are amenable to the oligomeric compounds of the present invention. Strategies that would be a hybrid of the above e.g. using a 5'-protecting group from one strategy with a 2'-O-protecting from another strategy is also contemplated herein.
[0342] Chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides can be synthesized according to U.S. Pat. No. 5,623,065.
[0343] Chimeric oligomeric compounds exhibiting enhanced cellular uptake and greater pharmacologic activity may be made in accordance to U.S. Pat. No. 8,501,703.
[0344] Another form of oligomeric compounds comprise tricyclo-DNA (tc-DNA) antisense oligonucleotides. Tricyclo-DNA nucleotides are nucleotides modified by the introduction of a cyclopropane ring to restrict conformational flexibility of the backbone and to optimize the backbone geometry of the torsion angle .gamma.. Homobasic adenine- and thymine-containing tc-DNAs form extraordinarily stable A-T base pairs with complementary RNAs. Antisense oligomeric compound that contains between 6-22 tricyclo nucleotides in length, in particular between 8-20 tricyclo nucleotides, more particularly between 10 and 18 or between 11 and 18 tricyclo nucleotides are suitable. See e.g. WO2010115993 for examples of tricyclo-DNA (tc-DNA) antisense oligonucleotides.
[0345] Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).
[0346] Antisense compounds can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The disclosure is not limited by the method of antisense compound synthesis.
[0347] Methods of oligonucleotide purification and analysis are known to those skilled in the art. Analysis methods include capillary electrophoresis (CE) and electrospray-mass spectroscopy. Such synthesis and analysis methods can be performed in multi-well plates. The methods described herein are not limited by the method of oligomer purification.
[0348] Optionally of the invention and/or embodiments thereof, the antisense compounds provided herein are resistant to RNase H degradation.
[0349] In one embodiment of the invention and/or embodiments thereof, the antisense compounds comprise at least one modified nucleotide. In another embodiment, the antisense compounds comprise a modified nucleotide at each position. In yet another embodiment, the antisense compounds are uniformly modified at each position.
[0350] Modulation of splicing can be assayed in a variety of ways known in the art. Target mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA by methods known in the art. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.
[0351] Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM(TM) 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
[0352] Levels of a protein encoded by a target mRNA can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to a protein encoded by a target mRNA can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.
[0353] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.
[0354] The effect of the oligomeric compounds of the present invention may be analysed by RT PCT, qPCR, flanking exon PCR and/or a method comprising
[0355] flanking exon PCR on each internal exon corresponding to the mRNA to obtain one or more flanking exon amplification products, and detecting the presence and length of the said flanking exon amplification products,
[0356] quantifying of each protein encoding exon of said mRNA.
[0357] The oligomeric compounds provided herein may be utilized for therapeutics or research. Furthermore, antisense compounds, which are able to inhibit gene expression or modulate splicing with specificity, may be used to elucidate the function of particular genes or gene products or to distinguish between functions of various members of a biological pathway. Optionally of the invention and/or embodiments thereof the oligomeric compounds are used for the treatment of Pompe disease. Optionally of the invention and/or embodiments thereof the oligomeric compounds are used in research of the function of the GAA gene.
[0358] Compounds described herein can be used to modulate splicing of a target mRNA in an metazoans, Optionally mammals Optionally human. In one non-limiting embodiment of the invention and/or embodiments thereof, the methods comprise the step of administering to said animal an effective amount of an antisense compound that modulates splicing of a target mRNA.
[0359] For example, modulation of splicing of a target mRNA can be measured by determining levels of mRNA splicing products in a bodily fluid, tissue, organ of cells of the animal. Bodily fluids include, but are not limited to, blood (serum or plasma), lymphatic fluid, cerebrospinal fluid, semen, urine, synovial fluid and saliva and can be obtained by methods routine to those skilled in the art. Tissues, organs or cells include, but are not limited to, blood (e.g., hematopoietic cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34+ cells CD4+ cells), lymphocytes and other blood lineage cells, skin, bone marrow, spleen, thymus, lymph node, brain, spinal cord, heart, skeletal muscle, liver, connective tissue, pancreas, prostate, kidney, lung, oral mucosa, esophagus, stomach, ilium, small intestine, colon, bladder, cervix, ovary, testis, mammary gland, adrenal gland, and adipose (white and brown). Samples of tissues, organs and cells can be routinely obtained by biopsy. In some alternative situations, samples of tissues or organs can be recovered from an animal after death. Optionally of the invention and/or embodiments thereof modulation of splicing is measured in fibroblast, Optionally primary fibroblasts, Optionally primary fibroblasts from patients suffering from Pompe disease.
[0360] The effects of treatment with the oligomeric compounds can be assessed by measuring biomarkers associated with modulation of splicing of a target mRNA in the aforementioned fluids, tissues or organs, collected from an animal contacted with one or more compounds, by routine clinical methods known in the art. These biomarkers include but are not limited to: glucose, cholesterol, lipoproteins, triglycerides, free fatty acids and other markers of glucose and lipid metabolism; liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine and other markers of kidney and liver function; interleukins, tumor necrosis factors, intracellular adhesion molecules, C-reactive protein and other markers of inflammation; testosterone, estrogen and other hormones; tumor markers; vitamins, minerals and electrolytes. Optionally of the invention and/or embodiments thereof the biomarker is glycogen.
[0361] The compounds disclosed herein can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. The compounds can also be used in the manufacture of a medicament for the treatment of diseases and disorders related to alterations in splicing. Optionally of the invention and/or embodiments thereof, the disease is Pompe disease.
[0362] Methods whereby bodily fluids, organs or tissues are contacted with an effective amount of one or more of the antisense compounds or compositions of the disclosure are also contemplated. Bodily fluids, organs or tissues can be contacted with one or more of the compounds of the disclosure resulting in modulation of splicing of target mRNA in the cells of bodily fluids, organs or tissues. An effective amount can be determined by monitoring the modulatory effect of the antisense compound or compounds or compositions on target nucleic acids or their products by methods routine to the skilled artisan. Further contemplated are ex vivo methods of treatment whereby cells or tissues are isolated from a subject, contacted with an effective amount of the antisense compound or compounds or compositions and reintroduced into the subject by routine methods known to those skilled in the art.
[0363] A sufficient amount of an antisense oligomeric compound to be administered will be an amount that is sufficient to induce amelioration of unwanted disease symptoms. Such an amount may vary inter alia depending on such factors as the gender, age, weight, overall physical condition, of the patient, etc. and may be determined on a case by case basis. The amount may also vary according to the type of condition being treated, and the other components of a treatment protocol (e.g. administration of other medicaments such as steroids, etc.). The amount may also vary according to the method of administration such as systemically or locally.
[0364] Typical dosage amounts of the antisense oligonucleotide molecules in pharmaceutical formulations may range from about 0.05 to 1000 mg/kg body weight, and in particular from about 5 to 500 mg/kg body weight. In one embodiment of the invention and/or embodiments thereof, the dosage amount is from about 50 to 300 mg/kg body weight once in 2 weeks, or once or twice a week, or any frequency required to achieve therapeutic effect. Optionally amounts are from 3-50 mg/kg, more Optionally 10-40 mg/kg, more Optionally 15-25 mg/kg.
[0365] The dosage administered will, of course, vary depending on the use and known factors such as the pharmacodynamic characteristics of the active ingredient; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. The recipient may be any type of mammal, but is Optionally a human. In one embodiment of the invention and/or embodiments thereof, dosage forms (compositions) of the inventive pharmaceutical composition may contain about 1 microgram to 50,000 micrograms of active ingredient per unit, and in particular, from about 10 to 10,000 micrograms of active ingredient per unit. (if here a unit means a vial or one package for one injection, then it will be much higher, up to 15 g if the weight of a patient is 50 kg) For intravenous delivery, a unit dose of the pharmaceutical formulation will generally contain from 0.5 to 500 micrograms per kg body weight and Optionally will contain from 5 to 300 micrograms, in particular 10, 15, 20, 30, 40, 50, 100, 200, or 300 micrograms per kg body weight ([mu]g/kg body weight) of the antisense oligonucleotide molecule. Preferred intravenous dosage ranges from 10 ng to 2000microg, Optionally 3 to 300 [mg, more Optionally 10 to 100 [mu]g of compound per kg of body weight. Alternatively the unit dose may contain from 2 to 20 milligrams of the antisense oligonucleotide molecule and be administered in multiples, if desired, to give the preceding daily dose. In these pharmaceutical compositions, the antisense oligonucleotide molecule will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
[0366] In one particular embodiment, it should be recognized that the dosage can be raised or lowered based on individual patient response. It will be appreciated that the actual amounts of antisense oligonucleotide molecule used will vary according to the specific antisense oligonucleotide molecule being utilized, the particular compositions formulated, the mode of application, and the particular site of administration.
[0367] Optionally the compounds are administered daily, once every 2 days, once every 3 days, once a week, once every two weeks, or once every month.
[0368] In another preferred embodiment the administration is only one time, e.g. when using a viral vector.
[0369] If a viral-based delivery of antisense oligomeric compounds is chosen, suitable doses will depend on different factors such as the viral strain that is employed, the route of delivery (intramuscular, intravenous, intra-arterial or other), Those of skill in the art will recognize that such parameters are normally worked out during clinical trials. Further, those of skill in the art will recognize that, while disease symptoms may be completely alleviated by the treatments described herein, this need not be the case. Even a partial or intermittent relief of symptoms may be of great benefit to the recipient. In addition, treatment of the patient is usually not a single event. Rather, the antisense oligomeric compounds of the invention will likely be administered on multiple occasions, that may be, depending on the results obtained, several days apart, several weeks apart, or several months apart, or even several years apart.
[0370] Those of skill in the art will recognize that there are many ways to determine or measure a level of functionality of a protein, and to determine a level of increase or decrease of functionality e.g. in response to a treatment protocol. Such methods include but are not limited to measuring or detecting an activity of the protein, etc. Such measurements are generally made in comparison to a standard or control or "normal" sample. In addition, when the protein's lack of functionality is involved in a disease process, disease symptoms may be monitored and/or measured in order to indirectly detect the presence or absence of a correctly functioning protein, or to gauge the success of a treatment protocol intended to remedy the lack of functioning of the protein. In preferred embodiment the functionality of the GAA protein is measured. This is Optionally performed with an enzymatic activity assays as is well known to a skilled person.
[0371] In a particular embodiment of the invention and/or embodiments thereof; antisense oligonucleotides of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide of the invention to the cells. Optionally, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, naked plasmids, non viral delivery systems (electroporation, sonoporation, cationic transfection agents, liposomes, etc. . . . ), phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: R A viruses such as a retrovirus (as for example moloney murine leukemia virus and lentiviral derived vectors), harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus. One can readily employ other vectors not named but known to the art.
[0372] Preferred viral vectors according to the invention include adenoviruses and adeno-associated (AAV) viruses, which are DNA viruses that have already been approved for human use in gene therapy. Actually 12 different AAV serotypes (AAV1 to 12) are known, each with different tissue tropisms (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAV are derived from the dependent parvovirus AAV (Choi, VW J Virol 2005; 79:6801-07). The adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316-27). It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.
[0373] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by, intranasal sprays or drops, rectal suppository and orally. Optionally, said DNA plasmid is injected intramuscular, or intravenous. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
[0374] Optionally of the invention and/or embodiments thereof, the antisense oligonucleotide nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
[0375] Optionally of the invention and/or embodiments thereof, the vector may code for more than one antisense oligomeric compound. Each antisense oligomeric compound is directed to different targets.
[0376] Pharmaceutical composition comprising the antisense compounds described herein may comprise any pharmaceutically acceptable salts, esters, or salts of such esters, or any other functional chemical equivalent which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the antisense compounds, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
[0377] The term "prodrug" indicates a therapeutic agent that is prepared in an inactive or less active form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes, chemicals, and/or conditions. In particular, prodrug versions of the oligonucleotides are prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed in WO 93/24510 or WO 94/26764. Prodrugs can also include antisense compounds wherein one or both ends comprise nucleotides that are cleaved (e.g., by incorporating phosphodiester backbone linkages at the ends) to produce the active compound.
[0378] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans. In another embodiment of the invention and/or embodiments thereof, sodium salts of dsRNA compounds are also provided.
[0379] The antisense compounds described herein may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds.
[0380] The present disclosure also includes pharmaceutical compositions and formulations which include the antisense compounds described herein. The pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Optionally of the invention and/or embodiments thereof, administration is intramuscular or intravenous.
[0381] The pharmaceutical formulations, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product (e.g., into a specific particle size for delivery). Optionally of the invention and/or embodiments thereof, the pharmaceutical formulations are prepared for intramuscular administration in an appropriate solvent, e.g., water or normal saline, possibly in a sterile formulation, with carriers or other agents.
[0382] A "pharmaceutical carrier" or "excipient" can be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal and are known in the art. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
[0383] Compositions provided herein may contain two or more antisense compounds. In another related embodiment, compositions may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions provided herein can contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Two or more combined compounds may be used together or sequentially. Compositions can also be combined with other non-antisense compound therapeutic agents.
[0384] The antisense oligomeric compound described herein may be in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. antisense oligomeric compound compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. Suspensions may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
[0385] The present disclosure also includes antisense oligomeric compound compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co., A. R. Gennaro edit., 1985). For example, preservatives and stabilizers can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used.
[0386] Pharmaceutical compositions of this disclosure can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxy ethylene sorbitan monooleate.
[0387] The antisense oligomeric compound of this disclosure may be administered to a patient by any standard means, with or without stabilizers, buffers, or the like, to form a composition suitable for treatment. When it is desired to use a liposome delivery mechanism, standard protocols for formation of liposomes can be followed. Thus the antisense oligomeric compound of the present disclosure may be administered in any form, for example intramuscular or by local, systemic, or intrathecal injection.
