Patent application title: SNPs OF APOLIPOPROTEIN B AND MODULATION OF THEIR EXPRESSION
Inventors:
Rosanne M. Crooke (Carlsbad, CA, US)
Mark J. Graham (Carlsbad, CA, US)
Steven Mah (San Diego, CA, US)
IPC8 Class: AC12N15113FI
USPC Class:
514 44 A
Class name: Nitrogen containing hetero ring polynucleotide (e.g., rna, dna, etc.) antisense or rna interference
Publication date: 2015-11-12
Patent application number: 20150322429
Abstract:
Compounds, compositions and methods are provided for modulating the
expression of apolipoprotein B. The compositions comprise
oligonucleotides, targeted to nucleic acid encoding apolipoprotein B.
Methods of using these compounds for modulation of apolipoprotein B
expression and for diagnosis and treatment of diseases and conditions
associated with expression of apolipoprotein B are provided.Claims:
1. An antisense compound 15 to 30 nucleobases in length which
specifically hybridizes with an allelic variant of a nucleic acid of SEQ
ID NO: 1 encoding human apolipoprotein B, wherein said compound inhibits
the expression of apolipoprotein B mRNA by at least 10% and wherein said
compound is targeted to a region that includes a cytosine (C) at position
27735 of SEQ ID NO: 1.
2. The antisense compound of claim 1 comprising a guanine (G) at position 15711 of SEQ ID NO: 2.
3. The antisense compound of claim 1 which is 20 nucleobases in length.
4. The antisense compound of claim 1 comprising an oligonucleotide.
5. The antisense compound of claim 4 comprising a DNA oligonucleotide.
6. The antisense compound of claim 4 comprising an RNA oligonucleotide.
7. The antisense compound of claim 4 comprising a chimeric oligonucleotide.
8. The antisense compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
9. The antisense compound of claim 1 having at least 80%, at least 85%, at least 90% or, at least 95% or at least 99% complementarity with said nucleic acid molecule encoding apolipoprotein B.
10. The antisense compound of claim 1 having one, two or more types of modifications, wherein the modification comprises a modified internucleoside linkage, sugar moiety, or nucleobase.
11. The antisense compound of claim 1 having at least one 2'-O-methoxyethyl sugar moiety.
12. The antisense compound of claim 1 having at least one phosphorothioate internucleoside linkage.
13. The antisense compound of claim 1 wherein at least one cytosine is a 5-methylcytosine.
14. A method of inhibiting the expression of apolipoprotein B in a cell or tissue comprising contacting said cell or tissue with an antisense compound so that expression of apolipoprotein B is inhibited, wherein the antisense compound is an antisense compound 15 to 30 nucleobases in length which specifically hybridizes with an allelic variant of a nucleic acid of SEQ ID NO: 1 encoding human apolipoprotein B, wherein said compound inhibits the expression of apolipoprotein B mRNA by at least 10% and wherein said compound is targeted to a region that includes a cytosine (C) at position 27735 of SEQ ID NO: 1.
15. (canceled)
16. (canceled)
17. A method of treating an animal having a disease or condition associated with apolipoprotein B comprising administering to said animal a therapeutically or prophylactically effective amount of an antisense compound so that expression of apolipoprotein B is inhibited, wherein the antisense compound is an antisense compound 15 to 30 nucleobases in length which specifically hybridizes with an allelic variant of a nucleic acid of SEQ ID NO: 1 encoding human apolipoprotein B, wherein said compound inhibits the expression of apolipoprotein B mRNA by at least 10% and wherein said compound is targeted to a region that includes a cytosine (C) at position 27735 of SEQ ID NO: 1.
18. The method of claim 17 wherein the disease or condition is a disorder of lipid metabolism.
19. The antisense compound of claim 7, wherein the antisense compound is a chimeric oligonucleotide consisting of: a gap segment consisting of from 10 to 18 linked 2'-deoxynucleosides; a 5' wing segment consisting of from 1 to 5 linked nucleosides; and a 3' wing segment consisting of from 1 to 5 linked nucleosides; wherein each nucleoside of the 5' wing segment is a sugar-modified nucleoside, each nucleoside of the 3' wing segment is a sugar-modified nucleoside, and each internucleoside linkage is a phosphorothioate internucleoside linkage.
20. The antisense compound of claim 19, wherein the sugar modification is a 2'-O-methoxyethyl sugar modification.
21. The antisense compound of claim 7 wherein the antisense compound is a chimeric oligonucleotide consisting of: a gap segment consisting of ten linked 2'-deoxynucleosides; a 5' wing segment consisting of five linked nucleosides; and a 3' wing segment consisting of five linked nucleosides; wherein each nucleoside of the 5' wing segment is a 2'-O-methoxyethyl nucleoside, each nucleoside of the 3' wing segment is a 2'-O-methoxyethyl nucleoside, and each internucleoside linkage is a phosphorothioate internucleoside linkage.
22. The antisense compound of claim 21 comprising at least one cytosine, wherein the cytosine is a 5-methylcytosine.
23. A pharmaceutical composition comprising the antisense compound of claim 1 and pharmaceutically acceptable excipient.
24. A pharmaceutical composition comprising the antisense compound of claim 19 and pharmaceutically acceptable excipient.
25. A pharmaceutical composition comprising the antisense compound of claim 21 and pharmaceutically acceptable excipient.
26. A pharmaceutical composition comprising the antisense compound of claim 22 and pharmaceutically acceptable excipient.
27. The antisense compound of claim 19, wherein the gap segment is flanked on both the 5' and 3' sides by wing segments of the same length.
28. The antisense compound of claim 19, wherein the gap segment is flanked on both the 5' and 3' sides by wing segments of different lengths.
29. The antisense compound of claim 27, wherein the antisense compound is 20 nucleobases in length, and wherein: (i) the gap segment is 8 nucleotides in length, and the 5' and 3' wing segments are each 6 nucleotides in length; (ii) the gap segment is 10 nucleotides in length, and the 5' and 3' wing segments are each 5 nucleotides in length; (iii) the gap segment is 12 nucleotides in length, and the 5' and 3' wing segments are each 4 nucleotides in length; (iv) the gap segment is 14 nucleotides in length, and the 5' and 3' wing segments are each 3 nucleotides in length; (v) the gap segment is 16 nucleotides in length, and the 5' and 3' wing segments are each 2 nucleotides in length; or (vi) the gap segment is 18 nucleotides in length, and the 5' and 3' wing segments are each 1 nucleotide in length.
30. The antisense compound of claim 28, wherein the antisense compound is 20 nucleobases in length, and wherein the gap segment is 10 nucleotides in length, flanked on one side by a wing segment 6 nucleotides in length and flanked on the other side by a wing segment 4 nucleotides in length.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser. No. 13/016,917 filed Jan. 28, 2011, which is a continuation application of U.S. Ser. No. 11/124,020 filed May 5, 2005 (abandoned), which claims the benefit of priority of U.S. Provisional Application Ser. No. 60/568,409 filed May 5, 2004, each of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The present specification is being filed with a computer readable form (CRF) copy of the Sequence Listing. The CRF entitled 10103-068-999_SEQLIST.txt, which was created on Dec. 1, 2014 and is 129,715 bytes in size, is identical to the paper copy of the Sequence Listing and is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention provides compositions and methods for modulating the expression of variants of apolipoprotein B (apo B). In particular, this invention relates to antisense compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding apolipoprotein B and containing SNPs.
BACKGROUND OF THE INVENTION
[0004] Natural genetic sequence variability exists between individuals in any and every population. Subtle alteration(s) in the primary nucleotide sequence of a gene encoding a pharmaceutically-important protein may be manifested as significant variation in expression, structure and/or function of the protein. Such alterations may explain the different response of individuals to therapy with a particular drug.
[0005] Variability in genetic sequence is particularly likely to cause a variable response to therapy when the therapeutic is an antisense compound that modulates the expression of protein through specific hybridization to the genetic sequence. In this case, changes in the sequence of the DNA or RNA can have a direct effect on the ability of such a compound to specifically hybridize.
[0006] Identification of polymorphisms among various populations is desirable to tailor design of suitable antisense therapeutics, select antisense therapeutics to administer to a particular population, and also predict responsiveness to therapeutics.
SUMMARY OF THE INVENTION
[0007] The present invention is directed to antisense compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding apolipoprotein B, and which modulate the expression of apolipoprotein B. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of apolipoprotein B and methods of modulating the expression of apolipoprotein B in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of apolipoprotein B are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.
DETAILED DESCRIPTION OF THE INVENTION
A. Overview of the Invention
[0008] The present invention employs antisense compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding apolipoprotein B. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding apolipoprotein B. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding apolipoprotein B" have been used for convenience to encompass DNA encoding apolipoprotein B, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as "antisense". Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition." Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.
[0009] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of apolipoprotein B. In the context of the present invention, "modulation" and "modulation of expression" mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.
[0010] In the context of this invention, "hybridization" means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.
[0011] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
[0012] In the present invention the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which a compound of the invention 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 in the context of this invention, "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.
[0013] "Complementary," as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.
[0014] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70%, or at least 75%, or at least 80%, or at least 85% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise at least 90% sequence complementarity and even more preferably comprise at least 95% or at least 99% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense 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).
[0015] 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). In some preferred embodiments, homology, sequence identity or complementarity, between the oligomeric and target is between about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is between about 60% to about 70%. In preferred embodiments, homology, sequence identity or complementarity, is between about 70% and about 80%. In more preferred embodiments, homology, sequence identity or complementarity, is between about 80% and about 90%. In some preferred embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.
B. Compounds of the Invention
[0016] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, siRNAs, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid.
[0017] One non-limiting example of such an enzyme is RNAse H, 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 RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.
[0018] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.
[0019] The first evidence that dsRNA, also known as small interfering RNAs (siRNAs) could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).
[0020] The antisense compounds of the present invention also include modified compounds in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenosine, modified compounds may be produced which contain thymidine, guanosine or cytidine at this position. This may be done at any of the positions of the antisense compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of apolipoprotein B mRNA.
[0021] In the context of this invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
[0022] While oligonucleotides are a preferred form of the antisense compounds of this invention, the present invention comprehends other families of antisense compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.
[0023] The antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies 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 nucleobases in length.
[0024] In one preferred embodiment, the antisense compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 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, or 50 nucleobases in length.
[0025] In another preferred embodiment, the antisense compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.
[0026] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.
[0027] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.
[0028] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5'-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3'-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). It is also understood that preferred antisense compounds may be represented by oligonucleotide sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred antisense compound, and may extend in either or both directions until the oligonucleotide contains about 8 to about 80 nucleobases.
[0029] One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.
C. Targets of the Invention
[0030] "Targeting" an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, 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. In the present invention, the target nucleic acid encodes apolipoprotein B.
[0031] 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. Within the context of the present invention, the term "region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. 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 positions within a target nucleic acid.
[0032] Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon". A minority of genes has a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding apolipoprotein B, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).
[0033] The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region" (or "translation initiation codon region") and the "stop codon region" (or "translation termination codon region") are all regions which may be targeted effectively with the antisense compounds of the present invention.
[0034] The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.
[0035] Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3' untranslated region (3 `UTR), known in the art to refer to the portion of an mRNA in the 3` direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5' cap region.
[0036] 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 sites where exons are joined. Targeting exon-exon junctions can be useful in situations where the overproduction of a normal splice product is implicated in disease, or where the overproduction of an aberrant splice product is implicated in disease. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may 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 preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.
[0037] 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.
[0038] 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.
[0039] 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. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.
[0040] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as "preferred target segments." As used herein the term "preferred target segment" is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.
[0041] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.
[0042] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.
[0043] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). It is also understood that preferred antisense target segments may be represented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred target segment, and may extend in either or both directions until the oligonucleotide contains about 8 to about 80 nucleobases. One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.
[0044] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
[0045] The oligomeric antisense compounds can also be targeted to regions of a target nucleobase sequence, such as those disclosed herein. All regions of the target nucleobase sequence to which an oligomeric antisense compound can be targeted, wherein the regions are greater than or equal to 8 and less than or equal to 80 nucleobases, are described as follows:
[0046] Let R(n, n+m-1) be a region from a target nucleobase sequence, where "n" is the 5'-most nucleobase position of the region, where "n+m-1" is the 3'-most nucleobase position of the region and where "m" is the length of the region. A set "S(m)", of regions of length "m" is defined as the regions where n ranges from 1 to L-m+1, where L is the length of the target nucleobase sequence and L>m. A set, "A", of all regions can be constructed as a union of the sets of regions for each length from where m is greater than or equal to 8 and is less than or equal to 80.
[0047] This set of regions can be represented using the following mathematical notation:
A = m S ( m ) where m .di-elect cons. N 8 ≦ m ≦ 80 ##EQU00001## and ##EQU00001.2## S ( m ) = { R n , n + m - 1 n .di-elect cons. ( 1 , 2 , 3 , , L - m + 1 } } ##EQU00001.3##
[0048] where the mathematical operator | indicates "such that",
[0049] where the mathematical operator ε indicates "a member of a set" (e.g. yεZ indicates that element y is a member of set Z),
[0050] where x is a variable,
[0051] where N indicates all natural numbers, defined as positive integers,
[0052] and where the mathematical operator ∪ indicates "the union of sets".
[0053] For example, the set of regions for m equal to 8, 9 and 80 can be constructed in the following manner. The set of regions, each 8 nucleobases in length, S(m=8), in a target nucleobase sequence 100 nucleobases in length (L=100), beginning at position 1 (n=1) of the target nucleobase sequence, can be created using the following expression:
S(8)={R1,8|nε{1,2,3, . . . , 93}}
and describes the set of regions comprising nucleobases 1-8, 2-9, 3-10, 4-11, 5-12, 6-13, 7-14, 8-15, 9-16, 10-17, 11-18, 12-19, 13-20, 14-21, 15-22, 16-23, 17-24, 18-25, 19-26, 20-27, 21-28, 22-29, 23-30, 24-31, 25-32, 26-33, 27-34, 28-35, 29-36, 30-37, 31-38, 32-39, 33-40, 34-41, 35-42, 36-43, 37-44, 38-45, 39-46, 40-47, 41-48, 42-49, 43-50, 44-51, 45-52, 46-53, 47-54, 48-55, 49-56, 50-57, 51-58, 52-59, 53-60, 54-61, 55-62, 56-63, 57-64, 58-65, 59-66, 60-67, 61-68, 62-69, 63-70, 64-71, 65-72, 66-73, 67-74, 68-75, 69-76, 70-77, 71-78, 72-79, 73-80, 74-81, 75-82, 76-83, 77-84, 78-85, 79-86, 80-87, 81-88, 82-89, 83-90, 84-91, 85-92, 86-93, 87-94, 88-95, 89-96, 90-97, 91-98, 92-99, 93-100.
[0054] An additional set for regions 20 nucleobases in length, in a target sequence 100 nucleobases in length, beginning at position 1 of the target nucleobase sequence, can be described using the following expression:
S(20)={R1,20|nε{1,2,3, . . . , 81}}
and describes the set of regions comprising nucleobases 1-20, 2-21, 3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42, 24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52, 34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72, 54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82, 64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92, 74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.
[0055] An additional set for regions 80 nucleobases in length, in a target sequence 100 nucleobases in length, beginning at position 1 of the target nucleobase sequence, can be described using the following expression:
S(80)={R1,80nε{1,2,3, . . . , 21}}
and describes the set of regions comprising nucleobases 1-80, 2-81, 3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92, 14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.
[0056] Thus, in this example, A would include regions 1-8, 2-9, 3-10 . . . 93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.
[0057] The union of these aforementioned example sets and other sets for lengths from 10 to 19 and 21 to 79 can be described using the mathematical expression
A = m S ( m ) ##EQU00002##
[0058] where ∪ represents the union of the sets obtained by combining all members of all sets.
[0059] The mathematical expressions described herein defines all possible target regions in a target nucleobase sequence of any length L, where the region is of length m, and where m is greater than or equal to 8 and less than or equal to 80 nucleobases and, and where m is less than L, and where n is less than L-m+1.
D. Screening and Target Validation
[0060] In a further embodiment, the "preferred target segments" identified herein may be employed in a screen for additional compounds that modulate the expression of apolipoprotein B. "Modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding apolipoprotein B and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding apolipoprotein B with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding apolipoprotein B. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding apolipoprotein B, the modulator may then be employed in further investigative studies of the function of apolipoprotein B, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.
[0061] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.
[0062] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).
[0063] The antisense compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between apolipoprotein B and a disease state, phenotype, or condition. These methods include detecting or modulating apolipoprotein B comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of apolipoprotein B and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.
E. Kits, Research Reagents, Diagnostics, and Therapeutics
[0064] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
[0065] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
[0066] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.
[0067] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).
[0068] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding apolipoprotein B. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective apolipoprotein B inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding apolipoprotein B and in the amplification of said nucleic acid molecules for detection or for use in further studies of apolipoprotein B. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding apolipoprotein B can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of apolipoprotein B in a sample may also be prepared.
[0069] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
[0070] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of apolipoprotein B is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of an apolipoprotein B inhibitor. The apolipoprotein B inhibitors of the present invention effectively inhibit the activity of the apolipoprotein B protein or inhibit the expression of the apolipoprotein B protein. In one embodiment, the activity or expression of apolipoprotein B in an animal is inhibited by about 10%. Preferably, the activity or expression of apolipoprotein B in an animal is inhibited by about 30%. More preferably, the activity or expression of apolipoprotein B in an animal is inhibited by 50% or more. Thus, the oligomeric antisense compounds modulate expression of apolipoprotein B mRNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.
[0071] For example, the reduction of the expression of apolipoprotein B may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding apolipoprotein B protein and/or the apolipoprotein B protein itself.
[0072] The antisense compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.
F. Modifications
[0073] 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. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded 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.
Modified Internucleoside Linkages (Backbones)
[0074] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
[0075] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriaminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 `-alkylene phosphonates, 5`-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 `-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, 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. Preferred 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.
[0076] 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.
[0077] Preferred modified 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.
[0078] 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.
Modified Sugar and Internucleoside Linkages-Mimetics
[0079] In other preferred antisense compounds, e.g., oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and 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 compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
[0080] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and 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 backbone is represented as --O--P--O--CH2--] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified Sugars
[0081] Modified antisense compounds may also contain one or more substituted sugar moieties. Preferred are antisense compounds, preferably antisense oligonucleotides, comprising 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. Particularly preferred 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 m are from 1 to about 10. Other preferred 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, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, 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. A preferred 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 preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH3)2, also described in examples hereinbelow.
[0082] Other preferred 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. A preferred 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; and 5,700,920.
[0083] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (--CH2--)n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
Natural and Modified Nucleobases
[0084] Antisense compounds may 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" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases 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≡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 nucleobases 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 nucleobases 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 nucleobases 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 nucleobases are particularly useful 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° C. and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
[0085] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases 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; 5,750,692; and 5,681,941.
Conjugates
[0086] Another modification of the antisense compounds of the invention involves chemically linking to the antisense compound one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. 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. No. 6,287,860, the entire disclosures of which are incorporated herein by reference. 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 triethylammonium 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. Antisense compounds of the invention may also be conjugated to active 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. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.
[0087] 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.
Chimeric Compounds
[0088] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
[0089] The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. Chimeric antisense oligonucleotides are thus a form of antisense compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
[0090] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Chimeric antisense compounds can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers". Such compounds have also been referred to in the art as hybrids. In a gapmer that is 20 nucleotides in length, a gap or wing can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 nucleotides in length. In one embodiment, a 20-nucleotide gapmer is comprised of a gap 8 nucleotides in length, flanked on both the 5' and 3' sides by wings 6 nucleotides in length. In another embodiment, a 20-nucleotide gapmer is comprised of a gap 10 nucleotides in length, flanked on both the 5' and 3' sides by wings 5 nucleotides in length. In another embodiment, a 20-nucleotide gapmer is comprised of a gap 12 nucleotides in length flanked on both the 5' and 3' sides by wings 4 nucleotides in length. In a further embodiment, a 20-nucleotide gapmer is comprised of a gap 14 nucleotides in length flanked on both the 5' and 3' sides by wings 3 nucleotides in length. In another embodiment, a 20-nucleotide gapmer is comprised of a gap 16 nucleotides in length flanked on both the 5' and 3' sides by wings 2 nucleotides in length. In a further embodiment, a 20-nucleotide gapmer is comprised of a gap 18 nucleotides in length flanked on both the 5' and 3' ends by wings 1 nucleotide in length. Alternatively, the wings are of different lengths, for example, a 20-nucleotide gapmer may be comprised of a gap 10 nucleotides in length, flanked by a 6-nucleotide wing on one side (5' or 3') and a 4-nucleotide wing on the other side (5' or 3'). In a hemimer, an "open end" chimeric antisense compound, 20 nucleotides in length, a gap segment, located at either the 5' or 3' terminus of the oligomeric compound, can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides in length. For example, a 20-nucleotide hemimer can have a gap segment of 10 nucleotides at the 5' end and a second segment of 10 nucleotides at the 3' end. Alternatively, a 20-nucleotide hemimer can have a gap segment of 10 nucleotides at the 3' end and a second segment of 10 nucleotides at the 5' end.
[0091] 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.
G. Formulations
[0092] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756.
[0093] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
[0094] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0095] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
[0096] The pharmaceutical formulations of the present invention, 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 or finely divided solid carriers or both, and then, if necessary, shaping the product.
[0097] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
[0098] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
[0099] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0100] Formulations of the present invention include liposomal formulations. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
[0101] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0102] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0103] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.
[0104] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.
[0105] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).
[0106] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.
[0107] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.
[0108] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
[0109] Oligonucleotides may be formulated for delivery in vivo in an acceptable dosage form, e.g. as parenteral or non-parenteral formulations. Parenteral formulations include intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intravitreal and intramuscular (IM) formulations, as well as formulations for delivery via pulmonary inhalation, intranasal administration, topical administration, etc. Non-parenteral formulations include formulations for delivery via the alimentary canal, e.g. oral administration, rectal administration, intrajejunal instillation, etc. Rectal administration includes administration as an enema or a suppository. Oral administration includes administration as a capsule, a gel capsule, a pill, an elixir, etc.
[0110] In some embodiments, an oligonucleotide may be administered to a subject via an oral route of administration. The subject may be an animal or a human (man). An animal subject may be a mammal, such as a mouse, a rat, a dog, a guinea pig, a non-human primate, a cat or a pig. Non-human primates include monkeys and chimpanzees. A suitable animal subject may be an experimental animal, such as a mouse, a rat, a dog, a non-human primate, a cat or a pig.
[0111] In some embodiments, the subject may be a human. In certain embodiments, the subject may be a human patient in need of therapeutic treatment as discussed in more detail herein. In certain embodiments, the subject may be in need of modulation of expression of one or more genes as discussed in more detail herein. In some particular embodiments, the subject may be in need of inhibition of expression of one or more genes as discussed in more detail herein. In particular embodiments, the subject may be in need of modulation, i.e. inhibition or enhancement, of hepatic lipase in order to obtain therapeutic indications discussed in more detail herein.
[0112] In some embodiments, non-parenteral (e.g. oral) oligonucleotide formulations according to the present invention result in enhanced bioavailability of the oligonucleotide. In this context, the term "bioavailability" refers to a measurement of that portion of an administered drug which reaches the circulatory system (e.g. blood, especially blood plasma) when a particular mode of administration is used to deliver the drug. Enhanced bioavailability refers to a particular mode of administration's ability to deliver oligonucleotide to the peripheral blood plasma of a subject relative to another mode of administration. For example, when a non-parenteral mode of administration (e.g. an oral mode) is used to introduce the drug into a subject, the bioavailability for that mode of administration may be compared to a different mode of administration, e.g. an IV mode of administration. In some embodiments, the area under a compound's blood plasma concentration curve (AUC0) after non-parenteral (e.g. oral, rectal, intrajejunal) administration may be divided by the area under the drug's plasma concentration curve after intravenous (i.v.) administration (AUCiv) to provide a dimensionless quotient (relative bioavailability, RB) that represents fraction of compound absorbed via the non-parenteral route as compared to the IV route. A composition's bioavailability is said to be enhanced in comparison to another composition's bioavailability when the first composition's relative bioavailability (RB1) is greater than the second composition's relative bioavailability (RB2).
[0113] In general, bioavailability correlates with therapeutic efficacy when a compound's therapeutic efficacy is related to the blood concentration achieved, even if the drug's ultimate site of action is intracellular (van Berge-Henegouwen et al., Gastroenterol., 1977, 73, 300). Bioavailability studies have been used to determine the degree of intestinal absorption of a drug by measuring the change in peripheral blood levels of the drug after an oral dose (DiSanto, Chapter 76 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 1451-1458).
[0114] In general, an oral composition's bioavailability is said to be "enhanced" when its relative bioavailability is greater than the bioavailability of a composition substantially consisting of pure oligonucleotide, i.e. oligonucleotide in the absence of a penetration enhancer.
[0115] Organ bioavailability refers to the concentration of compound in an organ. Organ bioavailability may be measured in test subjects by a number of means, such as by whole-body radiography. Organ bioavailability may be modified, e.g. enhanced, by one or more modifications to the oligonucleotide, by use of one or more carrier compounds or excipients, etc. as discussed in more detail herein. In general, an increase in bioavailability will result in an increase in organ bioavailability.
[0116] Oral oligonucleotide compositions according to the present invention may comprise one or more "mucosal penetration enhancers," also known as "absorption enhancers" or simply as "penetration enhancers." Accordingly, some embodiments of the invention comprise at least one oligonucleotide in combination with at least one penetration enhancer. In general, a penetration enhancer is a substance that facilitates the transport of a drug across mucous membrane(s) associated with the desired mode of administration, e.g. intestinal epithelial membranes. Accordingly it is desirable to select one or more penetration enhancers that facilitate the uptake of an oligonucleotide, without interfering with the activity of the oligonucleotide, and in a such a manner the oligonucleotide can be introduced into the body of an animal without unacceptable side-effects such as toxicity, irritation or allergic response.
[0117] Embodiments of the present invention provide compositions comprising one or more pharmaceutically acceptable penetration enhancers, and methods of using such compositions, which result in the improved bioavailability of oligonucleotides administered via non-parenteral modes of administration. Heretofore, certain penetration enhancers have been used to improve the bioavailability of certain drugs. See Muranishi, Crit. Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that the uptake and delivery of oligonucleotides, relatively complex molecules which are known to be difficult to administer to animals and man, can be greatly improved even when administered by non-parenteral means through the use of a number of different classes of penetration enhancers.
[0118] In some embodiments, compositions for non-parenteral administration include one or more modifications from naturally-occurring oligonucleotides (i.e. full-phosphodiester deoxyribosyl or full-phosphodiester ribosyl oligonucleotides). Such modifications may increase binding affinity, nuclease stability, cell or tissue permeability, tissue distribution, or other biological or pharmacokinetic property. Modifications may be made to the base, the linker, or the sugar, in general, as discussed in more detail herein with regards to oligonucleotide chemistry. In some embodiments of the invention, compositions for administration to a subject, and in particular oral compositions for administration to an animal or human subject, will comprise modified oligonucleotides having one or more modifications for enhancing affinity, stability, tissue distribution, or other biological property.
[0119] Suitable modified linkers include phosphorothioate linkers. In some embodiments according to the invention, the oligonucleotide has at least one phosphorothioate linker. Phosphorothioate linkers provide nuclease stability as well as plasma protein binding characteristics to the oligonucleotide. Nuclease stability is useful for increasing the in vivo lifetime of oligonucleotides, while plasma protein binding decreases the rate of first pass clearance of oligonucleotide via renal excretion. In some embodiments according to the present invention, the oligonucleotide has at least two phosphorothioate linkers. In some embodiments, wherein the oligonucleotide has exactly n nucleosides, the oligonucleotide has from one to n-1 phosphorothioate linkages. In some embodiments, wherein the oligonucleotide has exactly n nucleosides, the oligonucleotide has n-1 phosphorothioate linkages. In other embodiments wherein the oligonucleotide has exactly n nucleoside, and n is even, the oligonucleotide has from 1 to n/2 phosphorothioate linkages, or, when n is odd, from 1 to (n-1)/2 phosphorothioate linkages. In some embodiments, the oligonucleotide has alternating phosphodiester (PO) and phosphorothioate (PS) linkages. In other embodiments, the oligonucleotide has at least one stretch of two or more consecutive PO linkages and at least one stretch of two or more PS linkages. In other embodiments, the oligonucleotide has at least two stretches of PO linkages interrupted by at least on PS linkage.