[0388] This disclosure also features the use of antisense oligomeric compound compositions comprising surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modif[iota]ed, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of antisense oligomeric compound in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated antisense oligomeric compound (Lasic et al, Chem. Rev. 95:2601-2627 (1995) and Ishiwata et al, Chem. Pharm. Bull. 43:1005-1011 (1995). Long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of antisense oligomeric compound, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem. 42:24864-24870 (1995); Choi et al, PCT Publication No. WO 96/10391; Ansell et al, PCT Publication No. WO 96/10390; Holland et al, PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect antisense oligomeric compound from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
[0389] Following administration of the antisense oligomeric compound compositions according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated, as compared to placebo-treated or other suitable control subjects.
Sequence CWU
1
1
1594155DNAArtificial SequenceSequence in cDNA to which AON anneals
1gctctgcact cccctgctgg agcttttctc gcccttcctt ctggccctct cccca
55225DNAArtificial Sequencesequence of AON (5'-> 3') 2tggggagagg
gccagaagga agggc
25325DNAArtificial Sequencesequence of AON (5'-> 3') 3ggggagaggg
ccagaaggaa gggcg
25425DNAArtificial Sequencesequence of AON (5'-> 3') 4gggagagggc
cagaaggaag ggcga
25525DNAArtificial Sequencesequence of AON (5'-> 3') 5ggagagggcc
agaaggaagg gcgag
25625DNAArtificial Sequencesequence of AON (5'-> 3') 6gagagggcca
gaaggaaggg cgaga
25725DNAArtificial Sequencesequence of AON (5'-> 3') 7agagggccag
aaggaagggc gagaa
25825DNAArtificial Sequencesequence of AON (5'-> 3') 8gagggccaga
aggaagggcg agaaa
25925DNAArtificial Sequencesequence of AON (5'-> 3') 9agggccagaa
ggaagggcga gaaaa
251025DNAArtificial Sequencesequence of AON (5'-> 3') 10gggccagaag
gaagggcgag aaaag
251125DNAArtificial Sequencesequence of AON (5'-> 3') 11ggccagaagg
aagggcgaga aaagc
251225DNAArtificial Sequencesequence of AON (5'-> 3') 12gccagaagga
agggcgagaa aagct
251325DNAArtificial Sequencesequence of AON (5'-> 3') 13ccagaaggaa
gggcgagaaa agctc
251425DNAArtificial Sequencesequence of AON (5'-> 3') 14cagaaggaag
ggcgagaaaa gctcc
251525DNAArtificial Sequencesequence of AON (5'-> 3') 15agaaggaagg
gcgagaaaag ctcca
251625DNAArtificial Sequencesequence of AON (5'-> 3') 16gaaggaaggg
cgagaaaagc tccag
251725DNAArtificial Sequencesequence of AON (5'-> 3') 17aaggaagggc
gagaaaagct ccagc
251825DNAArtificial Sequencesequence of AON (5'-> 3') 18aggaagggcg
agaaaagctc cagca
251925DNAArtificial Sequencesequence of AON (5'-> 3') 19ggaagggcga
gaaaagctcc agcag
252025DNAArtificial Sequencesequence of AON (5'-> 3') 20gaagggcgag
aaaagctcca gcagg
252125DNAArtificial Sequencesequence of AON (5'-> 3') 21aagggcgaga
aaagctccag caggg
252225DNAArtificial Sequencesequence of AON (5'-> 3') 22agggcgagaa
aagctccagc agggg
252325DNAArtificial Sequencesequence of AON (5'-> 3') 23gggcgagaaa
agctccagca gggga
252425DNAArtificial Sequencesequence of AON (5'-> 3') 24ggcgagaaaa
gctccagcag gggag
252525DNAArtificial Sequencesequence of AON (5'-> 3') 25gcgagaaaag
ctccagcagg ggagt
252625DNAArtificial Sequencesequence of AON (5'-> 3') 26cgagaaaagc
tccagcaggg gagtg
252725DNAArtificial Sequencesequence of AON (5'-> 3') 27gagaaaagct
ccagcagggg agtgc
252825DNAArtificial Sequencesequence of AON (5'-> 3') 28agaaaagctc
cagcagggga gtgca
252925DNAArtificial Sequencesequence of AON (5'-> 3') 29gaaaagctcc
agcaggggag tgcag
253025DNAArtificial Sequencesequence of AON (5'-> 3') 30aaaagctcca
gcaggggagt gcaga
253125DNAArtificial Sequencesequence of AON (5'-> 3') 31aaagctccag
caggggagtg cagag
253225DNAArtificial Sequencesequence of AON (5'-> 3') 32aagctccagc
aggggagtgc agagc
253321DNAArtificial Sequencesequence of AON (5'-> 3') 33ccagaaggaa
gggcgagaaa a
213424DNAArtificial Sequencefake sequence to maintain correct numbering
34aaaaaaaaaa aaaaaaaaaa aaaa
243524DNAArtificial Sequencefake sequence to maintain correct numbering
35aaaaaaaaaa aaaaaaaaaa aaaa
243624DNAArtificial Sequencefake sequence to maintain correct numbering
36aaaaaaaaaa aaaaaaaaaa aaaa
243745DNAArtificial SequenceSequence in cDNA to which AON anneals
37gctctgcact cccctgctgg agcttttctc gcccttcctt ctggc
453831DNAArtificial SequenceSequence in cDNA to which AON anneals
38tgcactcccc tgctggagct tttctcgccc t
313936DNAArtificial SequenceSequence in cDNA to which AON anneals
39tgcactcccc tgctggagct tttctcgccc ttcctt
364031DNAArtificial SequenceSequence in cDNA to which AON anneals
40tcccctgctg gagcttttct cgcccttcct t
314120DNAArtificial SequenceAON sequence 5' -> 3' 41ccaaacagct
gtcgcctggg
204220DNAArtificial SequenceAON sequence 5' -> 3' 42aggtagacac
ttgaaacagg
204320DNAArtificial SequenceAON sequence 5' -> 3' 43cccaggaaga
ccagcaaggc
204420DNAArtificial SequenceAON sequence 5' -> 3' 44tcaaacacgc
ttagaatgtc
204520DNAArtificial SequenceAON sequence 5' -> 3' 45gtctgctaaa
atgttacaaa
204620DNAArtificial SequenceAON sequence 5' -> 3' 46gagtgcagag
cacttgcaca
204720DNAArtificial SequenceAON sequence 5' -> 3' 47cgagaaaagc
tccagcaggg
204820DNAArtificial SequenceAON sequence 5' -> 3' 48gagagggcca
gaaggaaggg
204920DNAArtificial SequenceAON sequence 5' -> 3' 49gccctgctgt
ctagactggg
205020DNAArtificial SequenceAON sequence 5' -> 3' 50aggtggccag
ggtgggtgtt
205120DNAArtificial SequenceAON sequence 5' -> 3' 51gcacccaggc
aggtggggta
205220DNAArtificial SequenceAON sequence 5' -> 3' 52caaccgcggc
tggcactgca
205320DNAArtificial SequenceAON sequence 5' -> 3' 53tcaaagcagc
tctgagacat
205420DNAArtificial SequenceAON sequence 5' -> 3' 54gggcggcact
cacggggctc
205520DNAArtificial SequenceAON sequence 5' -> 3' 55gctcagcagg
gaggcgggag
205620DNAArtificial SequenceAON sequence 5' -> 3' 56cctgcgggag
aagaaagcgg
205720DNAArtificial SequenceAON sequence 5' -> 3' 57gcctggacag
ctcctacagg
205820DNAArtificial SequenceAON sequence 5' -> 3' 58cactcccatg
gttggagatg
205920DNAArtificial SequenceAON sequence 5' -> 3' 59tgggagcagg
gcgggtgcct
206020DNAArtificial SequenceAON sequence 5' -> 3' 60cgcagacggc
caggagccgg
206120DNAArtificial SequenceAON sequence 5' -> 3' 61ggttgccaag
gacacgaggg
206220DNAArtificial SequenceAON sequence 5' -> 3' 62atgtgcccca
ggagtgcagc
206320DNAArtificial SequenceAON sequence 5' -> 3' 63gcaggaaatc
atggagtagg
206420DNAArtificial SequenceAON sequence 5' -> 3' 64actcagctct
cggggaacca
206520DNAArtificial SequenceAON sequence 5' -> 3' 65tccaggactg
gggaggagcc
206620DNAArtificial SequenceAON sequence 5' -> 3' 66ggtgagctgg
gtgagtctcc
206720DNAArtificial SequenceAON sequence 5' -> 3' 67tggtctgctg
gctccctgct
206820DNAArtificial SequenceAON sequence 5' -> 3' 68gcctgggcat
cccggggccc
206920DNAArtificial SequenceAON sequence 5' -> 3' 69ctctgggacg
gccggggtgt
207020DNAArtificial SequenceAON sequence 5' -> 3' 70gtcgcactgt
gtgggcactg
207120DNAArtificial SequenceAON sequence 5' -> 3' 71aagcggctgt
tgggggggac
207220DNAArtificial SequenceAON sequence 5' -> 3' 72ccttgtcagg
ggcgcaatcg
207320DNAArtificial SequenceAON sequence 5' -> 3' 73gcactgttcc
tgggtgatgg
207420DNAArtificial SequenceAON sequence 5' -> 3' 74tagcaacagc
cgcgggcctc
207520DNAArtificial SequenceAON sequence 5' -> 3' 75gcccctgctt
tgcagggatg
207620DNAArtificial SequenceAON sequence 5' -> 3' 76ccccatctgg
gctccctgca
207720DNAArtificial SequenceAON sequence 5' -> 3' 77gggaagaagc
accagggctg
207820DNAArtificial SequenceAON sequence 5' -> 3' 78tgtagctggg
gtagctgggt
207920DNAArtificial SequenceAON sequence 5' -> 3' 79ggagctcagg
ttctccagct
208020DNAArtificial SequenceAON sequence 5' -> 3' 80gccgtgtagc
ccatttcaga
208120DNAArtificial SequenceAON sequence 5' -> 3' 81gggtggtacg
ggtcagggtg
208220DNAArtificial SequenceAON sequence 5' -> 3' 82gtccttgggg
aagaaggtgg
208320DNAArtificial SequenceAON sequence 5' -> 3' 83tccagccgca
gggtcaggat
208420DNAArtificial SequenceAON sequence 5' -> 3' 84tctcagtctc
catcatcacg
208516DNAArtificial SequenceAON sequence 5' -> 3' 85gtgaagtgga ggcggt
168620DNAArtificial
SequenceAON sequence 5' -> 3' 86agagcacttg cacagtctgc
208720DNAArtificial SequenceAON sequence
5' -> 3' 87gcagagcact tgcacagtct
208820DNAArtificial SequenceAON sequence 5' -> 3'
88gtgcagagca cttgcacagt
208920DNAArtificial SequenceAON sequence 5' -> 3' 89gggagtgcag
agcacttgca
209020DNAArtificial SequenceAON sequence 5' -> 3' 90aggggagtgc
agagcacttg
209120DNAArtificial SequenceAON sequence 5' -> 3' 91gcaggggagt
gcagagcact
209220DNAArtificial SequenceAON sequence 5' -> 3' 92gccagaagga
agggcgagaa
209320DNAArtificial SequenceAON sequence 5' -> 3' 93gggccagaag
gaagggcgag
209420DNAArtificial SequenceAON sequence 5' -> 3' 94gagggccaga
aggaagggcg
209520DNAArtificial SequenceAON sequence 5' -> 3' 95gggagagggc
cagaaggaag
209620DNAArtificial SequenceAON sequence 5' -> 3' 96tggggagagg
gccagaagga
209720DNAArtificial SequenceAON sequence 5' -> 3' 97actggggaga
gggccagaag
209825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 98cacccaggca
ggtggggtaa ggtgg
259925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 99agcacccagg
caggtggggt aaggt
2510025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 100gcagcaccca
ggcaggtggg gtaag
2510125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 101ctgcagcacc
caggcaggtg gggta
2510225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 102cactgcagca
cccaggcagg tgggg
2510325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 103ggcactgcag
cacccaggca ggtgg
2510425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 104ctggcactgc
agcacccagg caggt
2510525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 105ggctggcact
gcagcaccca ggcag
2510625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 106gcggctggca
ctgcagcacc caggc
2510725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 107ccgcggctgg
cactgcagca cccag
2510825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 108tcaaccgcgg
ctggcactgc agcac
2510925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 109acccaggcag
gtggggtaag gtggc
2511025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 110gcacccaggc
aggtggggta aggtg
2511125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 111cagcacccag
gcaggtgggg taagg
2511225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 112tgcagcaccc
aggcaggtgg ggtaa
2511325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 113actgcagcac
ccaggcaggt ggggt
2511425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 114gcactgcagc
acccaggcag gtggg
2511525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 115tggcactgca
gcacccaggc aggtg
2511625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 116gctggcactg
cagcacccag gcagg
2511725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 117cggctggcac
tgcagcaccc aggca
2511825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 118cgcggctggc
actgcagcac ccagg
2511925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 119accgcggctg
gcactgcagc accca
2512025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 120caaccgcggc
tggcactgca gcacc
2512125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 121atcaaccgcg
gctggcactg cagca
2512225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 122ggctctcaaa
gcagctctga gacat
2512325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 123ggggctctca
aagcagctct gagac
2512425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 124acggggctct
caaagcagct ctgag
2512525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 125tcacggggct
ctcaaagcag ctctg
2512625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 126actcacgggg
ctctcaaagc agctc
2512725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 127gcactcacgg
ggctctcaaa gcagc
2512825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 128cggcactcac
ggggctctca aagca
2512925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 129ggcggcactc
acggggctct caaag
2513025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 130ggggcggcac
tcacggggct ctcaa
2513125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 131gaggggcggc
actcacgggg ctctc
2513225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 132gggaggggcg
gcactcacgg ggctc
2513325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 133gcgggagggg
cggcactcac ggggc
2513425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 134aggcgggagg
ggcggcactc acggg
2513525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 135ggaggcggga
ggggcggcac tcacg
2513625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 136agggaggcgg
gaggggcggc actca
2513725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 137gcagggaggc
gggaggggcg gcact
2513825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 138cagcagggag
gcgggagggg cggca
2513925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 139ctcagcaggg
aggcgggagg ggcgg
2514025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 140ggctcagcag
ggaggcggga ggggc
2514125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 141cgggctcagc
agggaggcgg gaggg
2514225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 142agcgggctca
gcagggaggc gggag
2514325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 143aaagcgggct
cagcagggag gcggg
2514425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 144agaaagcggg
ctcagcaggg aggcg
2514525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 145gaagaaagcg
ggctcagcag ggagg
2514625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 146gagaagaaag
cgggctcagc aggga
2514725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 147gggagaagaa
agcgggctca gcagg
2514825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 148gcgggagaag
aaagcgggct cagca
2514925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 149ctgcgggaga
agaaagcggg ctcag
2515025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 150gcctgcggga
gaagaaagcg ggctc
2515125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 151aggcctgcgg
gagaagaaag cgggc
2515225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 152actcccatgg
ttggagatgg cctgg
2515325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 153tcactcccat
ggttggagat ggcct
2515425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 154cctcactccc
atggttggag atggc
2515525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 155tgcctcactc
ccatggttgg agatg
2515625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 156ggtgcctcac
tcccatggtt ggaga
2515725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 157cgggtgcctc
actcccatgg ttgga
2515825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 158ggcgggtgcc
tcactcccat ggttg
2515925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 159agggcgggtg
cctcactccc atggt
2516025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 160gcagggcggg
tgcctcactc ccatg
2516125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 161gagcagggcg
ggtgcctcac tccca
2516225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 162gggagcaggg
cgggtgcctc actcc
2516325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 163gtgggagcag
ggcgggtgcc tcact
2516425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 164cggtgggagc
agggcgggtg cctca
2516525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 165gccggtggga
gcagggcggg tgcct
2516625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 166gagccggtgg
gagcagggcg ggtgc
2516725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 167aggagccggt
gggagcaggg cgggt
2516825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 168ccaggagccg
gtgggagcag ggcgg
2516925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 169ggccaggagc
cggtgggagc agggc
2517025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 170acggccagga
gccggtggga gcagg
2517125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 171agacggccag
gagccggtgg gagca
2517225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 172gcagacggcc
aggagccggt gggag
2517325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 173gcgcagacgg
ccaggagccg gtggg
2517425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 174gggcgcagac
ggccaggagc cggtg
2517525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 175gagggcgcag
acggccagga gccgg
2517625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 176acgagggcgc
agacggccag gagcc
2517725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 177acacgagggc
gcagacggcc aggag
2517825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 178ggacacgagg
gcgcagacgg ccagg
2517925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 179aaggacacga
gggcgcagac ggcca
2518025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 180ccaaggacac
gagggcgcag acggc
2518125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 181tgccaaggac
acgagggcgc agacg
2518225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 182gctctcaaag
cagctctgag acatc
2518325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 183gggctctcaa
agcagctctg agaca
2518425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 184ctcacggggc
tctcaaagca gctct
2518525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 185cactcacggg
gctctcaaag cagct
2518625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 186ggcactcacg
gggctctcaa agcag
2518725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 187gcggcactca
cggggctctc aaagc
2518825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 188gggcggcact
cacggggctc tcaaa
2518925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 189aggggcggca
ctcacggggc tctca
2519025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 190ggaggggcgg
cactcacggg gctct
2519125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 191cgggaggggc
ggcactcacg gggct
2519225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 192ggcgggaggg
gcggcactca cgggg
2519325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 193gaggcgggag
gggcggcact cacgg
2519425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 194gggaggcggg
aggggcggca ctcac
2519525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 195cagggaggcg
ggaggggcgg cactc
2519625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 196agcagggagg
cgggaggggc ggcac
2519725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 197tcagcaggga
ggcgggaggg gcggc
2519825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 198gctcagcagg
gaggcgggag gggcg
2519925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 199gggctcagca
gggaggcggg agggg
2520025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 200gcgggctcag
cagggaggcg ggagg
2520125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 201aagcgggctc
agcagggagg cggga
2520225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 202gaaagcgggc
tcagcaggga ggcgg
2520325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 203aagaaagcgg
gctcagcagg gaggc
2520425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 204agaagaaagc
gggctcagca gggag
2520525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 205ggagaagaaa
gcgggctcag caggg
2520625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 206cgggagaaga
aagcgggctc agcag
2520725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 207tgcgggagaa
gaaagcgggc tcagc
2520825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 208cctgcgggag
aagaaagcgg gctca
2520925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 209ggcctgcggg
agaagaaagc gggct
2521025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 210caggcctgcg
ggagaagaaa gcggg
2521125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 211cggggctctc
aaagcagctc tgaga
2521225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 212cacggggctc
tcaaagcagc tctga
2521325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 213ctcccatggt