[0120] In some embodiments, at least one of the nucleosides is modified on the ribosyl sugar unit by a modification that imparts nuclease stability, binding affinity or some other beneficial biological property to the sugar. In some cases, the sugar modification includes a 2'-modification, e.g. the 2'-OH of the ribosyl sugar is replaced or substituted. Suitable replacements for 2'-OH include 2'-F and 2'-arabino-F. Suitable substitutions for OH include 2'-O-alkyl, e.g. 2-O-methyl, and 2'-O-substituted alkyl, e.g. 2'-O-methoxyethyl, 2'-O-aminopropyl, etc. In some embodiments, the oligonucleotide contains at least one 2'-modification. In some embodiments, the oligonucleotide contains at least 2 2'-modifications. In some embodiments, the oligonucleotide has at least one 2'-modification at each of the termini (i.e. the 3'- and 5'-terminal nucleosides each have the same or different 2'-modifications). In some embodiments, the oligonucleotide has at least two sequential 2'-modifications at each end of the oligonucleotide. In some embodiments, oligonucleotides further comprise at least one deoxynucleoside. In particular embodiments, oligonucleotides comprise a stretch of deoxynucleosides such that the stretch is capable of activating RNase (e.g. RNase H) cleavage of an RNA to which the oligonucleotide is capable of hybridizing. In some embodiments, a stretch of deoxynucleosides capable of activating RNase-mediated cleavage of RNA comprises about 6 to about 16, e.g. about 8 to about 16 consecutive deoxynucleosides.
[0121] Oral compositions for administration of non-parenteral oligonucleotide compositions of the present invention may be formulated in various dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The term "alimentary delivery" encompasses e.g. oral, rectal, endoscopic and sublingual/buccal administration. A common requirement for these modes of administration is absorption over some portion or all of the alimentary tract and a need for efficient mucosal penetration of the nucleic acid(s) so administered.
[0122] Delivery of a drug via the oral mucosa, as in the case of buccal and sublingual administration, has several desirable features, including, in many instances, a more rapid rise in plasma concentration of the drug than via oral delivery (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711).
[0123] Endoscopy may be used for drug delivery directly to an interior portion of the alimentary tract. For example, endoscopic retrograde cystopancreatography (ERCP) takes advantage of extended gastroscopy and permits selective access to the biliary tract and the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591). Pharmaceutical compositions, including liposomal formulations, can be delivered directly into portions of the alimentary canal, such as, e.g., the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastric submucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) via endoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs, 1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington et al., Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for direct alimentary delivery of pharmaceutical compositions.
[0124] In some embodiments, oligonucleotide formulations may be administered through the anus into the rectum or lower intestine. Rectal suppositories, retention enemas or rectal catheters can be used for this purpose and may be preferred when patient compliance might otherwise be difficult to achieve (e.g., in pediatric and geriatric applications, or when the patient is vomiting or unconscious). Rectal administration can result in more prompt and higher blood levels than the oral route. (Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Because about 50% of the drug that is absorbed from the rectum will bypass the liver, administration by this route significantly reduces the potential for first-pass metabolism (Benet et al., Chapter 1 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996).
[0125] One advantageous method of non-parenteral administration oligonucleotide compositions is oral delivery. Some embodiments employ various penetration enhancers in order to effect transport of oligonucleotides and other nucleic acids across mucosal and epithelial membranes. Penetration enhancers may be classified as belonging to one of five broad categories--surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Accordingly, some embodiments comprise oral oligonucleotide compositions comprising at least one member of the group consisting of surfactants, fatty acids, bile salts, chelating agents, and non-chelating surfactants. Further embodiments comprise oral oligonucleotide comprising at least one fatty acid, e.g. capric or lauric acid, or combinations or salts thereof. Other embodiments comprise methods of enhancing the oral bioavailability of an oligonucleotide, the method comprising co-administering the oligonucleotide and at least one penetration enhancer.
[0126] Other excipients that may be added to oral oligonucleotide compositions include surfactants (or "surface-active agents"), which are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the alimentary mucosa and other epithelial membranes is enhanced. In addition to bile salts and fatty acids, surfactants include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and perfluorohemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol., 1988, 40, 252).
[0127] Fatty acids and their derivatives which act as penetration enhancers and may be used in compositions of the present invention include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines and mono- and di-glycerides thereof and/or physiologically acceptable salts thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651).
[0128] In some embodiments, oligonucleotide compositions for oral delivery comprise at least two discrete phases, which phases may comprise particles, capsules, gel-capsules, microspheres, etc. Each phase may contain one or more oligonucleotides, penetration enhancers, surfactants, bioadhesives, effervescent agents, or other adjuvant, excipient or diluent. In some embodiments, one phase comprises at least one oligonucleotide and at lease one penetration enhancer. In some embodiments, a first phase comprises at least one oligonucleotide and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer. In some embodiments, a first phase comprises at least one oligonucleotide and at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and substantially no oligonucleotide. In some embodiments, at least one phase is compounded with at least one degradation retardant, such as a coating or a matrix, which delays release of the contents of that phase. In some embodiments, a first phase comprises at least one oligonucleotide, at least one penetration enhancer, while a second phase comprises at least one penetration enhancer and a release-retardant. In particular embodiments, an oral oligonucleotide comprises a first phase comprising particles containing an oligonucleotide and a penetration enhancer, and a second phase comprising particles coated with a release-retarding agent and containing penetration enhancer.
[0129] A variety of bile salts also function as penetration enhancers to facilitate the uptake and bioavailability of drugs. The physiological roles of bile include the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579).
[0130] In some embodiments, penetration enhancers useful in some embodiments of present invention are mixtures of penetration enhancing compounds. One such penetration enhancer is a mixture of UDCA (and/or CDCA) with capric and/or lauric acids or salts thereof e.g. sodium. Such mixtures are useful for enhancing the delivery of biologically active substances across mucosal membranes, in particular intestinal mucosa. Other penetration enhancer mixtures comprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with 5-95% capric and/or lauric acid. Particular penetration enhancers are mixtures of the sodium salts of UDCA, capric acid and lauric acid in a ratio of about 1:2:2 respectively. Anther such penetration enhancer is a mixture of capric and lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio (mole basis). In particular embodiments capric acid and lauric acid are present in molar ratios of e.g. about 0.1:1 to about 1:0.1, in particular about 0.5:1 to about 1:0.5.
[0131] Other excipients include chelating agents, i.e. compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucelotides through the alimentary and other mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents of the invention include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. Control Rel., 1990, 14, 43).
[0132] As used herein, non-chelating non-surfactant penetration enhancers may be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary and other mucosal membranes (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1). This class of penetration enhancers includes, but is not limited to, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).
[0133] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), can be used.
[0134] Some oral oligonucleotide compositions also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which may be inert (i.e., does not possess biological activity per se) or may be necessary for transport, recognition or pathway activation or mediation, or is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177).
[0135] A "pharmaceutical carrier" or "excipient" may be a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. 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. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB); and wetting agents (e.g., sodium lauryl sulphate, etc.).
[0136] Oral oligonucleotide compositions may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipuritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
[0137] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethyl-melamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
[0138] In another related embodiment, compositions of the invention 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 of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.
H. Dosing
[0139] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, from 0.1 μg to 10 g per kg of body weight, from 1.0 μg to 1 g per kg of body weight, from 10.0 μg to 100 mg per kg of body weight, from 100 μg to 10 mg per kg of body weight, or from 1 mg to 5 mg per kg of body weight and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
[0140] The effects of treatments with therapeutic compositions can be assessed following collection of tissues or fluids from a patient or subject receiving said treatments. It is known in the art that a biopsy sample can be procured from certain tissues without resulting in detrimental effects to a patient or subject. In certain embodiments, a tissue and its constituent cells comprise, but are not limited to, blood (e.g., hematopoietic cells, such as human hematopoietic progenitor cells, human hematopoietic stem cells, CD34.sup.+ cells CD4.sup.+ cells), lymphocytes and other blood lineage cells, bone marrow, breast, cervix, colon, esophagus, lymph node, muscle, peripheral blood, oral mucosa and skin. In other embodiments, a fluid and its constituent cells comprise, but are not limited to, blood, urine, semen, synovial fluid, lymphatic fluid and cerebro-spinal fluid. Tissues or fluids procured from patients can be evaluated for expression levels of the target mRNA or protein by techniques known in the art. Additionally, the mRNA or protein expression levels of other genes known or suspected to be associated with the specific disease state, condition or phenotype, or levels of biological markers associated with the disease state, condition or phenotype, can similarly be assessed. Target or associated gene mRNA levels can be measured or evaluated by real-time PCR, Northern blot, in situ hybridization or DNA array analysis. Target or associated protein levels or biomarkers can be measured or evaluated by ELISA, immunoblotting, quantitative protein assays, protein activity assays (for example, caspase activity assays) immunohistochemistry, immunocytochemistry or routine clinical analysis.
I. Polymorphisms
[0141] One common allelic genomic sequence for apolipoprotein B is set forth in SEQ ID NO: 1 and is referred to herein as the "wild-type" sequence. Novel polymorphic sites have been identified in this gene at positions 27751, 27735, 27685, 27683, 27679, 27634, 27627 and 27618 of SEQ ID NO: 1 (and corresponding positions 15695, 15711, 15761, 15763, 15767, 15812, 15819 and 15828 of the reverse complement, SEQ ID NO: 2).
[0142] A polymorphism is a sequence variation in the gene observed within the population, and can include nucleotide substitutions (single nucleotide polymorphisms or SNPs), insertions, or deletions. Polymorphisms may or may not result in detectable differences in gene expression, protein structure, or protein function. A polymorphism may alter one or more properties of the gene or gene products, including DNA or RNA stability, binding of transcriptional or translation factors to the DNA or RNA, interactions of the DNA or RNA with other parts of the nuclear or cytosolic cell machinery, or may confer a change upon the encoded polypeptide sequence which in turn may alter the polypeptide's biological activity. Identification of polymorphisms among various populations is desirable to tailor design of suitable antisense therapeutics, select antisense therapeutics to administer to a particular population, and also predict responsiveness to therapeutics.
[0143] A "polymorphic site" is a position within a genetic locus at which at least one alternative nucleotide sequence variation (e.g., substitution, insertion, or deletion) has been observed in a population, and includes the position on both complementary strands at the polymorphic site. The first identified form of the nucleotide sequence at the polymorphic site is sometimes called the reference sequence, and the alternative forms are called alternative or variant alleles (or "allelic variant"). The most commonly occurring form of the nucleotide sequence at the polymorphic site is also sometimes called the wild type allele. Polymorphic sites of the invention are listed in the following table along with their approximate frequency.
TABLE-US-00001 Approximate frequency of Position in SNP detection out of 213 Sequence variation SEQ ID NO: 1 samples of diverse ancestry Substitution of A to G 27751 15% Substitution of C to G 27735 <1% Substitution of T to C 27685 27% Substitution of T to C 27683 Substitution of T to C 27679 40% Substitution of C to T 27634 <1% Substitution of G to A 27627 2% Substitution of T to C 27618 <1%
[0144] The invention provides a variety of polynucleotides, including reverse complements, single or double stranded polynucleotides, RNA, DNA or mimetics thereof, and antisense compounds, as defined above, that either contain or specifically hybridize to a polymorphic sequence at one or more of these polymorphic sites. Such "sequence-specific" polynucleotides can be used, alone or linked to other moieties, in a variety of areas. For example, the polynucleotides may be useful as therapeutic products for inhibiting gene expression, as probes or primers for detecting the polymorphic sequence as part of genotyping, diagnostic, pharmacogenomics and/or treatment methods, as part of arrays for screening, as part of diagnostic or therapeutic kits, or as tools for producing recombinant protein in host cells or transgenic organisms.
[0145] Methods of producing polynucleotides are well-known in the art, including chemical synthesis, cloning, and PCR amplification. The polymorphic polynucleotide sequences of the invention are preferably isolated, meaning in a form other than as part of an intact naturally occurring chromosome. Usually the polynucleotide sequences will also be purified, meaning at least about 50%, 75%, 80% or 90% pure or substantially free of other polynucleotide sequences that do not include an apolipoprotein B polynucleotide sequence or fragment thereof. The polynucleotide sequences can also be "recombinant", meaning flanked by one or more nucleotides with which they are not normally associated on a naturally occurring chromosome.
[0146] As noted elsewhere herein, polynucleotides or oligonucleotides may include modifications, including but not limited to modifications to the internucleoside linkages, modifications to the sugar moieties, and modified nucleobases, so long as the modified polynucleotides retain the ability to hybridize specifically to the target polymorphic site.
[0147] Such polynucleotides of the invention may be linked to a second moiety such as an additional nucleotide sequence for stabilization purposes or for directing transcription or translation, a moiety which facilitates linkage to a solid support (such as a microarray or microparticle), or a label to facilitate detection of the polynucleotide. Such labels include, without limitation, a radioactive label, a fluorescent label, a chemiluminescent label, a paramagnetic label, an enzymatic label, one member of a high affinity binding partner pair (such as biotin/avidin) or other labels known in the art. The second moiety may be attached to any position of the polynucleotide, so long as the polynucleotide retains its ability to hybridize to the polymorphic sites described herein. The second moiety may be linked to the polynucleotide after it has been generated, or may be linked to a component nucleobase that is then incorporated into the polynucleotide during synthesis or assembly. Polynucleotides of the invention can also be attached to the surface of a solid support through means not involving direct chemical linkage.
[0148] As used herein, "sequence-specific" means that the polynucleotide, oligonucleotide or antisense compound specifically hybridizes to one nucleotide sequence at a polymorphic site compared to another, e.g. preferentially hybridizes more strongly to the one sequence than to an alternative nucleotide sequence that has been observed in some individuals at that polymorphic site.
[0149] Sequence-specific polynucleotides when used for sequence detection must be capable of hybridizing to the polymorphic sites under conditions of stringency such as those employed in hybridization-based sequence determination methods, primer extension-based sequence determination methods, restriction site analysis, polynucleotide amplification methods, ligase-based sequencing methods, methods based on enzymatic detection of mismatches, microarray-based sequence determination methods, and other sequence determination methods known in the art. In a related embodiment, the invention also contemplates primer pairs comprising an oligonucleotide useful for amplification of a polymorphic site in the gene. Such primer pairs may comprise the polymorphic site or may surround it. Kits comprising such oligonucleotides and primer pairs are also contemplated.
[0150] In some embodiments, the invention provides sequence-specific polynucleotides comprising at least 15 contiguous nucleotides of SEQ ID NO: 1, or comprising at least 15 continguous nucleotides of an allelic variant of SEQ ID NO: 1, said polynucleotide including at least one of:
C at position 27751 of SEQ ID NO: 1; C at position 27735 of SEQ ID NO: 1; G at position 27685 of SEQ ID NO: 1; G at position 27683 of SEQ ID NO: 1; G at position 27679 of SEQ ID NO: 1; A at position 27634 of SEQ ID NO: 1; T/U at position 27627 of SEQ ID NO: 1; or G at position 27618 of SEQ ID NO: 1, wherein G is guanine, C is cytosine, T is thymine, U is uracil, and A is adenine.
[0151] In related embodiments, the invention further provides sequence-specific polynucleotides comprising at least 15 contiguous nucleotides of SEQ ID NO: 2, or comprising at least 15 contiguous nucleotides of an allelic variant of SEQ ID NO: 2, which is the reverse complement of SEQ ID NO: 1, said polynucleotide including at least one of:
G at position 15695 of SEQ ID NO: 2; G at position 15711 of SEQ ID NO: 2; C at position 15761 of SEQ ID NO: 2; C at position 15763 of SEQ ID NO: 2; C at position 15767 of SEQ ID NO: 2; T/U at position 15812 of SEQ ID NO: 2; A at position 15819 of SEQ ID NO: 2; or C at position 15828 of SEQ ID NO: 2.
[0152] Such polynucleotides, including reverse complements, or single or double stranded polynucleotides, may range in length, for example, from at least 8, 12, 15, or 20 bases, such as 12-20, 15-30, 15-50, 50-100, 8-80, 8-30, 8-50, 12-50, or 12-30 contiguous bases, or may correspond to the full length of the encoding cDNA.
[0153] As part of these above aspects of the invention, the invention contemplates a sequence-specific oligonucleotide or antisense compound of no more than 100 nucleobases in length that hybridize to a portion of SEQ ID NO: 1 including at least one polymorphic site selected from the group consisting of:
C at position 27751 of SEQ ID NO: 1; C at position 27735 of SEQ ID NO: 1; G at position 27685 of SEQ ID NO: 1; G at position 27683 of SEQ ID NO: 1; G at position 27679 of SEQ ID NO: 1; A at position 27634 of SEQ ID NO: 1; T/U at position 27627 of SEQ ID NO: 1; or G at position 27618 of SEQ ID NO: 1.
[0154] The invention also contemplates a sequence-specific oligonucleotides or antisense compound of no more than 100 nucleobases in length that hybridizes to a portion of SEQ ID NO: 2, which is the reverse complement of SEQ ID NO: 1, including at least one polymorphic site selected from the group consisting of:
G at position 15695 of SEQ ID NO: 2; G at position 15711 of SEQ ID NO: 2; C at position 15761 of SEQ ID NO: 2; C at position 15763 of SEQ ID NO: 2; C at position 15767 of SEQ ID NO: 2; T/U at position 15812 of SEQ ID NO: 2; A at position 15819 of SEQ ID NO: 2; or C at position 15828 of SEQ ID NO: 2.
[0155] The invention further contemplates an oligonucleotide comprising about 15 to 30 contiguous nucleobases of an allelic variant of SEQ ID NO: 1, said allelic variant comprising at least one of:
C at position 27751 of SEQ ID NO: 1; C at position 27735 of SEQ ID NO: 1; G at position 27685 of SEQ ID NO: 1; G at position 27683 of SEQ ID NO: 1; G at position 27679 of SEQ ID NO: 1; A at position 27634 of SEQ ID NO: 1; T/U at position 27627 of SEQ ID NO: 1; or G at position 27618 of SEQ ID NO: 1.
[0156] The invention also contemplates an oligonucleotide comprising about 15 to 30 contiguous nucleobases of an allelic variant of SEQ ID NO: 2, said allelic variant comprising at least one of:
G at position 15695 of SEQ ID NO: 2; G at position 15711 of SEQ ID NO: 2; C at position 15761 of SEQ ID NO: 2; C at position 15763 of SEQ ID NO: 2; C at position 15767 of SEQ ID NO: 2; T/U at position 15812 of SEQ ID NO: 2; A at position 15819 of SEQ ID NO: 2; or C at position 15828 of SEQ ID NO: 2.
[0157] As noted above, such oligonucleotides or antisense compounds may be single or double stranded, may include reverse complements, may be RNA, DNA or mimetics, may be chemically modified, and may range in length, for example, from at least 8, 12, 15, or 20 bases, such as 12-20, 15-30, 15-50, 50-100, 8-80, 8-30, 8-50, 12-50, or 12-30 contiguous bases. In particular, an oligonucleotide or antisense compound that specifically hybridizes with a polymorphic sequence of a polymorphic site identified herein is useful for antisense therapy as described in other sections herein.
[0158] Where the polymorphism results in a change in the encoded amino acid sequence, expression vectors, host cells and recombinant organisms useful for producing the encoded protein are additionally contemplated. Polypeptides of limited length may also be prepared using chemical synthesis methods. Expression vectors may include nucleotide sequences that regulate transcription and/or translation, which may be inducible or constitutive, and which are preferably operably linked to coding sequence. Host cells include any prokaryotic, eukaryotic host cells known in the art, including bacteria, yeast, insect and mammalian cells. A large variety of techniques for expressing and purifying recombinant protein in host cell systems are known in the art. The polynucleotides of the invention can also be used to generate genetically modified (or transgenic) non-human animals or site specific gene modifications in cell lines using techniques known in the art. Such transgenic animals or cell lines include those in which the polymorphic gene is deleted or knocked out, those in which an exogenous polynucleotide comprising the polymorphism is stably inserted and transmitted to progeny, or those in which an endogenous polynucleotide comprising the polymorphism is operably linked to an exogenous regulatory sequence. Homologous recombination techniques are well known, and may utilize nucleic acid alone or as part of a suitable vector, such as viral vectors.
[0159] The variant polypeptides encoded by polynucleotides comprising one or more polymorphisms are also of interest, as are fragments thereof particularly antigenic epitopes, functional domains, binding sites, and other regions of interest, and including fusion proteins thereof. Polypeptides thus expressed are useful for protein structure analysis, for drug binding studies, and for screening candidate drugs to treat diseases related to apolipoprotein B activity. Antibodies specific for the variant polypeptides that differentiate between variant polypeptides and wild type polypeptide, including monoclonal antibodies and humanized or human antibodies, are also contemplated.
[0160] Expression assays can be used to detect differences in expression of polymorphisms with respect to tissue specificity, expression level, or expression in response to exposure to various substrates, and/or timing of expression during development. Expression assays may be performed in cell-free extracts, or by transforming cells with a suitable vector. Alterations in expression may occur in the basal level that is expressed in one or more cell types, or in the effect that an expression modifier has on the ability of the gene to be inhibited or induced. Expression levels of variant alleles are compared by various methods known in the art.
[0161] Screening can also be performed to determine if the polymorphisms described herein are genetically linked to other polymorphisms, to microsatellite markers, or to a phenotypic variant in apolipoprotein B activity or expression. Two polymorphisms may be in linkage disequilibrium, i.e. where alleles show non-random associations between genes even though individual loci are in Hardy-Weinberg equilibrium. Association of a polymorphism with a phenotypic trait (risk of a disease, severity or staging of a disease, or response to a drug) can also be identified by comparing the frequency of the polymorphism in a population exhibiting the trait to the frequency in a reference population; a higher frequency occurrence of the polymorphism in the population exhibiting the trait indicates that the trait is associated with the polymorphism. When such an association is established, the risk of disease, severity or staging of a disease, or response of an individual to a drug can then be predicted by determining the patient's genotype with respect to the polymorphism. Where there is a differential distribution of a polymorphism by racial background, guidelines for drug administration can be generally tailored to a particular ethnic group.
[0162] Identifying the presence or absence of a SNP is useful in methods of genotyping a human comprising the step of determining the identity of a nucleotide at a particular polymorphic site, in either the sense strand or its complement. The genotyping method may comprise identifying the nucleotide pair that is present at one or more polymorphic sites described herein. Genotyping compositions or kits of the invention comprises an oligonucleotide probe or primer which is designed to specifically hybridize to a target region containing, or adjacent to, one of these novel polymorphic sites. A genotyping kit of the invention may further comprise a set of oligonucleotides designed to genotype other polymorphic sites.
[0163] Detection of the polymorphism can be performed by DNA or RNA sequence analysis of any patient sample that contains genetic material, including biopsied tissue, blood, skin, or other cell samples. The sample polynucleotide or desired segment thereof can be amplified or cloned by methods known in the art. The presence or absence of the polymorphism in question can be determined in a variety of ways known in the art. For example, the sequence of the sample polynucleotide may be determined by dideoxy sequencing or other conventional chemical analytical methods. Hybridization-based methods include Southern blots or dot blots, detecting a pattern of hybridization to sets of probes, ligase-based methods, primer extension-based methods, allele-specific amplification, Taqman, and other PCR-based methods. Other methods such as single strand conformational polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), and heteroduplex analysis in gel matrices are used to detect conformational changes created by DNA sequence variation as alterations in electrophoretic mobility. If a polymorphism creates or destroys a recognition site for a restriction endonuclease (restriction fragment length polymorphism, RFLP), polymorphic sequence can be detected by digesting the sample with that endonuclease, and separating the products by size (e.g. using gel or capillary electrophoresis) to determine whether the fragment was digested. Mismatch cleavage detection using enzymes or chemical cleavage agents followed by detecting product size using electrophoretic or mass spectrometry methods can also be carried out. Moreover, in cases where the polymorphism of the invention is linked to another marker (such as another polymorphism or a microsatellite marker) then detecting the presence of the marker serves to detect the presence of the polymorphism.
[0164] In one preferred embodiment, the invention provides a method of analyzing a patient's polynucleotides for the presence or absence of a mutation comprising: (a) providing a test sample comprising polynucleotides or replicas thereof from a biological sample obtained from the patient; (b) contacting the test sample with a probe comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or the complement thereof, the probe comprising at least one of the nucleotides at a polymorphic site in SEQ ID NO: 1 or the complement thereof; and (c) determining if the test sample comprises a polynucleotide that specifically hybridizes to the probe.
[0165] In another preferred embodiment, the invention provides a method of analyzing a patient's polynucleotides for the presence or absence of a mutation using PCR comprising: (a) providing a test sample comprising polynucleotides or replicas thereof from a biological sample obtained from the patient; (b) contacting the test sample with at least one primer comprising at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1 or its complement, and a polymerase, wherein the primer comprises at least one of the nucleotides at a polymorphic site in SEQ ID NO: 1 or the complement thereof; and (c) determining if a PCR product of the appropriate size is amplified.
[0166] Such analysis methods and related kits are useful for diagnostic, prognostic or pharmacogenomic purposes. Thus, the invention provides methods of (1) predicting risk of developing a disease condition (2) diagnosing a condition, and/or (3) predicting prognosis of a condition comprising: (a) analyzing a patient's polynucleotides to determine the identity of at least one of the nucleotides at a polymorphic site in SEQ ID NO: 1, wherein the presence or absence of the nucleotide correlates with a higher likelihood of developing said condition. The correlation may be based on statistically associating either the presence or absence of a single polymorphism (or multiple polymorphisms) with risk of developing a disease or condition, or with the diagnosis of a disease or condition, or with prognosis or staging of a disease or condition.
[0167] The invention further provides a method for selecting a treatment for a patient suffering from a disease or condition by determining whether or not a gene or genes in cells of the patient contain at least one polymorphism which is correlated to the effectiveness of the treatment of the disease or condition. The selection may be the selection of a method or methods which is/are more or less effective, safer, or toxic than certain other therapeutic regimens. The selection may involve either choice of a treatment to use or avoidance of a treatment. For example, a contra-indicated treatment should be avoided if it will not result in a therapeutic benefit, or if it will result in an excessive level of undesirable side effects. Thus, the frequency of the polymorphism itself may be correlated to the frequency of a beneficial therapeutic response to a drug or unresponsiveness to the drug, or it may be correlated to the frequency of an adverse event resulting from administration of the drug. Even where there the frequency of the polymorphism does not correspond closely with the frequency of a beneficial or adverse response, the polymorphism may still be useful for identifying a patient subset with high response or toxicity incidence. Preferably, the drug will be effective in more than 20%, 40% or 60% of individuals with one or more specific polymorphisms. Alternatively, the drug will be toxic or create clinically unacceptable side effects in more than 10%, 30%, 50%, 70% or 90% of individuals with one or more specific polymorphisms.
[0168] The invention thus provides a method of predicting a beneficial treatment for a patient comprising: (a) analyzing a patient's polynucleotides to determine the identity of at least one of the nucleotides at a polymorphic site in SEQ ID NO: 1 wherein the presence or absence of the nucleotide correlates with a prediction that the treatment will be beneficial. The method may further include selecting a suitable dosage amount and/or frequency of administration.
[0169] Similarly, the invention provides a method of predicting a contraindicated treatment for a patient comprising: (a) analyzing a patient's polynucleotides to determine the identity of at least one of the nucleotides at a polymorphic site in SEQ ID NO: 1, wherein the presence or absence of the nucleotide correlates with a prediction that the treatment will not be effective or will have significantly adverse effects. The correlation may be based on statistically associating either the presence or absence of a single polymorphism (or multiple polymorphisms) with a beneficial effect or contraindicated effect resulting from drug treatment.
[0170] One aspect of the invention specifically provides methods of treatment with sequence-specific antisense compounds comprising the step of detecting the presence of a polymorphism of the invention in the patient's sample prior to treatment with the desired sequence-specific compound, where detection of the polymorphism guides selection of the proper sequence-specific compound. For example, the presence of a polymorphism in a patient's genes may indicate that treatment with a compound that specifically hybridizes to the polymorphism may be beneficial. Similarly, the absence of a polymorphism in the patient's genes may mean that treatment with a compound that specifically hybridizes to the polymorphism is contraindicated.
[0171] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. Each of the references, GENBANK® accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.
EXAMPLES
Example 1
Synthesis of Nucleoside Phosphoramidites
[0172] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5'-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5'-O-Dimethoxytrityl-2'-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N4-benzoyl-5-methylcy- tidin-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)-N4-benzoyl-5-methyl-cytid- ine penultimate intermediate, [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N4-benzo- yl-5-methylcytidin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N6-benzo- yladenosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su- p.4-isobutyrylguanosin-3'-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidit- e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and 2'-O-(dimethylaminooxyethyl) nucleoside amidites, 2'-(Dimethylaminooxyethoxy) nucleoside amidites, 5'-O-tert-Butyldiphenylsilyl-O2-2'-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-met- hyluridine, 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'-[(2-cyanoethyl)-N,N-diisopropylphosphora- midite], 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside amidites, 2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethyl-aminoethoxy)-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.
Example 2
Oligonucleotide and Oligonucleoside Synthesis
[0173] The antisense compounds used in accordance with this invention may 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.
[0174] Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.
[0175] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH4OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270.
[0176] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863.
[0177] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.
[0178] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
[0179] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).
[0180] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925.
[0181] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243.
[0182] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0183] Oligonucleosides: 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 are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.
[0184] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564.
[0185] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618.
Example 3
RNA Synthesis
[0186] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5'-hydroxyl in combination with an acid-labile orthoester protecting group on the 2'-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2' hydroxyl.
[0187] Following this procedure for the sequential protection of the 5'-hydroxyl in combination with protection of the 2'-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.
[0188] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3'- to 5'-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3'-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5'-end of the first nucleoside. The support is washed and any unreacted 5'-hydroxyl groups are capped with acetic anhydride to yield 5'-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5'-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.
[0189] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S2Na2) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2'-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.
[0190] The 2'-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.
[0191] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).
[0192] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid, or for diagnostic or therapeutic purposes.
Example 4
Synthesis of Chimeric Compounds
[0193] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the "gap" segment of linked nucleosides is positioned between 5' and 3' "wing" segments of linked nucleosides and a second "open end" type wherein the "gap" segment is located at either the 3' or the 5' terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as "gapmers" or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as "hemimers" or "wingmers".
[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate Oligonucleotides
[0194] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3' wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH4OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.
[2'-O-(2-Methoxyethyl)]-[2'-deoxy]-[2'-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides
[0195] [2'-O-(2-methoxyethyl)]-[2'-deoxy]-[2'-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2'-O-methyl chimeric oligonucleotide, with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites.
[2'-O-(2-Methoxyethyl)Phosphodiester]-[2'-deoxy Phosphorothioate]-[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides
[0196] [2'-O-(2-methoxyethyl phosphodiester]-[2'-deoxy phosphorothioate]-[2'-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2'-O-methyl chimeric oligonucleotide with the substitution of 2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.
[0197] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065.
Example 5
Design and Screening of Duplexed Antisense Compounds Targeting Apolipoprotein B
[0198] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target apolipoprotein B. The nucleobase sequence of the antisense strand of the duplex comprises at least an 8-nucleobase portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.
[0199] In one embodiment, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 3), can be prepared with blunt ends (no single stranded overhang) as shown:
TABLE-US-00002 cgagaggcggacgggaccg Antisense (SEQ ID NO: 3) ||||||||||||||||||| Strand gctctccgcctgccctggc Complement (SEQ ID NO: 4)
[0200] In another embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 3) and having a two-nucleobase overhang of deoxythymidine (dT) would have the following structure:
TABLE-US-00003 cgagaggcggacgggaccgTT Antisense (SEQ ID NO: 5) ||||||||||||||||||| Strand TTgctctccgcctgccctggc Complement (SEQ ID NO: 6)
[0201] Overhangs can range from 2 to 6 nucleobases and these nucleobases may or may not be complementary to the target nucleic acid. In another embodiment, the duplexes can have an overhang on only one terminus.
[0202] The RNA duplex can be unimolecular or bimolecular; i.e, the two strands can be part of a single molecule or may be separate molecules.
[0203] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (-20° C.) and freeze-thawed up to 5 times.
[0204] Once prepared, the duplexed compounds are evaluated for their ability to modulate apolipoprotein B expression. When cells reach approximately 80% confluency, they are treated with duplexed compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM® 1 reduced-serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM® 1 containing 12 μg/mL LIPOFECTIN® (Invitrogen Life Technologies, Carlsbad, Calif.) and the desired duplex antisense compound (e.g. 200 nM) at a ratio of 6 μg/mL LIPOFECTIN® per 100 nM duplex antisense compound. After approximately 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested approximately 16 hours after treatment, at which time RNA is isolated and target reduction measured by real-time PCR.
Example 6
Oligonucleotide Isolation
[0205] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH4OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the -16 amu product (+/-32 +/-48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.
Example 7
Oligonucleotide Synthesis
96 Well Plate Format
[0206] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.
[0207] Oligonucleotides were cleaved from support and deprotected with concentrated NH4OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.
Example 8
Oligonucleotide Analysis
96-Well Plate Format
[0208] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE® MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE® 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
[0209] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.
HepG2 Cells:
[0210] The human hepatoblastoma cell line HepG2 is available from the American Type Culture Collection (Manassas, Va.). HepG2 cells are routinely cultured in Eagle's MEM supplemented with 10% fetal bovine serum, non-essential amino acids, and 1 mM sodium pyruvate (Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #3872, BD Biosciences, Bedford, Mass.) at a density of approximately 7000 cells/well for use in antisense oligonucleotide transfection experiments. For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
T-24 Cells:
[0211] The human transitional cell bladder carcinoma cell line T-24 is available from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells are routinely cultured in complete McCoy's 5A basal media supplemented with 10% fetal bovine serum, 100 units per mL penicillin, and 100 ug per mL streptomycin (media and supplements from Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 7000 cells/well for use in antisense oligonucleotide transfection experiments. For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
A549 Cells:
[0212] The human lung carcinoma cell line A549 is available from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells are routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal bovine serum, 100 units per mL penicillin, and 100 ug per mL streptomycin (media and supplements from Invitrogen Life Technologies, Carlsbad, Calif.). Cells are routinely passaged by trypsinization and dilution when they reach approximately 90% confluence. Cells are seeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a density of approximately 7000 cells/well for use in antisense oligonucleotide transfection experiments. For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.
NHDF Cells:
[0213] Human neonatal dermal fibroblast (NHDF) are available from the Clonetics Corporation (Walkersville, Md.). NHDFs are routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells are maintained for up to 10 passages as recommended by the supplier.
HEK Cells:
[0214] Human embryonic keratinocytes (HEK) are available from the Clonetics Corporation (Walkersville, Md.). HEKs are routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells are routinely maintained for up to 10 passages as recommended by the supplier.
Treatment with Antisense Compounds:
[0215] When cells reached 65-75% confluency, they are treated with oligonucleotide. Oligonucleotide is mixed with LIPOFECTIN® Invitrogen Life Technologies, Carlsbad, Calif.) in OPTI-MEM® 1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desired concentration of oligonucleotide and a LIPOFECTIN® concentration of 2.5 or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture is incubated at room temperature for approximately 0.5 hours. For cells grown in 96-well plates, wells are washed once with 100 μL OPTI-MEM® 1 and then treated with 130 μL of the transfection mixture. Cells grown in 24-well plates or other standard tissue culture plates are treated similarly, using appropriate volumes of medium and oligonucleotide. Cells are treated and data are obtained in duplicate or triplicate. After approximately 4-7 hours of treatment at 37° C., the medium containing the transfection mixture is replaced with fresh culture medium. Cells are harvested 16-24 hours after oligonucleotide treatment.
[0216] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 7) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 8) which is targeted to human Jun-N-terminal kinase-2 (JNK2). ISIS 13920 is a chimeric oligonucleotide having a 9 nucleotide gap segment composed of 2'-deoxynucleotides, which is flanked on the 5' side and 3' sides by 3 nucleotide and 8 nucleotide wing segments, respectively. ISIS 18078 is a chimeric oligonucleotide having a 5 nucleotide gap segment composed of 2'-deoxynucleotides, which is flanked on the 5' and 3' sides by 5 nucleotide and 6 nucleotide wing segments, respectively. The wings are composed of 2'-O-methoxyethyl nucleotides. Both compounds have phosphorothioate internucleoside (backbone) linkages, and cytidines in the wing segments are 5-methylcytidines. For mouse or rat cells the positive control oligonucleotide is ISIS 15770 (ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 9), a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in bold), which is which is targeted to both mouse and rat c-raf. ISIS 15770 is a chimeric oligonucleotide having a 10 nucleotide gap segment composed of 2'-deoxynucleotides, which is flanked on the 5' side and 3' sides by 5 nucleotide wing segments. The wings are composed of 2'-O-methoxyethyl nucleotides. Internucleoside (backbone) linkages are phosphorothioate and cytidines in the wing segments are 5-methylcytidines. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.
Example 10
Analysis of Oligonucleotide Inhibition of Apolipoprotein B Expression
[0217] Antisense modulation of apolipoprotein B expression can be assayed in a variety of ways known in the art. For example, apolipoprotein B mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR. Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. 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. 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® 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.
[0218] Protein levels of apolipoprotein B 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 apolipoprotein B 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. 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.
Example 11
Poly(A)+ mRNA Isolation
[0219] Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C. was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.
[0220] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.
Example 12
Total RNA Isolation
[0221] Total RNA was isolated using an RNEASY® 96 kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY® 96 well plate attached to a QIAvac manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY® 96 plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY® 96 plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 minutes. The plate was then removed from the QIAvac manifold and blotted dry on paper towels. The plate was then re-attached to the QIAvac manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.
[0222] The repetitive pipetting and elution steps may be automated using a QIAGEN® Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.
Example 13
Real-Time Quantitative PCR Analysis of Apolipoprotein B mRNA Levels
[0223] Quantitation of apolipoprotein B mRNA levels was determined by real-time quantitative PCR using the ABI PRISM® 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE®, FAM®, or VIC®, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5' end of the probe and a quencher dye (e.g., TAMRA®, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 3' end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3' quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM® 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.
[0224] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be "multiplexed" with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only ("single-plexing"), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.
[0225] After isolation the RNA is subjected to sequential reverse transcriptase (RT) reaction and real-time PCR, both of which are performed in the same well. RT and PCR reagents were obtained from Invitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR was carried out in the same by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).
[0226] Gene target quantities obtained by real time PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RIBOGREEN® (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN® RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J., et al, Analytical Biochemistry, 1998, 265, 368-374.
[0227] In this assay, 175 μL of RIBOGREEN® working reagent (RIBOGREEN® reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.
[0228] Probes and primers to human apolipoprotein B were designed to hybridize to a human apolipoprotein B sequence, using published sequence information (GENBANK® accession number NM--000384.1, incorporated herein as SEQ ID NO: 10). For human apolipoprotein B the PCR primers were:
[0229] forward primer: TGCTAAAGGCACATATGGCCT (SEQ ID NO: 11)
[0230] reverse primer: CTCAGGTTGGACTCTCCATTGAG (SEQ ID NO: 12) and the PCR probe was: FAM-CTTGTCAGAGGGATCCTAACACTGGCCG-TAMRA (SEQ ID NO: 13) where FAM® (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA® (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.
[0231] For human GAPDH the PCR primers were:
[0232] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 14)
[0233] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 15) and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3' (SEQ ID NO: 16) where JOE® (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA® (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.
[0234] Probes and primers to mouse apolipoprotein B were designed to hybridize to a mouse apolipoprotein B sequence, using published sequence information (GENBANK® accession number M35186, incorporated herein as SEQ ID NO: 17). For mouse apolipoprotein B the PCR primers were:
[0235] forward primer: CGTGGGCTCCAGCATTCTA (SEQ ID NO: 18)
[0236] reverse primer: AGTCATTTCTGCCTTTGCGTC (SEQ ID NO: 19) and the PCR probe was: 5' FAM-CCAATGGTCGGGCACTGCTCAA-TAMRA 3'(SEQ ID NO: 20) where FAM® (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA® (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For mouse GAPDH the PCR primers were:
[0237] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 21)
[0238] reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 22) and the PCR probe was: 5' JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3' (SEQ ID NO: 23) where JOE® (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA® (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.
Example 14
Northern Blot Analysis of Apolipoprotein B mRNA Levels
[0239] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL® (TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND®-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST "B" Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER® UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robed using QUICKHYB® hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.
[0240] To detect human apolipoprotein B, a human apolipoprotein B specific probe was prepared by PCR using the forward primer TGCTAAAGGCACATATGGCCT (SEQ ID NO: 11) and the reverse primer CTCAGGTTGGACTCTCCATTGAG (SEQ ID NO: 12). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
[0241] To detect mouse apolipoprotein B, a human apolipoprotein B specific probe was prepared by PCR using the forward primer CGTGGGCTCCAGCATTCTA (SEQ ID NO: 18) and the reverse primer AGTCATTTCTGCCTTTGCGTC (SEQ ID NO: 19). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).
[0242] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER® and IMAGEQUANT® Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.
Example 15
Western Blot Analysis of Apolipoprotein B Protein Levels
[0243] Western blot analysis (immunoblot analysis) was carried out using standard methods. Cells were harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels were run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to apolipoprotein B was used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands were visualized using a PHOSPHORIMAGER® (Molecular Dynamics, Sunnyvale Calif.) or the ECL PLUS® chemiluminescent detection system (Amersham Biosciences, Piscataway, N.J.).
Example 16
Antisense Inhibition of Apolipoprotein B Expression
[0244] U.S. Application Publication No. 20040214325 published Oct. 28, 2004 and International Patent Publication WO2004044181 published May 27, 2004, the disclosures and particularly the examples of which are hereby incorporated by reference in their entirety, describe the activity in vitro and in vivo of a variety of different antisense compounds, including dsRNA and chimeric phosphorothioate oligonucleotides, designed to target different regions of the human, mouse, rabbit and monkey apolipoprotein B RNA.
[0245] A number of different compounds demonstrated at least 10%, at least 30%, and/or at least 50% inhibition of apolipoprotein B expression. The target regions to which these preferred sequences are complementary are herein referred to as "preferred target segments" and are therefore preferred for targeting by compounds of the present invention. Where sequences are shown to contain thymine (T) one of skill in the art will appreciate that thymine (T) is generally replaced by uracil (U) in RNA sequences.
[0246] As these "preferred target segments" have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of apolipoprotein B.
[0247] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.
Example 17
Design of Phenotypic Assays for the Use of Apolipoprotein B Inhibitors
[0248] Once apolipoprotein B inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of apolipoprotein B in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).
[0249] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with apolipoprotein B inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.
[0250] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.
[0251] Measurement of the expression of one or more of the genes of the cell after treatment is also used as an indicator of the efficacy or potency of the apolipoprotein B inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.
Example 18
Activity in Animal Models
[0252] U.S. Application Publication No. 20040214325 published Oct. 28, 2004 and International Patent Publication WO2004044181 published May 27, 2004, the disclosures and particularly the examples of which are hereby incorporated by reference in their entirety, also describe the in vitro and in vivo biological effects of antisense inhibition of apolipoprotein B expression in mice and monkeys.
[0253] Treatment with ISIS 147764, a mouse-specific oligonucleotide, lowered cholesterol as well as LDL and HDL lipoproteins and serum glucose in both lean and high fat mice. The effects demonstrated are, in fact, due to the inhibition of apolipoprotein B expression as supported by the decrease in mRNA levels. No significant changes in liver enzyme levels were observed, indicating that the antisense oligonucleotide was not toxic to either treatment group.
[0254] Treatment of high fat fed mice with ISIS 147764 decreased apolipoprotein B protein expression in liver in a dose-dependent manner, reduced serum cholesterol and triglycerides, lowered levels of serum HDL, LDL and VLDL lipoproteins, reduced serum glucose levels, and decreased fat pad weight.
[0255] Treatment of apo E knockout mice with ISIS 147764 lowered glucose and cholesterol as well as serum HDL, LDL and VLDL lipoproteins. Further, these decreases correlated with a decrease in both protein and RNA levels of apolipoprotein B, demonstrating an antisense mechanism of action. No significant changes in liver enzyme levels were observed, indicating that the antisense oligonucleotide was not toxic to either treatment group.
[0256] LDL receptor-deficient mice (LDLr(-/-)mice), a strain that cannot edit the apolipoprotein B mRNA and therefore synthesize exclusively apolipoprotein B-100, have markedly elevated LDL cholesterol and apolipoprotein B-100 levels and develop extensive atherosclerosis. ISIS 147764 was able to lower cholesterol, triglycerides, and mRNA levels in a dose-dependent manner in both male and female LDLr(-/-) mice while the 4-base mismatch ISIS 270906 was not able to do this.
[0257] C57BL/6NTac-TgN(APOB100) transgenic mice have the human apolipoprotein B gene "knocked-in". These mice express high levels of human apolipoprotein B100 resulting in mice with elevated serum levels of LDL cholesterol. Treatment with either of these oligonucleotides targeted to the human apolipoprotein B which is expressed in these mice markedly decreased the mRNA levels of the human apolipoprotein, while the levels of the endogenous mouse apolipoprotein B were unaffected, indicating that these oligonucleotides exhibit specificity for the human apolipoprotein B. Immunoblot analysis of liver protein samples reveals a reduction in the expression of both forms of human apolipoprotein B, apolipoprotein B-100 and apolipoprotein B-48. Mouse apolipoprotein B levels in liver were not significantly changed. LDL-cholesterol levels were significantly reduced.
[0258] ob/ob mice have a mutation in the leptin gene which results in obesity and hyperglycemia. Treatment of ob/ob mice receiving a high fat and cholesterol diet with ISIS 147483 and 147764 were both able to lower apolipoprotein B mRNA levels, as well as glucose, cholesterol, and triglyceride levels.
[0259] Toxicity studies in mice revealed no severe toxic effects. In vitro assays showed that ISIS 301012 does not possess immunostimulatory activity.
[0260] In Cynomolgus monkeys, antisense inhibition by ISIS 301012 was compared to that of ISIS 326358, which is a perfect match to the Cynomolgus monkey apolipoprotein B sequence to which ISIS 301012 hybridizes. These data demonstrate that both ISIS 326359 and ISIS 301012 (despite two mismatches with the Cynomolgus monkey apolipoprotein B sequence) can inhibit the expression of apolipoprotein B mRNA in Cynomolgus monkey primary hepatocytes, in a dose- and time-dependent manner.
TABLE-US-00004 TABLE 1 Effects of antisense inhibition by ISIS 301012 in lean Cynomolgus monkeys Intravenous delivery Subcutaneous injection 2 mg/kg 4 mg/kg 12 mg/kg 3.5 mg/kg 20 mg/kg apolipoprotein B expression -45 -76 -96 N.D. -94 % change normalized to saline antisense oligonucleotide 92 179 550 N.D. 855 concentration μg/g Lipid parameters, % change normalized to untreated baseline value Saline 2 mg/kg 4 mg/kg 12 mg/kg 3.5 mg/kg 20 mg/kg Total cholesterol +1 -6 -2 -2 +5 -5 LDL-cholesterol +17 +15 +9 +3 -4 -16 HDL-cholesterol -11 -23 -15 -8 +13 +5 LDL/HDL +62 +94 +38 +44 -15 -19 Total cholesterol/HDL +30 +44 +22 +21 -7 -10 Triglyceride +37 +26 +32 +15 +1 -3 LDL Particle concentration +15 +8 +8 -11 -14 -21
[0261] These data show that ISIS 301012 inhibits apolipoprotein B expression in a dose-dependent manner in a primate species and concomitantly lowers lipid levels at higher doses of ISIS 301012. Furthermore, these results demonstrate that antisense oligonucleotide accumulates in the liver in a dose-dependent manner.
Example 19
Identification of SNPs
[0262] Polymorphisms were discovered by comparing the apolipoprotein B genomic sequences of 213 DNA samples. An initial analysis of 23 DNA samples and a followup analysis of an additional 190 DNA samples was conducted to identify SNPs in the target region of ISIS 301012 (exon 20, boundary of intron 20). The 190 DNA samples came from individuals self-identified as belonging to one of four major population groups: Caucasian (47 individuals), African descent (48 individuals), Asian (47 individuals), or Hispanic (48 individuals). All samples were analyzed in replicates using SEQUENOM's MASSARRAY® approach including MASSCLEAVE® biochemistry.
[0263] Seven previously unknown SNPs with varying frequencies were discovered in an approximately 541 bp portion of the ISIS 301012 target region, none in the antisense or exon regions. The SNPs and their positions and frequencies are set forth in Table 2 below.
TABLE-US-00005 TABLE 2 Identification of SNPs Approximate frequency of Position in SNP detection out of 213 Sequence variation SEQ ID NO: 1 samples of diverse ancestry Substitution of A to G 27751 15% Substitution of C to G 27735 <1% Substitution of T to C 27685 27% Substitution of T to C 27683 Substitution of T to C 27679 40% Substitution of C to T 27634 <1% Substitution of G to A 27627 2% Substitution of T to C 27618 <1%
[0264] The frequency of the T/C substitution at position 27679 and the T/C substitution at 27685 was significantly lower in Asian DNA samples compared to other ethnic groups. See table below.
TABLE-US-00006 Position in African SEQ ID NO: 1 Asian American Hispanic Caucasian 27685 9.8% 32.4% 42.2% 32.6% 27679 19.7% 45.6% 43.8% 48.6%
[0265] The C/T substitution at position 27634 was only present in Asian samples. It was detected in one heterozygote Asian sample and one homozygote Asian sample.
[0266] The distribution of the A/G substitution at position 27751 does not match the Hardy-Weinberg disequilibrium; all samples carrying this sequence variation are heterozygote.
[0267] The G/A substitution at position 27627 was found only in four samples of African American origin.
[0268] The T/C substitution at position 27618 was found in one African American sample.