tggagatggc ctgga
2521425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 214cactcccatg
gttggagatg gcctg
2521525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 215ctcactccca
tggttggaga tggcc
2521625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 216gcctcactcc
catggttgga gatgg
2521725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 217gtgcctcact
cccatggttg gagat
2521825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 218gggtgcctca
ctcccatggt tggag
2521925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 219gcgggtgcct
cactcccatg gttgg
2522025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 220gggcgggtgc
ctcactccca tggtt
2522125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 221cagggcgggt
gcctcactcc catgg
2522225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 222agcagggcgg
gtgcctcact cccat
2522325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 223ggagcagggc
gggtgcctca ctccc
2522425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 224tgggagcagg
gcgggtgcct cactc
2522525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 225ggtgggagca
gggcgggtgc ctcac
2522625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 226ccggtgggag
cagggcgggt gcctc
2522725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 227agccggtggg
agcagggcgg gtgcc
2522825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 228ggagccggtg
ggagcagggc gggtg
2522925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 229caggagccgg
tgggagcagg gcggg
2523025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 230gccaggagcc
ggtgggagca gggcg
2523125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 231cggccaggag
ccggtgggag caggg
2523225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 232gacggccagg
agccggtggg agcag
2523325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 233cagacggcca
ggagccggtg ggagc
2523425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 234cgcagacggc
caggagccgg tggga
2523525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 235ggcgcagacg
gccaggagcc ggtgg
2523625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 236agggcgcaga
cggccaggag ccggt
2523725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 237cgagggcgca
gacggccagg agccg
2523825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 238cacgagggcg
cagacggcca ggagc
2523925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 239gacacgaggg
cgcagacggc cagga
2524025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 240aggacacgag
ggcgcagacg gccag
2524125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 241caaggacacg
agggcgcaga cggcc
2524225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 242gccaaggaca
cgagggcgca gacgg
2524325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 243ttgccaagga
cacgagggcg cagac
2524425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 244ggatgtgccc
caggagtgca gcggt
2524525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 245taggatgtgc
cccaggagtg cagcg
2524625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 246agtaggatgt
gccccaggag tgcag
2524725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 247ggagtaggat
gtgccccagg agtgc
2524825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 248atggagtagg
atgtgcccca ggagt
2524925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 249tcatggagta
ggatgtgccc cagga
2525025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 250aatcatggag
taggatgtgc cccag
2525125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 251gaaatcatgg
agtaggatgt gcccc
2525225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 252aggaaatcat
ggagtaggat gtgcc
2525325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 253gcaggaaatc
atggagtagg atgtg
2525425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 254cagcaggaaa
tcatggagta ggatg
2525525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 255accagcagga
aatcatggag tagga
2525625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 256gaaccagcag
gaaatcatgg agtag
2525725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 257gggaaccagc
aggaaatcat ggagt
2525825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 258cggggaacca
gcaggaaatc atgga
2525925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 259ctcggggaac
cagcaggaaa tcatg
2526025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 260ctctcgggga
accagcagga aatca
2526125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 261agctctcggg
gaaccagcag gaaat
2526225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 262tcagctctcg
gggaaccagc aggaa
2526325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 263actcagctct
cggggaacca gcagg
2526425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 264ccactcagct
ctcggggaac cagca
2526525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 265agccactcag
ctctcgggga accag
2526625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 266ggagccactc
agctctcggg gaacc
2526725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 267gaggagccac
tcagctctcg gggaa
2526825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 268gggaggagcc
actcagctct cgggg
2526925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 269tggggaggag
ccactcagct ctcgg
2527025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 270actggggagg
agccactcag ctctc
2527125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 271ggactgggga
ggagccactc agctc
2527225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 272caggactggg
gaggagccac tcagc
2527325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 273tccaggactg
gggaggagcc actca
2527425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 274cctccaggac
tggggaggag ccact
2527525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 275ctcctccagg
actggggagg agcca
2527625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 276gtctcctcca
ggactgggga ggagc
2527725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 277gagtctcctc
caggactggg gagga
2527825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 278gtgagtctcc
tccaggactg gggag
2527925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 279gggtgagtct
cctccaggac tgggg
2528025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 280ctgggtgagt
ctcctccagg actgg
2528125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 281agctgggtga
gtctcctcca ggact
2528225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 282tgagctgggt
gagtctcctc cagga
2528325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 283ggtgagctgg
gtgagtctcc tccag
2528425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 284ctggtgagct
gggtgagtct cctcc
2528525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 285tgctggtgag
ctgggtgagt ctcct
2528625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 286cctgctggtg
agctgggtga gtctc
2528725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 287tccctgctgg
tgagctgggt gagtc
2528825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 288gctccctgct
ggtgagctgg gtgag
2528925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 289tggctccctg
ctggtgagct gggtg
2529025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 290gctggctccc
tgctggtgag ctggg
2529125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 291ctgctggctc
cctgctggtg agctg
2529225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 292gtctgctggc
tccctgctgg tgagc
2529325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 293gatgtgcccc
aggagtgcag cggtt
2529425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 294aggatgtgcc
ccaggagtgc agcgg
2529525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 295gtaggatgtg
ccccaggagt gcagc
2529625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 296gagtaggatg
tgccccagga gtgca
2529725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 297tggagtagga
tgtgccccag gagtg
2529825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 298catggagtag
gatgtgcccc aggag
2529925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 299atcatggagt
aggatgtgcc ccagg
2530025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 300aaatcatgga
gtaggatgtg cccca
2530125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 301ggaaatcatg
gagtaggatg tgccc
2530225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 302caggaaatca
tggagtagga tgtgc
2530325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 303agcaggaaat
catggagtag gatgt
2530425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 304ccagcaggaa
atcatggagt aggat
2530525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 305aaccagcagg
aaatcatgga gtagg
2530625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 306ggaaccagca
ggaaatcatg gagta
2530725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 307ggggaaccag
caggaaatca tggag
2530825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 308tcggggaacc
agcaggaaat catgg
2530925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 309tctcggggaa
ccagcaggaa atcat
2531025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 310gctctcgggg
aaccagcagg aaatc
2531125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 311cagctctcgg
ggaaccagca ggaaa
2531225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 312ctcagctctc
ggggaaccag cagga
2531325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 313cactcagctc
tcggggaacc agcag
2531425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 314gccactcagc
tctcggggaa ccagc
2531525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 315gagccactca
gctctcgggg aacca
2531625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 316aggagccact
cagctctcgg ggaac
2531725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 317ggaggagcca
ctcagctctc gggga
2531825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 318ggggaggagc
cactcagctc tcggg
2531925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 319ctggggagga
gccactcagc tctcg
2532025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 320gactggggag
gagccactca gctct
2532125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 321aggactgggg
aggagccact cagct
2532225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 322ccaggactgg
ggaggagcca ctcag
2532325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 323ctccaggact
ggggaggagc cactc
2532425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 324tcctccagga
ctggggagga gccac
2532525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 325tctcctccag
gactggggag gagcc
2532625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 326agtctcctcc
aggactgggg aggag
2532725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 327tgagtctcct
ccaggactgg ggagg
2532825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 328ggtgagtctc
ctccaggact gggga
2532925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 329tgggtgagtc
tcctccagga ctggg
2533025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 330gctgggtgag
tctcctccag gactg
2533125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 331gagctgggtg
agtctcctcc aggac
2533225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 332gtgagctggg
tgagtctcct ccagg
2533325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 333tggtgagctg
ggtgagtctc ctcca
2533425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 334gctggtgagc
tgggtgagtc tcctc
2533525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 335ctgctggtga
gctgggtgag tctcc
2533625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 336ccctgctggt
gagctgggtg agtct
2533725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 337ctccctgctg
gtgagctggg tgagt
2533825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 338ggctccctgc
tggtgagctg ggtga
2533925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 339ctggctccct
gctggtgagc tgggt
2534025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 340tgctggctcc
ctgctggtga gctgg
2534125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 341tctgctggct
ccctgctggt gagct
2534225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 342ggtctgctgg
ctccctgctg gtgag
2534325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 343agcccctgct
ttgcagggat gtagc
2534425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 344gcagcccctg
ctttgcaggg atgta
2534525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 345ctgcagcccc
tgctttgcag ggatg
2534625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 346ccctgcagcc
cctgctttgc aggga
2534725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 347ctccctgcag
cccctgcttt gcagg
2534825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 348ggctccctgc
agcccctgct ttgca
2534925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 349tgggctccct
gcagcccctg ctttg
2535025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 350tctgggctcc
ctgcagcccc tgctt
2535125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 351catctgggct
ccctgcagcc cctgc
2535225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 352cccatctggg
ctccctgcag cccct
2535325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 353gccccatctg
ggctccctgc agccc
2535425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 354ctgccccatc
tgggctccct gcagc
2535525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 355ggctgcccca
tctgggctcc ctgca
2535625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 356agggctgccc
catctgggct ccctg
2535725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 357ccagggctgc
cccatctggg ctccc
2535825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 358caccagggct
gccccatctg ggctc
2535925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 359agcaccaggg
ctgccccatc tgggc
2536025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 360gaagcaccag
ggctgcccca tctgg
2536125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 361aagaagcacc
agggctgccc catct
2536225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 362ggaagaagca
ccagggctgc cccat
2536325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 363tgggaagaag
caccagggct gcccc
2536425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 364ggtgggaaga
agcaccaggg ctgcc
2536525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 365tgggtgggaa
gaagcaccag ggctg
2536625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 366gctgggtggg
aagaagcacc agggc
2536725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 367gcccctgctt
tgcagggatg tagca
2536825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 368cagcccctgc
tttgcaggga tgtag
2536925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 369tgcagcccct
gctttgcagg gatgt
2537025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 370cctgcagccc
ctgctttgca gggat
2537125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 371tccctgcagc
ccctgctttg caggg
2537225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 372gctccctgca
gcccctgctt tgcag
2537325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 373gggctccctg
cagcccctgc tttgc
2537425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 374ctgggctccc
tgcagcccct gcttt
2537525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 375atctgggctc
cctgcagccc ctgct
2537625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 376ccatctgggc
tccctgcagc ccctg
2537725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 377ccccatctgg
gctccctgca gcccc
2537825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 378tgccccatct
gggctccctg cagcc
2537925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 379gctgccccat
ctgggctccc tgcag
2538025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 380gggctgcccc
atctgggctc cctgc
2538125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 381cagggctgcc
ccatctgggc tccct
2538225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 382accagggctg
ccccatctgg gctcc
2538325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 383gcaccagggc
tgccccatct gggct
2538425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 384aagcaccagg
gctgccccat ctggg
2538525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 385agaagcacca
gggctgcccc atctg
2538625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 386gaagaagcac
cagggctgcc ccatc
2538725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 387gggaagaagc
accagggctg cccca
2538825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 388gtgggaagaa
gcaccagggc tgccc
2538925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 389gggtgggaag
aagcaccagg gctgc
2539025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 390ctgggtggga
agaagcacca gggct
2539125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 391agctgggtgg
gaagaagcac caggg
2539225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 392cagcttgtag
ctggggtagc tgggt
2539325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 393tccagcttgt
agctggggta gctgg
2539425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 394tctccagctt
gtagctgggg tagct
2539525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 395gttctccagc
ttgtagctgg ggtag
2539625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 396aggttctcca
gcttgtagct ggggt
2539725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 397tcaggttctc
cagcttgtag ctggg
2539825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 398gctcaggttc
tccagcttgt agctg
2539925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 399gagctcaggt
tctccagctt gtagc
2540025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 400aggagctcag
gttctccagc ttgta
2540125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 401agaggagctc
aggttctcca gcttg
2540225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 402tcagaggagc
tcaggttctc cagct
2540325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 403tttcagagga
gctcaggttc tccag
2540425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 404agcttgtagc
tggggtagct gggtg
2540525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 405ccagcttgta
gctggggtag ctggg
2540625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 406ctccagcttg
tagctggggt agctg
2540725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 407ttctccagct
tgtagctggg gtagc
2540825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 408ggttctccag
cttgtagctg gggta
2540925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 409caggttctcc
agcttgtagc tgggg
2541025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 410ctcaggttct
ccagcttgta gctgg
2541125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 411agctcaggtt
ctccagcttg tagct
2541225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 412ggagctcagg
ttctccagct tgtag
2541325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 413gaggagctca
ggttctccag cttgt
2541425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 414cagaggagct
caggttctcc agctt
2541525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 415ttcagaggag
ctcaggttct ccagc
2541625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 416atttcagagg
agctcaggtt ctcca
2541725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 417ggggtggtac
gggtcagggt ggccg
2541825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 418tgggggtggt
acgggtcagg gtggc
2541925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 419ggtgggggtg
gtacgggtca gggtg
2542025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 420aaggtggggg