Sequence CWU
1
1
23143445DNAArtificial Sequenceallelic genomic sequence of apolipoprotein B
1accaagacag cgctcaggac tggttctcct cgtggctccc aattcagtcc aggagaagca
60gagattttgt ccccatggtg ggtcatctga agaaggcacc cctggtcagg gcaggcttct
120cagaccctga ggcgctggcc atggccccac tgagacacag gaagggccgc gccagagcac
180tgaagacgct tggggaaggg aacccacctg ggacccagcc cctggtggct gcggctgcat
240cccaggtggg ccccctcccc gaggctcttc aaggctcaaa gagaagccag tgtagaaaag
300caaacaggtc aggcccggga ggcgcccttt ggaccttttg caatcctggc gctcttgcag
360cctgggcttc ctataaatgg ggtgcgggcg ccggccgcgc attcccaccg ggacctgcgg
420ggctgagtgc ccttctcggt tgctgccgct gaggagcccg cccagccagc cagggccgcg
480aggccgaggc caggccgcag cccaggagcc gccccaccgc agctggcgat ggacccgccg
540aggcccgcgc tgctggcgct gctggcgctg cctgcgctgc tgctgctgct gctggcgggc
600gccagggccg gtgagtgcgc ggccgctctg cgggcgcaga gggagcggga gggagccggc
660ggcacgaggt tggccggggc agcctgggcc taggccagag ggagggcagc cacagggtcc
720agggcgagtg gggggattgg accagctggc ggcccctgca ggctcaggat ggggggcgcg
780ggatggaggg gctgaggagg gggtctccgg agcctgcctc cctcctgaaa ggtgaaacct
840gtgccggtgg tccccctgtc gggccctagc acccgctggg aagacgtggg aagctcacag
900atttctttct cctgtcttac agaagaggaa atgctggaaa atgtcagcct ggtctgtcca
960agtaaggcat ctgcgcatgg ggcgtggaag ggcgcccagc cccgtgcact ctcctacacc
1020cgggtccctg agggcctccc actctacagg gctgagatgg catcgtggtg tgccttgctc
1080tgaccccagg aagcaagttc cctgagcctc tgcccacacc caagggatgc caactctctt
1140ctacctggcc ttctgttctg tcccaaaagt tcagcctggg ggcgggggag ggaagggatt
1200gtctctccgc tggcctgtgc acactttgaa gaaacatcac tgtcctgttt atcagtgact
1260agtcattgat tcgaagcatg tgagggtgag gaaatactga ctttaacctt tgtgaagaaa
1320tcgaacctcc acccccttcc tatttacctg acccctgggg gttaaaggaa ctggcctcca
1380agcgcgaccc tgtgtgctgg agccgcgggg cggacttctg atggggcagc accgccatct
1440agtggccgtc tgtcatcact gcagctggac tcaggaccca gatgttcttt ttcttcaatt
1500gttcagaaaa ttcctctcaa ctacagtgga aacctccaga aattcttttc taggagtttg
1560ttaagttagt tacgcttaat gcttaatgaa ctttgcctta agtatttggt agtcttagag
1620tcacggaatt acggcgtgtt caagctaaaa aagcattaga gatagtacta tttgcgtaat
1680gttgtcatct cttaatttgc cagagggtct ctcatgcaga ttttctgagc cccattactt
1740gacacttgtc actcccttcc ctgtgcctca gatgagatat tcaagacatg ccagccaatt
1800taaacattag cctcagcaaa aacataatgg agaagtcaaa tctataaagg aaaattaagt
1860ataaagtcaa ttaaaaaata atttgagttg aattaccatt tttaattctc tatgccactg
1920cccctctctg cccagaattg gctgtccttg ggagagctat ttctgctatg tggctgacgt
1980atttctcccc acgttagaag atgcgacccg attcaagcac ctccggaagt acacatacaa
2040ctatgaggct gagagttcca gtggagtccc tgggactgct gattcaagaa gtgccaccag
2100gatcaactgc aaggtatgga ggatgcaggc aggagggacc tagagcccac agctttcccc
2160cagccctgtt ccagcgggcg cccaacacgc gaccttcccg gagggtgtgt actgagcaaa
2220cgcagaacat cccagaactg ttgtaatctg atcaaagcac tgggactttg cctctgtttg
2280taagtcagcc acattgctga gatgtggtct gcccccacca aatttcgcaa gtcagaagta
2340ttttcccgtt aacttcccag atgcaatagg aatccatgat ctagattagc agcagtgtgg
2400gtctgtagat ttcagcgtga gagaggccca gtaggtgagc tatgggaggc aggcaactcg
2460gaatcgcact gtgaaatgca gtttttataa tttaagtcaa acagaatctg ttgctgaaaa
2520atgaatggaa agaagaaaaa aatataaaca tacagtttgt tctaaaataa aactttgctt
2580attattgaga ctggttgtac tcatgttaca tacatgtgga gcagatctac aggctgctat
2640tggggtttgg gtggggaaga gaagtcaagc tgagcagtca ccttttttta gagagtaccg
2700tagctcttgt atgtgctgtc caatatggta gacatgagcc acattgggct atttaaatgg
2760aatgaaatta aaaattcata ttcgttgtca cattagctgc atttcaactg ctcaacagcc
2820accctggcta ctggctccca tattgaacag cacacatgta caacatttct ataaagttat
2880ttgaatagtg ctggataata agtaggaatc cgttgaaact ccagctatat gcaaagctct
2940aaataggccc taatagatat aaccagtttt ttgggtgaca ttaaggagac atttgctgtg
3000gaaacgaagg atggccctct tcctgctttc tgtttttctt cttcactttc actcctagtc
3060tgcagcgctt ctatttaacc acagctcttt ataattaaag tgagtaactt tagaaccaat
3120aaaaggacat cctccttccc atgcctaggg gcaaacttaa gaaatgtgtt acccgggagg
3180gggaaaacgt cagcaatagg actaagtcta ggttggtgca cagagaaccc aggaggcatg
3240ttgataaggc atgtggtgtt gaggcgcagg cagtggtgtt cccagcacca ttccctttgg
3300tgctctgatt agagattaag ccctgggctt caggggccac ctctcattct tgatagacaa
3360cctcaatgct ctgctaccct gaattctcag gttgagctgg aggttcccca gctctgcagc
3420ttcatcctga agaccagcca gtgcaccctg aaagaggtgt atggcttcaa ccctgagggc
3480aaagccttgc tgaagaaaac caagaactct gaggagtttg ctgcagccat gtccaggtaa
3540gtcatgttgt acatgagcac acgcatgtgt gtgtgtccgc tgaggtatga acttgtgtgt
3600ttgcaccagg cacggatgtg actgtaagta tttgtattcc gtatccatcg tggatcaggg
3660aattactgag ttttcacaat catcaaaaag agagaagcat tagttaacct tccctagtta
3720ggttccttta attatcattt tcatgtgttt ctaaaaatct catgctttaa acttcttgag
3780attataaaac tgagatgctt tgtttaaaca agtgaattct tatttaaaga actagtcaag
3840actagtgctt ggtggtcttt ggtgtggggt cccagaggca ctggctgctg tggccggcac
3900atggcggggc agggtctgtt caccgcaggg cagaggagca ccaaggcttc ggtggctccc
3960cctcctaggc tggcattcag ccactgcacg ctgatcggcc actgcagctg catctctgct
4020gactggtcag ggcccatgtc gcacccattg taaatatttt caacatcacc cctgcctcat
4080cctcaatcac agtttgtagg gtcctaggtg tgtatgaata caggcaggat agagttgtta
4140acttggtagc atcagaaaac tctgtctgta ttagtctgtt ttcatgctgc tgataaagac
4200atacctgaga ctgggcaatt tacaaaagaa aggtttattg gactcacagt tccacgtggc
4260tggggaggtc tcacaatcat ggcggaaggt gagggacagc aagtcacatc ttatgtagat
4320ggtggctggc aaagagagct tgtgcagaga aactcctgtt tttagaacca tcagatctcc
4380cgacacccat ctgcaatcac gagaacagca cgggaaagac ctgcccccat gattcaatca
4440cctccccccg ggtccctccc acaacacgtg ggaattatga gagctacgag acgaaatttg
4500ggtggggacg cagagccaaa ccatatcacc atccttgccc atttttcagt tttgctaaac
4560attagattca gatgccagtc ctttcttgcc aaaataggct gtgaggcttc tttctttcct
4620atgctttatt ttctccaaga cttaactgta tatgagggag aggggtatgg tggcaggagg
4680aaagagtggt ttattttttg gtccttggtc ttctccaaat acagaagaga ctcctgttct
4740tgaaaaggag ggctttccat gtttgcatct tcatgacttt aactgtcttt tttaaaaatt
4800gacatacaat aattatacat atttattgag aacatagtga tattttgata catgtaatgt
4860atggtgatca gatcagagta attagcatac ccatcatctc aaacatttat catttcttcg
4920tgttgggaac tttctgagag agtgtaggct gtgggagata agtccgtcac cttttcctcc
4980tgatgtaacc agagtggctg cagccaggtc ctcagaaact cagagagtac ccagtgggaa
5040atccctaaga ccaaagtcag catgggcttc agccatggcc tgacaccata caaaagaatg
5100actgtccaac aagtgtatga aaataagctc caattcactg gtagtcaaga aatgcgaatt
5160aatgtaacaa caagatattt atctgctttt acccatcata ctgcaaaact ggaaaacagt
5220gatagcacct gttgctggca ggccagtgag gaaaagtgtg ctgtcctgag ctgctggtgg
5280aaacgagagc catcaggcaa tatctactgt aatttaaaat acttaatacc ctttgacaca
5340gatattttag tctttgggac tctagcccat gaaaataaaa gcagtaatgt gtgaagatag
5400gcacataagg atgtttgttt tggtattgtt tgtgtggttt aaaaaaaatc cagaaagaga
5460gagggcaaat gccatcaaat ggggcaatgt gtgaataaat tatatttagc catggaatgg
5520aatgttctgc atgcagcttt taaaaaaatc tgttagagct gtaccaagtg actcagaagg
5580atttttgtga agtataatta agtgagaaaa acaagataaa agtatgcata atacaatgcc
5640acttgtataa aacaaacaat ggcaaaatct ttgtatgact ctgtttgcac tcacccatgt
5700ttacagagga ttgtatgagt gtgcagaaac aaatggaaca accactcggg tgtccgtatg
5760gggaggatgg gcaaagagac tgatatgggt ggagaacaga gcagggctgg atgagccaag
5820caaaaaaagt taaaacacag ctggacctgg tggctcatgc ctgtagtccc agcactttgg
5880gaggccgagg agggagaatc acctgaggtc aggagtttga gaccagcctg gccaacatgg
5940tgaaaactgt ctctactaaa aatacaaaaa ttagctgggt gtgatggcac atgccagtag
6000tcctagctac tccggaggct gaggcaggag aatcacttga tcccaggagg tggaggttgc
6060agtgagctga ggttgcgcca ttgcactcca gcccgggcga ccgagcgaga ctccatttca
6120aaaaaagaaa aagaaaaaag aaaaaaagaa aaaaaaagaa tcaccaaaac ttatgtatat
6180gtgcatactt ttttgaaaat gtatgtctat gtgtagctat attctatatt tacaaataaa
6240tgatgtcaga agaacaattg gttaaaaaaa tatgagaaaa gaaacttcag tgccacccag
6300cttacttcca gcaagttgta atggagaagg acatttccgt gaccatcctc tctctgggac
6360aggtatgagc tcaagctggc cattccagaa gggaagcagg ttttccttta cccggagaaa
6420gatgaaccta cttacatcct gaacatcaag aggggcatca tttctgccct cctggttccc
6480ccagagacag aagaagccaa gcaagtgttg tttctggtga ggatttagaa agctgatagc
6540agtggccctt gaaactcatc ttcatgtgtt agagaccagt cctaccatat acaaagcaga
6600tcactgagtc agctccatga ctagttacat aggaagccct ggattggcgt gaaatactgg
6660tgcccgaggt tcctcctgcc ccttaggctc actgacagat catcccaagc aggcttatca
6720ggttgggtct aattttaaaa cagtcattga ggagtcctgg ccaccccacc cctgcttttg
6780tttgatgctt cacctgtgtt tgctgggtta tggtgtacac agtaaatcct gtgtgtattt
6840taaacaccaa aaataatggg atctgttgct ggtctctttt acgaatttca ggtttcactg
6900tgagacagaa ttcatttcac ctcagtccca tgagcacttt tgtgtgttct aatttctcta
6960cgacaccata atgggagaag acaccgatgc aacctgcgga ggcctttctg cagacccacc
7020tttaactggt tttctctctc ccaacttggg ctggccaggc actagcaaga ccacactctg
7080cataggaaga aaaagaaagt ccctcccaaa gctagattcc ttctgctttt tctttcacga
7140tccccacccc atccctccca agtacccaag gatgttgccc gtgttgaata catgtggttg
7200catcttcttc ctccatagga taccgtgtat ggaaactgct ccactcactt taccgtcaag
7260acgaggaagg gcaatgtggc aacagaaata tccactgaaa gagacctggg gcagtgtgat
7320cgcttcaagc ccatccgcac aggcatcagc ccacttgctc tcatcaaagg catggtaagt
7380cccatgtcag cactgtcgtg cacagcaagg agcatcctct tattaataca attccagaac
7440ttttgagcta gtgggcacct ttgaggacag cctgccctgg ctgtttttta tacagactag
7500agataggacc ctgagcaggc acgggaaggt ctgcccaggc ttcacggcct gggatcagtt
7560gagccaaggc ttgagtcagg ctcctccctc ccagcccaga gctctgtctt tcctcctgtc
7620cttctgtcac tggcaccaaa ctgcctctaa tctcatcact tgagagtaat gactactcac
7680ctctgagaag gttccgggga tggatgtagg gcagcaaaac caccttctgt tcttttctgc
7740acaaggactc cttgtgccag ctccaagcct ctggcctttg aagaagtccc aagacctgtg
7800ttctccccct ctccctcatc ccatgaagtg gagtgactta gagtgctcca gcttcttgtc
7860cttccacccc cagtaccacc ctgaccaaac atggccccac tgccaccggc ctggagcacc
7920ctctcctctc tgttaactgg ggccatggag caccatatta cctgagcctg cctgacccct
7980gcaacatctt ccctgatatg agccccagcc tgtctcagtg aacatgaata acttgggcaa
8040tcactgtcat gctgggcgct gttcctggtc attgtcctta gggttgaaaa cagggagtct
8100gatgaccatg agtgccacag tcagaagagg ataatgcact ggcttagggg tcttttctga
8160gcatctgctg tttgctcaac cccactctgg gcagcaccaa ggaagggaca gtggcagatg
8220aaccatggac cttcccctca ggatgcttcc agtctaatgc aggagccagg tcaataaagt
8280atacgtggta tactcaataa ggtgataagc tgaacagtgc agacaagaag tcctgggcct
8340gaccaggaag gagaaagaat tattcatgta gctcagcggg caacatttca tggaagatgt
8400ggagcaggaa cccaaaaaat gcaaagaata tgtaaatgaa agagacatgt aagaatgggc
8460ttttgggcaa agaaaagtta ctgagcaggt gtgtgagggg ctatgtggtg ggatgggcat
8520gtggaggata caaagtttag acattgtcca gtgagggtgg aaaaagagga gtctacagct
8580tgactcagct ttggggatgc cgacttgttg caccccctgg tctaaatgtc aagtacccag
8640ttatcttctt tctctgagtt tatctagtgg tacaggactc ctgctccctt ctaccttgaa
8700ggtaaatgct tttaacagaa gatacaggga ctgatcaaaa tgctcgtctc caatctcttt
8760catagacccg ccccttgtca actctgatca gcagcagcca gtcctgtcag tacacactgg
8820acgctaagag gaagcatgtg gcagaagcca tctgcaagga gcaacacctc ttcctgcctt
8880tctcctacaa gtaggtcatg tgatgcaccc ctgatttgtc atttaatggg tcagtgtgaa
8940ctgaacactt ctcaagtgct ctgttccagg caaacctgtg cctgggaggg aggaatggag
9000agggataaaa tgccgcccct ccctgtcccc ctttttaagc gaacaggcca tttggcagaa
9060aagtcctagg catgcaaaac aatccaagac caacaaaaga tatctaagac ccattcttta
9120agggctgtag atccagaaaa cctgaggatc actgcagggt accctggtta gaaaaggttt
9180catggaagat ttgggatact gactggaaac ttgtgtatcc aaatccactt tgaaaactga
9240taatcaatga atatatattg agtaactgcc atattcttgg ctctatgttg tggaagatac
9300gaaagaattt tgagacattg cactagttcc tacctctggc cactccagac tagtggagag
9360tataaggcac gcatgtcttt ttgatgggag gataactagc gtgaccagga agaggtggat
9420gttattcatt cagggccaac aatggctgga tttacccatg ctttgaaaga tgggcaggac
9480ttgggtagat gcagagacag ggaaaacctt caacatggaa agaatagtat gttctggcca
9540tccgtgacat ggtgtgcttc cttggttacc aggaataagt atgggatggt agcacaagtg
9600acacagactt tgaaacttga agacacacca aagatcaaca gccgcttctt tggtgaaggt
9660aagagtttct gtccacatag ttgctggaaa atctactcaa gatgtgccta tcatggctta
9720gccacttgct gagccctgtt aaatgtctgc tgactaacaa gtgatacaga cactggtgtt
9780ctggctacct ctagtgagaa agcaaactca tttcatgatg tcaagttgca atggcataaa
9840ggaaaagaag ttcccaaagc tacttaggca tttgtaaata gaaaactgga atcctaagtt
9900taacatgaca tatttgatag aactgacatc acccatcctg tgataagatc cagagctgtc
9960ccagacgagg tggaccaagt gggagagaac cttcagagtc tggccagata gtaacctcag
10020gagtcagtct ttagaggtag aaggaactct aacaatctca agtccaaccc ttacccagta
10080ttgtattgta tttatatctg tccaaattcc ttcttgtaca ttacctcatt gtcctttttg
10140ctcatagcaa cctgtgatgt caggtggtag agatgtgatt ttatacctat tctacagagg
10200agacagtgac acagagaggc ttagagtttg atgtagtcaa ggccgcagaa tattagaggg
10260gggaaaataa gtgccaggtt gtaatctaag ccaggactat tctcattaca ccacatttcc
10320atgatgactt ttacctctct tcctggcata ggtcacagta ggtggtggag aggatacaaa
10380agtgtctccc ctccccacaa gctgctggta gacccaatta gaagaaatgg tgataagcac
10440ccatgtgcct ggtcccagtt gtaaccatgt caacagtagc acctcctcac caattatttc
10500aagctaaggg taacctgatg atagactcag acaagtctgg attccacttt agctctacct
10560cttagaccct gagagctctt gggaaaccta agttgctcat ctctgggtca cacttcctca
10620tctctgggtc tcatctcttt gtctcatctc tgggactcag agctgagatc cagggatgag
10680caatttacat ggcccaaaaa ctctgtgggt ctcagaagca gggctgaatt tatcattaaa
10740ttgaacaata atgccacccc acagggatag gatgatgagt cagtgaaaac aagtcaatca
10800cctatggcag agccagatct agcaggcatt gaatacagga tagtttcttt cccttttccc
10860ctgtgctgat actccacaat ttccagcttc cagtagacaa agatatggtt gagatgaaga
10920aagctagagt tcctttgaca ctttccatct tccaggtact aagaagatgg gcctcgcatt
10980tgagagcacc aaatccacat cacctccaaa gcaggccgaa gctgttttga agactctcca
11040ggaactgaaa aaactaacca tctctgagca aaatatccag agagctaatc tcttcaataa
11100gctggttact gagctgagag gcctcagtga tgaagcagtc acatctctct tgccacagct
11160gattgaggtg tccaggtatc taatggttac agctcaactt tttataaaac tgatggtaac
11220tgactgaact ttcaaacctt ggccaaatgg agaatctcag ggaccatttg gatatcaatc
11280cagttaatca attagtcaat cagttcatga ttgctggata gagaactatc agctgctgcg
11340ctgagttcca tgaaacacac acgcgcatac tgtgttcaag gcagctatgt atttgtgtgt
11400taaaacagaa ggagaatagt tcccacattt tgatgggtaa cttttaattc ctaggtctat
11460tgcaggtgct ctccagaagc ttataggctg gtggagagag aactcagacg aaaaatataa
11520tatgatttct ctacccttca aggcactggc tttaagtgct atgaaggtga gagaagggac
11580tgaggccagg aatgagaccc agctaatgtt ggccaggcat attctgtgtg ctggccaaag
11640gactgtgata acagtcttct tgttgctaca gatccacagt cccctcttgg aacttttctc
11700gattgggctt cttctgtggg taatattcct aaggaaagca tcatggttct gagctccaag
11760ttgggttttg aagttagatt tgaatagtga atgaggtgat taagggctct cctggcagag
11820gacacaccat gagcaatatt ttatgtgccc tgaaggtggt ctgtataact ttatccatgt
11880ctttcttctc agccccatca ctttacaagc cttggttcag tgtggacagc ctcagtgctc
11940cactcacatc ctccagtggc tgaaacgtgt gcatgccaac ccccttctga tagatgtggt
12000cacctacctg gtggccctga tccccgagcc ctcagcacag cagctgcgag agatcttcaa
12060catggcgagg gatcagcgca gccgagccac cttgtatgcg ctgagccacg cggtcaacaa
12120gtgagtttcc acactgtatt tctcctccta ggagcagagg aacatcttgc acctctgtgc
12180atctctgtat taaaactgaa cccctccttc cactttcaaa ctctgctcct tactcttgtg
12240ttttttcttg atcatttttg gggtaatgac ttgaaataag aaatcagcaa acacaaattg
12300aatttttaaa aatattttct ctacattata ttataaaagt ttttgaacat agcaaagttg
12360acagaatttc acagggaaaa cccctagaaa accagctatc tcctactatt taagtgttat
12420tatatttgct ttatcacata tacatccatc cattaattca tcttattttc tgaagcattt
12480caaagtaaat tgcaaacatc aacacacttt cccctaagta ttacagcttg catattatta
12540acttcagttc aatattagtt agcagttttt tcctctgaat ttttttgttt gtttgttttg
12600tttttttttg ttgttgttgt ttttttgaga tggtctcact gtgtcaccca ggctggagtg
12660cagtgatgca gtcacggctc actgaagcct caaattcctg ggctgaagtg atcctcccac
12720ctcagcctcc tgagtagctg ggaccacagg tgcatgctac catgccctgg ctaatttttg
12780tattcttggt agatacaggg tttcaccatg ttgctcaggc tagcaggttt ttcctttgat
12840gaaatttttt ggctttttct tttttacatt tttatataaa tttatgtgga acaagtgtaa
12900ttttgttaca tgaatagatt gtgcagtagt taagtcaggg ctttcagggt atccatcacc
12960cagacaacat atagtgtacc cactaagtaa tttctcacca tccatctccc tccacttcca
13020caccttctga gtctcaattg tctatcattc cacacactat gtccttgtgt gcacattatt
13080tcactcccac ttataaatga caacacgcaa tatttgtctt tctgtgactg tcctgtttca
13140cttaagacaa tgacctccag ttccatccat gttgctgcaa atgacatgat tttattcttt
13200ttatggccga atagtatttt attgcctata catttcacat ttttaatcca atcgtccatt
13260gatagacact taggttgatt ccatgtcttt gctattgtga atagtgctgt gataaacata
13320tgggtgcagg tttcctttgg atataatgat ttcttttcct ttaggtatat acccagtaat
13380gggattgttg gatttattgg tagttctatt tttagttctt tgagaaatct ctgtattgtt
13440ttccatagtg gttgtactta tttacaatcc catcaacagt gattaactgt ttccttttct
13500ctgtatcctc accaacaact gttatttttt gtcttttgaa taatggccct cctgactctt
13560gtaagatgtt atctcattgt ggttttaatt tacatttctc taatgattag taatgttatg
13620cattttttca tatgcctatt gccatttgta tgtcttcttt tgaaaaaaat gtctattcat
13680gtcctttgcc tactttttaa tgggattatt tgggggattt ttttgttgag ttgtttgaat
13740tgcttgtaca ttccggatat tagtacccca ttggatgaat agtttgcaaa tattttctcc
13800cattctgcag gttaccaccc tgttgattat ttgttttact gtgcagaaac tttttacttt
13860aattaagttc tatttgtcta ttttttgttt ttgttgtctt tgcctttgag gtcttattca
13920cgaattcttt gtctaggcca atgtccagag aagttttccc taggttttct tcttgcattt
13980ttatagtctc aggtcttata tttaagtctt tgatccatct tgagttgatt tttttatatg
14040gtgacagata ggagtccagt tttattcttc tgcatatggc aatccatctt tcccagcacc
14100acttattgaa aagggtgtcc tttccctagt gtatgttttt gtcaattttg tcaaagatcc
14160gttgactgta agtatgtgac tttatttctg ggttcagtat tctgttccat tgatctatgt
14220gtctattttt atgccagtac catgctgttt agattactat agccttgttg tataatctga
14280agtcaggtaa tgtgatgcct ccagctatgt tctttttgct taaaattgct tcagctattc
14340aggctctttt tggattccat atgaatttta taattatttt ttctaattca caagtttggg
14400ttttaagaca aacctaactg gggttaccaa gtcctgactc tcttctctta ttctgtagct
14460atcataagac aaaccctaca gggacccagg agctgctgga cattgctaat tacctgatgg
14520aacagattca agatgactgc actggggatg aagattacac ctatttgatt ctgcgggtaa
14580tctcagtctt ttatatgaca tacatcattt cagaagcact tttcctggac accttttact
14640tccctctcct gcaccctgat gggttcttgt ttcttttctt caatgcaggt cattggaaat
14700atgggccaaa ccatggagca gttaactcca gaactcaagt cttcaatcct gaaatgtgtc
14760caaagtacaa agccatcact gatgatccag aaagctgcca tccaggctct gcggaaaatg
14820gagcctaaag acaaggtaaa gtccacaaga agaggtctga aagtgaaagt ttattaacaa
14880ggatttggaa ggtactaggg gaatgagact ctagatttca tctactgact ttattctgct
14940gtttctttcc tttccttcct tccttccttc cttccttcct ccctccctcc ctttcttctt
15000tccttccttc cttccttctt tcgagatgga atctcactct attgcccagg ctggagtgca
15060gtggcatgat ctcggctcac tgcaacttct gcctcctggg ttcaagcaat tctctctgcc
15120tcagcctcct gagtaactgg gattacaggc atgtgccatt acacccagct aatttttgta
15180ttttttagta gagatggagt tttgccatgt tggccaggct ggtcttgagc tcctgacctc
15240aggtgatccg cctgcctcag ccttgcaaag tgctgggatt acaggcgtga gccactgcac
15300ctggcctcta ctgttttcta attgcaaatt tcaacaagcc tattgacttg actgcctagc
15360agtatgtgac gtgagagaaa tacttgactt tgctgctatg tcaacatgca gaacgtgaga
15420tgtttttgct tcctaccgtc cacctaccag attgaccatc cctctcatca tggaaaaaca
15480tgcttaattt tcccccaata agcttaggct aggatagcca acttggcccc ctcttaggtg
15540caaagactcc agaactttgg aaactaccct atttattagc cccaaactct tactacccct
15600tctcatcttt atcctcacat taaaataact tacgttaaaa caacttgatt ttcacttagt
15660ggtggatctc caaacaaatc acaacttggc cataatttat gtgttttaat ggaattgaat
15720tcaacaggca ttccacaggc tttttctggg aacccttact tgatagtgct ctaggaaaca
15780ctggcaagaa gattcaatac cagcatttga agaacgatta cagagaaatt agacctgtgc
15840ttaagaaaga gctagcagac aatgccagtg tttgccaggc atgttctgtg ttctgaccac
15900aggacagtga taaccatctc ctcttttgac tgcaggacca ggaggttctt cttcagactt
15960tccttgatga tgcttctccg ggagataagc gactggctgc ctatcttatg ttgatgagga
16020gtccttcaca ggcagatatt aacaaaattg tccaaattct accatgggaa cagaatgagc
16080aagtgaagaa ctttgtggct tcccatattg ccaatatctt gaactcagaa gaattggata
16140tccaagagta agtaagagct attcacccca tataccactg agggccctga gctggaattc
16200caaccctagg ttttggcata gccactgtct gcccttgctt ctgaaacaaa cacttgtgca
16260aatgtgtagc agatctagac ccaaagactt agggtcaatg aaatcaagac attttggtag
16320tgattggaaa tccatattta cttggggtgc aagagtcaaa ggataataac atggtgtgtc
16380agctcaaaat atacttcttc ttatctagtc tgaaaaagtt agtgaaagaa gctctgaaag
16440aatctcaact tccaactgtc atggacttca gaaaattctc tcggaactat caactctaca
16500aatctgtttc tcttccatca cttgacccag cctcagccaa aatagaaggg aatcttatat
16560ttgatccaaa taactacctt cctaaagaaa gcatgctgaa aactaccctc actgcctttg
16620gatttgcttc agctgacctc atcgaggtaa gtgtgaagag tttgaggttc tctagcccat
16680tttgtacagc atcataaaca gagagtccct gggagccagg agctacccag aggaaaacta
16740agaaccacca ggcacttcct accatgattc tgaggctttc ttctttccct ccttccccgc
16800cttcctctct ccccgctagg ggtcacctga agcatgactt cttaacatta atagaaatgc
16860aggcctggcg aggtggctca ctcctgtaat cccagcactt tgggaggccg aggcgggtgg
16920atcatgaggt caggatatcg acaccatcct ggctaacacg gtgaaagccc atctctacta
16980aaaatacaaa aaattagccg ggcgtggtgg caggcacctg tagtcccagc tacttgggag
17040gatgaggcag gagaatggcg tgaacccagg aggctgagct tgcagtgagc cgagagattg
17100cgccactgcg ctccagcctg ggcgacagag caagactcca tctcaaaaaa aaaaaaaaaa
17160aaaaaaattg aaatgcaaat gtctcgtctt taagtcccaa agccaaggaa gcatatgtgc
17220tgcctagtca gatctgcttc aaatctcaaa tcactcccaa ctctgaatcc tttgttgaat
17280tatttgtcct atctgaacct tagctgcctc ttctagaaaa aagcaagtaa taaggtcaag
17340attctagtga gattttaata aagcagctcc tgtgaaatgc taaggtcagc tcctggcctg