tggtacgggt caggg
2542125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 421agaaggtggg
ggtggtacgg gtcag
2542225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 422gaagaaggtg
ggggtggtac gggtc
2542325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 423gggaagaagg
tgggggtggt acggg
2542425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 424tggggaagaa
ggtgggggtg gtacg
2542525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 425cttggggaag
aaggtggggg tggta
2542625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 426tccttgggga
agaaggtggg ggtgg
2542725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 427tgtccttggg
gaagaaggtg ggggt
2542825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 428gatgtccttg
gggaagaagg tgggg
2542925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 429aggatgtcct
tggggaagaa ggtgg
2543025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 430tcaggatgtc
cttggggaag aaggt
2543125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 431ggtcaggatg
tccttgggga agaag
2543225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 432agggtcagga
tgtccttggg gaaga
2543325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 433gcagggtcag
gatgtccttg gggaa
2543425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 434ccgcagggtc
aggatgtcct tgggg
2543525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 435agccgcaggg
tcaggatgtc cttgg
2543625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 436gggtggtacg
ggtcagggtg gccgt
2543725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 437gggggtggta
cgggtcaggg tggcc
2543825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 438gtgggggtgg
tacgggtcag ggtgg
2543925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 439aggtgggggt
ggtacgggtc agggt
2544025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 440gaaggtgggg
gtggtacggg tcagg
2544125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 441aagaaggtgg
gggtggtacg ggtca
2544225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 442ggaagaaggt
gggggtggta cgggt
2544325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 443ggggaagaag
gtgggggtgg tacgg
2544425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 444ttggggaaga
aggtgggggt ggtac
2544525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 445ccttggggaa
gaaggtgggg gtggt
2544625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 446gtccttgggg
aagaaggtgg gggtg
2544725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 447atgtccttgg
ggaagaaggt ggggg
2544825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 448ggatgtcctt
ggggaagaag gtggg
2544925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 449caggatgtcc
ttggggaaga aggtg
2545025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 450gtcaggatgt
ccttggggaa gaagg
2545125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 451gggtcaggat
gtccttgggg aagaa
2545225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 452cagggtcagg
atgtccttgg ggaag
2545325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 453cgcagggtca
ggatgtcctt gggga
2545425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 454gccgcagggt
caggatgtcc ttggg
2545525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 455cagccgcagg
gtcaggatgt ccttg
2545625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 456cgtccagccg
cagggtcagg atgtc
2545725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 457cacgtccagc
cgcagggtca ggatg
2545825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 458atcacgtcca
gccgcagggt cagga
2545925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 459tcatcacgtc
cagccgcagg gtcag
2546025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 460catcatcacg
tccagccgca gggtc
2546125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 461tccatcatca
cgtccagccg caggg
2546225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 462tctccatcat
cacgtccagc cgcag
2546325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 463agtctccatc
atcacgtcca gccgc
2546425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 464tcagtctcca
tcatcacgtc cagcc
2546525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 465tctcagtctc
catcatcacg tccag
2546625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 466gttctcagtc
tccatcatca cgtcc
2546725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 467cggttctcag
tctccatcat cacgt
2546825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 468ggcggttctc
agtctccatc atcac
2546925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 469gaggcggttc
tcagtctcca tcatc
2547025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 470tggaggcggt
tctcagtctc catca
2547125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 471agtggaggcg
gttctcagtc tccat
2547225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 472gaagtggagg
cggttctcag tctcc
2547325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 473gtccagccgc
agggtcagga tgtcc
2547425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 474acgtccagcc
gcagggtcag gatgt
2547525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 475tcacgtccag
ccgcagggtc aggat
2547625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 476catcacgtcc
agccgcaggg tcagg
2547725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 477atcatcacgt
ccagccgcag ggtca
2547825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 478ccatcatcac
gtccagccgc agggt
2547925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 479ctccatcatc
acgtccagcc gcagg
2548025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 480gtctccatca
tcacgtccag ccgca
2548125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 481cagtctccat
catcacgtcc agccg
2548225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 482ctcagtctcc
atcatcacgt ccagc
2548325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 483ttctcagtct
ccatcatcac gtcca
2548425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 484ggttctcagt
ctccatcatc acgtc
2548525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 485gcggttctca
gtctccatca tcacg
2548625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 486aggcggttct
cagtctccat catca
2548725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 487ggaggcggtt
ctcagtctcc atcat
2548825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 488gtggaggcgg
ttctcagtct ccatc
2548925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 489aagtggaggc
ggttctcagt ctcca
2549025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 490tgaagtggag
gcggttctca gtctc
2549125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 491tgccctgccc
accgtgaagt ggagg
2549225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 492cctgccctgc
ccaccgtgaa gtgga
2549325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 493cccctgccct
gcccaccgtg aagtg
2549425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 494cgcccctgcc
ctgcccaccg tgaag
2549525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 495cccgcccctg
ccctgcccac cgtga
2549625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 496gccctgccca
ccgtgaagtg gaggc
2549725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 497ctgccctgcc
caccgtgaag tggag
2549825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 498ccctgccctg
cccaccgtga agtgg
2549925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 499gcccctgccc
tgcccaccgt gaagt
2550025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 500ccgcccctgc
cctgcccacc gtgaa
2550125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 501ccccgcccct
gccctgccca ccgtg
2550225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 502gcccccgccc
ctgccctgcc caccg
2550325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 503ccgcccccgc
ccctgccctg cccac
2550425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 504cgccgccccc
gcccctgccc tgccc
2550525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 505gccgccgccc
ccgcccctgc cctgc
2550625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 506tggccgccgc
ccccgcccct gccct
2550725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 507cctggccgcc
gcccccgccc ctgcc
2550825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 508gccctggccg
ccgcccccgc ccctg
2550925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 509ctgccctggc
cgccgccccc gcccc
2551025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 510ctctgccctg
gccgccgccc ccgcc
2551125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 511ccctctgccc
tggccgccgc ccccg
2551225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 512caccctctgc
cctggccgcc gcccc
2551325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 513cgcaccctct
gccctggccg ccgcc
2551425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 514cgcgcaccct
ctgccctggc cgccg
2551525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 515cccccgcccc
tgccctgccc accgt
2551625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 516cgcccccgcc
cctgccctgc ccacc
2551725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 517gccgcccccg
cccctgccct gccca
2551825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 518ccgccgcccc
cgcccctgcc ctgcc
2551925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 519ggccgccgcc
cccgcccctg ccctg
2552025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 520ctggccgccg
cccccgcccc tgccc
2552125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 521ccctggccgc
cgcccccgcc cctgc
2552225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 522tgccctggcc
gccgcccccg cccct
2552325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 523tctgccctgg
ccgccgcccc cgccc
2552425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 524cctctgccct
ggccgccgcc cccgc
2552525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 525accctctgcc
ctggccgccg ccccc
2552625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 526gcaccctctg
ccctggccgc cgccc
2552725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 527gcgcaccctc
tgccctggcc gccgc
2552825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 528agagatgggg
gtttattgat gttcc
2552925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 529gaagagatgg
gggtttattg atgtt
2553025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 530tagaagagat
gggggtttat tgatg
2553125DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 531tctagaagag
atgggggttt attga
2553225DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 532gatctagaag
agatgggggt ttatt
2553325DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 533ttgatctaga
agagatgggg gttta
2553425DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 534ctttgatcta
gaagagatgg gggtt
2553525DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 535atctttgatc
tagaagagat ggggg
2553625DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 536ggatctttga
tctagaagag atggg
2553725DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 537ctggatcttt
gatctagaag agatg
2553825DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 538agctggatct
ttgatctaga agaga
2553925DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 539ttagctggat
ctttgatcta gaaga
2554025DNAArtificial SequenceAON sequence designed to block the region
surrounding the identified splice element (5' -> 3') 540tgttagctgg
atctttgatc tagaa
2554125DNAArtificial SequenceAON sequence 5' -> 3' 541ctggaaggga
agcagctctg gggtt
2554225DNAArtificial SequenceAON sequence 5' -> 3' 542tctggaaggg
aagcagctct ggggt
2554325DNAArtificial SequenceAON sequence 5' -> 3' 543atctggaagg
gaagcagctc tgggg
2554425DNAArtificial SequenceAON sequence 5' -> 3' 544catctggaag
ggaagcagct ctggg
2554525DNAArtificial SequenceAON sequence 5' -> 3' 545acatctggaa
gggaagcagc tctgg
2554625DNAArtificial SequenceAON sequence 5' -> 3' 546cacatctgga
agggaagcag ctctg
2554725DNAArtificial SequenceAON sequence 5' -> 3' 547ccacatctgg
aagggaagca gctct
2554825DNAArtificial SequenceAON sequence 5' -> 3' 548accacatctg
gaagggaagc agctc
2554925DNAArtificial SequenceAON sequence 5' -> 3' 549gaccacatct
ggaagggaag cagct
2555025DNAArtificial SequenceAON sequence 5' -> 3' 550ggaccacatc
tggaagggaa gcagc
2555125DNAArtificial SequenceAON sequence 5' -> 3' 551aggaccacat
ctggaaggga agcag
2555225DNAArtificial SequenceAON sequence 5' -> 3' 552caggaccaca
tctggaaggg aagca
2555325DNAArtificial SequenceAON sequence 5' -> 3' 553gcaggaccac
atctggaagg gaagc
2555425DNAArtificial SequenceAON sequence 5' -> 3' 554tgcaggacca
catctggaag ggaag
2555525DNAArtificial SequenceAON sequence 5' -> 3' 555ctgcaggacc
acatctggaa gggaa
2555625DNAArtificial SequenceAON sequence 5' -> 3' 556gctgcaggac
cacatctgga aggga
2555725DNAArtificial SequenceAON sequence 5' -> 3' 557ggctgcagga
ccacatctgg aaggg
2555825DNAArtificial SequenceAON sequence 5' -> 3' 558cggctgcagg
accacatctg gaagg
2555925DNAArtificial SequenceAON sequence 5' -> 3' 559tcggctgcag
gaccacatct ggaag
2556025DNAArtificial SequenceAON sequence 5' -> 3' 560ctcggctgca
ggaccacatc tggaa
2556125DNAArtificial SequenceAON sequence 5' -> 3' 561gctcggctgc
aggaccacat ctgga
2556225DNAArtificial SequenceAON sequence 5' -> 3' 562ggctcggctg
caggaccaca tctgg
2556325DNAArtificial SequenceAON sequence 5' -> 3' 563gggctcggct
gcaggaccac atctg
2556425DNAArtificial SequenceAON sequence 5' -> 3' 564agggctcggc
tgcaggacca catct
2556525DNAArtificial SequenceAON sequence 5' -> 3' 565cagggctcgg
ctgcaggacc acatc
2556625DNAArtificial SequenceAON sequence 5' -> 3' 566gcagggctcg
gctgcaggac cacat
2556725DNAArtificial SequenceAON sequence 5' -> 3' 567ggcagggctc
ggctgcagga ccaca
2556825DNAArtificial SequenceAON sequence 5' -> 3' 568gggcagggct
cggctgcagg accac
2556925DNAArtificial SequenceAON sequence 5' -> 3' 569agggcagggc
tcggctgcag gacca
2557025DNAArtificial SequenceAON sequence 5' -> 3' 570aagggcaggg
ctcggctgca ggacc
2557125DNAArtificial SequenceAON sequence 5' -> 3' 571taagggcagg
gctcggctgc aggac
2557225DNAArtificial SequenceAON sequence 5' -> 3' 572ctaagggcag
ggctcggctg cagga
2557325DNAArtificial SequenceAON sequence 5' -> 3' 573gctaagggca
gggctcggct gcagg
2557425DNAArtificial SequenceAON sequence 5' -> 3' 574agctaagggc
agggctcggc tgcag
2557525DNAArtificial SequenceAON sequence 5' -> 3' 575cagctaaggg
cagggctcgg ctgca
2557625DNAArtificial SequenceAON sequence 5' -> 3' 576ccagctaagg
gcagggctcg gctgc
2557725DNAArtificial SequenceAON sequence 5' -> 3' 577tccagctaag
ggcagggctc ggctg
2557825DNAArtificial SequenceAON sequence 5' -> 3' 578ctccagctaa
gggcagggct cggct
2557925DNAArtificial SequenceAON sequence 5' -> 3' 579cctccagcta
agggcagggc tcggc
2558025DNAArtificial SequenceAON sequence 5' -> 3' 580acctccagct
aagggcaggg ctcgg
2558125DNAArtificial SequenceAON sequence 5' -> 3' 581gacctccagc
taagggcagg gctcg
2558225DNAArtificial SequenceAON sequence 5' -> 3' 582gacctccagc
taagggcagg gctcg
2558325DNAArtificial SequenceAON sequence 5' -> 3' 583tcgacctcca
gctaagggca gggct
2558425DNAArtificial SequenceAON sequence 5' -> 3' 584gtcgacctcc
agctaagggc agggc
2558525DNAArtificial SequenceAON sequence 5' -> 3' 585tgtcgacctc
cagctaaggg caggg
2558625DNAArtificial SequenceAON sequence 5' -> 3' 586ctgtcgacct
ccagctaagg gcagg
2558725DNAArtificial SequenceAON sequence 5' -> 3' 587cctgtcgacc
tccagctaag ggcag
2558825DNAArtificial SequenceAON sequence 5' -> 3' 588acctgtcgac
ctccagctaa gggca
2558925DNAArtificial SequenceAON sequence 5' -> 3' 589cacctgtcga
cctccagcta agggc
2559025DNAArtificial SequenceAON sequence 5' -> 3' 590ccacctgtcg
acctccagct aaggg
2559125DNAArtificial SequenceAON sequence 5' -> 3' 591cccacctgtc
gacctccagc taagg
2559225DNAArtificial SequenceAON sequence 5' -> 3' 592tcccacctgt
cgacctccag ctaag
2559325DNAArtificial SequenceAON sequence 5' -> 3' 593atcccacctg
tcgacctcca gctaa
2559425DNAArtificial SequenceAON sequence 5' -> 3' 594gatcccacct
gtcgacctcc agcta
2559525DNAArtificial SequenceAON sequence 5' -> 3' 595ggatcccacc
tgtcgacctc cagct
2559625DNAArtificial SequenceAON sequence 5' -> 3' 596aggatcccac
ctgtcgacct ccagc
2559725DNAArtificial SequenceAON sequence 5' -> 3' 597caggatccca
cctgtcgacc tccag
2559825DNAArtificial SequenceAON sequence 5' -> 3' 598ccaggatccc
acctgtcgac ctcca
2559925DNAArtificial SequenceAON sequence 5' -> 3' 599tccaggatcc
cacctgtcga cctcc
2560025DNAArtificial SequenceAON sequence 5' -> 3' 600atccaggatc
ccacctgtcg acctc
2560125DNAArtificial SequenceAON sequence 5' -> 3' 601catccaggat
cccacctgtc gacct
2560225DNAArtificial SequenceAON sequence 5' -> 3' 602acatccagga
tcccacctgt cgacc
2560325DNAArtificial SequenceAON sequence 5' -> 3' 603gacatccagg
atcccacctg tcgac
2560425DNAArtificial SequenceAON sequence 5' -> 3' 604agacatccag
gatcccacct gtcga
2560525DNAArtificial SequenceAON sequence 5' -> 3' 605tagacatcca
ggatcccacc tgtcg
2560625DNAArtificial SequenceAON sequence 5' -> 3' 606gtagacatcc
aggatcccac ctgtc
2560725DNAArtificial SequenceAON sequence 5' -> 3' 607tgtagacatc
caggatccca cctgt
2560825DNAArtificial SequenceAON sequence 5' -> 3' 608atgtagacat
ccaggatccc acctg
2560925DNAArtificial SequenceAON sequence 5' -> 3' 609gatgtagaca
tccaggatcc cacct
2561025DNAArtificial SequenceAON sequence 5' -> 3' 610agatgtagac
atccaggatc ccacc
2561125DNAArtificial SequenceAON sequence 5' -> 3' 611aagatgtaga
catccaggat cccac
2561225DNAArtificial SequenceAON sequence 5' -> 3' 612gaagatgtag
acatccagga tccca
2561325DNAArtificial SequenceAON sequence 5' -> 3' 613ggaagatgta
gacatccagg atccc
2561425DNAArtificial SequenceAON sequence 5' -> 3' 614aggaagatgt
agacatccag gatcc
2561525DNAArtificial SequenceAON sequence 5' -> 3' 615caggaagatg
tagacatcca ggatc
2561625DNAArtificial SequenceAON sequence 5' -> 3' 616ccaggaagat
gtagacatcc aggat
2561725DNAArtificial SequenceAON sequence 5' -> 3' 617cccaggaaga
tgtagacatc cagga
2561825DNAArtificial SequenceAON sequence 5' -> 3' 618gcccaggaag
atgtagacat ccagg
2561925DNAArtificial SequenceAON sequence 5' -> 3' 619ggcccaggaa
gatgtagaca tccag
2562025DNAArtificial SequenceAON sequence 5' -> 3' 620gggcccagga
agatgtagac atcca
2562125DNAArtificial SequenceAON sequence 5' -> 3' 621tgggcccagg
aagatgtaga catcc
2562225DNAArtificial SequenceAON sequence 5' -> 3' 622ctgggcccag
gaagatgtag acatc
2562325DNAArtificial SequenceAON sequence 5' -> 3' 623tctgggccca
ggaagatgta gacat
2562425DNAArtificial SequenceAON sequence 5' -> 3' 624ctctgggccc
aggaagatgt agaca
2562525DNAArtificial SequenceAON sequence 5' -> 3' 625gctctgggcc
caggaagatg tagac
2562625DNAArtificial SequenceAON sequence 5' -> 3' 626ggctctgggc
ccaggaagat gtaga
2562725DNAArtificial SequenceAON sequence 5' -> 3' 627gggctctggg
cccaggaaga tgtag
2562825DNAArtificial SequenceAON sequence 5' -> 3' 628tgggctctgg
gcccaggaag atgta
2562925DNAArtificial SequenceAON sequence 5' -> 3' 629ttgggctctg
ggcccaggaa gatgt
2563025DNAArtificial SequenceAON sequence 5' -> 3' 630cttgggctct
gggcccagga agatg
2563125DNAArtificial SequenceAON sequence 5' -> 3' 631tcttgggctc
tgggcccagg aagat
2563225DNAArtificial SequenceAON sequence 5' -> 3' 632ctcttgggct
ctgggcccag gaaga
2563325DNAArtificial SequenceAON sequence 5' -> 3' 633gctcttgggc
tctgggccca ggaag
2563425DNAArtificial SequenceAON sequence 5' -> 3' 634cgctcttggg
ctctgggccc aggaa
2563525DNAArtificial SequenceAON sequence 5' -> 3' 635acgctcttgg
gctctgggcc cagga
2563625DNAArtificial SequenceAON sequence 5' -> 3' 636cacgctcttg
ggctctgggc ccagg
2563725DNAArtificial SequenceAON sequence 5' -> 3' 637ccacgctctt
gggctctggg cccag
2563825DNAArtificial SequenceAON sequence 5' -> 3' 638accacgctct
tgggctctgg gccca
2563925DNAArtificial SequenceAON sequence 5' -> 3' 639caccacgctc
ttgggctctg ggccc
2564025DNAArtificial SequenceAON sequence 5' -> 3' 640gcaccacgct
cttgggctct gggcc
2564125DNAArtificial SequenceAON sequence 5' -> 3' 641tgcaccacgc
tcttgggctc tgggc
2564225DNAArtificial SequenceAON sequence 5' -> 3' 642ctgcaccacg
ctcttgggct ctggg
2564325DNAArtificial SequenceAON sequence 5' -> 3' 643gctgcaccac
gctcttgggc tctgg
2564425DNAArtificial SequenceAON sequence 5' -> 3' 644tgctgcacca
cgctcttggg ctctg
2564525DNAArtificial SequenceAON sequence 5' -> 3' 645ctgctgcacc
acgctcttgg gctct
2564625DNAArtificial SequenceAON sequence 5' -> 3' 646actgctgcac
cacgctcttg ggctc
2564725DNAArtificial SequenceAON sequence 5' -> 3' 647tactgctgca
ccacgctctt gggct
2564825DNAArtificial SequenceAON sequence 5' -> 3' 648gtactgctgc
accacgctct tgggc
2564925DNAArtificial SequenceAON sequence 5' -> 3' 649ggtactgctg
caccacgctc ttggg
2565025DNAArtificial SequenceAON sequence 5' -> 3' 650aggtactgct
gcaccacgct cttgg
2565125DNAArtificial SequenceAON sequence 5' -> 3' 651caggtactgc
tgcaccacgc tcttg
2565225DNAArtificial SequenceAON sequence 5' -> 3' 652ccaggtactg
ctgcaccacg ctctt
2565325DNAArtificial SequenceAON sequence 5' -> 3' 653tccaggtact
gctgcaccac gctct
2565425DNAArtificial SequenceAON sequence 5' -> 3' 654gtccaggtac
tgctgcacca cgctc
2565525DNAArtificial SequenceAON sequence 5' -> 3' 655cgtccaggta
ctgctgcacc acgct
2565625DNAArtificial SequenceAON sequence 5' -> 3' 656acgtccaggt
actgctgcac cacgc
2565725DNAArtificial SequenceAON sequence 5' -> 3' 657aacgtccagg
tactgctgca ccacg
2565825DNAArtificial SequenceAON sequence 5' -> 3' 658caacgtccag
gtactgctgc accac
2565925DNAArtificial SequenceAON sequence 5' -> 3' 659acaacgtcca
ggtactgctg cacca
2566025DNAArtificial SequenceAON sequence 5' -> 3' 660cacaacgtcc
aggtactgct gcacc
2566125DNAArtificial SequenceAON sequence 5' -> 3' 661ccacaacgtc
caggtactgc tgcac
2566225DNAArtificial SequenceAON sequence 5' -> 3' 662cccacaacgt
ccaggtactg ctgca
2566325DNAArtificial SequenceAON sequence 5' -> 3' 663acccacaacg
tccaggtact gctgc
2566425DNAArtificial SequenceAON sequence 5' -> 3' 664tacccacaac
gtccaggtac tgctg
2566525DNAArtificial SequenceAON sequence 5' -> 3' 665ctacccacaa
cgtccaggta ctgct
2566625DNAArtificial SequenceAON sequence 5' -> 3' 666cctacccaca
acgtccaggt actgc
2566725DNAArtificial SequenceAON sequence 5' -> 3' 667ccctacccac
aacgtccagg tactg
2566825DNAArtificial SequenceAON sequence 5' -> 3' 668gccctaccca
caacgtccag gtact
2566925DNAArtificial SequenceAON sequence 5' -> 3' 669ggccctaccc
acaacgtcca ggtac
2567025DNAArtificial SequenceAON sequence 5' -> 3' 670aggccctacc
cacaacgtcc aggta
2567125DNAArtificial SequenceAON sequence 5' -> 3' 671caggccctac
ccacaacgtc caggt
2567225DNAArtificial SequenceAON sequence 5' -> 3' 672gcaggcccta
cccacaacgt ccagg
2567325DNAArtificial SequenceAON sequence 5' -> 3' 673agcaggccct
acccacaacg tccag
2567425DNAArtificial SequenceAON sequence 5' -> 3' 674gagcaggccc
tacccacaac gtcca
2567525DNAArtificial SequenceAON sequence 5' -> 3' 675ggagcaggcc
ctacccacaa cgtcc
2567625DNAArtificial SequenceAON sequence 5' -> 3' 676gggagcaggc
cctacccaca acgtc
2567725DNAArtificial SequenceAON sequence 5' -> 3' 677agggagcagg
ccctacccac aacgt
2567825DNAArtificial SequenceAON sequence 5' -> 3' 678cagggagcag
gccctaccca caacg
2567925DNAArtificial SequenceAON sequence 5' -> 3' 679ccagggagca
ggccctaccc acaac
2568025DNAArtificial SequenceAON sequence 5' -> 3' 680gccagggagc
aggccctacc cacaa
2568125DNAArtificial SequenceAON sequence 5' -> 3' 681ggccagggag
caggccctac ccaca
2568225DNAArtificial SequenceAON sequence 5' -> 3' 682cggccaggga
gcaggcccta cccac
2568325DNAArtificial SequenceAON sequence 5' -> 3' 683gcggccaggg
agcaggccct accca
2568425DNAArtificial SequenceAON sequence 5' -> 3' 684cgcggccagg
gagcaggccc taccc
2568525DNAArtificial SequenceAON sequence 5' -> 3' 685ccgcggccag
ggagcaggcc ctacc
2568625DNAArtificial SequenceAON sequence 5' -> 3' 686gccgcggcca
gggagcaggc cctac
2568725DNAArtificial SequenceAON sequence 5' -> 3' 687ggccgcggcc
agggagcagg cccta
2568825DNAArtificial SequenceAON sequence 5' -> 3' 688gggccgcggc
cagggagcag gccct
2568925DNAArtificial SequenceAON sequence 5' -> 3' 689ggggccgcgg
ccagggagca ggccc
2569025DNAArtificial SequenceAON sequence 5' -> 3' 690gggggccgcg
gccagggagc aggcc
2569125DNAArtificial SequenceAON sequence 5' -> 3' 691cgggggccgc
ggccagggag caggc
2569225DNAArtificial SequenceAON sequence 5' -> 3' 692gcgggggccg
cggccaggga gcagg
2569325DNAArtificial SequenceAON sequence 5' -> 3' 693ggcgggggcc
gcggccaggg agcag
2569425DNAArtificial SequenceAON sequence 5' -> 3' 694gggcgggggc
cgcggccagg gagca
2569525DNAArtificial SequenceAON sequence 5' -> 3' 695ggggcggggg
ccgcggccag ggagc
2569625DNAArtificial SequenceAON sequence 5' -> 3' 696tggggcgggg
gccgcggcca gggag
2569725DNAArtificial SequenceAON sequence 5' -> 3' 697ttggggcggg
ggccgcggcc aggga
2569825DNAArtificial SequenceAON sequence 5' -> 3' 698cttggggcgg
gggccgcggc caggg
2569925DNAArtificial SequenceAON sequence 5' -> 3' 699ccttggggcg
ggggccgcgg ccagg
2570025DNAArtificial SequenceAON sequence 5' -> 3' 700gccttggggc
gggggccgcg gccag
2570125DNAArtificial SequenceAON sequence 5' -> 3' 701agccttgggg
cgggggccgc ggcca
2570225DNAArtificial SequenceAON sequence 5' -> 3' 702gagccttggg
gcgggggccg cggcc
2570325DNAArtificial SequenceAON sequence 5' -> 3' 703ggagccttgg
ggcgggggcc gcggc
2570425DNAArtificial SequenceAON sequence 5' -> 3' 704gggagccttg
gggcgggggc cgcgg
2570525DNAArtificial SequenceAON sequence 5' -> 3' 705agggagcctt
ggggcggggg ccgcg
2570625DNAArtificial SequenceAON sequence 5' -> 3' 706gagggagcct
tggggcgggg gccgc
2570725DNAArtificial SequenceAON sequence 5' -> 3' 707ggagggagcc
ttggggcggg ggccg
2570825DNAArtificial SequenceAON sequence 5' -> 3' 708aggagggagc
cttggggcgg gggcc
2570925DNAArtificial SequenceAON sequence 5' -> 3' 709gaggagggag
ccttggggcg ggggc
2571025DNAArtificial SequenceAON sequence 5' -> 3' 710ggaggaggga
gccttggggc ggggg
2571125DNAArtificial SequenceAON sequence 5' -> 3' 711gggaggaggg
agccttgggg cgggg
2571225DNAArtificial SequenceAON sequence 5' -> 3' 712agggaggagg
gagccttggg gcggg
2571325DNAArtificial SequenceAON sequence 5' -> 3' 713gagggaggag
ggagccttgg ggcgg
2571425DNAArtificial SequenceAON sequence 5' -> 3' 714ggagggagga
gggagccttg gggcg
2571525DNAArtificial SequenceAON sequence 5' -> 3' 715gggagggagg
agggagcctt ggggc
2571625DNAArtificial SequenceAON sequence 5' -> 3' 716agggagggag
gagggagcct tgggg
2571725DNAArtificial SequenceAON sequence 5' -> 3' 717gagggaggga
ggagggagcc ttggg
2571825DNAArtificial SequenceAON sequence 5' -> 3' 718tgagggaggg
aggagggagc cttgg
2571925DNAArtificial SequenceAON sequence 5' -> 3' 719atgagggagg
gaggagggag ccttg
2572025DNAArtificial SequenceAON sequence 5' -> 3' 720catgagggag
ggaggaggga gcctt
2572125DNAArtificial SequenceAON sequence 5' -> 3' 721tcatgaggga
gggaggaggg agcct
2572225DNAArtificial SequenceAON sequence 5' -> 3' 722ttcatgaggg
agggaggagg gagcc
2572325DNAArtificial SequenceAON sequence 5' -> 3' 723cttcatgagg
gagggaggag ggagc
2572425DNAArtificial SequenceAON sequence 5' -> 3' 724acttcatgag
ggagggagga gggag
2572525DNAArtificial SequenceAON sequence 5' -> 3' 725gacttcatga
gggagggagg aggga
2572625DNAArtificial SequenceAON sequence 5' -> 3' 726cgacttcatg
agggagggag gaggg
2572725DNAArtificial SequenceAON sequence 5' -> 3' 727ccgacttcat
gagggaggga ggagg
2572825DNAArtificial SequenceAON sequence 5' -> 3' 728gccgacttca
tgagggaggg aggag
2572925DNAArtificial SequenceAON sequence 5' -> 3' 729cgccgacttc
atgagggagg gagga
2573025DNAArtificial SequenceAON sequence 5' -> 3' 730acgccgactt
catgagggag ggagg
2573125DNAArtificial SequenceAON sequence 5' -> 3' 731aacgccgact
tcatgaggga gggag
2573225DNAArtificial SequenceAON sequence 5' -> 3' 732caacgccgac
ttcatgaggg aggga
2573325DNAArtificial SequenceAON sequence 5' -> 3' 733ccaacgccga
cttcatgagg gaggg
2573425DNAArtificial SequenceAON sequence 5' -> 3' 734gccaacgccg
acttcatgag ggagg
2573525DNAArtificial SequenceAON sequence 5' -> 3' 735ggccaacgcc
gacttcatga gggag
2573625DNAArtificial SequenceAON sequence 5' -> 3' 736aggccaacgc
cgacttcatg aggga
2573725DNAArtificial SequenceAON sequence 5' -> 3' 737caggccaacg
ccgacttcat gaggg
2573825DNAArtificial SequenceAON sequence 5' -> 3' 738gcaggccaac
gccgacttca tgagg
2573925DNAArtificial SequenceAON sequence 5' -> 3' 739tgcaggccaa
cgccgacttc atgag
2574025DNAArtificial SequenceAON sequence 5' -> 3' 740ctgcaggcca
acgccgactt catga
2574125DNAArtificial SequenceAON sequence 5' -> 3' 741cctgcaggcc
aacgccgact tcatg
2574225DNAArtificial SequenceAON sequence 5' -> 3' 742tcctgcaggc
caacgccgac ttcat
2574325DNAArtificial SequenceAON sequence 5' -> 3' 743atcctgcagg
ccaacgccga cttca
2574425DNAArtificial SequenceAON sequence 5' -> 3' 744tatcctgcag
gccaacgccg acttc
2574525DNAArtificial SequenceAON sequence 5' -> 3' 745gtatcctgca
ggccaacgcc gactt
2574625DNAArtificial SequenceAON sequence 5' -> 3' 746ggtatcctgc
aggccaacgc cgact
2574725DNAArtificial SequenceAON sequence 5' -> 3' 747gggtatcctg
caggccaacg ccgac
2574825DNAArtificial SequenceAON sequence 5' -> 3' 748cgggtatcct
gcaggccaac gccga
2574925DNAArtificial SequenceAON sequence 5' -> 3' 749acgggtatcc
tgcaggccaa cgccg
2575025DNAArtificial SequenceAON sequence 5' -> 3' 750aacgggtatc
ctgcaggcca acgcc
2575125DNAArtificial SequenceAON sequence 5' -> 3' 751gaacgggtat
cctgcaggcc aacgc
2575225DNAArtificial SequenceAON sequence 5' -> 3' 752tgaacgggta
tcctgcaggc caacg
2575325DNAArtificial SequenceAON sequence 5' -> 3' 753atgaacgggt
atcctgcagg ccaac
2575425DNAArtificial SequenceAON sequence 5' -> 3' 754catgaacggg
tatcctgcag gccaa
2575525DNAArtificial SequenceAON sequence 5' -> 3' 755gcatgaacgg
gtatcctgca ggcca
2575625DNAArtificial SequenceAON sequence 5' -> 3' 756ggcatgaacg
ggtatcctgc aggcc
2575725DNAArtificial SequenceAON sequence 5' -> 3' 757cggcatgaac
gggtatcctg caggc
2575825DNAArtificial SequenceAON sequence 5' -> 3' 758gcggcatgaa
cgggtatcct gcagg
2575925DNAArtificial SequenceAON sequence 5' -> 3' 759ggcggcatga
acgggtatcc tgcag
2576025DNAArtificial SequenceAON sequence 5' -> 3' 760tggcggcatg
aacgggtatc ctgca
2576125DNAArtificial SequenceAON sequence 5' -> 3' 761atggcggcat
gaacgggtat cctgc
2576225DNAArtificial SequenceAON sequence 5' -> 3' 762tatggcggca
tgaacgggta tcctg
2576325DNAArtificial SequenceAON sequence 5' -> 3' 763gtatggcggc
atgaacgggt atcct
2576425DNAArtificial SequenceAON sequence 5' -> 3' 764agtatggcgg
catgaacggg tatcc
2576525DNAArtificial SequenceAON sequence 5' -> 3' 765cagtatggcg
gcatgaacgg gtatc
2576625DNAArtificial SequenceAON sequence 5' -> 3' 766ccagtatggc
ggcatgaacg ggtat
2576725DNAArtificial SequenceAON sequence 5' -> 3' 767cccagtatgg
cggcatgaac gggta
2576825DNAArtificial SequenceAON sequence 5' -> 3' 768ccccagtatg
gcggcatgaa cgggt
2576925DNAArtificial SequenceAON sequence 5' -> 3' 769gccccagtat
ggcggcatga acggg
2577025DNAArtificial SequenceAON sequence 5' -> 3' 770ggccccagta
tggcggcatg aacgg
2577125DNAArtificial SequenceAON sequence 5' -> 3' 771aggccccagt
atggcggcat gaacg
2577225DNAArtificial SequenceAON sequence 5' -> 3' 772caggccccag
tatggcggca tgaac
2577325DNAArtificial SequenceAON sequence 5' -> 3' 773ccaggcccca
gtatggcggc atgaa
2577425DNAArtificial SequenceAON sequence 5' -> 3' 774cccaggcccc
agtatggcgg catga
2577525DNAArtificial SequenceAON sequence 5' -> 3' 775gcccaggccc
cagtatggcg gcatg
2577625DNAArtificial SequenceAON sequence 5' -> 3' 776agcccaggcc
ccagtatggc ggcat
2577725DNAArtificial SequenceAON sequence 5' -> 3' 777aagcccaggc
cccagtatgg cggca
2577825DNAArtificial SequenceAON sequence 5' -> 3' 778gaagcccagg
ccccagtatg gcggc
2577925DNAArtificial SequenceAON sequence 5' -> 3' 779ggaagcccag
gccccagtat ggcgg
2578025DNAArtificial SequenceAON sequence 5' -> 3' 780tggaagccca
ggccccagta tggcg
2578125DNAArtificial SequenceAON sequence 5' -> 3' 781gtggaagccc
aggccccagt atggc
2578225DNAArtificial SequenceAON sequence 5' -> 3' 782ggtggaagcc
caggccccag tatgg
2578325DNAArtificial SequenceAON sequence 5' -> 3' 783aggtggaagc
ccaggcccca gtatg
2578425DNAArtificial SequenceAON sequence 5' -> 3' 784caggtggaag
cccaggcccc agtat
2578525DNAArtificial SequenceAON sequence 5' -> 3' 785acaggtggaa
gcccaggccc cagta
2578625DNAArtificial SequenceAON sequence 5' -> 3' 786cacaggtgga
agcccaggcc ccagt
2578725DNAArtificial SequenceAON sequence 5' -> 3' 787gcacaggtgg
aagcccaggc cccag
2578825DNAArtificial SequenceAON sequence 5' -> 3' 788ggcacaggtg
gaagcccagg cccca
2578925DNAArtificial SequenceAON sequence 5' -> 3' 789cggcacaggt
ggaagcccag gcccc
2579025DNAArtificial SequenceAON sequence 5' -> 3' 790gcggcacagg
tggaagccca ggccc
2579125DNAArtificial SequenceAON sequence 5' -> 3' 791agcggcacag
gtggaagccc aggcc
2579225DNAArtificial SequenceAON sequence 5' -> 3' 792cagcggcaca
ggtggaagcc caggc
2579325DNAArtificial SequenceAON sequence 5' -> 3' 793ccagcggcac
aggtggaagc ccagg
2579425DNAArtificial SequenceAON sequence 5' -> 3' 794cccagcggca
caggtggaag cccag
2579525DNAArtificial SequenceAON sequence 5' -> 3' 795ccccagcggc
acaggtggaa gccca
2579625DNAArtificial SequenceAON sequence 5' -> 3' 796gccccagcgg
cacaggtgga agccc
2579725DNAArtificial SequenceAON sequence 5' -> 3' 797agccccagcg
gcacaggtgg aagcc
2579825DNAArtificial SequenceAON sequence 5' -> 3' 798tagccccagc
ggcacaggtg gaagc
2579925DNAArtificial SequenceAON sequence 5' -> 3' 799gtagccccag
cggcacaggt ggaag
2580025DNAArtificial SequenceAON sequence 5' -> 3' 800agtagcccca
gcggcacagg tggaa
2580125DNAArtificial SequenceAON sequence 5' -> 3' 801gagtagcccc
agcggcacag gtgga
2580225DNAArtificial SequenceAON sequence 5' -> 3' 802ggagtagccc
cagcggcaca ggtgg
2580325DNAArtificial SequenceAON sequence 5' -> 3' 803aggagtagcc
ccagcggcac aggtg
2580425DNAArtificial SequenceAON sequence 5' -> 3' 804gaggagtagc
cccagcggca caggt
2580525DNAArtificial SequenceAON sequence 5' -> 3' 805ggaggagtag
ccccagcggc acagg
2580625DNAArtificial SequenceAON sequence 5' -> 3' 806tggaggagta
gccccagcgg cacag
2580725DNAArtificial SequenceAON sequence 5' -> 3' 807gtggaggagt
agccccagcg gcaca
2580825DNAArtificial SequenceAON sequence 5' -> 3' 808ggtggaggag
tagccccagc ggcac
2580925DNAArtificial SequenceAON sequence 5' -> 3' 809cggtggagga
gtagccccag cggca
2581025DNAArtificial SequenceAON sequence 5' -> 3' 810gcggtggagg
agtagcccca gcggc
2581125DNAArtificial SequenceAON sequence 5' -> 3' 811agcggtggag
gagtagcccc agcgg
2581225DNAArtificial SequenceAON sequence 5' -> 3' 812tagcggtgga
ggagtagccc cagcg
2581325DNAArtificial SequenceAON sequence 5' -> 3' 813atagcggtgg
aggagtagcc ccagc
2581425DNAArtificial SequenceAON sequence 5' -> 3' 814gatagcggtg
gaggagtagc cccag
2581525DNAArtificial SequenceAON sequence 5' -> 3' 815tgatagcggt
ggaggagtag cccca
2581625DNAArtificial SequenceAON sequence 5' -> 3' 816gtgatagcgg
tggaggagta gcccc
2581725DNAArtificial SequenceAON sequence 5' -> 3' 817ggtgatagcg
gtggaggagt agccc
2581825DNAArtificial SequenceAON sequence 5' -> 3' 818gggtgatagc
ggtggaggag tagcc
2581925DNAArtificial SequenceAON sequence 5' -> 3' 819cgggtgatag
cggtggagga gtagc
2582025DNAArtificial SequenceAON sequence 5' -> 3' 820gcgggtgata
gcggtggagg agtag
2582125DNAArtificial SequenceAON sequence 5' -> 3' 821ggcgggtgat
agcggtggag gagta
2582225DNAArtificial SequenceAON sequence 5' -> 3' 822tggcgggtga
tagcggtgga ggagt
2582325DNAArtificial SequenceAON sequence 5' -> 3' 823ctggcgggtg
atagcggtgg aggag
2582425DNAArtificial SequenceAON sequence 5' -> 3' 824cctggcgggt
gatagcggtg gagga
2582525DNAArtificial SequenceAON sequence 5' -> 3' 825acctggcggg
tgatagcggt ggagg
2582625DNAArtificial SequenceAON sequence 5' -> 3' 826cacctggcgg
gtgatagcgg tggag
2582725DNAArtificial SequenceAON sequence 5' -> 3' 827ccacctggcg
ggtgatagcg gtgga
2582825DNAArtificial SequenceAON sequence 5' -> 3' 828accacctggc
gggtgatagc ggtgg
2582925DNAArtificial SequenceAON sequence 5' -> 3' 829caccacctgg
cgggtgatag cggtg
2583025DNAArtificial SequenceAON sequence 5' -> 3' 830ccaccacctg
gcgggtgata gcggt
2583125DNAArtificial SequenceAON sequence 5' -> 3' 831tccaccacct
ggcgggtgat agcgg
2583225DNAArtificial SequenceAON sequence 5' -> 3' 832ctccaccacc
tggcgggtga tagcg
2583325DNAArtificial SequenceAON sequence 5' -> 3' 833tctccaccac
ctggcgggtg atagc
2583425DNAArtificial SequenceAON sequence 5' -> 3' 834ttctccacca
cctggcgggt gatag
2583525DNAArtificial SequenceAON sequence 5' -> 3' 835gttctccacc
acctggcggg tgata
2583625DNAArtificial SequenceAON sequence 5' -> 3' 836tgttctccac
cacctggcgg gtgat
2583725DNAArtificial SequenceAON sequence 5' -> 3' 837atgttctcca
ccacctggcg ggtga
2583825DNAArtificial SequenceAON sequence 5' -> 3' 838catgttctcc
accacctggc gggtg
2583925DNAArtificial SequenceAON sequence 5' -> 3' 839tcatgttctc
caccacctgg cgggt
2584025DNAArtificial SequenceAON sequence 5' -> 3' 840gtcatgttct
ccaccacctg gcggg
2584125DNAArtificial SequenceAON sequence 5' -> 3' 841ggtcatgttc
tccaccacct ggcgg
2584225DNAArtificial SequenceAON sequence 5' -> 3' 842tggtcatgtt
ctccaccacc tggcg
2584325DNAArtificial SequenceAON sequence 5' -> 3' 843ctggtcatgt
tctccaccac ctggc
2584425DNAArtificial SequenceAON sequence 5' -> 3' 844cctggtcatg
ttctccacca cctgg
2584525DNAArtificial SequenceAON sequence 5' -> 3' 845ccctggtcat
gttctccacc acctg
2584625DNAArtificial SequenceAON sequence 5' -> 3' 846gccctggtca
tgttctccac cacct
2584725DNAArtificial SequenceAON sequence 5' -> 3' 847ggccctggtc
atgttctcca ccacc
2584825DNAArtificial SequenceAON sequence 5' -> 3' 848gggccctggt
catgttctcc accac
2584925DNAArtificial SequenceAON sequence 5' -> 3' 849tgggccctgg
tcatgttctc cacca
2585025DNAArtificial SequenceAON sequence 5' -> 3' 850gtgggccctg
gtcatgttct ccacc
2585125DNAArtificial SequenceAON sequence 5' -> 3' 851agtgggccct
ggtcatgttc tccac
2585225DNAArtificial SequenceAON sequence 5' -> 3' 852aagtgggccc
tggtcatgtt ctcca
2585325DNAArtificial SequenceAON sequence 5' -> 3' 853gaagtgggcc
ctggtcatgt tctcc
2585425DNAArtificial SequenceAON sequence 5' -> 3' 854ggaagtgggc
cctggtcatg ttctc
2585525DNAArtificial SequenceAON sequence 5' -> 3' 855gggaagtggg
ccctggtcat gttct
2585625DNAArtificial SequenceAON sequence 5' -> 3' 856ggggaagtgg
gccctggtca tgttc
2585725DNAArtificial SequenceAON sequence 5' -> 3' 857gggggaagtg
ggccctggtc atgtt
2585825DNAArtificial SequenceAON sequence 5' -> 3' 858agggggaagt
gggccctggt catgt
2585925DNAArtificial SequenceAON sequence 5' -> 3' 859cagggggaag
tgggccctgg tcatg
2586025DNAArtificial SequenceAON sequence 5' -> 3' 860ccagggggaa
gtgggccctg gtcat
2586125DNAArtificial SequenceAON sequence 5' -> 3' 861accaggggga
agtgggccct ggtca
2586225DNAArtificial SequenceAON sequence 5' -> 3' 862caccaggggg
aagtgggccc tggtc
2586325DNAArtificial SequenceAON sequence 5' -> 3' 863tcaccagggg
gaagtgggcc ctggt
2586425DNAArtificial SequenceAON sequence 5' -> 3' 864ctcaccaggg
ggaagtgggc cctgg
2586525DNAArtificial SequenceAON sequence 5' -> 3' 865actcaccagg
gggaagtggg ccctg
2586625DNAArtificial SequenceAON sequence 5' -> 3' 866aactcaccag
ggggaagtgg gccct
2586725DNAArtificial SequenceAON sequence 5' -> 3' 867caactcacca
gggggaagtg ggccc
2586825DNAArtificial SequenceAON sequence 5' -> 3' 868ccaactcacc
agggggaagt gggcc
2586925DNAArtificial SequenceAON sequence 5' -> 3' 869cccaactcac
cagggggaag tgggc