17400tggtattcaa atacttgttt agataaatgg acatcaagag tggggactac taggctggca
17460tacaacaaag aaacctgatg ccattttctt gtctgatttt ctttctcaga ttggcttgga
17520aggaaaaggc tttgagccaa cattggaagc tctttttggg aagcaaggat ttttcccaga
17580cagtgtcaac aaagctttgt actgggttaa tggtcaagtt cctgatggtg tctctaaggt
17640cttagtggac cactttggct ataccaaaga tgataaacat gagcaggtgt gtatttgtga
17700agtatcttct taaggaaagc tttgggtctc aatgcaaaaa caattctttt ctaagcatgg
17760aagtcctcaa aatactatct aactgaaggg ataactatgg tttttatcaa ccagacctgc
17820tggggtaagg gccagtatcc tctgcagtta aagatctcct gaattcagtg tgcccagaaa
17880ccagactcac aataagtact ctaggataac aagagtatga actctgggct gggtgtggtg
17940gttcatgcct gtaatcccag cactttggga ggccaaggtg ggcagatcac aaggtcagga
18000atttgagacc agcctggcca acatactgaa accccgtctc tactaaaaat acaaaaaaac
18060tagctgggca tggtagtggg tgcctgtaat cctagctact cgggaggctg agacaggaga
18120attgcttgaa cccgggaggt ggaggttgca gtgagccgag atcacgccgt tacactccag
18180cccgggtgac agtgtgagac tgtatcttaa aaaaaaaaaa agtatgaact ctgggcatag
18240atttaattct aacttccctg tcttgaagct gtgcgcactt ggggaagttg gttgatatta
18300tgtgtatctg tttctgtctg tatcccagac tactaataac agtccaaacc tcacaaggtt
18360atttaaagac aatgaaataa ggcatctaaa atgccaagca cagtgcctga tgctggcatt
18420ggttgttcaa taagcagaca ctattacgag ttctaaatta atattttcat tattattaac
18480tgctgtcttt ggctctcact cccatcagtg cactagcaaa tgagaccaaa cttccacttt
18540gaagctagca atgagccccc atttaaggag ggaaataggt tgtatgatct ggagcttatt
18600cttgaatttt ttgctaccca aagtgtggtc tggtcagaaa tacagcttct catgcttcac
18660ccacaatcta ctgaatcaga agcgcatttt agcaagacct catgtgactt gtatgcacat
18720tcaactttgc agagcaaggc agtaatttac ccctccaggc tcactgttga gcacgagctc
18780catcttctaa tttcctgacc cccacttgag gccgaggatc tttgatctgc tttgagtctg
18840tcagtttcac attttttttt tcccaatgcc tgggcatcca tctctgagat tcttcttctc
18900tctgagaaga acttgtctag gatcaagtgt ttttcaaact tctggtgaat ttatataaca
18960gctacatttt cttaagaaac accttgtagt cttcactggt caaagaagag aaggctaagc
19020agggaacggg tgggggatag aggatcttct aatcttgagg atcctggcat actggagaat
19080agggacccct cctctcatcc caccacatct tactatgtct acagattttt taattaagaa
19140tagctttagg agtgccacta tccctgacaa gaccttagtt ctttaatctc tgcttagagg
19200aattagcctg gacttcagtg tctccctgtt cctcacctgg agcatttttt aggcccatcc
19260tggctgcatc agacaggtcc cacattggga actgaaaggt gtttgacatt gctgacatct
19320cactggccat tttattacta aactctcagg atatggtaaa tggaataatg ctcagtgttg
19380agaagctgat taaagatttg aaatccaaag aagtcccgga agccagagcc tacctccgca
19440tcttgggaga ggagcttggt tttgccagtc tccatgacct ccagctcctg ggaaagctgc
19500ttctgatggg tgcccgcact ctgcagggga tcccccagat ggtaagtcag caggccccac
19560tgggggccca tgagaccaga cgttggtttt tttttagatc gcccagactc ccttacgatc
19620ccagctgcac aagcccgaaa agatgcttgt actttcttca gagatggagg tttgccttga
19680atttcactga agatgactct tggatcacat ggaaatgtta acatttagaa attaagctat
19740tcataatgtt agctgtattt ttaagagcat taatttattc atctggaaaa caatgttcgg
19800tataccttcc tctacctttg ctgaaggtcc ttttattttt atttttattt ttttaatttt
19860ttgagatgga gtcttgctcc caggctggag tgcagtgata caatctcggc tcactgcaac
19920tctgccttcc gggttcaagc aattctcctg cctcagcctc ccaagtagct gggactgtgg
19980acgtgcacca gcatgcccgg ctaatttgtg tatctttagt agagacaagc ctgttgacaa
20040ccatgtcagg ctggtttcga actcctgacc tcaagtgatc ctccagcctg ggcctcccac
20100agtgctggaa taacaggtgt gagccactgc acctgacctg aaggtccttt taagattgaa
20160atgatacaat gattataaaa gaaagtattt ggcaaactat aattcactat ctaaatatgc
20220tataattttt attattaatt cataaaagga aatatataaa tgtactccta tggcttgatt
20280aaaaaaatgt tgactttaag aaaacaggtc tcaagctatt ttattgaaat attatttaaa
20340aaataaaacc caatgcaaat tgatatgtac atcatctcaa taggcctttg gtttcaaaaa
20400attgatttta tcataatata atacatttca agtacacctt cacttacagt cagactccag
20460aacaccagaa ttaagccatg gcatatatga tacttaaagt ccataaagct ctgaggccca
20520gcaatattct taagagcctt ctgagtccac ttgaaaatga catgatatct atctagtgaa
20580atttcttata tcctgattca ctgaaaacgg taaaaacatc agtttgatct ttatttatca
20640aactattcag ctcatcaaaa tatgctagtc cttcctttcc agataaagag gaattactct
20700ccaatgtatg ggaggttgta attaacaaaa ccgactttaa aaagacttac ttttatttgc
20760tctcccttgt tgggtctaca gattggagag gtcatcagga agggctcaaa gaatgacttt
20820tttcttcact acatcttcat ggagaatgcc tttgaactcc ccactggagc tggattacag
20880ttgcaaatat cttcatctgg agtcattgct cccggagcca aggctggagt aaaactggaa
20940gtagccaacg taagattctg tttgcctttt gatttcttag gttattactt tcttccaggg
21000tgcatttctt gttaaaacat atttaaaaat gtgtttccac ttcaagacaa aatgcttcat
21060cattgtaatc acctcattat ttttttatga aaaacttcaa gcttccacca gaatgcacta
21120cctcactagc tccagtagtg gtatggccat aagacaagaa ctcagttctc tcaacaaatg
21180agtattccta tcatcttttt aatctggttt tgcctcacgt taactcaggt gctttctagt
21240tctgggtagt atactccaac tctagagaac tgagaactcg ctttccttct tccaaacaaa
21300tcccagtaat gtttccaaag gtctgagtta tccaggaaat ctttgcccgg aggtgagaaa
21360gggtggttga tctgactgac aggggactga agtatttaat gaatctgaat aggttgtttt
21420ctgacttata gatgcaggct gaactggtgg caaaaccctc cgtgtctgtg gagtttgtga
21480caaatatggg catcatcatt ccggacttcg ctaggagtgg ggtccagatg aacaccaact
21540tcttccacga gtcgggtctg gaggctcatg ttgccctaaa agctgggaag ctgaagttta
21600tcattccttc cccaaagaga ccagtcaagc tgctcagtgg agggtaattc tttcagccaa
21660gtctgcctag ccagtttgaa agagagaaca gagaatgtac ctgcagaatt ttgccaggct
21720aaacagttga ttgagatcat tcaggtcctg aggaagcagg agaggagtag aaaggaaaga
21780ttccgggtta cctattttaa ttctagccta gacttactac ataactacat aattaccttt
21840cttctacttt tcacatttta ctaaactgtc ctttatcttt ctgctttgag acttattaag
21900acctactgct taattagttt ttattaagtt gtgatttttt gttatctatt tgttttgaga
21960atgaagaaac aatagctctg gagagatcat ctttggaaaa ttaatatttt cccccccaaa
22020aaatacctaa gaacatattg atttgaggta gctaggtagg taaagcatga aactcctaac
22080ctcgtgataa tggaatacag cctcttttgg agagttccat tttaagtggc accctcaacc
22140attgatttgc cttagttttc atattttaga cacattcatg tgttcattca aaaataatat
22200ttaattggcc agccacggtg gttcatgcct gtaatcctag cactttggga gccccaggtg
22260gatggatcgc ttgagccctg gtgtttggat accagcctgg gcaacatggc aaaaccccat
22320ctctacaaaa aaaattaaat aaataacaaa attagccagt cgtggtggca catgcctgta
22380gctccagcta ctcagaaggc tgagatggga ggatcaactg agcccaagag ttcaagcctt
22440cagtgaacca tgcttgcacc actgcactcc agcctgggag acagagcaag atcctgtctc
22500acaaaaaaca aaaaatagta tatttaattg cctaatatat accacgtatg ttgagtgaga
22560cacacaaggt ccctgacctt tgaacgctta cattttataa gggagacaca caattaagca
22620agcagtaatc atagagtaag ggctaagtta tagaaagtat tagagtacca tgaaatttta
22680tatcatgtag cctgtgctag tcagggaatg cattctgaag caagtgtact tgacctgata
22740actgaggact gtgtcagagt catttaggca aaggagaaag gagtgagtgt tccaggcaaa
22800aggaaaagca tgtaatggcc tgaaggtaaa ggaatatggt tcaaggaact ggaagaagtg
22860cagaatggta aggggctcag agatgatggg gagaggtagg caggggagag agcatgccca
22920gctgcgaaag ccatcctaag gagtttggac tcttttgaag gcacaggagt tgaaaagggg
22980agcagaaata agataggggt gatgttttag aagaaatact ctgactctag tgtggaagat
23040gggtgagaag gaggcacagc tggacacgaa gagaccattg gacatctctt acgatcctat
23100gtggctaaga gctgataatg gcctgcagtg gagaaaagcc aggtatagaa aggagtgagc
23160agattctaca actttctaag aggcagaatc ataagtactg ggtgattaac tgggtatggg
23220gacaaggcaa aagaaagaag aaaagaggaa ggaggcgccc ttcattttaa taagaactac
23280agtgggagag cttctggttt caaggaaagt gacaaattca gttttggatg tgctgtattt
23340gatgtcctcc tatgaaacaa ccagtttaga aatctagctg tcaaatagac ctatggatct
23400gagcccagta aagaggcttg ggctccacat atggatttgg gaatcattag tatacagagg
23460ttgttgtggt taaacagcaa ctggtataga gtgagacatg agagatgagg acagaaatat
23520ggagaagaca aacatataaa ggaagaaggg gaataaccag caatgagtta gaagaagtga
23580ccagagaagc agaaggagaa ccaaagccat aaaaggtcac agaagccaaa gagcagccac
23640aggggagatc accccatggg taggcgaaag ctggcattag gactccagca catcagcaaa
23700gcttggtctt gtggcacccc caacttggag aaacaatact tggaggaaaa tgtgctattt
23760caaagaaagc atccttagaa aaaaccaggc caatgttgaa ctttcttaca tgtactaagt
23820ttttaagtac acacttggaa ggaaggtgcc atcatctctt cagatgtgag aggctccagc
23880gtcttagtct ggtcatgagt gcgcaactct atggaaggct tctgggaggt caaggaagat
23940gaaacctaaa tatgcccatt ggatgtagga gcaaggaggg cattagagac attgatgaaa
24000gcattttcag gagatggagt gagcagtcag agcacattgg gaggaagtag agactgcaaa
24060ggcagacaac tcttgatggt gaggaagatg agaaagcaag aaaagaaaga aaggagcata
24120ggggaggggc acaggggaag agacttgagc gtgcttaatg caggtggaag gaagcaggta
24180gagagtagga gatttcatat gaaagagaca gtttctcttg ccctgcattg taggaaggaa
24240ggggcacact gaagttcagc cccagtgatc agctatttaa catctctgag cctctgcttc
24300tgtaaaatga gaaccataag cctactgttg tggggattac aggtaacaga tggaaagaac
24360tcagccagaa gcttcagagt cactctcatg gcttgtcatg ttgatgttct ttctaatatt
24420atttgtttct cagtaaatta aatagttaga gataggtgtg gactgaggga agacaggagg
24480ataagggggt atttgcaccc tgagaatttg tgatgtccat tttgattcat gacttggcaa
24540taactcaggt atttttgttc ttcaccagca acacattaca tttggtctct accaccaaaa
24600cggaggtgat cccacctctc attgagaaca ggcagtcctg gtcagtttgc aagcaagtct
24660ttcctggcct gaattactgc acctcaggcg cttactccaa cgccagctcc acagactccg
24720cctcctacta tccgctgacc ggggacacca ggttagagat gctcagtgcc tgacccagca
24780ttttctcacc ttccacatca tggccaccta gcatggcaca ggaaaaaata ctctgtgttg
24840taagaccctg tcactagcct tctgggtttg caccatcttt gggtatttaa agcagggtcc
24900tctggccaac acattgggtg tcaccttttg cttccttgtg catgggatgg gatcacagca
24960cagatcccaa tttgctccta attcagtgtc catgtttctg agcctccaga cccatcgcta
25020tgagcttcct ggagcccacc aatgtgcttg aagccttcac cgtacttagg tggctccctg
25080tcttcagccc ccaagttcca gtgcttgttc tcagctttgc tgaaacaacc agccaactcc
25140tgctctgctt gtccaaagtc ttgggaatcc tggtgtctgc ccttgccttg ggttcttgta
25200ggactgaggg atcaaaaaga tcatcttagt taagggcaag agacaatgtt aaaataagga
25260ccatattttt gttgcatttg aggctgaatt gttttgggaa cataatcacc atccttgaaa
25320gctctaacat tatgcactgt cttcattgta atgtctttag attagagctg gaactgaggc
25380ctacaggaga gattgagcag tattctgtca gcgcaaccta tgagctccag agagaggaca
25440gagccttggt ggataccctg aagtttgtaa ctcaagcaga aggtgagtat tcaaaacaca
25500gctgcctcat ctctgctcgc agtctcaggt tcagaattca tgaggagaag acatgtaatt
25560taacctattt aacaaatagg ttaactgagt acccactaag cggcaggcct attctaagac
25620ctgggttaac tgagtaccca ataagcggca ggcctattct aagacctggg gctagaacag
25680tgaacaatgg agtctctgcc ttcatggaag ttacagtgaa caaccaaaca agttaatatt
25740tggaatatca gataagtact gaggaggaaa acagagcgta gactggtcta tggagggcta
25800ggagtaggag ggaggaagaa gggcagggaa agcagtgcat ttggaataat aagggaaagt
25860ctccctggta aagtgagcat aaggagacct atcagaaata agaggagaag ccgtgtggta
25920agactgttaa caggcagagg gaccagcaag tgcaaaggcc ctgaggctga cacactacta
25980ccatgtttca aggaaaggaa ggaagacagt atggctggag cagaaagacc agggagaaaa
26040gaggtagaag atgaggacag agagatatgg agaggtgaag gaaggataat ctcataggcc
26100atggtaagaa ctttggcttt ttctatgaat taaacgaaag ccattgggga gtcctcatga
26160tttgatttat gtttatgttg agaaaagact atgggcagac aagggcagag aaactaatat
26220gtaggttatc acaataatcc aggcaggaat cagtgttgtt ttggatcagg gcaatggcag
26280aagagatatg agaaggggat ggattctggc catattttga agattaggct gacaagattt
26340gctgatacag tggatgttga gtgtaagagg aaaaggggaa tgaagacaaa cctaaggttt
26400ttggcccggg caactgaaaa atggaacttc catttattga gatggaaagg gctactggag
26460gagcaggttt tagggaatgg gagaaattta ggtgttcact ttggaaaaaa aattatatag
26520ggatagcgag gagcaggttt tagggaatgg ggcacattta ggtgttcact ttggaaaaat
26580ttttatatag ggatagcata tcacagaatt aaactaggaa gaaaatccca tgatagaaag
26640cactggagga gcagggcacg ctggggaaat agtgtttggt aaacattgtt ttacgaagga
26700tataaaatgg accagcctat ggattgaagg acgcccggga atcttgttac aaagaaaggg
26760ggagttgggg agatggagcc cagggcaagg gcagcaagga accaggacag gcatcttggg
26820tagaaagtaa tatagagatg tcgtgtcttc ctggcccaga agggctgcga gcctttgctg
26880ttccacaaac aagctaagtg ctccccattt cagggccttt gcattcctga ccttctgcct
26940ggaatgtgct cctcccagaa ctcagcgtgg ctccaacctc ttttcattct ggtctctgcc
27000cacatgtgcc cttatcagag agaatttctc tgaccaccaa gtatgaaata acacttcttc
27060tatccctttc ttttatcctt gtatccagtt ttactcttct tcataacatt cattaccatc
27120tgacatgagc aagttacttg tttattgcct gtacacctcc cccactagaa ggtaagcccc
27180atgaaagcaa ggattcccca gtaccaagag cagtgcccag cacacaatag gctcataaca
27240ggcaatccat aaagacttgc atacatgaac acaactgagt ttaaaattat cagtaaatga
27300gacccattaa aaaattttaa tgagaaaaaa aaaattcagt aaaatcctga actgtgtttt
27360tgtttaagca cattgattcc ttggagtttc tctacctttt cctctctttc cttccaaaac
27420atagcttctt tatttattta tttatttatt tgtttgttta tttatttatt tatttattta
27480tttatttttt gagatggagt ctcgctcttt tgcccaggct gcagtgcagt ggtgccatct
27540cggctcactg caagccccgc ctcccgggtt catgccattc tcctgcctca gcctcctgag
27600tagctgggac tacaggcacc caccaacgcg cccggctaat tttttgtatt tttagtagag
27660acggggtttc accatgttag ccagaatggt cttgatctcc tgacctcatg atctgcccgc
27720cttggcctcc caaagtgctg ggattacagg tgtgagccac cgcacccggc ccaaaacata
27780gcttcttacc acacatctct tgattctctt atacactcgt ccaggtgcga agcagactga
27840ggctaccatg acattcaaat ataatcggca gagtatgacc ttgtccagtg aagtccaaat
27900tccggatttt gatgttgacc tcggaacaat cctcagagtt aatgatgaat ctactgaggg
27960caaaacgtct tacagactca ccctggacat tcagaacaag aaaattactg aggtcgccct
28020catgggccac ctaaggtaaa gaaggccgag ggtcatctga cctgcactgc aggcctgggt
28080ggttcttttc attattcctc ttccacttca tacctgacca agccatgttc tcccctagtc
28140tacaatcaga gtggcagaga gagccctcaa caattttttt tttttttgag atggagtctc
28200actctgtcac caggctggag tgcagtggca caatctcggc tcactgcaac ctccgcctcc
28260cgagttcaag tgattctcct gcttaagcct cccaaggagc tggaactata ggtgcatgcc
28320accacaccca gctaattttt atatttttag tagagacagg gtttcaccat attgaccagg
28380atggtctcga tctcctgacc tcgtgatcca cctgccttgg cctcccaaag tgctgggatt
28440acaggtgtaa gccactgcac ccggccaagc tctcaacatt ttaaccctct gcgcatgtcc
28500agttggattt tcctaccatt tatcaggcac ttactattca tgtatcaagc acagtgctgg
28560gtgctttaaa gaaattatct cggtcctcac aataaactgc gaggtcactg tgagttttcc
28620tgtttcatgg ataaggaaat ggtagctcag aggggttaaa tcatttggtc aaaatcacag
28680agctagtaaa tagcagagca ggattcaaac agttttcaaa aaacttctct ttctcctaaa
28740cctgtttgca aagtccttaa tttgtgctga atgttggctt tagaagttga tgagtttgat
28800ctgtggctgt ttctctgaac catccttgta tctggttttg atcaccacaa atggaacttc
28860tgtttaatcc tgcatatctc cattgaaagg acaaaatcat tggtgccaac tgattttctt
28920taccatagtt gtgacacaaa ggaagaaaga aaaatcaagg gtgttatttc cataccccgt
28980ttgcaagcag aagccagaag tgagatcctc gcccactggt cgcctgccaa actgcttctc
29040caaatggact catctgctac agcttatggc tccacagttt ccaagagggt ggcatggcat
29100tatggtatgt gtctcttccc ctgtgtgagc acttccaaag taatgcaggt gttgagacct
29160gtggttacag gctgaactag taccattcac aactatttcc tacgtatttt cagatgaaga
29220gaagattgaa tttgaatgga acacaggcac caatgtagat accaaaaaaa tgacttccaa
29280tttccctgtg gatctctccg attatcctaa gagcttgcat atgtatgcta atagactcct
29340ggatcacaga gtccctcaaa cagacatgac tttccggcac gtgggttcca aattaatagt
29400tgtaagtatg agtctgccag tcaataaata catggatata agtgctaatt acatcctcaa
29460ctctgagcta ggtgcaggaa ggtttccaaa gatgtataag gcatgcttcc ttccccccag
29520ggaattcttg gggagaaaaa aaaactttca caagtgtgta gttacccagt tacacaaagc
29580tgaatgtgat acatatcaaa gagatgctac taagtagaac agttctttgc ctagtggtat
29640caaaggaagc ttcaggacac cagctaggag gctgactatg ttagacattc cttttataaa
29700tatggacagt gatcagtgac tggcaacgaa gattcataat tttctgttat ttatttttaa
29760ctttcagtgc attgtccagc ttaataatta acttgtcaaa tcggtatttt tgcctaatgt
29820tcattgctct ttgaggctca tccaagccca ttaccttaaa aatctcctgt cattttgtag
29880gcaatgagct catggcttca gaaggcatct gggagtcttc cttataccca gactttgcaa
29940gaccacctca atagcctgaa ggagttcaac ctccagaaca tgggattgcc agacttccac
30000atcccagaaa acctcttctt aaaaaggtaa aagaagaaag cagcaaggct tcttgaacca
30060tgcaaagtaa atgaaagatt ttacatagca tgatttagac atttttttaa atttttaaag
30120gaaataattt aagcatttta aggagattaa taactatagc acaaacactg tggcatcttt
30180gcattagtaa acatgagaac accaaccctg tcaggaagaa tctaagaaag tcattagagg
30240attctggtac tttcacccta agatatttta ttcagtacaa cctgttataa gcaaattctc
30300cctctgactg tgaagaattc agaatggcta gaggcgttat tgactacagg cttgctgtta
30360agctagagag agtcagaaca gccattgagc actaaatgga ggcagcattc tgagaaaata
30420ctttaaccca ggcttactga cttccatacc tatgttcttt ccacaaatca agttgtctca
30480attcagttta gcaaatttgt atcaagtatc ccctatgtgc aaaatgctag actaggtaca
30540gtgagaagat agaaactggg taaggtatag ccttttcttt caagaagata ccatggagac
30600atcaacaaat gagaaataat taattatata agcaaaatta tgacatgctc tttgagaaag
30660gtgcaaggga ctatgtaact gtaagaatga gacaaattgg ctatgactta ggtgggatgg
30720taatgataag gagtggccct tagaagagct ttgtcaggat ttgagtgttt gacaggtgga
30780ggtaaaagca aaggggtcca ggcataggag tagcacaaag aaaagtgcag agtggctttg
30840ggaatggggc aagtacaata ttgttgtgaa ggtcagaggc agagaacttt gaatgactga
30900tgtctgactg tggggatgtt atctttgttg ttcatttcag cgatggccgg gtcaaatata
30960ccttgaacaa gaacagtttg aaaattgaga ttcctttgcc ttttggtggc aaatcctcca
31020gagatctaaa gatgttagag actgttagga caccagccct ccacttcaag tctgtgggat
31080tccatctgcc atctcgagag ttccaagtcc ctacttttac cattcccaag ttgtatcaac
31140tgcaagtgcc tctcctgggt gttctagacc tctccacgaa tgtctacagc aacttgtaca
31200actggtccgc ctcctacagt ggtggcaaca ccagcacaga ccatttcagc cttcgggctc
31260gttaccacat gaaggctgac tctgtggttg acctgctttc ctacaatgtg caaggtgagc
31320tatgctcagg taaagggtgc accgggctag ttcatggcag gctctaagag gagagcctcc
31380tccagggagg aaaggacttt ggctttctag cagataatct tccttgctac ttggaagtct
31440tttattttat tcaacaaata gaaatattta ttaaacatat cacgtgtatt aaatattcta
31500gtaggcagta acagaaagta gacagataag ccagcaatta taattcagtg tgagaggtgc
31560tatgataaag tgtagtatat aagtataagg tagagtggaa gcactcaaca agggaaccta
31620aacaaagcct gtggtggtca ggcaaggctt cctggaggaa tgccttttgc tatcagattt
31680tatctttgca ttacagatgg aggagtctat tgcacaattg gcccagaaaa atggggcttt
31740attattgaaa gactttcaac atagagattg ctctggaaat gtactgctta atttaaccaa
31800tgtcttttca tttttatgtt aggatctgga gaaacaacat atgaccacaa gaatacgttc
31860acactatcat atgatgggtc tctacgccac aaatttctag attcgaatat caaattcagt
31920catgtagaaa aacttggaaa caacccagtc tcaaaaggtt tactaatatt cgatgcatct
31980agttcctggg gaccacagat gtctgcttca gttcatttgg actccaaaaa gaaacagcat
32040ttgtttgtca aagaagtcaa gattgatggg cagttcagag tctcttcgtt ctatgctaaa
32100ggcacatatg gcctgtcttg tcagagggat cctaacactg gccggctcaa tggagagtcc
32160aacctgaggt ttaactcctc ctacctccaa ggcaccaacc agataacagg aagatatgaa
32220gatggaaccc tctccctcac ctccacctct gatctgcaaa gtggcatcat taaaaatact
32280gcttccctaa agtatgagaa ctacgagctg actttaaaat ctgacaccaa tgggaagtat
32340aagaactttg ccacttctaa caagatggat atgaccttct ctaagcaaaa tgcactgctg
32400cgttctgaat atcaggctga ttacgagtca ttgaggttct tcagcctgct ttctggatca
32460ctaaattccc atggtcttga gttaaatgct gacatcttag gcactgacaa aattaatagt
32520ggtgctcaca aggcgacact aaggattggc caagatggaa tatctaccag tgcaacgacc
32580aacttgaagt gtagtctcct ggtgctggag aatgagctga atgcagagct tggcctctct
32640ggggcatcta tgaaattaac aacaaatggc cgcttcaggg aacacaatgc aaaattcagt
32700ctggatggga aagccgccct cacagagcta tcactgggaa gtgcttatca ggccatgatt
32760ctgggtgtcg acagcaaaaa cattttcaac ttcaaggtca gtcaagaagg acttaagctc
32820tcaaatgaca tgatgggctc atatgctgaa atgaaatttg accacacaaa cagtctgaac
32880attgcaggct tatcactgga cttctcttca aaacttgaca acatttacag ctctgacaag
32940ttttataagc aaactgttaa tttacagcta cagccctatt ctctggtaac tactttaaac
33000agtgacctga aatacaatgc tctggatctc accaacaatg ggaaactacg gctagaaccc
33060ctgaagctgc atgtggctgg taacctaaaa ggagcctacc aaaataatga aataaaacac
33120atctatgcca tctcttctgc tgccttatca gcaagctata aagcagacac tgttgctaag
33180gttcagggtg tggagtttag ccatcggctc aacacagaca tcgctgggct ggcttcagcc
33240attgacatga gcacaaacta taattcagac tcactgcatt tcagcaatgt cttccgttct
33300gtaatggccc cgtttaccat gaccatcgat gcacatacaa atggcaatgg gaaactcgct
33360ctctggggag aacatactgg gcagctgtat agcaaattcc tgttgaaagc agaacctctg
33420gcatttactt tctctcatga ttacaaaggc tccacaagtc atcatctcgt gtctaggaaa
33480agcatcagtg cagctcttga acacaaagtc agtgccctgc ttactccagc tgagcagaca
33540ggcacctgga aactcaagac ccaatttaac aacaatgaat acagccagga cttggatgct
33600tacaacacta aagataaaat tggcgtggag cttactggac gaactctggc tgacctaact
33660ctactagact ccccaattaa agtgccactt ttactcagtg agcccatcaa tatcattgat
33720gctttagaga tgagagatgc cgttgagaag ccccaagaat ttacaattgt tgcttttgta
33780aagtatgata aaaaccaaga tgttcactcc attaacctcc cattttttga gaccttgcaa
33840gaatattttg agaggaatcg acaaaccatt atagttgtac tggaaaacgt acagagaaac
33900ctgaagcaca tcaatattga tcaatttgta agaaaataca gagcagccct gggaaaactc
33960ccacagcaag ctaatgatta tctgaattca ttcaattggg agagacaagt ttcacatgcc
34020aaggagaaac tgactgctct cacaaaaaag tatagaatta cagaaaatga tatacaaatt
34080gcattagatg atgccaaaat caactttaat gaaaaactat ctcaactgca gacatatatg
34140atacaatttg atcagtatat taaagatagt tatgatttac atgatttgaa aatagctatt
34200gctaatatta ttgatgaaat cattgaaaaa ttaaaaagtc ttgatgagca ctatcatatc
34260cgtgtaaatt tagtaaaaac aatccatgat ctacatttgt ttattgaaaa tattgatttt
34320aacaaaagtg gaagtagtac tgcatcctgg attcaaaatg tggatactaa gtaccaaatc
34380agaatccaga tacaagaaaa actgcagcag cttaagagac acatacagaa tatagacatc
34440cagcacctag ctggaaagtt aaaacaacac attgaggcta ttgatgttag agtgctttta
34500gatcaattgg gaactacaat ttcatttgaa agaataaatg acattcttga gcatgtcaaa
34560cactttgtta taaatcttat tggggatttt gaagtagctg agaaaatcaa tgccttcaga
34620gccaaagtcc atgagttaat cgagaggtat gaagtagacc aacaaatcca ggttttaatg
34680gataaattag tagagttggc ccaccaatac aagttgaagg agactattca gaagctaagc
34740aatgtcctac aacaagttaa gataaaagat tactttgaga aattggttgg atttattgat