2587025DNAArtificial SequenceAON sequence 5' -> 3' 870ccccaactca
ccagggggaa gtggg
2587125DNAArtificial SequenceAON sequence 5' -> 3' 871accccaactc
accaggggga agtgg
2587225DNAArtificial SequenceAON sequence 5' -> 3' 872caccccaact
caccaggggg aagtg
2587325DNAArtificial SequenceAON sequence 5' -> 3' 873ccaccccaac
tcaccagggg gaagt
2587425DNAArtificial SequenceAON sequence 5' -> 3' 874accaccccaa
ctcaccaggg ggaag
2587525DNAArtificial SequenceAON sequence 5' -> 3' 875caccacccca
actcaccagg gggaa
2587625DNAArtificial SequenceAON sequence 5' -> 3' 876ccaccacccc
aactcaccag gggga
2587725DNAArtificial SequenceAON sequence 5' -> 3' 877gccaccaccc
caactcacca ggggg
2587825DNAArtificial SequenceAON sequence 5' -> 3' 878tgccaccacc
ccaactcacc agggg
2587925DNAArtificial SequenceAON sequence 5' -> 3' 879ctgccaccac
cccaactcac caggg
2588025DNAArtificial SequenceAON sequence 5' -> 3' 880cctgccacca
ccccaactca ccagg
2588125DNAArtificial SequenceAON sequence 5' -> 3' 881ccctgccacc
accccaactc accag
2588225DNAArtificial SequenceAON sequence 5' -> 3' 882cccctgccac
caccccaact cacca
2588325DNAArtificial SequenceAON sequence 5' -> 3' 883tcccctgcca
ccaccccaac tcacc
2588425DNAArtificial SequenceAON sequence 5' -> 3' 884ctcccctgcc
accaccccaa ctcac
2588521DNAArtificial SequenceAON sequence 5' -> 3' 885aagggaagca
gctctggggt t
2188621DNAArtificial SequenceAON sequence 5' -> 3' 886gaagggaagc
agctctgggg t
2188721DNAArtificial SequenceAON sequence 5' -> 3' 887ggaagggaag
cagctctggg g
2188821DNAArtificial SequenceAON sequence 5' -> 3' 888tggaagggaa
gcagctctgg g
2188921DNAArtificial SequenceAON sequence 5' -> 3' 889ctggaaggga
agcagctctg g
2189021DNAArtificial SequenceAON sequence 5' -> 3' 890tctggaaggg
aagcagctct g
2189121DNAArtificial SequenceAON sequence 5' -> 3' 891atctggaagg
gaagcagctc t
2189221DNAArtificial SequenceAON sequence 5' -> 3' 892catctggaag
ggaagcagct c
2189321DNAArtificial SequenceAON sequence 5' -> 3' 893acatctggaa
gggaagcagc t
2189421DNAArtificial SequenceAON sequence 5' -> 3' 894cacatctgga
agggaagcag c
2189521DNAArtificial SequenceAON sequence 5' -> 3' 895ccacatctgg
aagggaagca g
2189621DNAArtificial SequenceAON sequence 5' -> 3' 896accacatctg
gaagggaagc a
2189721DNAArtificial SequenceAON sequence 5' -> 3' 897gaccacatct
ggaagggaag c
2189821DNAArtificial SequenceAON sequence 5' -> 3' 898ggaccacatc
tggaagggaa g
2189921DNAArtificial SequenceAON sequence 5' -> 3' 899aggaccacat
ctggaaggga a
2190021DNAArtificial SequenceAON sequence 5' -> 3' 900caggaccaca
tctggaaggg a
2190121DNAArtificial SequenceAON sequence 5' -> 3' 901gcaggaccac
atctggaagg g
2190221DNAArtificial SequenceAON sequence 5' -> 3' 902tgcaggacca
catctggaag g
2190321DNAArtificial SequenceAON sequence 5' -> 3' 903ctgcaggacc
acatctggaa g
2190421DNAArtificial SequenceAON sequence 5' -> 3' 904gctgcaggac
cacatctgga a
2190521DNAArtificial SequenceAON sequence 5' -> 3' 905ggctgcagga
ccacatctgg a
2190621DNAArtificial SequenceAON sequence 5' -> 3' 906cggctgcagg
accacatctg g
2190721DNAArtificial SequenceAON sequence 5' -> 3' 907tcggctgcag
gaccacatct g
2190821DNAArtificial SequenceAON sequence 5' -> 3' 908ctcggctgca
ggaccacatc t
2190921DNAArtificial SequenceAON sequence 5' -> 3' 909gctcggctgc
aggaccacat c
2191021DNAArtificial SequenceAON sequence 5' -> 3' 910ggctcggctg
caggaccaca t
2191121DNAArtificial SequenceAON sequence 5' -> 3' 911gggctcggct
gcaggaccac a
2191221DNAArtificial SequenceAON sequence 5' -> 3' 912agggctcggc
tgcaggacca c
2191321DNAArtificial SequenceAON sequence 5' -> 3' 913cagggctcgg
ctgcaggacc a
2191421DNAArtificial SequenceAON sequence 5' -> 3' 914gcagggctcg
gctgcaggac c
2191521DNAArtificial SequenceAON sequence 5' -> 3' 915ggcagggctc
ggctgcagga c
2191621DNAArtificial SequenceAON sequence 5' -> 3' 916gggcagggct
cggctgcagg a
2191721DNAArtificial SequenceAON sequence 5' -> 3' 917agggcagggc
tcggctgcag g
2191821DNAArtificial SequenceAON sequence 5' -> 3' 918aagggcaggg
ctcggctgca g
2191921DNAArtificial SequenceAON sequence 5' -> 3' 919taagggcagg
gctcggctgc a
2192021DNAArtificial SequenceAON sequence 5' -> 3' 920ctaagggcag
ggctcggctg c
2192121DNAArtificial SequenceAON sequence 5' -> 3' 921gctaagggca
gggctcggct g
2192221DNAArtificial SequenceAON sequence 5' -> 3' 922agctaagggc
agggctcggc t
2192321DNAArtificial SequenceAON sequence 5' -> 3' 923cagctaaggg
cagggctcgg c
2192421DNAArtificial SequenceAON sequence 5' -> 3' 924ccagctaagg
gcagggctcg g
2192521DNAArtificial SequenceAON sequence 5' -> 3' 925tccagctaag
ggcagggctc g
2192621DNAArtificial SequenceAON sequence 5' -> 3' 926ctccagctaa
gggcagggct c
2192721DNAArtificial SequenceAON sequence 5' -> 3' 927cctccagcta
agggcagggc t
2192821DNAArtificial SequenceAON sequence 5' -> 3' 928acctccagct
aagggcaggg c
2192921DNAArtificial SequenceAON sequence 5' -> 3' 929gacctccagc
taagggcagg g
2193021DNAArtificial SequenceAON sequence 5' -> 3' 930cgacctccag
ctaagggcag g
2193121DNAArtificial SequenceAON sequence 5' -> 3' 931tcgacctcca
gctaagggca g
2193221DNAArtificial SequenceAON sequence 5' -> 3' 932gtcgacctcc
agctaagggc a
2193321DNAArtificial SequenceAON sequence 5' -> 3' 933tgtcgacctc
cagctaaggg c
2193421DNAArtificial SequenceAON sequence 5' -> 3' 934ctgtcgacct
ccagctaagg g
2193521DNAArtificial SequenceAON sequence 5' -> 3' 935cctgtcgacc
tccagctaag g
2193621DNAArtificial SequenceAON sequence 5' -> 3' 936acctgtcgac
ctccagctaa g
2193721DNAArtificial SequenceAON sequence 5' -> 3' 937cacctgtcga
cctccagcta a
2193821DNAArtificial SequenceAON sequence 5' -> 3' 938ccacctgtcg
acctccagct a
2193921DNAArtificial SequenceAON sequence 5' -> 3' 939cccacctgtc
gacctccagc t
2194021DNAArtificial SequenceAON sequence 5' -> 3' 940tcccacctgt
cgacctccag c
2194121DNAArtificial SequenceAON sequence 5' -> 3' 941atcccacctg
tcgacctcca g
2194221DNAArtificial SequenceAON sequence 5' -> 3' 942gatcccacct
gtcgacctcc a
2194321DNAArtificial SequenceAON sequence 5' -> 3' 943ggatcccacc
tgtcgacctc c
2194421DNAArtificial SequenceAON sequence 5' -> 3' 944aggatcccac
ctgtcgacct c
2194521DNAArtificial SequenceAON sequence 5' -> 3' 945caggatccca
cctgtcgacc t
2194621DNAArtificial SequenceAON sequence 5' -> 3' 946ccaggatccc
acctgtcgac c
2194721DNAArtificial SequenceAON sequence 5' -> 3' 947tccaggatcc
cacctgtcga c
2194821DNAArtificial SequenceAON sequence 5' -> 3' 948atccaggatc
ccacctgtcg a
2194921DNAArtificial SequenceAON sequence 5' -> 3' 949catccaggat
cccacctgtc g
2195021DNAArtificial SequenceAON sequence 5' -> 3' 950acatccagga
tcccacctgt c
2195121DNAArtificial SequenceAON sequence 5' -> 3' 951gacatccagg
atcccacctg t
2195221DNAArtificial SequenceAON sequence 5' -> 3' 952agacatccag
gatcccacct g
2195321DNAArtificial SequenceAON sequence 5' -> 3' 953tagacatcca
ggatcccacc t
2195421DNAArtificial SequenceAON sequence 5' -> 3' 954gtagacatcc
aggatcccac c
2195521DNAArtificial SequenceAON sequence 5' -> 3' 955tgtagacatc
caggatccca c
2195621DNAArtificial SequenceAON sequence 5' -> 3' 956atgtagacat
ccaggatccc a
2195721DNAArtificial SequenceAON sequence 5' -> 3' 957gatgtagaca
tccaggatcc c
2195821DNAArtificial SequenceAON sequence 5' -> 3' 958agatgtagac
atccaggatc c
2195921DNAArtificial SequenceAON sequence 5' -> 3' 959aagatgtaga
catccaggat c
2196021DNAArtificial SequenceAON sequence 5' -> 3' 960gaagatgtag
acatccagga t
2196121DNAArtificial SequenceAON sequence 5' -> 3' 961ggaagatgta
gacatccagg a
2196221DNAArtificial SequenceAON sequence 5' -> 3' 962aggaagatgt
agacatccag g
2196321DNAArtificial SequenceAON sequence 5' -> 3' 963caggaagatg
tagacatcca g
2196421DNAArtificial SequenceAON sequence 5' -> 3' 964ccaggaagat
gtagacatcc a
2196521DNAArtificial SequenceAON sequence 5' -> 3' 965cccaggaaga
tgtagacatc c
2196621DNAArtificial SequenceAON sequence 5' -> 3' 966gcccaggaag
atgtagacat c
2196721DNAArtificial SequenceAON sequence 5' -> 3' 967ggcccaggaa
gatgtagaca t
2196821DNAArtificial SequenceAON sequence 5' -> 3' 968gggcccagga
agatgtagac a
2196921DNAArtificial SequenceAON sequence 5' -> 3' 969tgggcccagg
aagatgtaga c
2197021DNAArtificial SequenceAON sequence 5' -> 3' 970ctgggcccag
gaagatgtag a
2197121DNAArtificial SequenceAON sequence 5' -> 3' 971tctgggccca
ggaagatgta g
2197221DNAArtificial SequenceAON sequence 5' -> 3' 972ctctgggccc
aggaagatgt a
2197321DNAArtificial SequenceAON sequence 5' -> 3' 973gctctgggcc
caggaagatg t
2197421DNAArtificial SequenceAON sequence 5' -> 3' 974ggctctgggc
ccaggaagat g
2197521DNAArtificial SequenceAON sequence 5' -> 3' 975gggctctggg
cccaggaaga t
2197621DNAArtificial SequenceAON sequence 5' -> 3' 976tgggctctgg
gcccaggaag a
2197721DNAArtificial SequenceAON sequence 5' -> 3' 977ttgggctctg
ggcccaggaa g
2197821DNAArtificial SequenceAON sequence 5' -> 3' 978cttgggctct
gggcccagga a
2197921DNAArtificial SequenceAON sequence 5' -> 3' 979tcttgggctc
tgggcccagg a
2198021DNAArtificial SequenceAON sequence 5' -> 3' 980ctcttgggct
ctgggcccag g
2198121DNAArtificial SequenceAON sequence 5' -> 3' 981gctcttgggc
tctgggccca g
2198221DNAArtificial SequenceAON sequence 5' -> 3' 982cgctcttggg
ctctgggccc a
2198321DNAArtificial SequenceAON sequence 5' -> 3' 983acgctcttgg
gctctgggcc c
2198421DNAArtificial SequenceAON sequence 5' -> 3' 984cacgctcttg
ggctctgggc c
2198521DNAArtificial SequenceAON sequence 5' -> 3' 985ccacgctctt
gggctctggg c
2198621DNAArtificial SequenceAON sequence 5' -> 3' 986accacgctct
tgggctctgg g
2198721DNAArtificial SequenceAON sequence 5' -> 3' 987caccacgctc
ttgggctctg g
2198821DNAArtificial SequenceAON sequence 5' -> 3' 988gcaccacgct
cttgggctct g
2198921DNAArtificial SequenceAON sequence 5' -> 3' 989tgcaccacgc
tcttgggctc t
2199021DNAArtificial SequenceAON sequence 5' -> 3' 990ctgcaccacg
ctcttgggct c
2199121DNAArtificial SequenceAON sequence 5' -> 3' 991gctgcaccac
gctcttgggc t
2199221DNAArtificial SequenceAON sequence 5' -> 3' 992tgctgcacca
cgctcttggg c
2199321DNAArtificial SequenceAON sequence 5' -> 3' 993ctgctgcacc
acgctcttgg g
2199421DNAArtificial SequenceAON sequence 5' -> 3' 994actgctgcac
cacgctcttg g
2199521DNAArtificial SequenceAON sequence 5' -> 3' 995tactgctgca
ccacgctctt g
2199621DNAArtificial SequenceAON sequence 5' -> 3' 996gtactgctgc
accacgctct t
2199721DNAArtificial SequenceAON sequence 5' -> 3' 997ggtactgctg
caccacgctc t
2199821DNAArtificial SequenceAON sequence 5' -> 3' 998aggtactgct
gcaccacgct c
2199921DNAArtificial SequenceAON sequence 5' -> 3' 999caggtactgc
tgcaccacgc t
21100021DNAArtificial SequenceAON sequence 5' -> 3' 1000ccaggtactg
ctgcaccacg c
21100121DNAArtificial SequenceAON sequence 5' -> 3' 1001tccaggtact
gctgcaccac g
21100221DNAArtificial SequenceAON sequence 5' -> 3' 1002gtccaggtac
tgctgcacca c
21100321DNAArtificial SequenceAON sequence 5' -> 3' 1003cgtccaggta
ctgctgcacc a
21100421DNAArtificial SequenceAON sequence 5' -> 3' 1004acgtccaggt
actgctgcac c
21100521DNAArtificial SequenceAON sequence 5' -> 3' 1005aacgtccagg
tactgctgca c
21100621DNAArtificial SequenceAON sequence 5' -> 3' 1006caacgtccag
gtactgctgc a
21100721DNAArtificial SequenceAON sequence 5' -> 3' 1007acaacgtcca
ggtactgctg c
21100821DNAArtificial SequenceAON sequence 5' -> 3' 1008cacaacgtcc
aggtactgct g
21100921DNAArtificial SequenceAON sequence 5' -> 3' 1009ccacaacgtc
caggtactgc t
21101021DNAArtificial SequenceAON sequence 5' -> 3' 1010cccacaacgt
ccaggtactg c
21101121DNAArtificial SequenceAON sequence 5' -> 3' 1011acccacaacg
tccaggtact g
21101221DNAArtificial SequenceAON sequence 5' -> 3' 1012tacccacaac
gtccaggtac t
21101321DNAArtificial SequenceAON sequence 5' -> 3' 1013ctacccacaa
cgtccaggta c
21101421DNAArtificial SequenceAON sequence 5' -> 3' 1014cctacccaca
acgtccaggt a
21101521DNAArtificial SequenceAON sequence 5' -> 3' 1015ccctacccac
aacgtccagg t
21101621DNAArtificial SequenceAON sequence 5' -> 3' 1016gccctaccca
caacgtccag g
21101721DNAArtificial SequenceAON sequence 5' -> 3' 1017ggccctaccc
acaacgtcca g
21101821DNAArtificial SequenceAON sequence 5' -> 3' 1018aggccctacc
cacaacgtcc a
21101921DNAArtificial SequenceAON sequence 5' -> 3' 1019caggccctac
ccacaacgtc c
21102021DNAArtificial SequenceAON sequence 5' -> 3' 1020gcaggcccta
cccacaacgt c
21102121DNAArtificial SequenceAON sequence 5' -> 3' 1021agcaggccct
acccacaacg t
21102221DNAArtificial SequenceAON sequence 5' -> 3' 1022gagcaggccc
tacccacaac g
21102321DNAArtificial SequenceAON sequence 5' -> 3' 1023ggagcaggcc
ctacccacaa c
21102421DNAArtificial SequenceAON sequence 5' -> 3' 1024gggagcaggc
cctacccaca a
21102521DNAArtificial SequenceAON sequence 5' -> 3' 1025agggagcagg
ccctacccac a
21102621DNAArtificial SequenceAON sequence 5' -> 3' 1026cagggagcag
gccctaccca c
21102721DNAArtificial SequenceAON sequence 5' -> 3' 1027ccagggagca
ggccctaccc a
21102821DNAArtificial SequenceAON sequence 5' -> 3' 1028gccagggagc
aggccctacc c
21102921DNAArtificial SequenceAON sequence 5' -> 3' 1029ggccagggag
caggccctac c
21103021DNAArtificial SequenceAON sequence 5' -> 3' 1030cggccaggga
gcaggcccta c
21103121DNAArtificial SequenceAON sequence 5' -> 3' 1031gcggccaggg
agcaggccct a
21103221DNAArtificial SequenceAON sequence 5' -> 3' 1032cgcggccagg
gagcaggccc t
21103321DNAArtificial SequenceAON sequence 5' -> 3' 1033ccgcggccag
ggagcaggcc c
21103421DNAArtificial SequenceAON sequence 5' -> 3' 1034gccgcggcca
gggagcaggc c
21103521DNAArtificial SequenceAON sequence 5' -> 3' 1035ggccgcggcc
agggagcagg c
21103621DNAArtificial SequenceAON sequence 5' -> 3' 1036gggccgcggc
cagggagcag g
21103721DNAArtificial SequenceAON sequence 5' -> 3' 1037ggggccgcgg
ccagggagca g
21103821DNAArtificial SequenceAON sequence 5' -> 3' 1038gggggccgcg
gccagggagc a
21103921DNAArtificial SequenceAON sequence 5' -> 3' 1039cgggggccgc
ggccagggag c
21104021DNAArtificial SequenceAON sequence 5' -> 3' 1040gcgggggccg
cggccaggga g
21104121DNAArtificial SequenceAON sequence 5' -> 3' 1041ggcgggggcc
gcggccaggg a
21104221DNAArtificial SequenceAON sequence 5' -> 3' 1042gggcgggggc
cgcggccagg g
21104321DNAArtificial SequenceAON sequence 5' -> 3' 1043ggggcggggg
ccgcggccag g
21104421DNAArtificial SequenceAON sequence 5' -> 3' 1044tggggcgggg
gccgcggcca g
21104521DNAArtificial SequenceAON sequence 5' -> 3' 1045ttggggcggg
ggccgcggcc a
21104621DNAArtificial SequenceAON sequence 5' -> 3' 1046cttggggcgg
gggccgcggc c
21104721DNAArtificial SequenceAON sequence 5' -> 3' 1047ccttggggcg
ggggccgcgg c
21104821DNAArtificial SequenceAON sequence 5' -> 3' 1048gccttggggc
gggggccgcg g
21104921DNAArtificial SequenceAON sequence 5' -> 3' 1049agccttgggg
cgggggccgc g
21105021DNAArtificial SequenceAON sequence 5' -> 3' 1050gagccttggg
gcgggggccg c
21105121DNAArtificial SequenceAON sequence 5' -> 3' 1051ggagccttgg
ggcgggggcc g
21105221DNAArtificial SequenceAON sequence 5' -> 3' 1052gggagccttg
gggcgggggc c
21105321DNAArtificial SequenceAON sequence 5' -> 3' 1053agggagcctt
ggggcggggg c
21105421DNAArtificial SequenceAON sequence 5' -> 3' 1054gagggagcct
tggggcgggg g
21105521DNAArtificial SequenceAON sequence 5' -> 3' 1055ggagggagcc
ttggggcggg g
21105621DNAArtificial SequenceAON sequence 5' -> 3' 1056aggagggagc
cttggggcgg g
21105721DNAArtificial SequenceAON sequence 5' -> 3' 1057gaggagggag
ccttggggcg g
21105821DNAArtificial SequenceAON sequence 5' -> 3' 1058ggaggaggga
gccttggggc g
21105921DNAArtificial SequenceAON sequence 5' -> 3' 1059gggaggaggg
agccttgggg c
21106021DNAArtificial SequenceAON sequence 5' -> 3' 1060agggaggagg
gagccttggg g
21106121DNAArtificial SequenceAON sequence 5' -> 3' 1061gagggaggag
ggagccttgg g
21106221DNAArtificial SequenceAON sequence 5' -> 3' 1062ggagggagga
gggagccttg g
21106321DNAArtificial SequenceAON sequence 5' -> 3' 1063gggagggagg
agggagcctt g
21106421DNAArtificial SequenceAON sequence 5' -> 3' 1064agggagggag
gagggagcct t
21106521DNAArtificial SequenceAON sequence 5' -> 3' 1065gagggaggga
ggagggagcc t
21106621DNAArtificial SequenceAON sequence 5' -> 3' 1066tgagggaggg
aggagggagc c
21106721DNAArtificial SequenceAON sequence 5' -> 3' 1067atgagggagg
gaggagggag c
21106821DNAArtificial SequenceAON sequence 5' -> 3' 1068catgagggag
ggaggaggga g
21106921DNAArtificial SequenceAON sequence 5' -> 3' 1069tcatgaggga
gggaggaggg a
21107021DNAArtificial SequenceAON sequence 5' -> 3' 1070ttcatgaggg
agggaggagg g
21107121DNAArtificial SequenceAON sequence 5' -> 3' 1071cttcatgagg
gagggaggag g
21107221DNAArtificial SequenceAON sequence 5' -> 3' 1072acttcatgag
ggagggagga g
21107321DNAArtificial SequenceAON sequence 5' -> 3' 1073gacttcatga
gggagggagg a
21107421DNAArtificial SequenceAON sequence 5' -> 3' 1074cgacttcatg
agggagggag g
21107521DNAArtificial SequenceAON sequence 5' -> 3' 1075ccgacttcat
gagggaggga g
21107621DNAArtificial SequenceAON sequence 5' -> 3' 1076gccgacttca
tgagggaggg a
21107721DNAArtificial SequenceAON sequence 5' -> 3' 1077cgccgacttc
atgagggagg g
21107821DNAArtificial SequenceAON sequence 5' -> 3' 1078acgccgactt
catgagggag g
21107921DNAArtificial SequenceAON sequence 5' -> 3' 1079aacgccgact
tcatgaggga g
21108021DNAArtificial SequenceAON sequence 5' -> 3' 1080caacgccgac
ttcatgaggg a
21108121DNAArtificial SequenceAON sequence 5' -> 3' 1081ccaacgccga
cttcatgagg g
21108221DNAArtificial SequenceAON sequence 5' -> 3' 1082gccaacgccg
acttcatgag g
21108321DNAArtificial SequenceAON sequence 5' -> 3' 1083ggccaacgcc
gacttcatga g
21108421DNAArtificial SequenceAON sequence 5' -> 3' 1084aggccaacgc
cgacttcatg a
21108521DNAArtificial SequenceAON sequence 5' -> 3' 1085caggccaacg
ccgacttcat g
21108621DNAArtificial SequenceAON sequence 5' -> 3' 1086gcaggccaac
gccgacttca t
21108721DNAArtificial SequenceAON sequence 5' -> 3' 1087tgcaggccaa
cgccgacttc a
21108821DNAArtificial SequenceAON sequence 5' -> 3' 1088ctgcaggcca
acgccgactt c
21108921DNAArtificial SequenceAON sequence 5' -> 3' 1089cctgcaggcc
aacgccgact t
21109021DNAArtificial SequenceAON sequence 5' -> 3' 1090tcctgcaggc
caacgccgac t
21109121DNAArtificial SequenceAON sequence 5' -> 3' 1091atcctgcagg
ccaacgccga c
21109221DNAArtificial SequenceAON sequence 5' -> 3' 1092tatcctgcag
gccaacgccg a
21109321DNAArtificial SequenceAON sequence 5' -> 3' 1093gtatcctgca
ggccaacgcc g
21109421DNAArtificial SequenceAON sequence 5' -> 3' 1094ggtatcctgc
aggccaacgc c
21109521DNAArtificial SequenceAON sequence 5' -> 3' 1095gggtatcctg
caggccaacg c
21109621DNAArtificial SequenceAON sequence 5' -> 3' 1096cgggtatcct
gcaggccaac g
21109721DNAArtificial SequenceAON sequence 5' -> 3' 1097acgggtatcc
tgcaggccaa c
21109821DNAArtificial SequenceAON sequence 5' -> 3' 1098aacgggtatc
ctgcaggcca a
21109921DNAArtificial SequenceAON sequence 5' -> 3' 1099gaacgggtat
cctgcaggcc a
21110021DNAArtificial SequenceAON sequence 5' -> 3' 1100tgaacgggta
tcctgcaggc c
21110121DNAArtificial SequenceAON sequence 5' -> 3' 1101atgaacgggt
atcctgcagg c
21110221DNAArtificial SequenceAON sequence 5' -> 3' 1102catgaacggg
tatcctgcag g
21110321DNAArtificial SequenceAON sequence 5' -> 3' 1103gcatgaacgg
gtatcctgca g
21110421DNAArtificial SequenceAON sequence 5' -> 3' 1104ggcatgaacg
ggtatcctgc a
21110521DNAArtificial SequenceAON sequence 5' -> 3' 1105cggcatgaac
gggtatcctg c
21110621DNAArtificial SequenceAON sequence 5' -> 3' 1106gcggcatgaa
cgggtatcct g
21110721DNAArtificial SequenceAON sequence 5' -> 3' 1107ggcggcatga
acgggtatcc t
21110821DNAArtificial SequenceAON sequence 5' -> 3' 1108tggcggcatg
aacgggtatc c
21110921DNAArtificial SequenceAON sequence 5' -> 3' 1109atggcggcat
gaacgggtat c
21111021DNAArtificial SequenceAON sequence 5' -> 3' 1110tatggcggca
tgaacgggta t
21111121DNAArtificial SequenceAON sequence 5' -> 3' 1111gtatggcggc
atgaacgggt a
21111221DNAArtificial SequenceAON sequence 5' -> 3' 1112agtatggcgg
catgaacggg t
21111321DNAArtificial SequenceAON sequence 5' -> 3' 1113cagtatggcg
gcatgaacgg g
21111421DNAArtificial SequenceAON sequence 5' -> 3' 1114ccagtatggc
ggcatgaacg g
21111521DNAArtificial SequenceAON sequence 5' -> 3' 1115cccagtatgg
cggcatgaac g
21111621DNAArtificial SequenceAON sequence 5' -> 3' 1116ccccagtatg
gcggcatgaa c
21111721DNAArtificial SequenceAON sequence 5' -> 3' 1117gccccagtat
ggcggcatga a
21111821DNAArtificial SequenceAON sequence 5' -> 3' 1118ggccccagta
tggcggcatg a
21111921DNAArtificial SequenceAON sequence 5' -> 3' 1119aggccccagt
atggcggcat g
21112021DNAArtificial SequenceAON sequence 5' -> 3' 1120caggccccag
tatggcggca t
21112121DNAArtificial SequenceAON sequence 5' -> 3' 1121ccaggcccca
gtatggcggc a
21112221DNAArtificial SequenceAON sequence 5' -> 3' 1122cccaggcccc
agtatggcgg c
21112321DNAArtificial SequenceAON sequence 5' -> 3' 1123gcccaggccc
cagtatggcg g
21112421DNAArtificial SequenceAON sequence 5' -> 3' 1124agcccaggcc
ccagtatggc g
21112521DNAArtificial SequenceAON sequence 5' -> 3' 1125aagcccaggc
cccagtatgg c
21112621DNAArtificial SequenceAON sequence 5' -> 3' 1126gaagcccagg
ccccagtatg g
21112721DNAArtificial SequenceAON sequence 5' -> 3' 1127ggaagcccag
gccccagtat g
21112821DNAArtificial SequenceAON sequence 5' -> 3' 1128tggaagccca
ggccccagta t
21112921DNAArtificial SequenceAON sequence 5' -> 3' 1129gtggaagccc
aggccccagt a
21113021DNAArtificial SequenceAON sequence 5' -> 3' 1130ggtggaagcc
caggccccag t
21113121DNAArtificial SequenceAON sequence 5' -> 3' 1131aggtggaagc
ccaggcccca g
21113221DNAArtificial SequenceAON sequence 5' -> 3' 1132caggtggaag
cccaggcccc a
21113321DNAArtificial SequenceAON sequence 5' -> 3' 1133acaggtggaa
gcccaggccc c
21113421DNAArtificial SequenceAON sequence 5' -> 3' 1134cacaggtgga
agcccaggcc c
21113521DNAArtificial SequenceAON sequence 5' -> 3' 1135gcacaggtgg
aagcccaggc c
21113621DNAArtificial SequenceAON sequence 5' -> 3' 1136ggcacaggtg
gaagcccagg c
21113721DNAArtificial SequenceAON sequence 5' -> 3' 1137cggcacaggt
ggaagcccag g
21113821DNAArtificial SequenceAON sequence 5' -> 3' 1138gcggcacagg
tggaagccca g
21113921DNAArtificial SequenceAON sequence 5' -> 3' 1139agcggcacag
gtggaagccc a
21114021DNAArtificial SequenceAON sequence 5' -> 3' 1140cagcggcaca
ggtggaagcc c
21114121DNAArtificial SequenceAON sequence 5' -> 3' 1141ccagcggcac
aggtggaagc c
21114221DNAArtificial SequenceAON sequence 5' -> 3' 1142cccagcggca
caggtggaag c
21114321DNAArtificial SequenceAON sequence 5' -> 3' 1143ccccagcggc
acaggtggaa g
21114421DNAArtificial SequenceAON sequence 5' -> 3' 1144gccccagcgg
cacaggtgga a
21114521DNAArtificial SequenceAON sequence 5' -> 3' 1145agccccagcg
gcacaggtgg a
21114621DNAArtificial SequenceAON sequence 5' -> 3' 1146tagccccagc
ggcacaggtg g
21114721DNAArtificial SequenceAON sequence 5' -> 3' 1147gtagccccag
cggcacaggt g
21114821DNAArtificial SequenceAON sequence 5' -> 3' 1148agtagcccca
gcggcacagg t
21114921DNAArtificial SequenceAON sequence 5' -> 3' 1149gagtagcccc
agcggcacag g
21115021DNAArtificial SequenceAON sequence 5' -> 3' 1150ggagtagccc
cagcggcaca g
21115121DNAArtificial SequenceAON sequence 5' -> 3' 1151aggagtagcc
ccagcggcac a
21115221DNAArtificial SequenceAON sequence 5' -> 3' 1152gaggagtagc
cccagcggca c
21115321DNAArtificial SequenceAON sequence 5' -> 3' 1153ggaggagtag
ccccagcggc a
21115421DNAArtificial SequenceAON sequence 5' -> 3' 1154tggaggagta
gccccagcgg c
21115521DNAArtificial SequenceAON sequence 5' -> 3' 1155gtggaggagt
agccccagcg g
21115621DNAArtificial SequenceAON sequence 5' -> 3' 1156ggtggaggag
tagccccagc g
21115721DNAArtificial SequenceAON sequence 5' -> 3' 1157cggtggagga
gtagccccag c
21115821DNAArtificial SequenceAON sequence 5' -> 3' 1158gcggtggagg
agtagcccca g
21115921DNAArtificial SequenceAON sequence 5' -> 3' 1159agcggtggag
gagtagcccc a
21116021DNAArtificial SequenceAON sequence 5' -> 3' 1160tagcggtgga
ggagtagccc c
21116121DNAArtificial SequenceAON sequence 5' -> 3' 1161atagcggtgg
aggagtagcc c
21116221DNAArtificial SequenceAON sequence 5' -> 3' 1162gatagcggtg
gaggagtagc c
21116321DNAArtificial SequenceAON sequence 5' -> 3' 1163tgatagcggt
ggaggagtag c
21116421DNAArtificial SequenceAON sequence 5' -> 3' 1164gtgatagcgg
tggaggagta g
21116521DNAArtificial SequenceAON sequence 5' -> 3' 1165ggtgatagcg
gtggaggagt a
21116621DNAArtificial SequenceAON sequence 5' -> 3' 1166gggtgatagc
ggtggaggag t
21116721DNAArtificial SequenceAON sequence 5' -> 3' 1167cgggtgatag
cggtggagga g
21116821DNAArtificial SequenceAON sequence 5' -> 3' 1168gcgggtgata
gcggtggagg a
21116921DNAArtificial SequenceAON sequence 5' -> 3' 1169ggcgggtgat
agcggtggag g
21117021DNAArtificial SequenceAON sequence 5' -> 3' 1170tggcgggtga
tagcggtgga g
21117121DNAArtificial SequenceAON sequence 5' -> 3' 1171ctggcgggtg
atagcggtgg a
21117221DNAArtificial SequenceAON sequence 5' -> 3' 1172cctggcgggt
gatagcggtg g
21117321DNAArtificial SequenceAON sequence 5' -> 3' 1173acctggcggg
tgatagcggt g
21117421DNAArtificial SequenceAON sequence 5' -> 3' 1174cacctggcgg
gtgatagcgg t
21117521DNAArtificial SequenceAON sequence 5' -> 3' 1175ccacctggcg
ggtgatagcg g
21117621DNAArtificial SequenceAON sequence 5' -> 3' 1176accacctggc
gggtgatagc g
21117721DNAArtificial SequenceAON sequence 5' -> 3' 1177caccacctgg
cgggtgatag c
21117821DNAArtificial SequenceAON sequence 5' -> 3' 1178ccaccacctg
gcgggtgata g
21117921DNAArtificial SequenceAON sequence 5' -> 3' 1179tccaccacct
ggcgggtgat a
21118021DNAArtificial SequenceAON sequence 5' -> 3' 1180ctccaccacc
tggcgggtga t
21118121DNAArtificial SequenceAON sequence 5' -> 3' 1181tctccaccac
ctggcgggtg a
21118221DNAArtificial SequenceAON sequence 5' -> 3' 1182ttctccacca
cctggcgggt g
21118321DNAArtificial SequenceAON sequence 5' -> 3' 1183gttctccacc
acctggcggg t
21118421DNAArtificial SequenceAON sequence 5' -> 3' 1184tgttctccac
cacctggcgg g
21118521DNAArtificial SequenceAON sequence 5' -> 3' 1185atgttctcca
ccacctggcg g
21118621DNAArtificial SequenceAON sequence 5' -> 3' 1186catgttctcc
accacctggc g
21118721DNAArtificial SequenceAON sequence 5' -> 3' 1187tcatgttctc
caccacctgg c
21118821DNAArtificial SequenceAON sequence 5' -> 3' 1188gtcatgttct
ccaccacctg g
21118921DNAArtificial SequenceAON sequence 5' -> 3' 1189ggtcatgttc
tccaccacct g
21119021DNAArtificial SequenceAON sequence 5' -> 3' 1190tggtcatgtt
ctccaccacc t
21119121DNAArtificial SequenceAON sequence 5' -> 3' 1191ctggtcatgt
tctccaccac c
21119221DNAArtificial SequenceAON sequence 5' -> 3' 1192cctggtcatg
ttctccacca c
21119321DNAArtificial SequenceAON sequence 5' -> 3' 1193ccctggtcat
gttctccacc a
21119421DNAArtificial SequenceAON sequence 5' -> 3' 1194gccctggtca
tgttctccac c
21119521DNAArtificial SequenceAON sequence 5' -> 3' 1195ggccctggtc
atgttctcca c
21119621DNAArtificial SequenceAON sequence 5' -> 3' 1196gggccctggt
catgttctcc a
21119721DNAArtificial SequenceAON sequence 5' -> 3' 1197tgggccctgg
tcatgttctc c
21119821DNAArtificial SequenceAON sequence 5' -> 3' 1198gtgggccctg
gtcatgttct c
21119921DNAArtificial SequenceAON sequence 5' -> 3' 1199agtgggccct
ggtcatgttc t
21120021DNAArtificial SequenceAON sequence 5' -> 3' 1200aagtgggccc
tggtcatgtt c
21120121DNAArtificial SequenceAON sequence 5' -> 3' 1201gaagtgggcc
ctggtcatgt t
21120221DNAArtificial SequenceAON sequence 5' -> 3' 1202ggaagtgggc
cctggtcatg t
21120321DNAArtificial SequenceAON sequence 5' -> 3' 1203gggaagtggg
ccctggtcat g
21120421DNAArtificial SequenceAON sequence 5' -> 3' 1204ggggaagtgg
gccctggtca t
21120521DNAArtificial SequenceAON sequence 5' -> 3' 1205gggggaagtg
ggccctggtc a
21120621DNAArtificial SequenceAON sequence 5' -> 3' 1206agggggaagt
gggccctggt c
21120721DNAArtificial SequenceAON sequence 5' -> 3' 1207cagggggaag
tgggccctgg t
21120821DNAArtificial SequenceAON sequence 5' -> 3' 1208ccagggggaa
gtgggccctg g
21120921DNAArtificial SequenceAON sequence 5' -> 3' 1209accaggggga
agtgggccct g
21121021DNAArtificial SequenceAON sequence 5' -> 3' 1210caccaggggg
aagtgggccc t
21121121DNAArtificial SequenceAON sequence 5' -> 3' 1211tcaccagggg
gaagtgggcc c
21121221DNAArtificial SequenceAON sequence 5' -> 3' 1212ctcaccaggg
ggaagtgggc c
21121321DNAArtificial SequenceAON sequence 5' -> 3' 1213actcaccagg
gggaagtggg c
21121421DNAArtificial SequenceAON sequence 5' -> 3' 1214aactcaccag
ggggaagtgg g
21121521DNAArtificial SequenceAON sequence 5' -> 3' 1215caactcacca
gggggaagtg g
21121621DNAArtificial SequenceAON sequence 5' -> 3' 1216ccaactcacc
agggggaagt g
21121721DNAArtificial SequenceAON sequence 5' -> 3' 1217cccaactcac
cagggggaag t
21121821DNAArtificial SequenceAON sequence 5' -> 3' 1218ccccaactca
ccagggggaa g
21121921DNAArtificial SequenceAON sequence 5' -> 3' 1219accccaactc
accaggggga a
21122021DNAArtificial SequenceAON sequence 5' -> 3' 1220caccccaact
caccaggggg a
21122121DNAArtificial SequenceAON sequence 5' -> 3' 1221ccaccccaac
tcaccagggg g
21122221DNAArtificial SequenceAON sequence 5' -> 3' 1222accaccccaa
ctcaccaggg g
21122321DNAArtificial SequenceAON sequence 5' -> 3' 1223caccacccca
actcaccagg g
21122421DNAArtificial SequenceAON sequence 5' -> 3' 1224ccaccacccc
aactcaccag g
21122521DNAArtificial SequenceAON sequence 5' -> 3' 1225gccaccaccc
caactcacca g
21122621DNAArtificial SequenceAON sequence 5' -> 3' 1226tgccaccacc
ccaactcacc a
21122721DNAArtificial SequenceAON sequence 5' -> 3' 1227ctgccaccac
cccaactcac c
21122821DNAArtificial SequenceAON sequence 5' -> 3' 1228cctgccacca
ccccaactca c
21122921DNAArtificial SequenceAON sequence 5' -> 3' 1229ccctgccacc
accccaactc a
21123021DNAArtificial SequenceAON sequence 5' -> 3' 1230cccctgccac
caccccaact c
21123121DNAArtificial SequenceAON sequence 5' -> 3' 1231tcccctgcca
ccaccccaac t
21123221DNAArtificial SequenceAON sequence 5' -> 3' 1232ctcccctgcc
accaccccaa c
21123318DNAArtificial SequenceAON sequence 5' -> 3' 1233ggaagcagct
ctggggtt
18123418DNAArtificial SequenceAON sequence 5' -> 3' 1234gggaagcagc
tctggggt
18123518DNAArtificial SequenceAON sequence 5' -> 3' 1235agggaagcag
ctctgggg
18123618DNAArtificial SequenceAON sequence 5' -> 3' 1236aagggaagca
gctctggg
18123718DNAArtificial SequenceAON sequence 5' -> 3' 1237gaagggaagc
agctctgg
18123818DNAArtificial SequenceAON sequence 5' -> 3' 1238ggaagggaag
cagctctg
18123918DNAArtificial SequenceAON sequence 5' -> 3' 1239tggaagggaa
gcagctct
18124018DNAArtificial SequenceAON sequence 5' -> 3' 1240ctggaaggga
agcagctc
18124118DNAArtificial SequenceAON sequence 5' -> 3' 1241tctggaaggg
aagcagct
18124218DNAArtificial SequenceAON sequence 5' -> 3' 1242atctggaagg
gaagcagc
18124318DNAArtificial SequenceAON sequence 5' -> 3' 1243catctggaag
ggaagcag
18124418DNAArtificial SequenceAON sequence 5' -> 3' 1244acatctggaa
gggaagca
18124518DNAArtificial SequenceAON sequence 5' -> 3' 1245cacatctgga
agggaagc
18124618DNAArtificial SequenceAON sequence 5' -> 3' 1246ccacatctgg
aagggaag
18124718DNAArtificial SequenceAON sequence 5' -> 3' 1247accacatctg
gaagggaa
18124818DNAArtificial SequenceAON sequence 5' -> 3' 1248gaccacatct
ggaaggga
18124918DNAArtificial SequenceAON sequence 5' -> 3' 1249ggaccacatc
tggaaggg
18125018DNAArtificial SequenceAON sequence 5' -> 3' 1250aggaccacat
ctggaagg
18125118DNAArtificial SequenceAON sequence 5' -> 3' 1251caggaccaca
tctggaag
18125218DNAArtificial SequenceAON sequence 5' -> 3' 1252gcaggaccac
atctggaa
18125318DNAArtificial SequenceAON sequence 5' -> 3' 1253tgcaggacca
catctgga
18125418DNAArtificial SequenceAON sequence 5' -> 3' 1254ctgcaggacc
acatctgg
18125518DNAArtificial SequenceAON sequence 5' -> 3' 1255gctgcaggac
cacatctg
18125618DNAArtificial SequenceAON sequence 5' -> 3' 1256ggctgcagga
ccacatct
18125718DNAArtificial SequenceAON sequence 5' -> 3' 1257cggctgcagg
accacatc
18125818DNAArtificial SequenceAON sequence 5' -> 3' 1258tcggctgcag
gaccacat
18125918DNAArtificial SequenceAON sequence 5' -> 3' 1259ctcggctgca
ggaccaca
18126018DNAArtificial SequenceAON sequence 5' -> 3' 1260gctcggctgc
aggaccac
18126118DNAArtificial SequenceAON sequence 5' -> 3' 1261ggctcggctg
caggacca
18126218DNAArtificial SequenceAON sequence 5' -> 3' 1262gggctcggct
gcaggacc
18126318DNAArtificial SequenceAON sequence 5' -> 3' 1263agggctcggc
tgcaggac
18126418DNAArtificial SequenceAON sequence 5' -> 3' 1264cagggctcgg
ctgcagga
18126518DNAArtificial SequenceAON sequence 5' -> 3' 1265gcagggctcg
gctgcagg
18126618DNAArtificial SequenceAON sequence 5' -> 3' 1266ggcagggctc
ggctgcag
18126718DNAArtificial SequenceAON sequence 5' -> 3' 1267gggcagggct
cggctgca
18126818DNAArtificial SequenceAON sequence 5' -> 3' 1268agggcagggc
tcggctgc
18126918DNAArtificial SequenceAON sequence 5' -> 3' 1269aagggcaggg
ctcggctg
18127018DNAArtificial SequenceAON sequence 5' -> 3' 1270taagggcagg
gctcggct
18127118DNAArtificial SequenceAON sequence 5' -> 3' 1271ctaagggcag
ggctcggc
18127218DNAArtificial SequenceAON sequence 5' -> 3' 1272gctaagggca
gggctcgg
18127318DNAArtificial SequenceAON sequence 5' -> 3' 1273agctaagggc
agggctcg
18127418DNAArtificial SequenceAON sequence 5' -> 3' 1274cagctaaggg
cagggctc
18127518DNAArtificial SequenceAON sequence 5' -> 3' 1275ccagctaagg
gcagggct
18127618DNAArtificial SequenceAON sequence 5' -> 3' 1276tccagctaag
ggcagggc
18127718DNAArtificial SequenceAON sequence 5' -> 3' 1277ctccagctaa
gggcaggg
18127818DNAArtificial SequenceAON sequence 5' -> 3' 1278cctccagcta
agggcagg
18127918DNAArtificial SequenceAON sequence 5' -> 3' 1279acctccagct
aagggcag
18128018DNAArtificial SequenceAON sequence 5' -> 3' 1280gacctccagc
taagggca
18128118DNAArtificial SequenceAON sequence 5' -> 3' 1281cgacctccag
ctaagggc
18128218DNAArtificial SequenceAON sequence 5' -> 3' 1282tcgacctcca
gctaaggg
18128318DNAArtificial SequenceAON sequence 5' -> 3' 1283gtcgacctcc
agctaagg
18128418DNAArtificial SequenceAON sequence 5' -> 3' 1284tgtcgacctc
cagctaag
18128518DNAArtificial SequenceAON sequence 5' -> 3' 1285ctgtcgacct
ccagctaa
18128618DNAArtificial SequenceAON sequence 5' -> 3' 1286cctgtcgacc
tccagcta
18128718DNAArtificial SequenceAON sequence 5' -> 3' 1287acctgtcgac
ctccagct
18128818DNAArtificial SequenceAON sequence 5' -> 3' 1288cacctgtcga
cctccagc
18128918DNAArtificial SequenceAON sequence 5' -> 3' 1289ccacctgtcg
acctccag
18129018DNAArtificial SequenceAON sequence 5' -> 3' 1290cccacctgtc
gacctcca
18129118DNAArtificial SequenceAON sequence 5' -> 3' 1291tcccacctgt
cgacctcc
18129218DNAArtificial SequenceAON sequence 5' -> 3' 1292atcccacctg
tcgacctc
18129318DNAArtificial SequenceAON sequence 5' -> 3' 1293gatcccacct
gtcgacct
18129418DNAArtificial SequenceAON sequence 5' -> 3' 1294ggatcccacc
tgtcgacc
18129518DNAArtificial SequenceAON sequence 5' -> 3' 1295aggatcccac
ctgtcgac
18129618DNAArtificial SequenceAON sequence 5' -> 3' 1296caggatccca
cctgtcga
18129718DNAArtificial SequenceAON sequence 5' -> 3' 1297ccaggatccc
acctgtcg
18129818DNAArtificial SequenceAON sequence 5' -> 3' 1298tccaggatcc
cacctgtc
18129918DNAArtificial SequenceAON sequence 5' -> 3' 1299atccaggatc
ccacctgt
18130018DNAArtificial SequenceAON sequence 5' -> 3' 1300catccaggat
cccacctg
18130118DNAArtificial SequenceAON sequence 5' -> 3' 1301acatccagga
tcccacct
18130218DNAArtificial SequenceAON sequence 5' -> 3' 1302gacatccagg
atcccacc
18130318DNAArtificial SequenceAON sequence 5' -> 3' 1303agacatccag
gatcccac
18130418DNAArtificial SequenceAON sequence 5' -> 3' 1304tagacatcca
ggatccca
18130518DNAArtificial SequenceAON sequence 5' -> 3' 1305gtagacatcc
aggatccc
18130618DNAArtificial SequenceAON sequence 5' -> 3' 1306tgtagacatc
caggatcc
18130718DNAArtificial SequenceAON sequence 5' -> 3' 1307atgtagacat
ccaggatc
18130818DNAArtificial SequenceAON sequence 5' -> 3' 1308gatgtagaca
tccaggat
18130918DNAArtificial SequenceAON sequence 5' -> 3' 1309agatgtagac
atccagga
18131018DNAArtificial SequenceAON sequence 5' -> 3' 1310aagatgtaga
catccagg
18131118DNAArtificial SequenceAON sequence 5' -> 3' 1311gaagatgtag
acatccag
18131218DNAArtificial SequenceAON sequence 5' -> 3' 1312ggaagatgta
gacatcca
18131318DNAArtificial SequenceAON sequence 5' -> 3' 1313aggaagatgt
agacatcc
18131418DNAArtificial SequenceAON sequence 5' -> 3' 1314caggaagatg
tagacatc
18131518DNAArtificial SequenceAON sequence 5' -> 3' 1315ccaggaagat
gtagacat
18131618DNAArtificial SequenceAON sequence 5' -> 3' 1316cccaggaaga
tgtagaca
18131718DNAArtificial SequenceAON sequence 5' -> 3' 1317gcccaggaag
atgtagac
18131818DNAArtificial SequenceAON sequence 5' -> 3' 1318ggcccaggaa
gatgtaga
18131918DNAArtificial SequenceAON sequence 5' -> 3' 1319gggcccagga
agatgtag
18132018DNAArtificial SequenceAON sequence 5' -> 3' 1320tgggcccagg
aagatgta
18132118DNAArtificial SequenceAON sequence 5' -> 3' 1321ctgggcccag
gaagatgt
18132218DNAArtificial SequenceAON sequence 5' -> 3' 1322tctgggccca
ggaagatg
18132318DNAArtificial SequenceAON sequence 5' -> 3' 1323ctctgggccc
aggaagat
18132418DNAArtificial SequenceAON sequence 5' -> 3' 1324gctctgggcc
caggaaga
18132518DNAArtificial SequenceAON sequence 5' -> 3' 1325ggctctgggc
ccaggaag
18132618DNAArtificial SequenceAON sequence 5' -> 3' 1326gggctctggg
cccaggaa
18132718DNAArtificial SequenceAON sequence 5' -> 3' 1327tgggctctgg
gcccagga
18132818DNAArtificial SequenceAON sequence 5' -> 3' 1328ttgggctctg
ggcccagg
18132918DNAArtificial SequenceAON sequence 5' -> 3' 1329cttgggctct
gggcccag
18133018DNAArtificial SequenceAON sequence 5' -> 3' 1330tcttgggctc
tgggccca
18133118DNAArtificial SequenceAON sequence 5' -> 3' 1331ctcttgggct
ctgggccc
18133218DNAArtificial SequenceAON sequence 5' -> 3' 1332gctcttgggc
tctgggcc
18133318DNAArtificial SequenceAON sequence 5' -> 3' 1333cgctcttggg
ctctgggc
18133418DNAArtificial SequenceAON sequence 5' -> 3' 1334acgctcttgg
gctctggg
18133518DNAArtificial SequenceAON sequence 5' -> 3' 1335cacgctcttg
ggctctgg
18133618DNAArtificial SequenceAON sequence 5' -> 3' 1336ccacgctctt
gggctctg
18133718DNAArtificial SequenceAON sequence 5' -> 3' 1337accacgctct
tgggctct
18133818DNAArtificial SequenceAON sequence 5' -> 3' 1338caccacgctc
ttgggctc
18133918DNAArtificial SequenceAON sequence 5' -> 3' 1339gcaccacgct
cttgggct
18134018DNAArtificial SequenceAON sequence 5' -> 3' 1340tgcaccacgc
tcttgggc
18134118DNAArtificial SequenceAON sequence 5' -> 3' 1341ctgcaccacg
ctcttggg
18134218DNAArtificial SequenceAON sequence 5' -> 3' 1342gctgcaccac
gctcttgg
18134318DNAArtificial SequenceAON sequence 5' -> 3' 1343tgctgcacca
cgctcttg
18134418DNAArtificial SequenceAON sequence 5' -> 3' 1344ctgctgcacc
acgctctt
18134518DNAArtificial SequenceAON sequence 5' -> 3' 1345actgctgcac
cacgctct
18134618DNAArtificial SequenceAON sequence 5' -> 3' 1346tactgctgca
ccacgctc
18134718DNAArtificial SequenceAON sequence 5' -> 3' 1347gtactgctgc
accacgct
18134818DNAArtificial SequenceAON sequence 5' -> 3' 1348ggtactgctg
caccacgc
18134918DNAArtificial SequenceAON sequence 5' -> 3' 1349aggtactgct
gcaccacg
18135018DNAArtificial SequenceAON sequence 5' -> 3' 1350caggtactgc
tgcaccac
18135118DNAArtificial SequenceAON sequence 5' -> 3' 1351ccaggtactg
ctgcacca
18135218DNAArtificial SequenceAON sequence 5' -> 3' 1352tccaggtact
gctgcacc
18135318DNAArtificial SequenceAON sequence 5' -> 3' 1353gtccaggtac
tgctgcac
18135418DNAArtificial SequenceAON sequence 5' -> 3' 1354cgtccaggta
ctgctgca
18135518DNAArtificial SequenceAON sequence 5' -> 3' 1355acgtccaggt