34800gatgctgtca agaagcttaa tgaattatct tttaaaacat tcattgaaga tgttaacaaa
34860ttccttgaca tgttgataaa gaaattaaag tcatttgatt accaccagtt tgtagatgaa
34920accaatgaca aaatccgtga ggtgactcag agactcaatg gtgaaattca ggctctggaa
34980ctaccacaaa aagctgaagc attaaaactg tttttagagg aaaccaaggc cacagttgca
35040gtgtatctgg aaagcctaca ggacaccaaa ataaccttaa tcatcaattg gttacaggag
35100gctttaagtt cagcatcttt ggctcacatg aaggccaaat tccgagagac cctagaagat
35160acacgagacc gaatgtatca aatggacatt cagcaggaac ttcaacgata cctgtctctg
35220gtaggccagg tttatagcac acttgtcacc tacatttctg attggtggac tcttgctgct
35280aagaacctta ctgactttgc agagcaatat tctatccaag attgggctaa acgtatgaaa
35340gcattggtag agcaagggtt cactgttcct gaaatcaaga ccatccttgg gaccatgcct
35400gcctttgaag tcagtcttca ggctcttcag aaagctacct tccagacacc tgattttata
35460gtccccctaa cagatttgag gattccatca gttcagataa acttcaaaga cttaaaaaat
35520ataaaaatcc catccaggtt ttccacacca gaatttacca tccttaacac cttccacatt
35580ccttccttta caattgactt tgtagaaatg aaagtaaaga tcatcagaac cattgaccag
35640atgctgaaca gtgagctgca gtggcccgtt ccagatatat atctcaggga tctgaaggtg
35700gaggacattc ctctagcgag aatcaccctg ccagacttcc gtttaccaga aatcgcaatt
35760ccagaattca taatcccaac tctcaacctt aatgattttc aagttcctga ccttcacata
35820ccagaattcc agcttcccca catctcacac acaattgaag tacctacttt tggcaagcta
35880tacagtattc tgaaaatcca atctcctctt ttcacattag atgcaaatgc tgacataggg
35940aatggaacca cctcagcaaa cgaagcaggt atcgcagctt ccatcactgc caaaggagag
36000tccaaattag aagttctcaa ttttgatttt caagcaaatg cacaactctc aaaccctaag
36060attaatccgc tggctctgaa ggagtcagtg aagttctcca gcaagtacct gagaacggag
36120catgggagtg aaatgctgtt ttttggaaat gctattgagg gaaaatcaaa cacagtggca
36180agtttacaca cagaaaaaaa tacactggag cttagtaatg gagtgattgt caagataaac
36240aatcagctta ccctggatag caacactaaa tacttccaca aattgaacat ccccaaactg
36300gacttctcta gtcaggctga cctgcgcaac gagatcaaga cactgttgaa agctggccac
36360atagcatgga cttcttctgg aaaagggtca tggaaatggg cctgccccag attctcagat
36420gagggaacac atgaatcaca aattagtttc accatagaag gacccctcac ttcctttgga
36480ctgtccaata agatcaatag caaacaccta agagtaaacc aaaacttggt ttatgaatct
36540ggctccctca acttttctaa acttgaaatt caatcacaag tcgattccca gcatgtgggc
36600cacagtgttc taactgctaa aggcatggca ctgtttggag aagggaaggc agagtttact
36660gggaggcatg atgctcattt aaatggaaag gttattggaa ctttgaaaaa ttctcttttc
36720ttttcagccc agccatttga gatcacggca tccacaaaca atgaagggaa tttgaaagtt
36780cgttttccat taaggttaac agggaagata gacttcctga ataactatgc actgtttctg
36840agtcccagtg cccagcaagc aagttggcaa gtaagtgcta ggttcaatca gtataagtac
36900aaccaaaatt tctctgctgg aaacaacgag aacattatgg aggcccatgt aggaataaat
36960ggagaagcaa atctggattt cttaaacatt cctttaacaa ttcctgaaat gcgtctacct
37020tacacaataa tcacaactcc tccactgaaa gatttctctc tatgggaaaa aacaggcttg
37080aaggaattct tgaaaacgac aaagcaatca tttgatttaa gtgtaaaagc tcagtataag
37140aaaaacaaac acaggcattc catcacaaat cctttggctg tgctttgtga gtttatcagt
37200cagagcatca aatcctttga caggcatttt gaaaaaaaca gaaacaatgc attagatttt
37260gtcaccaaat cctataatga aacaaaaatt aagtttgata agtacaaagc tgaaaaatct
37320cacgacgagc tccccaggac ctttcaaatt cctggataca ctgttccagt tgtcaatgtt
37380gaagtgtctc cattcaccat agagatgtcg gcattcggct atgtgttccc aaaagcagtc
37440agcatgccta gtttctccat cctaggttct gacgtccgtg tgccttcata cacattaatc
37500ctgccatcat tagagctgcc agtccttcat gtccctagaa atctcaagct ttctcttcca
37560gatttcaagg aattgtgtac cataagccat atttttattc ctgccatggg caatattacc
37620tatgatttct cctttaaatc aagtgtcatc acactgaata ccaatgctga actttttaac
37680cagtcagata ttgttgctca tctcctttct tcatcttcat ctgtcattga tgcactgcag
37740tacaaattag agggcaccac aagattgaca agaaaaaggg gattgaagtt agccacagct
37800ctgtctctga gcaacaaatt tgtggagggt agtcataaca gtactgtgag cttaaccacg
37860aaaaatatgg aagtgtcagt ggcaacaacc acaaaagccc aaattccaat tttgagaatg
37920aatttcaagc aagaacttaa tggaaatacc aagtcaaaac ctactgtctc ttcctccatg
37980gaatttaagt atgatttcaa ttcttcaatg ctgtactcta ccgctaaagg agcagttgac
38040cacaagctta gcttggaaag cctcacctct tacttttcca ttgagtcatc taccaaagga
38100gatgtcaagg gttcggttct ttctcgggaa tattcaggaa ctattgctag tgaggccaac
38160acttacttga attccaagag cacacggtct tcagtgaagc tgcagggcac ttccaaaatt
38220gatgatatct ggaaccttga agtaaaagaa aattttgctg gagaagccac actccaacgc
38280atatattccc tctgggagca cagtacgaaa aaccacttac agctagaggg cctctttttc
38340accaacggag aacatacaag caaagccacc ctggaactct ctccatggca aatgtcagct
38400cttgttcagg tccatgcaag tcagcccagt tccttccatg atttccctga ccttggccag
38460gaagtggccc tgaatgctaa cactaagaac cagaagatca gatggaaaaa tgaagtccgg
38520attcattctg ggtctttcca gagccaggtc gagctttcca atgaccaaga aaaggcacac
38580cttgacattg caggatcctt agaaggacac ctaaggttcc tcaaaaatat catcctacca
38640gtctatgaca agagcttatg ggatttccta aagctggatg taaccaccag cattggtagg
38700agacagcatc ttcgtgtttc aactgccttt gtgtacacca aaaaccccaa tggctattca
38760ttctccatcc ctgtaaaagt tttggctgat aaattcatta ttcctgggct gaaactaaat
38820gatctaaatt cagttcttgt catgcctacg ttccatgtcc catttacaga tcttcaggtt
38880ccatcgtgca aacttgactt cagagaaata caaatctata agaagctgag aacttcatca
38940tttgccctca acctaccaac actccccgag gtaaaattcc ctgaagttga tgtgttaaca
39000aaatattctc aaccagaaga ctccttgatt cccttttttg agataaccgt gcctgaatct
39060cagttaactg tgtcccagtt cacgcttcca aaaagtgttt cagatggcat tgctgctttg
39120gatctaaatg cagtagccaa caagatcgca gactttgagt tgcccaccat catcgtgcct
39180gagcagacca ttgagattcc ctccattaag ttctctgtac ctgctggaat tgtcattcct
39240tcctttcaag cactgactgc acgctttgag gtagactctc ccgtgtataa tgccacttgg
39300agtgccagtt tgaaaaacaa agcagattat gttgaaacag tcctggattc cacatgcagc
39360tcaaccgtac agttcctaga atatgaacta aatggtaaga aatatcctgc ctcctctcct
39420agatactgta tattttcaat gagagttatg agtaaataat tatgtattta gttgtgagta
39480gatgtacaat tactcaatgt cacaaaattt taagtaagaa aagagataca tgtataccct
39540acacgtaaaa accaaactgt agaaaatcta gtgtcattca agacaaacag ctttaaagaa
39600aatggatttt tctgtaatta ttttaggact aacaatgtct tttaactatt tattttaaaa
39660taagtgtgag ctgtacattg catattttaa acacaagtga aatatctggt taggatagaa
39720ttctcccagt tttcacaatg aaaacatcaa cgtcctactg ttatgaatct aataaaatac
39780aaaatctctc ctatacagtt ttgggaacac acaaaatcga agatggtacg ttagcctcta
39840agactaaagg aacatttgca caccgtgact tcagtgcaga atatgaagaa gatggcaaat
39900atgaaggact tcagtatgga gcttttattg aattgaaacc ttataccttt tgaaaactca
39960ttgtgatttt cttcatctcc ataccccttt cgtgatagct catctgtttt tctgctttca
40020gggaatggga aggaaaagcg cacctcaata tcaaaagccc agcgttcacc gatctccatc
40080tgcgctacca gaaagacaag aaaggcatct ccacctcagc agcctcccca gccgtaggca
40140ccgtgggcat ggatatggat gaagatgacg acttttctaa atggaacttc tactacagcc
40200ctcaggtaaa taccacctaa tgagtgacac gcccccaaga gcgagtggag aattggggca
40260gatacattta attcaggacc aaatattcag agattcccca aactaggtga aagacaggcg
40320gtaagcaact tcttctctga ggaaatattc tctagaaagt attacaatga gtccttgatt
40380gattttaatg tttagatgca cacatgacat cccatcagca ctattattta ttaattctgg
40440gcaaatccag gaagatgagg gttatacctc atcatctaaa tcataggcaa gctcagccat
40500aggcagggta tatttttcag agaggactgg tttctgtagt atttaaaact ttaaaattct
40560tccccacaat agaattgcta gatgagatac atcaaattcc tctcatgtca tttacaagct
40620ctgccagggc caaatcaagg gtgacattac cagaggagaa gaccaaacat ggttctatga
40680ctgttactaa aagtttgtca tgggcttgga gaatgcgtac tgatgttggg attctgggtc
40740tctgcagggt gggctccaac ttgccttttt tgctatttct tcttttccta tctgtcattt
40800cctgactctt cttctctctc ctcttctttc tcttcccccc actcctcttc cagttttcag
40860tcctaggaag gctttaattt taagtgtcac aatgtaaatg acaaacagca agcgtttttg
40920ttaaatcctt tctggggcat gtgataaaga gaaattaaca acagtagact tatttaacca
40980taaaacaaac acatgaactg acatatgaaa gataaatccc tttcagtata tgaaagattc
41040tctgatcttt atttttaact gctaatgaag ttttagtgta ctatattgtg taattggagt
41100aattgaaaac atgttatttt tttttttctc tctgtttagt cctctccaga taaaaaactc
41160accatattca aaactgagtt gagggtccgg gaatctgatg aggaaactca gatcaaagtt
41220aattgggaag aagaggcagc ttctggcttg ctaacctctc tgaaagacaa cgtgcccaag
41280gccacagggg tcctttatga ttatgtcaac aagtaccact gggaacacac agggctcacc
41340ctgagagaag tgtcttcaaa gctgagaaga aatctgcaga acaatgctga gtgggtttat
41400caaggggcca ttaggcaaat tgatgatatc gacgtgaggt tccagaaagc agccagtggc
41460accactggga cctaccaaga gtggaaggac aaggcccaga atctgtacca ggaactgttg
41520actcaggaag gccaagccag tttccaggga ctcaaggata acgtgtttga tggcttggta
41580cgagttactc aagaattcca tatgaaagtc aagcatctga ttgactcact cattgatttt
41640ctgaacttcc ccagattcca gtttccgggg aaacctggga tatacactag ggaggaactt
41700tgcactatgt tcataaggga ggtagggacg gtactgtccc aggtatattc gaaagtccat
41760aatggttcag aaatactgtt ttcctatttc caagacctag tgattacact tcctttcgag
41820ttaaggaaac ataaactaat agatgtaatc tcgatgtata gggaactgtt gaaagattta
41880tcaaaagaag cccaagaggt atttaaagcc attcagtctc tcaagaccac agaggtgcta
41940cgtaatcttc aggacctttt acaattcatt ttccaactaa tagaagataa cattaaacag
42000ctgaaagaga tgaaatttac ttatcttatt aattatatcc aagatgagat caacacaatc
42060ttcagtgatt atatcccata tgtttttaaa ttgttgaaag aaaacctatg ccttaatctt
42120cataagttca atgaatttat tcaaaacgag cttcaggaag cttctcaaga gttacagcag
42180atccatcaat acattatggc ccttcgtgaa gaatattttg atccaagtat agttggctgg
42240acagtgaaat attatgaact tgaagaaaag atagtcagtc tgatcaagaa cctgttagtt
42300gctcttaagg acttccattc tgaatatatt gtcagtgcct ctaactttac ttcccaactc
42360tcaagtcaag ttgagcaatt tctgcacaga aatattcagg aatatcttag catccttacc
42420gatccagatg gaaaagggaa agagaagatt gcagagcttt ctgccactgc tcaggaaata
42480attaaaagcc aggccattgc gacgaagaaa ataatttctg attaccacca gcagtttaga
42540tataaactgc aagatttttc agaccaactc tctgattact atgaaaaatt tattgctgaa
42600tccaaaagat tgattgacct gtccattcaa aactaccaca catttctgat atacatcacg
42660gagttactga aaaagctgca atcaaccaca gtcatgaacc cctacatgaa gcttgctcca
42720ggagaactta ctatcatcct ctaatttttt aaaagaaatc ttcatttatt cttcttttcc
42780aattgaactt tcacatagca cagaaaaaat tcaaactgcc tatattgata aaaccataca
42840gtgagccagc cttgcagtag gcagtagact ataagcagaa gcacatatga actggacctg
42900caccaaagct ggcaccaggg ctcggaaggt ctctgaactc agaaggatgg cattttttgc
42960aagttaaaga aaatcaggat ctgagttatt ttgctaaact tgggggagga ggaacaaata
43020aatggagtct ttattgtgta tcataccact gaatgtggct catttgtatt gaaagacagt
43080gaaacgaggg cattgataaa atgttctggc acagcaaaac ctctagaaca catagtgtga
43140tttaagtaac agaataaaaa tggaaacgga gaaattatgg agggaaatat tttgcaaaaa
43200tatttaaaaa gatgaggtaa ttgtgttttt ataattaaat attttataat taaaatattt
43260ataattaaaa tatttataat taaatatttt ataattaaaa tatttataat taaatatttt
43320ataattaaag tatttataat taaatatttt ataattaaaa tatttataat taaatatttt
43380ataattaaaa tatttataat taaatatttt ataattaaaa tatttataat taaatatttt
43440ataat
43445243445DNAArtificial Sequencereverse complement sequence of SEQ ID
NO. 1 2attataaaat atttaattat aaatatttta attataaaat atttaattat aaatatttta
60attataaaat atttaattat aaatatttta attataaaat atttaattat aaatacttta
120attataaaat atttaattat aaatatttta attataaaat atttaattat aaatatttta
180attataaata ttttaattat aaaatattta attataaaaa cacaattacc tcatcttttt
240aaatattttt gcaaaatatt tccctccata atttctccgt ttccattttt attctgttac
300ttaaatcaca ctatgtgttc tagaggtttt gctgtgccag aacattttat caatgccctc
360gtttcactgt ctttcaatac aaatgagcca cattcagtgg tatgatacac aataaagact
420ccatttattt gttcctcctc ccccaagttt agcaaaataa ctcagatcct gattttcttt
480aacttgcaaa aaatgccatc cttctgagtt cagagacctt ccgagccctg gtgccagctt
540tggtgcaggt ccagttcata tgtgcttctg cttatagtct actgcctact gcaaggctgg
600ctcactgtat ggttttatca atataggcag tttgaatttt ttctgtgcta tgtgaaagtt
660caattggaaa agaagaataa atgaagattt cttttaaaaa attagaggat gatagtaagt
720tctcctggag caagcttcat gtaggggttc atgactgtgg ttgattgcag ctttttcagt
780aactccgtga tgtatatcag aaatgtgtgg tagttttgaa tggacaggtc aatcaatctt
840ttggattcag caataaattt ttcatagtaa tcagagagtt ggtctgaaaa atcttgcagt
900ttatatctaa actgctggtg gtaatcagaa attattttct tcgtcgcaat ggcctggctt
960ttaattattt cctgagcagt ggcagaaagc tctgcaatct tctctttccc ttttccatct
1020ggatcggtaa ggatgctaag atattcctga atatttctgt gcagaaattg ctcaacttga
1080cttgagagtt gggaagtaaa gttagaggca ctgacaatat attcagaatg gaagtcctta
1140agagcaacta acaggttctt gatcagactg actatctttt cttcaagttc ataatatttc
1200actgtccagc caactatact tggatcaaaa tattcttcac gaagggccat aatgtattga
1260tggatctgct gtaactcttg agaagcttcc tgaagctcgt tttgaataaa ttcattgaac
1320ttatgaagat taaggcatag gttttctttc aacaatttaa aaacatatgg gatataatca
1380ctgaagattg tgttgatctc atcttggata taattaataa gataagtaaa tttcatctct
1440ttcagctgtt taatgttatc ttctattagt tggaaaatga attgtaaaag gtcctgaaga
1500ttacgtagca cctctgtggt cttgagagac tgaatggctt taaatacctc ttgggcttct
1560tttgataaat ctttcaacag ttccctatac atcgagatta catctattag tttatgtttc
1620cttaactcga aaggaagtgt aatcactagg tcttggaaat aggaaaacag tatttctgaa
1680ccattatgga ctttcgaata tacctgggac agtaccgtcc ctacctccct tatgaacata
1740gtgcaaagtt cctccctagt gtatatccca ggtttccccg gaaactggaa tctggggaag
1800ttcagaaaat caatgagtga gtcaatcaga tgcttgactt tcatatggaa ttcttgagta
1860actcgtacca agccatcaaa cacgttatcc ttgagtccct ggaaactggc ttggccttcc
1920tgagtcaaca gttcctggta cagattctgg gccttgtcct tccactcttg gtaggtccca
1980gtggtgccac tggctgcttt ctggaacctc acgtcgatat catcaatttg cctaatggcc
2040ccttgataaa cccactcagc attgttctgc agatttcttc tcagctttga agacacttct
2100ctcagggtga gccctgtgtg ttcccagtgg tacttgttga cataatcata aaggacccct
2160gtggccttgg gcacgttgtc tttcagagag gttagcaagc cagaagctgc ctcttcttcc
2220caattaactt tgatctgagt ttcctcatca gattcccgga ccctcaactc agttttgaat
2280atggtgagtt ttttatctgg agaggactaa acagagagaa aaaaaaaaat aacatgtttt
2340caattactcc aattacacaa tatagtacac taaaacttca ttagcagtta aaaataaaga
2400tcagagaatc tttcatatac tgaaagggat ttatctttca tatgtcagtt catgtgtttg
2460ttttatggtt aaataagtct actgttgtta atttctcttt atcacatgcc ccagaaagga
2520tttaacaaaa acgcttgctg tttgtcattt acattgtgac acttaaaatt aaagccttcc
2580taggactgaa aactggaaga ggagtggggg gaagagaaag aagaggagag agaagaagag
2640tcaggaaatg acagatagga aaagaagaaa tagcaaaaaa ggcaagttgg agcccaccct
2700gcagagaccc agaatcccaa catcagtacg cattctccaa gcccatgaca aacttttagt
2760aacagtcata gaaccatgtt tggtcttctc ctctggtaat gtcacccttg atttggccct
2820ggcagagctt gtaaatgaca tgagaggaat ttgatgtatc tcatctagca attctattgt
2880ggggaagaat tttaaagttt taaatactac agaaaccagt cctctctgaa aaatataccc
2940tgcctatggc tgagcttgcc tatgatttag atgatgaggt ataaccctca tcttcctgga
3000tttgcccaga attaataaat aatagtgctg atgggatgtc atgtgtgcat ctaaacatta
3060aaatcaatca aggactcatt gtaatacttt ctagagaata tttcctcaga gaagaagttg
3120cttaccgcct gtctttcacc tagtttgggg aatctctgaa tatttggtcc tgaattaaat
3180gtatctgccc caattctcca ctcgctcttg ggggcgtgtc actcattagg tggtatttac
3240ctgagggctg tagtagaagt tccatttaga aaagtcgtca tcttcatcca tatccatgcc
3300cacggtgcct acggctgggg aggctgctga ggtggagatg cctttcttgt ctttctggta
3360gcgcagatgg agatcggtga acgctgggct tttgatattg aggtgcgctt ttccttccca
3420ttccctgaaa gcagaaaaac agatgagcta tcacgaaagg ggtatggaga tgaagaaaat
3480cacaatgagt tttcaaaagg tataaggttt caattcaata aaagctccat actgaagtcc
3540ttcatatttg ccatcttctt catattctgc actgaagtca cggtgtgcaa atgttccttt
3600agtcttagag gctaacgtac catcttcgat tttgtgtgtt cccaaaactg tataggagag
3660attttgtatt ttattagatt cataacagta ggacgttgat gttttcattg tgaaaactgg
3720gagaattcta tcctaaccag atatttcact tgtgtttaaa atatgcaatg tacagctcac
3780acttatttta aaataaatag ttaaaagaca ttgttagtcc taaaataatt acagaaaaat
3840ccattttctt taaagctgtt tgtcttgaat gacactagat tttctacagt ttggttttta
3900cgtgtagggt atacatgtat ctcttttctt acttaaaatt ttgtgacatt gagtaattgt
3960acatctactc acaactaaat acataattat ttactcataa ctctcattga aaatatacag
4020tatctaggag aggaggcagg atatttctta ccatttagtt catattctag gaactgtacg
4080gttgagctgc atgtggaatc caggactgtt tcaacataat ctgctttgtt tttcaaactg
4140gcactccaag tggcattata cacgggagag tctacctcaa agcgtgcagt cagtgcttga
4200aaggaaggaa tgacaattcc agcaggtaca gagaacttaa tggagggaat ctcaatggtc
4260tgctcaggca cgatgatggt gggcaactca aagtctgcga tcttgttggc tactgcattt
4320agatccaaag cagcaatgcc atctgaaaca ctttttggaa gcgtgaactg ggacacagtt
4380aactgagatt caggcacggt tatctcaaaa aagggaatca aggagtcttc tggttgagaa
4440tattttgtta acacatcaac ttcagggaat tttacctcgg ggagtgttgg taggttgagg
4500gcaaatgatg aagttctcag cttcttatag atttgtattt ctctgaagtc aagtttgcac
4560gatggaacct gaagatctgt aaatgggaca tggaacgtag gcatgacaag aactgaattt
4620agatcattta gtttcagccc aggaataatg aatttatcag ccaaaacttt tacagggatg
4680gagaatgaat agccattggg gtttttggtg tacacaaagg cagttgaaac acgaagatgc
4740tgtctcctac caatgctggt ggttacatcc agctttagga aatcccataa gctcttgtca
4800tagactggta ggatgatatt tttgaggaac cttaggtgtc cttctaagga tcctgcaatg
4860tcaaggtgtg ccttttcttg gtcattggaa agctcgacct ggctctggaa agacccagaa
4920tgaatccgga cttcattttt ccatctgatc ttctggttct tagtgttagc attcagggcc
4980acttcctggc caaggtcagg gaaatcatgg aaggaactgg gctgacttgc atggacctga
5040acaagagctg acatttgcca tggagagagt tccagggtgg ctttgcttgt atgttctccg
5100ttggtgaaaa agaggccctc tagctgtaag tggtttttcg tactgtgctc ccagagggaa
5160tatatgcgtt ggagtgtggc ttctccagca aaattttctt ttacttcaag gttccagata
5220tcatcaattt tggaagtgcc ctgcagcttc actgaagacc gtgtgctctt ggaattcaag
5280taagtgttgg cctcactagc aatagttcct gaatattccc gagaaagaac cgaacccttg
5340acatctcctt tggtagatga ctcaatggaa aagtaagagg tgaggctttc caagctaagc
5400ttgtggtcaa ctgctccttt agcggtagag tacagcattg aagaattgaa atcatactta
5460aattccatgg aggaagagac agtaggtttt gacttggtat ttccattaag ttcttgcttg
5520aaattcattc tcaaaattgg aatttgggct tttgtggttg ttgccactga cacttccata
5580tttttcgtgg ttaagctcac agtactgtta tgactaccct ccacaaattt gttgctcaga
5640gacagagctg tggctaactt caatcccctt tttcttgtca atcttgtggt gccctctaat
5700ttgtactgca gtgcatcaat gacagatgaa gatgaagaaa ggagatgagc aacaatatct
5760gactggttaa aaagttcagc attggtattc agtgtgatga cacttgattt aaaggagaaa
5820tcataggtaa tattgcccat ggcaggaata aaaatatggc ttatggtaca caattccttg
5880aaatctggaa gagaaagctt gagatttcta gggacatgaa ggactggcag ctctaatgat
5940ggcaggatta atgtgtatga aggcacacgg acgtcagaac ctaggatgga gaaactaggc
6000atgctgactg cttttgggaa cacatagccg aatgccgaca tctctatggt gaatggagac
6060acttcaacat tgacaactgg aacagtgtat ccaggaattt gaaaggtcct ggggagctcg
6120tcgtgagatt tttcagcttt gtacttatca aacttaattt ttgtttcatt ataggatttg
6180gtgacaaaat ctaatgcatt gtttctgttt ttttcaaaat gcctgtcaaa ggatttgatg
6240ctctgactga taaactcaca aagcacagcc aaaggatttg tgatggaatg cctgtgtttg
6300tttttcttat actgagcttt tacacttaaa tcaaatgatt gctttgtcgt tttcaagaat
6360tccttcaagc ctgttttttc ccatagagag aaatctttca gtggaggagt tgtgattatt
6420gtgtaaggta gacgcatttc aggaattgtt aaaggaatgt ttaagaaatc cagatttgct
6480tctccattta ttcctacatg ggcctccata atgttctcgt tgtttccagc agagaaattt
6540tggttgtact tatactgatt gaacctagca cttacttgcc aacttgcttg ctgggcactg
6600ggactcagaa acagtgcata gttattcagg aagtctatct tccctgttaa ccttaatgga
6660aaacgaactt tcaaattccc ttcattgttt gtggatgccg tgatctcaaa tggctgggct
6720gaaaagaaaa gagaattttt caaagttcca ataacctttc catttaaatg agcatcatgc
6780ctcccagtaa actctgcctt cccttctcca aacagtgcca tgcctttagc agttagaaca
6840ctgtggccca catgctggga atcgacttgt gattgaattt caagtttaga aaagttgagg
6900gagccagatt cataaaccaa gttttggttt actcttaggt gtttgctatt gatcttattg
6960gacagtccaa aggaagtgag gggtccttct atggtgaaac taatttgtga ttcatgtgtt
7020ccctcatctg agaatctggg gcaggcccat ttccatgacc cttttccaga agaagtccat
7080gctatgtggc cagctttcaa cagtgtcttg atctcgttgc gcaggtcagc ctgactagag
7140aagtccagtt tggggatgtt caatttgtgg aagtatttag tgttgctatc cagggtaagc
7200tgattgttta tcttgacaat cactccatta ctaagctcca gtgtattttt ttctgtgtgt
7260aaacttgcca ctgtgtttga ttttccctca atagcatttc caaaaaacag catttcactc
7320ccatgctccg ttctcaggta cttgctggag aacttcactg actccttcag agccagcgga
7380ttaatcttag ggtttgagag ttgtgcattt gcttgaaaat caaaattgag aacttctaat
7440ttggactctc ctttggcagt gatggaagct gcgatacctg cttcgtttgc tgaggtggtt
7500ccattcccta tgtcagcatt tgcatctaat gtgaaaagag gagattggat tttcagaata
7560ctgtatagct tgccaaaagt aggtacttca attgtgtgtg agatgtgggg aagctggaat
7620tctggtatgt gaaggtcagg aacttgaaaa tcattaaggt tgagagttgg gattatgaat
7680tctggaattg cgatttctgg taaacggaag tctggcaggg tgattctcgc tagaggaatg
7740tcctccacct tcagatccct gagatatata tctggaacgg gccactgcag ctcactgttc
7800agcatctggt caatggttct gatgatcttt actttcattt ctacaaagtc aattgtaaag
7860gaaggaatgt ggaaggtgtt aaggatggta aattctggtg tggaaaacct ggatgggatt
7920tttatatttt ttaagtcttt gaagtttatc tgaactgatg gaatcctcaa atctgttagg
7980gggactataa aatcaggtgt ctggaaggta gctttctgaa gagcctgaag actgacttca
8040aaggcaggca tggtcccaag gatggtcttg atttcaggaa cagtgaaccc ttgctctacc
8100aatgctttca tacgtttagc ccaatcttgg atagaatatt gctctgcaaa gtcagtaagg
8160ttcttagcag caagagtcca ccaatcagaa atgtaggtga caagtgtgct ataaacctgg
8220cctaccagag acaggtatcg ttgaagttcc tgctgaatgt ccatttgata cattcggtct
8280cgtgtatctt ctagggtctc tcggaatttg gccttcatgt gagccaaaga tgctgaactt
8340aaagcctcct gtaaccaatt gatgattaag gttattttgg tgtcctgtag gctttccaga
8400tacactgcaa ctgtggcctt ggtttcctct aaaaacagtt ttaatgcttc agctttttgt
8460ggtagttcca gagcctgaat ttcaccattg agtctctgag tcacctcacg gattttgtca
8520ttggtttcat ctacaaactg gtggtaatca aatgacttta atttctttat caacatgtca
8580aggaatttgt taacatcttc aatgaatgtt ttaaaagata attcattaag cttcttgaca
8640gcatcatcaa taaatccaac caatttctca aagtaatctt ttatcttaac ttgttgtagg
8700acattgctta gcttctgaat agtctccttc aacttgtatt ggtgggccaa ctctactaat
8760ttatccatta aaacctggat ttgttggtct acttcatacc tctcgattaa ctcatggact
8820ttggctctga aggcattgat tttctcagct acttcaaaat ccccaataag atttataaca
8880aagtgtttga catgctcaag aatgtcattt attctttcaa atgaaattgt agttcccaat
8940tgatctaaaa gcactctaac atcaatagcc tcaatgtgtt gttttaactt tccagctagg
9000tgctggatgt ctatattctg tatgtgtctc ttaagctgct gcagtttttc ttgtatctgg
9060attctgattt ggtacttagt atccacattt tgaatccagg atgcagtact acttccactt
9120ttgttaaaat caatattttc aataaacaaa tgtagatcat ggattgtttt tactaaattt
9180acacggatat gatagtgctc atcaagactt tttaattttt caatgatttc atcaataata
9240ttagcaatag ctattttcaa atcatgtaaa tcataactat ctttaatata ctgatcaaat
9300tgtatcatat atgtctgcag ttgagatagt ttttcattaa agttgatttt ggcatcatct
9360aatgcaattt gtatatcatt ttctgtaatt ctatactttt ttgtgagagc agtcagtttc
9420tccttggcat gtgaaacttg tctctcccaa ttgaatgaat tcagataatc attagcttgc
9480tgtgggagtt ttcccagggc tgctctgtat tttcttacaa attgatcaat attgatgtgc
9540ttcaggtttc tctgtacgtt ttccagtaca actataatgg tttgtcgatt cctctcaaaa
9600tattcttgca aggtctcaaa aaatgggagg ttaatggagt gaacatcttg gtttttatca
9660tactttacaa aagcaacaat tgtaaattct tggggcttct caacggcatc tctcatctct
9720aaagcatcaa tgatattgat gggctcactg agtaaaagtg gcactttaat tggggagtct
9780agtagagtta ggtcagccag agttcgtcca gtaagctcca cgccaatttt atctttagtg
9840ttgtaagcat ccaagtcctg gctgtattca ttgttgttaa attgggtctt gagtttccag
9900gtgcctgtct gctcagctgg agtaagcagg gcactgactt tgtgttcaag agctgcactg
9960atgcttttcc tagacacgag atgatgactt gtggagcctt tgtaatcatg agagaaagta
10020aatgccagag gttctgcttt caacaggaat ttgctataca gctgcccagt atgttctccc
10080cagagagcga gtttcccatt gccatttgta tgtgcatcga tggtcatggt aaacggggcc
10140attacagaac ggaagacatt gctgaaatgc agtgagtctg aattatagtt tgtgctcatg
10200tcaatggctg aagccagccc agcgatgtct gtgttgagcc gatggctaaa ctccacaccc
10260tgaaccttag caacagtgtc tgctttatag cttgctgata aggcagcaga agagatggca
10320tagatgtgtt ttatttcatt attttggtag gctcctttta ggttaccagc cacatgcagc
10380ttcaggggtt ctagccgtag tttcccattg ttggtgagat ccagagcatt gtatttcagg
10440tcactgttta aagtagttac cagagaatag ggctgtagct gtaaattaac agtttgctta
10500taaaacttgt cagagctgta aatgttgtca agttttgaag agaagtccag tgataagcct
10560gcaatgttca gactgtttgt gtggtcaaat ttcatttcag catatgagcc catcatgtca
10620tttgagagct taagtccttc ttgactgacc ttgaagttga aaatgttttt gctgtcgaca
10680cccagaatca tggcctgata agcacttccc agtgatagct ctgtgagggc ggctttccca
10740tccagactga attttgcatt gtgttccctg aagcggccat ttgttgttaa tttcatagat
10800gccccagaga ggccaagctc tgcattcagc tcattctcca gcaccaggag actacacttc
10860aagttggtcg ttgcactggt agatattcca tcttggccaa tccttagtgt cgccttgtga
10920gcaccactat taattttgtc agtgcctaag atgtcagcat ttaactcaag accatgggaa
10980tttagtgatc cagaaagcag gctgaagaac ctcaatgact cgtaatcagc ctgatattca
11040gaacgcagca gtgcattttg cttagagaag gtcatatcca tcttgttaga agtggcaaag
11100ttcttatact tcccattggt gtcagatttt aaagtcagct cgtagttctc atactttagg
11160gaagcagtat ttttaatgat gccactttgc agatcagagg tggaggtgag ggagagggtt
11220ccatcttcat atcttcctgt tatctggttg gtgccttgga ggtaggagga gttaaacctc
11280aggttggact ctccattgag ccggccagtg ttaggatccc tctgacaaga caggccatat
11340gtgcctttag catagaacga agagactctg aactgcccat caatcttgac ttctttgaca
11400aacaaatgct gtttcttttt ggagtccaaa tgaactgaag cagacatctg tggtccccag
11460gaactagatg catcgaatat tagtaaacct tttgagactg ggttgtttcc aagtttttct
11520acatgactga atttgatatt cgaatctaga aatttgtggc gtagagaccc atcatatgat
11580agtgtgaacg tattcttgtg gtcatatgtt gtttctccag atcctaacat aaaaatgaaa
11640agacattggt taaattaagc agtacatttc cagagcaatc tctatgttga aagtctttca
11700ataataaagc cccatttttc tgggccaatt gtgcaataga ctcctccatc tgtaatgcaa
11760agataaaatc tgatagcaaa aggcattcct ccaggaagcc ttgcctgacc accacaggct
11820ttgtttaggt tcccttgttg agtgcttcca ctctacctta tacttatata ctacacttta
11880tcatagcacc tctcacactg aattataatt gctggcttat ctgtctactt tctgttactg
11940cctactagaa tatttaatac acgtgatatg tttaataaat atttctattt gttgaataaa
12000ataaaagact tccaagtagc aaggaagatt atctgctaga aagccaaagt cctttcctcc
12060ctggaggagg ctctcctctt agagcctgcc atgaactagc ccggtgcacc ctttacctga
12120gcatagctca ccttgcacat tgtaggaaag caggtcaacc acagagtcag ccttcatgtg
12180gtaacgagcc cgaaggctga aatggtctgt gctggtgttg ccaccactgt aggaggcgga
12240ccagttgtac aagttgctgt agacattcgt ggagaggtct agaacaccca ggagaggcac
12300ttgcagttga tacaacttgg gaatggtaaa agtagggact tggaactctc gagatggcag
12360atggaatccc acagacttga agtggagggc tggtgtccta acagtctcta acatctttag
12420atctctggag gatttgccac caaaaggcaa aggaatctca attttcaaac tgttcttgtt
12480caaggtatat ttgacccggc catcgctgaa atgaacaaca aagataacat ccccacagtc
12540agacatcagt cattcaaagt tctctgcctc tgaccttcac aacaatattg tacttgcccc
12600attcccaaag ccactctgca cttttctttg tgctactcct atgcctggac ccctttgctt
12660ttacctccac ctgtcaaaca ctcaaatcct gacaaagctc ttctaagggc cactccttat
12720cattaccatc ccacctaagt catagccaat ttgtctcatt cttacagtta catagtccct
12780tgcacctttc tcaaagagca tgtcataatt ttgcttatat aattaattat ttctcatttg
12840ttgatgtctc catggtatct tcttgaaaga aaaggctata ccttacccag tttctatctt
12900ctcactgtac ctagtctagc attttgcaca taggggatac ttgatacaaa tttgctaaac
12960tgaattgaga caacttgatt tgtggaaaga acataggtat ggaagtcagt aagcctgggt
13020taaagtattt tctcagaatg ctgcctccat ttagtgctca atggctgttc tgactctctc
13080tagcttaaca gcaagcctgt agtcaataac gcctctagcc attctgaatt cttcacagtc
13140agagggagaa tttgcttata acaggttgta ctgaataaaa tatcttaggg tgaaagtacc
13200agaatcctct aatgactttc ttagattctt cctgacaggg ttggtgttct catgtttact
13260aatgcaaaga tgccacagtg tttgtgctat agttattaat ctccttaaaa tgcttaaatt
13320atttccttta aaaatttaaa aaaatgtcta aatcatgcta tgtaaaatct ttcatttact
13380ttgcatggtt caagaagcct tgctgctttc ttcttttacc tttttaagaa gaggttttct
13440gggatgtgga agtctggcaa tcccatgttc tggaggttga actccttcag gctattgagg
13500tggtcttgca aagtctgggt ataaggaaga ctcccagatg ccttctgaag ccatgagctc
13560attgcctaca aaatgacagg agatttttaa ggtaatgggc ttggatgagc ctcaaagagc
13620aatgaacatt aggcaaaaat accgatttga caagttaatt attaagctgg acaatgcact
13680gaaagttaaa aataaataac agaaaattat gaatcttcgt tgccagtcac tgatcactgt
13740ccatatttat aaaaggaatg tctaacatag tcagcctcct agctggtgtc ctgaagcttc
13800ctttgatacc actaggcaaa gaactgttct acttagtagc atctctttga tatgtatcac
13860attcagcttt gtgtaactgg gtaactacac acttgtgaaa gttttttttt ctccccaaga
13920attccctggg gggaaggaag catgccttat acatctttgg aaaccttcct gcacctagct
13980cagagttgag gatgtaatta gcacttatat ccatgtattt attgactggc agactcatac
14040ttacaactat taatttggaa cccacgtgcc ggaaagtcat gtctgtttga gggactctgt
14100gatccaggag tctattagca tacatatgca agctcttagg ataatcggag agatccacag
14160ggaaattgga agtcattttt ttggtatcta cattggtgcc tgtgttccat tcaaattcaa
14220tcttctcttc atctgaaaat acgtaggaaa tagttgtgaa tggtactagt tcagcctgta
14280accacaggtc tcaacacctg cattactttg gaagtgctca cacaggggaa gagacacata
14340ccataatgcc atgccaccct cttggaaact gtggagccat aagctgtagc agatgagtcc
14400atttggagaa gcagtttggc aggcgaccag tgggcgagga tctcacttct ggcttctgct
14460tgcaaacggg gtatggaaat aacacccttg atttttcttt cttcctttgt gtcacaacta
14520tggtaaagaa aatcagttgg caccaatgat tttgtccttt caatggagat atgcaggatt
14580aaacagaagt tccatttgtg gtgatcaaaa ccagatacaa ggatggttca gagaaacagc
14640cacagatcaa actcatcaac ttctaaagcc aacattcagc acaaattaag gactttgcaa
14700acaggtttag gagaaagaga agttttttga aaactgtttg aatcctgctc tgctatttac
14760tagctctgtg attttgacca aatgatttaa cccctctgag ctaccatttc cttatccatg
14820aaacaggaaa actcacagtg acctcgcagt ttattgtgag gaccgagata atttctttaa
14880agcacccagc actgtgcttg atacatgaat agtaagtgcc tgataaatgg taggaaaatc
14940caactggaca tgcgcagagg gttaaaatgt tgagagcttg gccgggtgca gtggcttaca
15000cctgtaatcc cagcactttg ggaggccaag gcaggtggat cacgaggtca ggagatcgag
15060accatcctgg tcaatatggt gaaaccctgt ctctactaaa aatataaaaa ttagctgggt
15120gtggtggcat gcacctatag ttccagctcc ttgggaggct taagcaggag aatcacttga
15180actcgggagg cggaggttgc agtgagccga gattgtgcca ctgcactcca gcctggtgac
15240agagtgagac tccatctcaa aaaaaaaaaa aattgttgag ggctctctct gccactctga
15300ttgtagacta ggggagaaca tggcttggtc aggtatgaag tggaagagga ataatgaaaa
15360gaaccaccca ggcctgcagt gcaggtcaga tgaccctcgg ccttctttac cttaggtggc
15420ccatgagggc gacctcagta attttcttgt tctgaatgtc cagggtgagt ctgtaagacg
15480ttttgccctc agtagattca tcattaactc tgaggattgt tccgaggtca acatcaaaat
15540ccggaatttg gacttcactg gacaaggtca tactctgccg attatatttg aatgtcatgg
15600tagcctcagt ctgcttcgca cctggacgag tgtataagag aatcaagaga tgtgtggtaa
15660gaagctatgt tttgggccgg gtgcggtggc tcacacctgt aatcccagca ctttgggagg
15720ccaaggcggg cagatcatga ggtcaggaga tcaagaccat tctggctaac atggtgaaac
15780cccgtctcta ctaaaaatac aaaaaattag ccgggcgcgt tggtgggtgc ctgtagtccc
15840agctactcag gaggctgagg caggagaatg gcatgaaccc gggaggcggg gcttgcagtg
15900agccgagatg gcaccactgc actgcagcct gggcaaaaga gcgagactcc atctcaaaaa
15960ataaataaat aaataaataa ataaataaac aaacaaataa ataaataaat aaataaagaa
16020gctatgtttt ggaaggaaag agaggaaaag gtagagaaac tccaaggaat caatgtgctt
16080aaacaaaaac acagttcagg attttactga attttttttt tctcattaaa attttttaat
16140gggtctcatt tactgataat tttaaactca gttgtgttca tgtatgcaag tctttatgga
16200ttgcctgtta tgagcctatt gtgtgctggg cactgctctt ggtactgggg aatccttgct
16260ttcatggggc ttaccttcta gtgggggagg tgtacaggca ataaacaagt aacttgctca
16320tgtcagatgg taatgaatgt tatgaagaag agtaaaactg gatacaagga taaaagaaag
16380ggatagaaga agtgttattt catacttggt ggtcagagaa attctctctg ataagggcac
16440atgtgggcag agaccagaat gaaaagaggt tggagccacg ctgagttctg ggaggagcac
16500attccaggca gaaggtcagg aatgcaaagg ccctgaaatg gggagcactt agcttgtttg
16560tggaacagca aaggctcgca gcccttctgg gccaggaaga cacgacatct ctatattact
16620ttctacccaa gatgcctgtc ctggttcctt gctgcccttg ccctgggctc catctcccca
16680actccccctt tctttgtaac aagattcccg ggcgtccttc aatccatagg ctggtccatt
16740ttatatcctt cgtaaaacaa tgtttaccaa acactatttc cccagcgtgc cctgctcctc
16800cagtgctttc tatcatggga ttttcttcct agtttaattc tgtgatatgc tatccctata
16860taaaaatttt tccaaagtga acacctaaat gtgccccatt ccctaaaacc tgctcctcgc
16920tatccctata taattttttt tccaaagtga acacctaaat ttctcccatt ccctaaaacc
16980tgctcctcca gtagcccttt ccatctcaat aaatggaagt tccatttttc agttgcccgg
17040gccaaaaacc ttaggtttgt cttcattccc cttttcctct tacactcaac atccactgta
17100tcagcaaatc ttgtcagcct aatcttcaaa atatggccag aatccatccc cttctcatat
17160ctcttctgcc attgccctga tccaaaacaa cactgattcc tgcctggatt attgtgataa
17220cctacatatt agtttctctg cccttgtctg cccatagtct tttctcaaca taaacataaa
17280tcaaatcatg aggactcccc aatggctttc gtttaattca tagaaaaagc caaagttctt
17340accatggcct atgagattat ccttccttca cctctccata tctctctgtc ctcatcttct
17400acctcttttc tccctggtct ttctgctcca gccatactgt cttccttcct ttccttgaaa
17460catggtagta gtgtgtcagc ctcagggcct ttgcacttgc tggtccctct gcctgttaac
17520agtcttacca cacggcttct cctcttattt ctgataggtc tccttatgct cactttacca
17580gggagacttt cccttattat tccaaatgca ctgctttccc tgcccttctt cctccctcct
17640actcctagcc ctccatagac cagtctacgc tctgttttcc tcctcagtac ttatctgata
17700ttccaaatat taacttgttt ggttgttcac tgtaacttcc atgaaggcag agactccatt
17760gttcactgtt ctagccccag gtcttagaat aggcctgccg cttattgggt actcagttaa
17820cccaggtctt agaataggcc tgccgcttag tgggtactca gttaacctat ttgttaaata
17880ggttaaatta catgtcttct cctcatgaat tctgaacctg agactgcgag cagagatgag
17940gcagctgtgt tttgaatact caccttctgc ttgagttaca aacttcaggg tatccaccaa
18000ggctctgtcc tctctctgga gctcataggt tgcgctgaca gaatactgct caatctctcc
18060tgtaggcctc agttccagct ctaatctaaa gacattacaa tgaagacagt gcataatgtt
18120agagctttca aggatggtga ttatgttccc aaaacaattc agcctcaaat gcaacaaaaa
18180tatggtcctt attttaacat tgtctcttgc ccttaactaa gatgatcttt ttgatccctc
18240agtcctacaa gaacccaagg caagggcaga caccaggatt cccaagactt tggacaagca
18300gagcaggagt tggctggttg tttcagcaaa gctgagaaca agcactggaa cttgggggct
18360gaagacaggg agccacctaa gtacggtgaa ggcttcaagc acattggtgg gctccaggaa
18420gctcatagcg atgggtctgg aggctcagaa acatggacac tgaattagga gcaaattggg
18480atctgtgctg tgatcccatc ccatgcacaa ggaagcaaaa ggtgacaccc aatgtgttgg
18540ccagaggacc ctgctttaaa tacccaaaga tggtgcaaac ccagaaggct agtgacaggg
18600tcttacaaca cagagtattt tttcctgtgc catgctaggt ggccatgatg tggaaggtga
18660gaaaatgctg ggtcaggcac tgagcatctc taacctggtg tccccggtca gcggatagta
18720ggaggcggag tctgtggagc tggcgttgga gtaagcgcct gaggtgcagt aattcaggcc
18780aggaaagact tgcttgcaaa ctgaccagga ctgcctgttc tcaatgagag gtgggatcac
18840ctccgttttg gtggtagaga ccaaatgtaa tgtgttgctg gtgaagaaca aaaatacctg
18900agttattgcc aagtcatgaa tcaaaatgga catcacaaat tctcagggtg caaatacccc
18960cttatcctcc tgtcttccct cagtccacac ctatctctaa ctatttaatt tactgagaaa
19020caaataatat tagaaagaac atcaacatga caagccatga gagtgactct gaagcttctg
19080gctgagttct ttccatctgt tacctgtaat ccccacaaca gtaggcttat ggttctcatt
19140ttacagaagc agaggctcag agatgttaaa tagctgatca ctggggctga acttcagtgt
19200gccccttcct tcctacaatg cagggcaaga gaaactgtct ctttcatatg aaatctccta
19260ctctctacct gcttccttcc acctgcatta agcacgctca agtctcttcc cctgtgcccc
19320tcccctatgc tcctttcttt cttttcttgc tttctcatct tcctcaccat caagagttgt
19380ctgcctttgc agtctctact tcctcccaat gtgctctgac tgctcactcc atctcctgaa
19440aatgctttca tcaatgtctc taatgccctc cttgctccta catccaatgg gcatatttag
19500gtttcatctt ccttgacctc ccagaagcct tccatagagt tgcgcactca tgaccagact
19560aagacgctgg agcctctcac atctgaagag atgatggcac cttccttcca agtgtgtact
19620taaaaactta gtacatgtaa gaaagttcaa cattggcctg gttttttcta aggatgcttt
19680ctttgaaata gcacattttc ctccaagtat tgtttctcca agttgggggt gccacaagac
19740caagctttgc tgatgtgctg gagtcctaat gccagctttc gcctacccat ggggtgatct
19800cccctgtggc tgctctttgg cttctgtgac cttttatggc tttggttctc cttctgcttc
19860tctggtcact tcttctaact cattgctggt tattcccctt cttcctttat atgtttgtct
19920tctccatatt tctgtcctca tctctcatgt ctcactctat accagttgct gtttaaccac
19980aacaacctct gtatactaat gattcccaaa tccatatgtg gagcccaagc ctctttactg
20040ggctcagatc cataggtcta tttgacagct agatttctaa actggttgtt tcataggagg
20100acatcaaata cagcacatcc aaaactgaat ttgtcacttt ccttgaaacc agaagctctc
20160ccactgtagt tcttattaaa atgaagggcg cctccttcct cttttcttct ttcttttgcc
20220ttgtccccat acccagttaa tcacccagta cttatgattc tgcctcttag aaagttgtag
20280aatctgctca ctcctttcta tacctggctt ttctccactg caggccatta tcagctctta
20340gccacatagg atcgtaagag atgtccaatg gtctcttcgt gtccagctgt gcctccttct
20400cacccatctt ccacactaga gtcagagtat ttcttctaaa acatcacccc tatcttattt
20460ctgctcccct tttcaactcc tgtgccttca aaagagtcca aactccttag gatggctttc
20520gcagctgggc atgctctctc ccctgcctac ctctccccat catctctgag ccccttacca
20580ttctgcactt cttccagttc cttgaaccat attcctttac cttcaggcca ttacatgctt
20640ttccttttgc ctggaacact cactcctttc tcctttgcct aaatgactct gacacagtcc
20700tcagttatca ggtcaagtac acttgcttca gaatgcattc cctgactagc acaggctaca
20760tgatataaaa tttcatggta ctctaatact ttctataact tagcccttac tctatgatta
20820ctgcttgctt aattgtgtgt ctcccttata aaatgtaagc gttcaaaggt cagggacctt
20880gtgtgtctca ctcaacatac gtggtatata ttaggcaatt aaatatacta ttttttgttt
20940tttgtgagac aggatcttgc tctgtctccc aggctggagt gcagtggtgc aagcatggtt
21000cactgaaggc ttgaactctt gggctcagtt gatcctccca tctcagcctt ctgagtagct
21060ggagctacag gcatgtgcca ccacgactgg ctaattttgt tatttattta attttttttg
21120tagagatggg gttttgccat gttgcccagg ctggtatcca aacaccaggg ctcaagcgat
21180ccatccacct ggggctccca aagtgctagg attacaggca tgaaccaccg tggctggcca
21240attaaatatt atttttgaat gaacacatga atgtgtctaa aatatgaaaa ctaaggcaaa
21300tcaatggttg agggtgccac ttaaaatgga actctccaaa agaggctgta ttccattatc
21360acgaggttag gagtttcatg ctttacctac ctagctacct caaatcaata tgttcttagg
21420tattttttgg gggggaaaat attaattttc caaagatgat ctctccagag ctattgtttc
21480ttcattctca aaacaaatag ataacaaaaa atcacaactt aataaaaact aattaagcag
21540taggtcttaa taagtctcaa agcagaaaga taaaggacag tttagtaaaa tgtgaaaagt
21600agaagaaagg taattatgta gttatgtagt aagtctaggc tagaattaaa ataggtaacc
21660cggaatcttt cctttctact cctctcctgc ttcctcagga cctgaatgat ctcaatcaac
21720tgtttagcct ggcaaaattc tgcaggtaca ttctctgttc tctctttcaa actggctagg
21780cagacttggc tgaaagaatt accctccact gagcagcttg actggtctct ttggggaagg
21840aatgataaac ttcagcttcc cagcttttag ggcaacatga gcctccagac ccgactcgtg
21900gaagaagttg gtgttcatct ggaccccact cctagcgaag tccggaatga tgatgcccat
21960atttgtcaca aactccacag acacggaggg ttttgccacc agttcagcct gcatctataa
22020gtcagaaaac aacctattca gattcattaa atacttcagt cccctgtcag tcagatcaac
22080caccctttct cacctccggg caaagatttc ctggataact cagacctttg gaaacattac
22140tgggatttgt ttggaagaag gaaagcgagt tctcagttct ctagagttgg agtatactac
22200ccagaactag aaagcacctg agttaacgtg aggcaaaacc agattaaaaa gatgatagga
22260atactcattt gttgagagaa ctgagttctt gtcttatggc cataccacta ctggagctag
22320tgaggtagtg cattctggtg gaagcttgaa gtttttcata aaaaaataat gaggtgatta
22380caatgatgaa gcattttgtc ttgaagtgga aacacatttt taaatatgtt ttaacaagaa
22440atgcaccctg gaagaaagta ataacctaag aaatcaaaag gcaaacagaa tcttacgttg
22500gctacttcca gttttactcc agccttggct ccgggagcaa tgactccaga tgaagatatt
22560tgcaactgta atccagctcc agtggggagt tcaaaggcat tctccatgaa gatgtagtga
22620agaaaaaagt cattctttga gcccttcctg atgacctctc caatctgtag acccaacaag
22680ggagagcaaa taaaagtaag tctttttaaa gtcggttttg ttaattacaa cctcccatac
22740attggagagt aattcctctt tatctggaaa ggaaggacta gcatattttg atgagctgaa
22800tagtttgata aataaagatc aaactgatgt ttttaccgtt ttcagtgaat caggatataa
22860gaaatttcac tagatagata tcatgtcatt ttcaagtgga ctcagaaggc tcttaagaat
22920attgctgggc ctcagagctt tatggacttt aagtatcata tatgccatgg cttaattctg
22980gtgttctgga gtctgactgt aagtgaaggt gtacttgaaa tgtattatat tatgataaaa
23040tcaatttttt gaaaccaaag gcctattgag atgatgtaca tatcaatttg cattgggttt
23100tattttttaa ataatatttc aataaaatag cttgagacct gttttcttaa agtcaacatt
23160tttttaatca agccatagga gtacatttat atatttcctt ttatgaatta ataataaaaa
23220ttatagcata tttagatagt gaattatagt ttgccaaata ctttctttta taatcattgt
23280atcatttcaa tcttaaaagg accttcaggt caggtgcagt ggctcacacc tgttattcca
23340gcactgtggg aggcccaggc tggaggatca cttgaggtca ggagttcgaa accagcctga
23400catggttgtc aacaggcttg tctctactaa agatacacaa attagccggg catgctggtg
23460cacgtccaca gtcccagcta cttgggaggc tgaggcagga gaattgcttg aacccggaag
23520gcagagttgc agtgagccga gattgtatca ctgcactcca gcctgggagc aagactccat
23580ctcaaaaaat taaaaaaata aaaataaaaa taaaaggacc ttcagcaaag gtagaggaag
23640gtataccgaa cattgttttc cagatgaata aattaatgct cttaaaaata cagctaacat
23700tatgaatagc ttaatttcta aatgttaaca tttccatgtg atccaagagt catcttcagt
23760gaaattcaag gcaaacctcc atctctgaag aaagtacaag catcttttcg ggcttgtgca
23820gctgggatcg taagggagtc tgggcgatct aaaaaaaaac caacgtctgg tctcatgggc
23880ccccagtggg gcctgctgac ttaccatctg ggggatcccc tgcagagtgc gggcacccat
23940cagaagcagc tttcccagga gctggaggtc atggagactg gcaaaaccaa gctcctctcc
24000caagatgcgg aggtaggctc tggcttccgg gacttctttg gatttcaaat ctttaatcag
24060cttctcaaca ctgagcatta ttccatttac catatcctga gagtttagta ataaaatggc
24120cagtgagatg tcagcaatgt caaacacctt tcagttccca atgtgggacc tgtctgatgc
24180agccaggatg ggcctaaaaa atgctccagg tgaggaacag ggagacactg aagtccaggc
24240taattcctct aagcagagat taaagaacta aggtcttgtc agggatagtg gcactcctaa
24300agctattctt aattaaaaaa tctgtagaca tagtaagatg tggtgggatg agaggagggg
24360tccctattct ccagtatgcc aggatcctca agattagaag atcctctatc ccccacccgt
24420tccctgctta gccttctctt ctttgaccag tgaagactac aaggtgtttc ttaagaaaat
24480gtagctgtta tataaattca ccagaagttt gaaaaacact tgatcctaga caagttcttc
24540tcagagagaa gaagaatctc agagatggat gcccaggcat tgggaaaaaa aaaatgtgaa
24600actgacagac tcaaagcaga tcaaagatcc tcggcctcaa gtgggggtca ggaaattaga
24660agatggagct cgtgctcaac agtgagcctg gaggggtaaa ttactgcctt gctctgcaaa
24720gttgaatgtg catacaagtc acatgaggtc ttgctaaaat gcgcttctga ttcagtagat
24780tgtgggtgaa gcatgagaag ctgtatttct gaccagacca cactttgggt agcaaaaaat
24840tcaagaataa gctccagatc atacaaccta tttccctcct taaatggggg ctcattgcta
24900gcttcaaagt ggaagtttgg tctcatttgc tagtgcactg atgggagtga gagccaaaga
24960cagcagttaa taataatgaa aatattaatt tagaactcgt aatagtgtct gcttattgaa
25020caaccaatgc cagcatcagg cactgtgctt ggcattttag atgccttatt tcattgtctt
25080taaataacct tgtgaggttt ggactgttat tagtagtctg ggatacagac agaaacagat
25140acacataata tcaaccaact tccccaagtg cgcacagctt caagacaggg aagttagaat
25200taaatctatg cccagagttc atactttttt tttttttaag atacagtctc acactgtcac
25260ccgggctgga gtgtaacggc gtgatctcgg ctcactgcaa cctccacctc ccgggttcaa
25320gcaattctcc tgtctcagcc tcccgagtag ctaggattac aggcacccac taccatgccc
25380agctagtttt tttgtatttt tagtagagac ggggtttcag tatgttggcc aggctggtct
25440caaattcctg accttgtgat ctgcccacct tggcctccca aagtgctggg attacaggca
25500tgaaccacca cacccagccc agagttcata ctcttgttat cctagagtac ttattgtgag
25560tctggtttct gggcacactg aattcaggag atctttaact gcagaggata ctggccctta
25620ccccagcagg tctggttgat aaaaaccata gttatccctt cagttagata gtattttgag
25680gacttccatg cttagaaaag aattgttttt gcattgagac ccaaagcttt ccttaagaag
25740atacttcaca aatacacacc tgctcatgtt tatcatcttt ggtatagcca aagtggtcca
25800ctaagacctt agagacacca tcaggaactt gaccattaac ccagtacaaa gctttgttga
25860cactgtctgg gaaaaatcct tgcttcccaa aaagagcttc caatgttggc tcaaagcctt
25920ttccttccaa gccaatctga gaaagaaaat cagacaagaa aatggcatca ggtttctttg
25980ttgtatgcca gcctagtagt ccccactctt gatgtccatt tatctaaaca agtatttgaa
26040taccacaggc caggagctga ccttagcatt tcacaggagc tgctttatta aaatctcact