actgctgc
18135618DNAArtificial SequenceAON sequence 5' -> 3' 1356aacgtccagg
tactgctg
18135718DNAArtificial SequenceAON sequence 5' -> 3' 1357caacgtccag
gtactgct
18135818DNAArtificial SequenceAON sequence 5' -> 3' 1358acaacgtcca
ggtactgc
18135918DNAArtificial SequenceAON sequence 5' -> 3' 1359cacaacgtcc
aggtactg
18136018DNAArtificial SequenceAON sequence 5' -> 3' 1360ccacaacgtc
caggtact
18136118DNAArtificial SequenceAON sequence 5' -> 3' 1361cccacaacgt
ccaggtac
18136218DNAArtificial SequenceAON sequence 5' -> 3' 1362acccacaacg
tccaggta
18136318DNAArtificial SequenceAON sequence 5' -> 3' 1363tacccacaac
gtccaggt
18136418DNAArtificial SequenceAON sequence 5' -> 3' 1364ctacccacaa
cgtccagg
18136518DNAArtificial SequenceAON sequence 5' -> 3' 1365cctacccaca
acgtccag
18136618DNAArtificial SequenceAON sequence 5' -> 3' 1366ccctacccac
aacgtcca
18136718DNAArtificial SequenceAON sequence 5' -> 3' 1367gccctaccca
caacgtcc
18136818DNAArtificial SequenceAON sequence 5' -> 3' 1368ggccctaccc
acaacgtc
18136918DNAArtificial SequenceAON sequence 5' -> 3' 1369aggccctacc
cacaacgt
18137018DNAArtificial SequenceAON sequence 5' -> 3' 1370caggccctac
ccacaacg
18137118DNAArtificial SequenceAON sequence 5' -> 3' 1371gcaggcccta
cccacaac
18137218DNAArtificial SequenceAON sequence 5' -> 3' 1372agcaggccct
acccacaa
18137318DNAArtificial SequenceAON sequence 5' -> 3' 1373gagcaggccc
tacccaca
18137418DNAArtificial SequenceAON sequence 5' -> 3' 1374ggagcaggcc
ctacccac
18137518DNAArtificial SequenceAON sequence 5' -> 3' 1375gggagcaggc
cctaccca
18137618DNAArtificial SequenceAON sequence 5' -> 3' 1376agggagcagg
ccctaccc
18137718DNAArtificial SequenceAON sequence 5' -> 3' 1377cagggagcag
gccctacc
18137818DNAArtificial SequenceAON sequence 5' -> 3' 1378ccagggagca
ggccctac
18137918DNAArtificial SequenceAON sequence 5' -> 3' 1379gccagggagc
aggcccta
18138018DNAArtificial SequenceAON sequence 5' -> 3' 1380ggccagggag
caggccct
18138118DNAArtificial SequenceAON sequence 5' -> 3' 1381cggccaggga
gcaggccc
18138218DNAArtificial SequenceAON sequence 5' -> 3' 1382gcggccaggg
agcaggcc
18138318DNAArtificial SequenceAON sequence 5' -> 3' 1383cgcggccagg
gagcaggc
18138418DNAArtificial SequenceAON sequence 5' -> 3' 1384ccgcggccag
ggagcagg
18138518DNAArtificial SequenceAON sequence 5' -> 3' 1385gccgcggcca
gggagcag
18138618DNAArtificial SequenceAON sequence 5' -> 3' 1386ggccgcggcc
agggagca
18138718DNAArtificial SequenceAON sequence 5' -> 3' 1387gggccgcggc
cagggagc
18138818DNAArtificial SequenceAON sequence 5' -> 3' 1388ggggccgcgg
ccagggag
18138918DNAArtificial SequenceAON sequence 5' -> 3' 1389gggggccgcg
gccaggga
18139018DNAArtificial SequenceAON sequence 5' -> 3' 1390cgggggccgc
ggccaggg
18139118DNAArtificial SequenceAON sequence 5' -> 3' 1391gcgggggccg
cggccagg
18139218DNAArtificial SequenceAON sequence 5' -> 3' 1392ggcgggggcc
gcggccag
18139318DNAArtificial SequenceAON sequence 5' -> 3' 1393gggcgggggc
cgcggcca
18139418DNAArtificial SequenceAON sequence 5' -> 3' 1394ggggcggggg
ccgcggcc
18139518DNAArtificial SequenceAON sequence 5' -> 3' 1395tggggcgggg
gccgcggc
18139618DNAArtificial SequenceAON sequence 5' -> 3' 1396ttggggcggg
ggccgcgg
18139718DNAArtificial SequenceAON sequence 5' -> 3' 1397cttggggcgg
gggccgcg
18139818DNAArtificial SequenceAON sequence 5' -> 3' 1398ccttggggcg
ggggccgc
18139918DNAArtificial SequenceAON sequence 5' -> 3' 1399gccttggggc
gggggccg
18140018DNAArtificial SequenceAON sequence 5' -> 3' 1400agccttgggg
cgggggcc
18140118DNAArtificial SequenceAON sequence 5' -> 3' 1401gagccttggg
gcgggggc
18140218DNAArtificial SequenceAON sequence 5' -> 3' 1402ggagccttgg
ggcggggg
18140318DNAArtificial SequenceAON sequence 5' -> 3' 1403gggagccttg
gggcgggg
18140418DNAArtificial SequenceAON sequence 5' -> 3' 1404agggagcctt
ggggcggg
18140518DNAArtificial SequenceAON sequence 5' -> 3' 1405gagggagcct
tggggcgg
18140618DNAArtificial SequenceAON sequence 5' -> 3' 1406ggagggagcc
ttggggcg
18140718DNAArtificial SequenceAON sequence 5' -> 3' 1407aggagggagc
cttggggc
18140818DNAArtificial SequenceAON sequence 5' -> 3' 1408gaggagggag
ccttgggg
18140918DNAArtificial SequenceAON sequence 5' -> 3' 1409ggaggaggga
gccttggg
18141018DNAArtificial SequenceAON sequence 5' -> 3' 1410gggaggaggg
agccttgg
18141118DNAArtificial SequenceAON sequence 5' -> 3' 1411agggaggagg
gagccttg
18141218DNAArtificial SequenceAON sequence 5' -> 3' 1412gagggaggag
ggagcctt
18141318DNAArtificial SequenceAON sequence 5' -> 3' 1413ggagggagga
gggagcct
18141418DNAArtificial SequenceAON sequence 5' -> 3' 1414gggagggagg
agggagcc
18141518DNAArtificial SequenceAON sequence 5' -> 3' 1415agggagggag
gagggagc
18141618DNAArtificial SequenceAON sequence 5' -> 3' 1416gagggaggga
ggagggag
18141718DNAArtificial SequenceAON sequence 5' -> 3' 1417tgagggaggg
aggaggga
18141818DNAArtificial SequenceAON sequence 5' -> 3' 1418atgagggagg
gaggaggg
18141918DNAArtificial SequenceAON sequence 5' -> 3' 1419catgagggag
ggaggagg
18142018DNAArtificial SequenceAON sequence 5' -> 3' 1420tcatgaggga
gggaggag
18142118DNAArtificial SequenceAON sequence 5' -> 3' 1421ttcatgaggg
agggagga
18142218DNAArtificial SequenceAON sequence 5' -> 3' 1422cttcatgagg
gagggagg
18142318DNAArtificial SequenceAON sequence 5' -> 3' 1423acttcatgag
ggagggag
18142418DNAArtificial SequenceAON sequence 5' -> 3' 1424gacttcatga
gggaggga
18142518DNAArtificial SequenceAON sequence 5' -> 3' 1425cgacttcatg
agggaggg
18142618DNAArtificial SequenceAON sequence 5' -> 3' 1426ccgacttcat
gagggagg
18142718DNAArtificial SequenceAON sequence 5' -> 3' 1427gccgacttca
tgagggag
18142818DNAArtificial SequenceAON sequence 5' -> 3' 1428cgccgacttc
atgaggga
18142918DNAArtificial SequenceAON sequence 5' -> 3' 1429acgccgactt
catgaggg
18143018DNAArtificial SequenceAON sequence 5' -> 3' 1430aacgccgact
tcatgagg
18143118DNAArtificial SequenceAON sequence 5' -> 3' 1431caacgccgac
ttcatgag
18143218DNAArtificial SequenceAON sequence 5' -> 3' 1432ccaacgccga
cttcatga
18143318DNAArtificial SequenceAON sequence 5' -> 3' 1433gccaacgccg
acttcatg
18143418DNAArtificial SequenceAON sequence 5' -> 3' 1434ggccaacgcc
gacttcat
18143518DNAArtificial SequenceAON sequence 5' -> 3' 1435aggccaacgc
cgacttca
18143618DNAArtificial SequenceAON sequence 5' -> 3' 1436caggccaacg
ccgacttc
18143718DNAArtificial SequenceAON sequence 5' -> 3' 1437gcaggccaac
gccgactt
18143818DNAArtificial SequenceAON sequence 5' -> 3' 1438tgcaggccaa
cgccgact
18143918DNAArtificial SequenceAON sequence 5' -> 3' 1439ctgcaggcca
acgccgac
18144018DNAArtificial SequenceAON sequence 5' -> 3' 1440cctgcaggcc
aacgccga
18144118DNAArtificial SequenceAON sequence 5' -> 3' 1441tcctgcaggc
caacgccg
18144218DNAArtificial SequenceAON sequence 5' -> 3' 1442atcctgcagg
ccaacgcc
18144318DNAArtificial SequenceAON sequence 5' -> 3' 1443tatcctgcag
gccaacgc
18144418DNAArtificial SequenceAON sequence 5' -> 3' 1444gtatcctgca
ggccaacg
18144518DNAArtificial SequenceAON sequence 5' -> 3' 1445ggtatcctgc
aggccaac
18144618DNAArtificial SequenceAON sequence 5' -> 3' 1446gggtatcctg
caggccaa
18144718DNAArtificial SequenceAON sequence 5' -> 3' 1447cgggtatcct
gcaggcca
18144818DNAArtificial SequenceAON sequence 5' -> 3' 1448acgggtatcc
tgcaggcc
18144918DNAArtificial SequenceAON sequence 5' -> 3' 1449aacgggtatc
ctgcaggc
18145018DNAArtificial SequenceAON sequence 5' -> 3' 1450gaacgggtat
cctgcagg
18145118DNAArtificial SequenceAON sequence 5' -> 3' 1451tgaacgggta
tcctgcag
18145218DNAArtificial SequenceAON sequence 5' -> 3' 1452atgaacgggt
atcctgca
18145318DNAArtificial SequenceAON sequence 5' -> 3' 1453catgaacggg
tatcctgc
18145418DNAArtificial SequenceAON sequence 5' -> 3' 1454gcatgaacgg
gtatcctg
18145518DNAArtificial SequenceAON sequence 5' -> 3' 1455ggcatgaacg
ggtatcct
18145618DNAArtificial SequenceAON sequence 5' -> 3' 1456cggcatgaac
gggtatcc
18145718DNAArtificial SequenceAON sequence 5' -> 3' 1457gcggcatgaa
cgggtatc
18145818DNAArtificial SequenceAON sequence 5' -> 3' 1458ggcggcatga
acgggtat
18145918DNAArtificial SequenceAON sequence 5' -> 3' 1459tggcggcatg
aacgggta
18146018DNAArtificial SequenceAON sequence 5' -> 3' 1460atggcggcat
gaacgggt
18146118DNAArtificial SequenceAON sequence 5' -> 3' 1461tatggcggca
tgaacggg
18146218DNAArtificial SequenceAON sequence 5' -> 3' 1462gtatggcggc
atgaacgg
18146318DNAArtificial SequenceAON sequence 5' -> 3' 1463agtatggcgg
catgaacg
18146418DNAArtificial SequenceAON sequence 5' -> 3' 1464cagtatggcg
gcatgaac
18146518DNAArtificial SequenceAON sequence 5' -> 3' 1465ccagtatggc
ggcatgaa
18146618DNAArtificial SequenceAON sequence 5' -> 3' 1466cccagtatgg
cggcatga
18146718DNAArtificial SequenceAON sequence 5' -> 3' 1467ccccagtatg
gcggcatg
18146818DNAArtificial SequenceAON sequence 5' -> 3' 1468gccccagtat
ggcggcat
18146918DNAArtificial SequenceAON sequence 5' -> 3' 1469ggccccagta
tggcggca
18147018DNAArtificial SequenceAON sequence 5' -> 3' 1470aggccccagt
atggcggc
18147118DNAArtificial SequenceAON sequence 5' -> 3' 1471caggccccag
tatggcgg
18147218DNAArtificial SequenceAON sequence 5' -> 3' 1472ccaggcccca
gtatggcg
18147318DNAArtificial SequenceAON sequence 5' -> 3' 1473cccaggcccc
agtatggc
18147418DNAArtificial SequenceAON sequence 5' -> 3' 1474gcccaggccc
cagtatgg
18147518DNAArtificial SequenceAON sequence 5' -> 3' 1475agcccaggcc
ccagtatg
18147618DNAArtificial SequenceAON sequence 5' -> 3' 1476aagcccaggc
cccagtat
18147718DNAArtificial SequenceAON sequence 5' -> 3' 1477gaagcccagg
ccccagta
18147818DNAArtificial SequenceAON sequence 5' -> 3' 1478ggaagcccag
gccccagt
18147918DNAArtificial SequenceAON sequence 5' -> 3' 1479tggaagccca
ggccccag
18148018DNAArtificial SequenceAON sequence 5' -> 3' 1480gtggaagccc
aggcccca
18148118DNAArtificial SequenceAON sequence 5' -> 3' 1481ggtggaagcc
caggcccc
18148218DNAArtificial SequenceAON sequence 5' -> 3' 1482aggtggaagc
ccaggccc
18148318DNAArtificial SequenceAON sequence 5' -> 3' 1483caggtggaag
cccaggcc
18148418DNAArtificial SequenceAON sequence 5' -> 3' 1484acaggtggaa
gcccaggc
18148518DNAArtificial SequenceAON sequence 5' -> 3' 1485cacaggtgga
agcccagg
18148618DNAArtificial SequenceAON sequence 5' -> 3' 1486gcacaggtgg
aagcccag
18148718DNAArtificial SequenceAON sequence 5' -> 3' 1487ggcacaggtg
gaagccca
18148818DNAArtificial SequenceAON sequence 5' -> 3' 1488cggcacaggt
ggaagccc
18148918DNAArtificial SequenceAON sequence 5' -> 3' 1489gcggcacagg
tggaagcc
18149018DNAArtificial SequenceAON sequence 5' -> 3' 1490agcggcacag
gtggaagc
18149118DNAArtificial SequenceAON sequence 5' -> 3' 1491cagcggcaca
ggtggaag
18149218DNAArtificial SequenceAON sequence 5' -> 3' 1492ccagcggcac
aggtggaa
18149318DNAArtificial SequenceAON sequence 5' -> 3' 1493cccagcggca
caggtgga
18149418DNAArtificial SequenceAON sequence 5' -> 3' 1494ccccagcggc
acaggtgg
18149518DNAArtificial SequenceAON sequence 5' -> 3' 1495gccccagcgg
cacaggtg
18149618DNAArtificial SequenceAON sequence 5' -> 3' 1496agccccagcg
gcacaggt
18149718DNAArtificial SequenceAON sequence 5' -> 3' 1497tagccccagc
ggcacagg
18149818DNAArtificial SequenceAON sequence 5' -> 3' 1498gtagccccag
cggcacag
18149918DNAArtificial SequenceAON sequence 5' -> 3' 1499agtagcccca
gcggcaca
18150018DNAArtificial SequenceAON sequence 5' -> 3' 1500gagtagcccc
agcggcac
18150118DNAArtificial SequenceAON sequence 5' -> 3' 1501ggagtagccc
cagcggca
18150218DNAArtificial SequenceAON sequence 5' -> 3' 1502aggagtagcc
ccagcggc
18150318DNAArtificial SequenceAON sequence 5' -> 3' 1503gaggagtagc
cccagcgg
18150418DNAArtificial SequenceAON sequence 5' -> 3' 1504ggaggagtag
ccccagcg
18150518DNAArtificial SequenceAON sequence 5' -> 3' 1505tggaggagta
gccccagc
18150618DNAArtificial SequenceAON sequence 5' -> 3' 1506gtggaggagt
agccccag
18150718DNAArtificial SequenceAON sequence 5' -> 3' 1507ggtggaggag
tagcccca
18150818DNAArtificial SequenceAON sequence 5' -> 3' 1508cggtggagga
gtagcccc
18150918DNAArtificial SequenceAON sequence 5' -> 3' 1509gcggtggagg
agtagccc
18151036DNAArtificial SequenceAON sequence 5' -> 3' 1510agcggtggag
gagtagccag cggtggagga gtagcc
36151118DNAArtificial SequenceAON sequence 5' -> 3' 1511tagcggtgga
ggagtagc
18151218DNAArtificial SequenceAON sequence 5' -> 3' 1512atagcggtgg
aggagtag
18151318DNAArtificial SequenceAON sequence 5' -> 3' 1513gatagcggtg
gaggagta
18151418DNAArtificial SequenceAON sequence 5' -> 3' 1514tgatagcggt
ggaggagt
18151518DNAArtificial SequenceAON sequence 5' -> 3' 1515gtgatagcgg
tggaggag
18151618DNAArtificial SequenceAON sequence 5' -> 3' 1516ggtgatagcg
gtggagga
18151718DNAArtificial SequenceAON sequence 5' -> 3' 1517gggtgatagc
ggtggagg
18151818DNAArtificial SequenceAON sequence 5' -> 3' 1518cgggtgatag
cggtggag
18151918DNAArtificial SequenceAON sequence 5' -> 3' 1519gcgggtgata
gcggtgga
18152018DNAArtificial SequenceAON sequence 5' -> 3' 1520ggcgggtgat
agcggtgg
18152118DNAArtificial SequenceAON sequence 5' -> 3' 1521tggcgggtga
tagcggtg
18152218DNAArtificial SequenceAON sequence 5' -> 3' 1522ctggcgggtg
atagcggt
18152318DNAArtificial SequenceAON sequence 5' -> 3' 1523cctggcgggt
gatagcgg
18152418DNAArtificial SequenceAON sequence 5' -> 3' 1524acctggcggg
tgatagcg
18152518DNAArtificial SequenceAON sequence 5' -> 3' 1525cacctggcgg
gtgatagc
18152618DNAArtificial SequenceAON sequence 5' -> 3' 1526ccacctggcg
ggtgatag
18152718DNAArtificial SequenceAON sequence 5' -> 3' 1527accacctggc
gggtgata
18152818DNAArtificial SequenceAON sequence 5' -> 3' 1528caccacctgg
cgggtgat
18152918DNAArtificial SequenceAON sequence 5' -> 3' 1529ccaccacctg
gcgggtga
18153018DNAArtificial SequenceAON sequence 5' -> 3' 1530tccaccacct
ggcgggtg
18153118DNAArtificial SequenceAON sequence 5' -> 3' 1531ctccaccacc
tggcgggt
18153218DNAArtificial SequenceAON sequence 5' -> 3' 1532tctccaccac
ctggcggg
18153318DNAArtificial SequenceAON sequence 5' -> 3' 1533ttctccacca
cctggcgg
18153418DNAArtificial SequenceAON sequence 5' -> 3' 1534gttctccacc
acctggcg
18153518DNAArtificial SequenceAON sequence 5' -> 3' 1535tgttctccac
cacctggc
18153618DNAArtificial SequenceAON sequence 5' -> 3' 1536atgttctcca
ccacctgg
18153718DNAArtificial SequenceAON sequence 5' -> 3' 1537catgttctcc
accacctg
18153818DNAArtificial SequenceAON sequence 5' -> 3' 1538tcatgttctc
caccacct
18153918DNAArtificial SequenceAON sequence 5' -> 3' 1539gtcatgttct
ccaccacc
18154018DNAArtificial SequenceAON sequence 5' -> 3' 1540ggtcatgttc
tccaccac
18154118DNAArtificial SequenceAON sequence 5' -> 3' 1541tggtcatgtt
ctccacca
18154218DNAArtificial SequenceAON sequence 5' -> 3' 1542ctggtcatgt
tctccacc
18154318DNAArtificial SequenceAON sequence 5' -> 3' 1543cctggtcatg
ttctccac
18154418DNAArtificial SequenceAON sequence 5' -> 3' 1544ccctggtcat
gttctcca
18154518DNAArtificial SequenceAON sequence 5' -> 3' 1545gccctggtca
tgttctcc
18154618DNAArtificial SequenceAON sequence 5' -> 3' 1546ggccctggtc
atgttctc
18154718DNAArtificial SequenceAON sequence 5' -> 3' 1547gggccctggt
catgttct
18154818DNAArtificial SequenceAON sequence 5' -> 3' 1548tgggccctgg
tcatgttc
18154918DNAArtificial SequenceAON sequence 5' -> 3' 1549gtgggccctg
gtcatgtt
18155018DNAArtificial SequenceAON sequence 5' -> 3' 1550agtgggccct
ggtcatgt
18155118DNAArtificial SequenceAON sequence 5' -> 3' 1551aagtgggccc
tggtcatg
18155218DNAArtificial SequenceAON sequence 5' -> 3' 1552gaagtgggcc
ctggtcat
18155318DNAArtificial SequenceAON sequence 5' -> 3' 1553ggaagtgggc
cctggtca
18155418DNAArtificial SequenceAON sequence 5' -> 3' 1554gggaagtggg
ccctggtc
18155518DNAArtificial SequenceAON sequence 5' -> 3' 1555ggggaagtgg
gccctggt
18155618DNAArtificial SequenceAON sequence 5' -> 3' 1556gggggaagtg
ggccctgg
18155718DNAArtificial SequenceAON sequence 5' -> 3' 1557agggggaagt
gggccctg
18155818DNAArtificial SequenceAON sequence 5' -> 3' 1558cagggggaag
tgggccct
18155918DNAArtificial SequenceAON sequence 5' -> 3' 1559ccagggggaa
gtgggccc
18156018DNAArtificial SequenceAON sequence 5' -> 3' 1560accaggggga
agtgggcc
18156118DNAArtificial SequenceAON sequence 5' -> 3' 1561caccaggggg
aagtgggc
18156218DNAArtificial SequenceAON sequence 5' -> 3' 1562tcaccagggg
gaagtggg
18156318DNAArtificial SequenceAON sequence 5' -> 3' 1563ctcaccaggg
ggaagtgg
18156418DNAArtificial SequenceAON sequence 5' -> 3' 1564actcaccagg
gggaagtg
18156518DNAArtificial SequenceAON sequence 5' -> 3' 1565aactcaccag
ggggaagt
18156618DNAArtificial SequenceAON sequence 5' -> 3' 1566caactcacca
gggggaag
18156718DNAArtificial SequenceAON sequence 5' -> 3' 1567ccaactcacc
agggggaa
18156818DNAArtificial SequenceAON sequence 5' -> 3' 1568cccaactcac
caggggga
18156918DNAArtificial SequenceAON sequence 5' -> 3' 1569ccccaactca
ccaggggg
18157018DNAArtificial SequenceAON sequence 5' -> 3' 1570accccaactc
accagggg
18157118DNAArtificial SequenceAON sequence 5' -> 3' 1571caccccaact
caccaggg
18157218DNAArtificial SequenceAON sequence 5' -> 3' 1572ccaccccaac
tcaccagg
18157318DNAArtificial SequenceAON sequence 5' -> 3' 1573accaccccaa
ctcaccag
18157418DNAArtificial SequenceAON sequence 5' -> 3' 1574caccacccca
actcacca
18157518DNAArtificial SequenceAON sequence 5' -> 3' 1575ccaccacccc
aactcacc
18157618DNAArtificial SequenceAON sequence 5' -> 3' 1576gccaccaccc
caactcac
18157718DNAArtificial SequenceAON sequence 5' -> 3' 1577tgccaccacc
ccaactca
18157818DNAArtificial SequenceAON sequence 5' -> 3' 1578ctgccaccac
cccaactc
18157918DNAArtificial SequenceAON sequence 5' -> 3' 1579cctgccacca
ccccaact
18158018DNAArtificial SequenceAON sequence 5' -> 3' 1580ccctgccacc
accccaac
18158118DNAArtificial SequenceAON sequence 5' -> 3' 1581cccctgccac
caccccaa
18158218DNAArtificial SequenceAON sequence 5' -> 3' 1582tcccctgcca
ccacccca
18158318DNAArtificial SequenceAON sequence 5' -> 3' 1583ctcccctgcc
accacccc
181584368DNAArtificial Sequencesequence of region (5'-> 3')
1584aaccccagag ctgcttccct tccagatgtg gtcctgcagc cgagccctgc ccttagctgg
60aggtcgacag gtgggatcct ggatgtctac atcttcctgg gcccagagcc caagagcgtg
120gtgcagcagt acctggacgt tgtgggtagg gcctgctccc tggccgcggc ccccgcccca
180aggctccctc ctccctccct catgaagtcg gcgttggcct gcaggatacc cgttcatgcc
240gccatactgg ggcctgggct tccacctgtg ccgctggggc tactcctcca ccgctatcac
300ccgccaggtg gtggagaaca tgaccagggc ccacttcccc ctggtgagtt ggggtggtgg
360caggggag
368158574DNAArtificial Sequencesequence of region (5'-> 3')
1585aaccccagag ctgcttccct tccagatgtg gtcctgcagc cgagccctgc ccttagctgg
60aggtcgacag gtgg
74158674DNAArtificial Sequencesequence of region (5'-> 3')
1586gatcctggat gtctacatct tcctgggccc agagcccaag agcgtggtgc agcagtacct
60ggacgttgtg ggta
74158774DNAArtificial Sequencesequence of region (5'-> 3')
1587gggcctgctc cctggccgcg gcccccgccc caaggctccc tcctccctcc ctcatgaagt
60cggcgttggc ctgc
74158874DNAArtificial Sequencesequence of region (5'-> 3')
1588aggatacccg ttcatgccgc catactgggg cctgggcttc cacctgtgcc gctggggcta
60ctcctccacc gcta
74158972DNAArtificial Sequencesequence of region (5'-> 3')
1589tcacccgcca ggtggtggag aacatgacca gggcccactt ccccctggtg agttggggtg
60gtggcagggg ag
72159025DNAArtificial SequenceAON sequence 5' -> 3' 1590tcagtcaagt
atctggaaag tacga
25159121DNAArtificial SequenceAON sequence 5' -> 3' 1591agtcaagtat
ctggaaagta c
21159225DNAArtificial SequenceAON sequence 5' -> 3' 1592ggaagtcccg
gaagccaacc ttgtt
25159321DNAArtificial SequenceAON sequence 5' -> 3' 1593tgactctgcc
cagagtgagg a
21159425DNAArtificial SequenceAON sequence 5' -> 3' 1594agctttctgg
gatgaggcag aggct 25
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