26100agaatcttga ccttattact tgcttttttc tagaagaggc agctaaggtt cagataggac
26160aaataattca acaaaggatt cagagttggg agtgatttga gatttgaagc agatctgact
26220aggcagcaca tatgcttcct tggctttggg acttaaagac gagacatttg catttcaatt
26280tttttttttt tttttttttt tgagatggag tcttgctctg tcgcccaggc tggagcgcag
26340tggcgcaatc tctcggctca ctgcaagctc agcctcctgg gttcacgcca ttctcctgcc
26400tcatcctccc aagtagctgg gactacaggt gcctgccacc acgcccggct aattttttgt
26460atttttagta gagatgggct ttcaccgtgt tagccaggat ggtgtcgata tcctgacctc
26520atgatccacc cgcctcggcc tcccaaagtg ctgggattac aggagtgagc cacctcgcca
26580ggcctgcatt tctattaatg ttaagaagtc atgcttcagg tgacccctag cggggagaga
26640ggaaggcggg gaaggaggga aagaagaaag cctcagaatc atggtaggaa gtgcctggtg
26700gttcttagtt ttcctctggg tagctcctgg ctcccaggga ctctctgttt atgatgctgt
26760acaaaatggg ctagagaacc tcaaactctt cacacttacc tcgatgaggt cagctgaagc
26820aaatccaaag gcagtgaggg tagttttcag catgctttct ttaggaaggt agttatttgg
26880atcaaatata agattccctt ctattttggc tgaggctggg tcaagtgatg gaagagaaac
26940agatttgtag agttgatagt tccgagagaa ttttctgaag tccatgacag ttggaagttg
27000agattctttc agagcttctt tcactaactt tttcagacta gataagaaga agtatatttt
27060gagctgacac accatgttat tatcctttga ctcttgcacc ccaagtaaat atggatttcc
27120aatcactacc aaaatgtctt gatttcattg accctaagtc tttgggtcta gatctgctac
27180acatttgcac aagtgtttgt ttcagaagca agggcagaca gtggctatgc caaaacctag
27240ggttggaatt ccagctcagg gccctcagtg gtatatgggg tgaatagctc ttacttactc
27300ttggatatcc aattcttctg agttcaagat attggcaata tgggaagcca caaagttctt
27360cacttgctca ttctgttccc atggtagaat ttggacaatt ttgttaatat ctgcctgtga
27420aggactcctc atcaacataa gataggcagc cagtcgctta tctcccggag aagcatcatc
27480aaggaaagtc tgaagaagaa cctcctggtc ctgcagtcaa aagaggagat ggttatcact
27540gtcctgtggt cagaacacag aacatgcctg gcaaacactg gcattgtctg ctagctcttt
27600cttaagcaca ggtctaattt ctctgtaatc gttcttcaaa tgctggtatt gaatcttctt
27660gccagtgttt cctagagcac tatcaagtaa gggttcccag aaaaagcctg tggaatgcct
27720gttgaattca attccattaa aacacataaa ttatggccaa gttgtgattt gtttggagat
27780ccaccactaa gtgaaaatca agttgtttta acgtaagtta ttttaatgtg aggataaaga
27840tgagaagggg tagtaagagt ttggggctaa taaatagggt agtttccaaa gttctggagt
27900ctttgcacct aagagggggc caagttggct atcctagcct aagcttattg ggggaaaatt
27960aagcatgttt ttccatgatg agagggatgg tcaatctggt aggtggacgg taggaagcaa
28020aaacatctca cgttctgcat gttgacatag cagcaaagtc aagtatttct ctcacgtcac
28080atactgctag gcagtcaagt caataggctt gttgaaattt gcaattagaa aacagtagag
28140gccaggtgca gtggctcacg cctgtaatcc cagcactttg caaggctgag gcaggcggat
28200cacctgaggt caggagctca agaccagcct ggccaacatg gcaaaactcc atctctacta
28260aaaaatacaa aaattagctg ggtgtaatgg cacatgcctg taatcccagt tactcaggag
28320gctgaggcag agagaattgc ttgaacccag gaggcagaag ttgcagtgag ccgagatcat
28380gccactgcac tccagcctgg gcaatagagt gagattccat ctcgaaagaa ggaaggaagg
28440aaggaaagaa gaaagggagg gagggaggaa ggaaggaagg aaggaaggaa ggaaaggaaa
28500gaaacagcag aataaagtca gtagatgaaa tctagagtct cattccccta gtaccttcca
28560aatccttgtt aataaacttt cactttcaga cctcttcttg tggactttac cttgtcttta
28620ggctccattt tccgcagagc ctggatggca gctttctgga tcatcagtga tggctttgta
28680ctttggacac atttcaggat tgaagacttg agttctggag ttaactgctc catggtttgg
28740cccatatttc caatgacctg cattgaagaa aagaaacaag aacccatcag ggtgcaggag
28800agggaagtaa aaggtgtcca ggaaaagtgc ttctgaaatg atgtatgtca tataaaagac
28860tgagattacc cgcagaatca aataggtgta atcttcatcc ccagtgcagt catcttgaat
28920ctgttccatc aggtaattag caatgtccag cagctcctgg gtccctgtag ggtttgtctt
28980atgatagcta cagaataaga gaagagagtc aggacttggt aaccccagtt aggtttgtct
29040taaaacccaa acttgtgaat tagaaaaaat aattataaaa ttcatatgga atccaaaaag
29100agcctgaata gctgaagcaa ttttaagcaa aaagaacata gctggaggca tcacattacc
29160tgacttcaga ttatacaaca aggctatagt aatctaaaca gcatggtact ggcataaaaa
29220tagacacata gatcaatgga acagaatact gaacccagaa ataaagtcac atacttacag
29280tcaacggatc tttgacaaaa ttgacaaaaa catacactag ggaaaggaca cccttttcaa
29340taagtggtgc tgggaaagat ggattgccat atgcagaaga ataaaactgg actcctatct
29400gtcaccatat aaaaaaatca actcaagatg gatcaaagac ttaaatataa gacctgagac
29460tataaaaatg caagaagaaa acctagggaa aacttctctg gacattggcc tagacaaaga
29520attcgtgaat aagacctcaa aggcaaagac aacaaaaaca aaaaatagac aaatagaact
29580taattaaagt aaaaagtttc tgcacagtaa aacaaataat caacagggtg gtaacctgca
29640gaatgggaga aaatatttgc aaactattca tccaatgggg tactaatatc cggaatgtac
29700aagcaattca aacaactcaa caaaaaaatc ccccaaataa tcccattaaa aagtaggcaa
29760aggacatgaa tagacatttt tttcaaaaga agacatacaa atggcaatag gcatatgaaa
29820aaatgcataa cattactaat cattagagaa atgtaaatta aaaccacaat gagataacat
29880cttacaagag tcaggagggc cattattcaa aagacaaaaa ataacagttg ttggtgagga
29940tacagagaaa aggaaacagt taatcactgt tgatgggatt gtaaataagt acaaccacta
30000tggaaaacaa tacagagatt tctcaaagaa ctaaaaatag aactaccaat aaatccaaca
30060atcccattac tgggtatata cctaaaggaa aagaaatcat tatatccaaa ggaaacctgc
30120acccatatgt ttatcacagc actattcaca atagcaaaga catggaatca acctaagtgt
30180ctatcaatgg acgattggat taaaaatgtg aaatgtatag gcaataaaat actattcggc
30240cataaaaaga ataaaatcat gtcatttgca gcaacatgga tggaactgga ggtcattgtc
30300ttaagtgaaa caggacagtc acagaaagac aaatattgcg tgttgtcatt tataagtggg
30360agtgaaataa tgtgcacaca aggacatagt gtgtggaatg atagacaatt gagactcaga
30420aggtgtggaa gtggagggag atggatggtg agaaattact tagtgggtac actatatgtt
30480gtctgggtga tggataccct gaaagccctg acttaactac tgcacaatct attcatgtaa
30540caaaattaca cttgttccac ataaatttat ataaaaatgt aaaaaagaaa aagccaaaaa
30600atttcatcaa aggaaaaacc tgctagcctg agcaacatgg tgaaaccctg tatctaccaa
30660gaatacaaaa attagccagg gcatggtagc atgcacctgt ggtcccagct actcaggagg
30720ctgaggtggg aggatcactt cagcccagga atttgaggct tcagtgagcc gtgactgcat
30780cactgcactc cagcctgggt gacacagtga gaccatctca aaaaaacaac aacaacaaaa
30840aaaaacaaaa caaacaaaca aaaaaattca gaggaaaaaa ctgctaacta atattgaact
30900gaagttaata atatgcaagc tgtaatactt aggggaaagt gtgttgatgt ttgcaattta
30960ctttgaaatg cttcagaaaa taagatgaat taatggatgg atgtatatgt gataaagcaa
31020atataataac acttaaatag taggagatag ctggttttct aggggttttc cctgtgaaat
31080tctgtcaact ttgctatgtt caaaaacttt tataatataa tgtagagaaa atatttttaa
31140aaattcaatt tgtgtttgct gatttcttat ttcaagtcat taccccaaaa atgatcaaga
31200aaaaacacaa gagtaaggag cagagtttga aagtggaagg aggggttcag ttttaataca
31260gagatgcaca gaggtgcaag atgttcctct gctcctagga ggagaaatac agtgtggaaa
31320ctcacttgtt gaccgcgtgg ctcagcgcat acaaggtggc tcggctgcgc tgatccctcg
31380ccatgttgaa gatctctcgc agctgctgtg ctgagggctc ggggatcagg gccaccaggt
31440aggtgaccac atctatcaga agggggttgg catgcacacg tttcagccac tggaggatgt
31500gagtggagca ctgaggctgt ccacactgaa ccaaggcttg taaagtgatg gggctgagaa
31560gaaagacatg gataaagtta tacagaccac cttcagggca cataaaatat tgctcatggt
31620gtgtcctctg ccaggagagc ccttaatcac ctcattcact attcaaatct aacttcaaaa
31680cccaacttgg agctcagaac catgatgctt tccttaggaa tattacccac agaagaagcc
31740caatcgagaa aagttccaag aggggactgt ggatctgtag caacaagaag actgttatca
31800cagtcctttg gccagcacac agaatatgcc tggccaacat tagctgggtc tcattcctgg
31860cctcagtccc ttctctcacc ttcatagcac ttaaagccag tgccttgaag ggtagagaaa
31920tcatattata tttttcgtct gagttctctc tccaccagcc tataagcttc tggagagcac
31980ctgcaataga cctaggaatt aaaagttacc catcaaaatg tgggaactat tctccttctg
32040ttttaacaca caaatacata gctgccttga acacagtatg cgcgtgtgtg tttcatggaa
32100ctcagcgcag cagctgatag ttctctatcc agcaatcatg aactgattga ctaattgatt
32160aactggattg atatccaaat ggtccctgag attctccatt tggccaaggt ttgaaagttc
32220agtcagttac catcagtttt ataaaaagtt gagctgtaac cattagatac ctggacacct
32280caatcagctg tggcaagaga gatgtgactg cttcatcact gaggcctctc agctcagtaa
32340ccagcttatt gaagagatta gctctctgga tattttgctc agagatggtt agttttttca
32400gttcctggag agtcttcaaa acagcttcgg cctgctttgg aggtgatgtg gatttggtgc
32460tctcaaatgc gaggcccatc ttcttagtac ctggaagatg gaaagtgtca aaggaactct
32520agctttcttc atctcaacca tatctttgtc tactggaagc tggaaattgt ggagtatcag
32580cacaggggaa aagggaaaga aactatcctg tattcaatgc ctgctagatc tggctctgcc
32640ataggtgatt gacttgtttt cactgactca tcatcctatc cctgtggggt ggcattattg
32700ttcaatttaa tgataaattc agccctgctt ctgagaccca cagagttttt gggccatgta
32760aattgctcat ccctggatct cagctctgag tcccagagat gagacaaaga gatgagaccc
32820agagatgagg aagtgtgacc cagagatgag caacttaggt ttcccaagag ctctcagggt
32880ctaagaggta gagctaaagt ggaatccaga cttgtctgag tctatcatca ggttaccctt
32940agcttgaaat aattggtgag gaggtgctac tgttgacatg gttacaactg ggaccaggca
33000catgggtgct tatcaccatt tcttctaatt gggtctacca gcagcttgtg gggaggggag
33060acacttttgt atcctctcca ccacctactg tgacctatgc caggaagaga ggtaaaagtc
33120atcatggaaa tgtggtgtaa tgagaatagt cctggcttag attacaacct ggcacttatt
33180ttcccccctc taatattctg cggccttgac tacatcaaac tctaagcctc tctgtgtcac
33240tgtctcctct gtagaatagg tataaaatca catctctacc acctgacatc acaggttgct
33300atgagcaaaa aggacaatga ggtaatgtac aagaaggaat ttggacagat ataaatacaa
33360tacaatactg ggtaagggtt ggacttgaga ttgttagagt tccttctacc tctaaagact
33420gactcctgag gttactatct ggccagactc tgaaggttct ctcccacttg gtccacctcg
33480tctgggacag ctctggatct tatcacagga tgggtgatgt cagttctatc aaatatgtca
33540tgttaaactt aggattccag ttttctattt acaaatgcct aagtagcttt gggaacttct
33600tttcctttat gccattgcaa cttgacatca tgaaatgagt ttgctttctc actagaggta
33660gccagaacac cagtgtctgt atcacttgtt agtcagcaga catttaacag ggctcagcaa
33720gtggctaagc catgataggc acatcttgag tagattttcc agcaactatg tggacagaaa
33780ctcttacctt caccaaagaa gcggctgttg atctttggtg tgtcttcaag tttcaaagtc
33840tgtgtcactt gtgctaccat cccatactta ttcctggtaa ccaaggaagc acaccatgtc
33900acggatggcc agaacatact attctttcca tgttgaaggt tttccctgtc tctgcatcta
33960cccaagtcct gcccatcttt caaagcatgg gtaaatccag ccattgttgg ccctgaatga
34020ataacatcca cctcttcctg gtcacgctag ttatcctccc atcaaaaaga catgcgtgcc
34080ttatactctc cactagtctg gagtggccag aggtaggaac tagtgcaatg tctcaaaatt
34140ctttcgtatc ttccacaaca tagagccaag aatatggcag ttactcaata tatattcatt
34200gattatcagt tttcaaagtg gatttggata cacaagtttc cagtcagtat cccaaatctt
34260ccatgaaacc ttttctaacc agggtaccct gcagtgatcc tcaggttttc tggatctaca
34320gcccttaaag aatgggtctt agatatcttt tgttggtctt ggattgtttt gcatgcctag
34380gacttttctg ccaaatggcc tgttcgctta aaaaggggga cagggagggg cggcatttta
34440tccctctcca ttcctccctc ccaggcacag gtttgcctgg aacagagcac ttgagaagtg
34500ttcagttcac actgacccat taaatgacaa atcaggggtg catcacatga cctacttgta
34560ggagaaaggc aggaagaggt gttgctcctt gcagatggct tctgccacat gcttcctctt
34620agcgtccagt gtgtactgac aggactggct gctgctgatc agagttgaca aggggcgggt
34680ctatgaaaga gattggagac gagcattttg atcagtccct gtatcttctg ttaaaagcat
34740ttaccttcaa ggtagaaggg agcaggagtc ctgtaccact agataaactc agagaaagaa
34800gataactggg tacttgacat ttagaccagg gggtgcaaca agtcggcatc cccaaagctg
34860agtcaagctg tagactcctc tttttccacc ctcactggac aatgtctaaa ctttgtatcc
34920tccacatgcc catcccacca catagcccct cacacacctg ctcagtaact tttctttgcc
34980caaaagccca ttcttacatg tctctttcat ttacatattc tttgcatttt ttgggttcct
35040gctccacatc ttccatgaaa tgttgcccgc tgagctacat gaataattct ttctccttcc
35100tggtcaggcc caggacttct tgtctgcact gttcagctta tcaccttatt gagtatacca
35160cgtatacttt attgacctgg ctcctgcatt agactggaag catcctgagg ggaaggtcca
35220tggttcatct gccactgtcc cttccttggt gctgcccaga gtggggttga gcaaacagca
35280gatgctcaga aaagacccct aagccagtgc attatcctct tctgactgtg gcactcatgg
35340tcatcagact ccctgttttc aaccctaagg acaatgacca ggaacagcgc ccagcatgac
35400agtgattgcc caagttattc atgttcactg agacaggctg gggctcatat cagggaagat
35460gttgcagggg tcaggcaggc tcaggtaata tggtgctcca tggccccagt taacagagag
35520gagagggtgc tccaggccgg tggcagtggg gccatgtttg gtcagggtgg tactgggggt
35580ggaaggacaa gaagctggag cactctaagt cactccactt catgggatga gggagagggg
35640gagaacacag gtcttgggac ttcttcaaag gccagaggct tggagctggc acaaggagtc
35700cttgtgcaga aaagaacaga aggtggtttt gctgccctac atccatcccc ggaaccttct
35760cagaggtgag tagtcattac tctcaagtga tgagattaga ggcagtttgg tgccagtgac
35820agaaggacag gaggaaagac agagctctgg gctgggaggg aggagcctga ctcaagcctt
35880ggctcaactg atcccaggcc gtgaagcctg ggcagacctt cccgtgcctg ctcagggtcc
35940tatctctagt ctgtataaaa aacagccagg gcaggctgtc ctcaaaggtg cccactagct
36000caaaagttct ggaattgtat taataagagg atgctccttg ctgtgcacga cagtgctgac
36060atgggactta ccatgccttt gatgagagca agtgggctga tgcctgtgcg gatgggcttg
36120aagcgatcac actgccccag gtctctttca gtggatattt ctgttgccac attgcccttc
36180ctcgtcttga cggtaaagtg agtggagcag tttccataca cggtatccta tggaggaaga
36240agatgcaacc acatgtattc aacacgggca acatccttgg gtacttggga gggatggggt
36300ggggatcgtg aaagaaaaag cagaaggaat ctagctttgg gagggacttt ctttttcttc
36360ctatgcagag tgtggtcttg ctagtgcctg gccagcccaa gttgggagag agaaaaccag
36420ttaaaggtgg gtctgcagaa aggcctccgc aggttgcatc ggtgtcttct cccattatgg
36480tgtcgtagag aaattagaac acacaaaagt gctcatggga ctgaggtgaa atgaattctg
36540tctcacagtg aaacctgaaa ttcgtaaaag agaccagcaa cagatcccat tatttttggt
36600gtttaaaata cacacaggat ttactgtgta caccataacc cagcaaacac aggtgaagca
36660tcaaacaaaa gcaggggtgg ggtggccagg actcctcaat gactgtttta aaattagacc
36720caacctgata agcctgcttg ggatgatctg tcagtgagcc taaggggcag gaggaacctc
36780gggcaccagt atttcacgcc aatccagggc ttcctatgta actagtcatg gagctgactc
36840agtgatctgc tttgtatatg gtaggactgg tctctaacac atgaagatga gtttcaaggg
36900ccactgctat cagctttcta aatcctcacc agaaacaaca cttgcttggc ttcttctgtc
36960tctgggggaa ccaggagggc agaaatgatg cccctcttga tgttcaggat gtaagtaggt
37020tcatctttct ccgggtaaag gaaaacctgc ttcccttctg gaatggccag cttgagctca
37080tacctgtccc agagagagga tggtcacgga aatgtccttc tccattacaa cttgctggaa
37140gtaagctggg tggcactgaa gtttcttttc tcatattttt ttaaccaatt gttcttctga
37200catcatttat ttgtaaatat agaatatagc tacacataga catacatttt caaaaaagta
37260tgcacatata cataagtttt ggtgattctt tttttttctt tttttctttt ttctttttct
37320ttttttgaaa tggagtctcg ctcggtcgcc cgggctggag tgcaatggcg caacctcagc
37380tcactgcaac ctccacctcc tgggatcaag tgattctcct gcctcagcct ccggagtagc
37440taggactact ggcatgtgcc atcacaccca gctaattttt gtatttttag tagagacagt
37500tttcaccatg ttggccaggc tggtctcaaa ctcctgacct caggtgattc tccctcctcg
37560gcctcccaaa gtgctgggac tacaggcatg agccaccagg tccagctgtg ttttaacttt
37620ttttgcttgg ctcatccagc cctgctctgt tctccaccca tatcagtctc tttgcccatc
37680ctccccatac ggacacccga gtggttgttc catttgtttc tgcacactca tacaatcctc
37740tgtaaacatg ggtgagtgca aacagagtca tacaaagatt ttgccattgt ttgttttata
37800caagtggcat tgtattatgc atacttttat cttgtttttc tcacttaatt atacttcaca
37860aaaatccttc tgagtcactt ggtacagctc taacagattt ttttaaaagc tgcatgcaga
37920acattccatt ccatggctaa atataattta ttcacacatt gccccatttg atggcatttg
37980ccctctctct ttctggattt tttttaaacc acacaaacaa taccaaaaca aacatcctta
38040tgtgcctatc ttcacacatt actgctttta ttttcatggg ctagagtccc aaagactaaa
38100atatctgtgt caaagggtat taagtatttt aaattacagt agatattgcc tgatggctct
38160cgtttccacc agcagctcag gacagcacac ttttcctcac tggcctgcca gcaacaggtg
38220ctatcactgt tttccagttt tgcagtatga tgggtaaaag cagataaata tcttgttgtt
38280acattaattc gcatttcttg actaccagtg aattggagct tattttcata cacttgttgg
38340acagtcattc ttttgtatgg tgtcaggcca tggctgaagc ccatgctgac tttggtctta
38400gggatttccc actgggtact ctctgagttt ctgaggacct ggctgcagcc actctggtta
38460catcaggagg aaaaggtgac ggacttatct cccacagcct acactctctc agaaagttcc
38520caacacgaag aaatgataaa tgtttgagat gatgggtatg ctaattactc tgatctgatc
38580accatacatt acatgtatca aaatatcact atgttctcaa taaatatgta taattattgt
38640atgtcaattt ttaaaaaaga cagttaaagt catgaagatg caaacatgga aagccctcct
38700tttcaagaac aggagtctct tctgtatttg gagaagacca aggaccaaaa aataaaccac
38760tctttcctcc tgccaccata cccctctccc tcatatacag ttaagtcttg gagaaaataa
38820agcataggaa agaaagaagc ctcacagcct attttggcaa gaaaggactg gcatctgaat
38880ctaatgttta gcaaaactga aaaatgggca aggatggtga tatggtttgg ctctgcgtcc
38940ccacccaaat ttcgtctcgt agctctcata attcccacgt gttgtgggag ggacccgggg
39000ggaggtgatt gaatcatggg ggcaggtctt tcccgtgctg ttctcgtgat tgcagatggg
39060tgtcgggaga tctgatggtt ctaaaaacag gagtttctct gcacaagctc tctttgccag
39120ccaccatcta cataagatgt gacttgctgt ccctcacctt ccgccatgat tgtgagacct
39180ccccagccac gtggaactgt gagtccaata aacctttctt ttgtaaattg cccagtctca
39240ggtatgtctt tatcagcagc atgaaaacag actaatacag acagagtttt ctgatgctac
39300caagttaaca actctatcct gcctgtattc atacacacct aggaccctac aaactgtgat
39360tgaggatgag gcaggggtga tgttgaaaat atttacaatg ggtgcgacat gggccctgac
39420cagtcagcag agatgcagct gcagtggccg atcagcgtgc agtggctgaa tgccagccta
39480ggagggggag ccaccgaagc cttggtgctc ctctgccctg cggtgaacag accctgcccc
39540gccatgtgcc ggccacagca gccagtgcct ctgggacccc acaccaaaga ccaccaagca
39600ctagtcttga ctagttcttt aaataagaat tcacttgttt aaacaaagca tctcagtttt
39660ataatctcaa gaagtttaaa gcatgagatt tttagaaaca catgaaaatg ataattaaag
39720gaacctaact agggaaggtt aactaatgct tctctctttt tgatgattgt gaaaactcag
39780taattccctg atccacgatg gatacggaat acaaatactt acagtcacat ccgtgcctgg
39840tgcaaacaca caagttcata cctcagcgga cacacacaca tgcgtgtgct catgtacaac
39900atgacttacc tggacatggc tgcagcaaac tcctcagagt tcttggtttt cttcagcaag
39960gctttgccct cagggttgaa gccatacacc tctttcaggg tgcactggct ggtcttcagg
40020atgaagctgc agagctgggg aacctccagc tcaacctgag aattcagggt agcagagcat
40080tgaggttgtc tatcaagaat gagaggtggc ccctgaagcc cagggcttaa tctctaatca
40140gagcaccaaa gggaatggtg ctgggaacac cactgcctgc gcctcaacac cacatgcctt
40200atcaacatgc ctcctgggtt ctctgtgcac caacctagac ttagtcctat tgctgacgtt
40260ttccccctcc cgggtaacac atttcttaag tttgccccta ggcatgggaa ggaggatgtc
40320cttttattgg ttctaaagtt actcacttta attataaaga gctgtggtta aatagaagcg
40380ctgcagacta ggagtgaaag tgaagaagaa aaacagaaag caggaagagg gccatccttc
40440gtttccacag caaatgtctc cttaatgtca cccaaaaaac tggttatatc tattagggcc
40500tatttagagc tttgcatata gctggagttt caacggattc ctacttatta tccagcacta
40560ttcaaataac tttatagaaa tgttgtacat gtgtgctgtt caatatggga gccagtagcc
40620agggtggctg ttgagcagtt gaaatgcagc taatgtgaca acgaatatga atttttaatt
40680tcattccatt taaatagccc aatgtggctc atgtctacca tattggacag cacatacaag
40740agctacggta ctctctaaaa aaaggtgact gctcagcttg acttctcttc cccacccaaa
40800ccccaatagc agcctgtaga tctgctccac atgtatgtaa catgagtaca accagtctca
40860ataataagca aagttttatt ttagaacaaa ctgtatgttt atattttttt cttctttcca
40920ttcatttttc agcaacagat tctgtttgac ttaaattata aaaactgcat ttcacagtgc
40980gattccgagt tgcctgcctc ccatagctca cctactgggc ctctctcacg ctgaaatcta
41040cagacccaca ctgctgctaa tctagatcat ggattcctat tgcatctggg aagttaacgg
41100gaaaatactt ctgacttgcg aaatttggtg ggggcagacc acatctcagc aatgtggctg
41160acttacaaac agaggcaaag tcccagtgct ttgatcagat tacaacagtt ctgggatgtt
41220ctgcgtttgc tcagtacaca ccctccggga aggtcgcgtg ttgggcgccc gctggaacag
41280ggctggggga aagctgtggg ctctaggtcc ctcctgcctg catcctccat accttgcagt
41340tgatcctggt ggcacttctt gaatcagcag tcccagggac tccactggaa ctctcagcct
41400catagttgta tgtgtacttc cggaggtgct tgaatcgggt cgcatcttct aacgtgggga
41460gaaatacgtc agccacatag cagaaatagc tctcccaagg acagccaatt ctgggcagag
41520aggggcagtg gcatagagaa ttaaaaatgg taattcaact caaattattt tttaattgac
41580tttatactta attttccttt atagatttga cttctccatt atgtttttgc tgaggctaat
41640gtttaaattg gctggcatgt cttgaatatc tcatctgagg cacagggaag ggagtgacaa
41700gtgtcaagta atggggctca gaaaatctgc atgagagacc ctctggcaaa ttaagagatg
41760acaacattac gcaaatagta ctatctctaa tgctttttta gcttgaacac gccgtaattc
41820cgtgactcta agactaccaa atacttaagg caaagttcat taagcattaa gcgtaactaa
41880cttaacaaac tcctagaaaa gaatttctgg aggtttccac tgtagttgag aggaattttc
41940tgaacaattg aagaaaaaga acatctgggt cctgagtcca gctgcagtga tgacagacgg
42000ccactagatg gcggtgctgc cccatcagaa gtccgccccg cggctccagc acacagggtc
42060gcgcttggag gccagttcct ttaaccccca ggggtcaggt aaataggaag ggggtggagg
42120ttcgatttct tcacaaaggt taaagtcagt atttcctcac cctcacatgc ttcgaatcaa
42180tgactagtca ctgataaaca ggacagtgat gtttcttcaa agtgtgcaca ggccagcgga
42240gagacaatcc cttccctccc ccgcccccag gctgaacttt tgggacagaa cagaaggcca
42300ggtagaagag agttggcatc ccttgggtgt gggcagaggc tcagggaact tgcttcctgg
42360ggtcagagca aggcacacca cgatgccatc tcagccctgt agagtgggag gccctcaggg
42420acccgggtgt aggagagtgc acggggctgg gcgcccttcc acgccccatg cgcagatgcc
42480ttacttggac agaccaggct gacattttcc agcatttcct cttctgtaag acaggagaaa
42540gaaatctgtg agcttcccac gtcttcccag cgggtgctag ggcccgacag ggggaccacc
42600ggcacaggtt tcacctttca ggagggaggc aggctccgga gaccccctcc tcagcccctc
42660catcccgcgc cccccatcct gagcctgcag gggccgccag ctggtccaat ccccccactc
42720gccctggacc ctgtggctgc cctccctctg gcctaggccc aggctgcccc ggccaacctc
42780gtgccgccgg ctccctcccg ctccctctgc gcccgcagag cggccgcgca ctcaccggcc
42840ctggcgcccg ccagcagcag cagcagcagc gcaggcagcg ccagcagcgc cagcagcgcg
42900ggcctcggcg ggtccatcgc cagctgcggt ggggcggctc ctgggctgcg gcctggcctc
42960ggcctcgcgg ccctggctgg ctgggcgggc tcctcagcgg cagcaaccga gaagggcact
43020cagccccgca ggtcccggtg ggaatgcgcg gccggcgccc gcaccccatt tataggaagc
43080ccaggctgca agagcgccag gattgcaaaa ggtccaaagg gcgcctcccg ggcctgacct
43140gtttgctttt ctacactggc ttctctttga gccttgaaga gcctcgggga gggggcccac
43200ctgggatgca gccgcagcca ccaggggctg ggtcccaggt gggttccctt ccccaagcgt
43260cttcagtgct ctggcgcggc ccttcctgtg tctcagtggg gccatggcca gcgcctcagg
43320gtctgagaag cctgccctga ccaggggtgc cttcttcaga tgacccacca tggggacaaa
43380atctctgctt ctcctggact gaattgggag ccacgaggag aaccagtcct gagcgctgtc
43440ttggt
43445319RNAArtificial SequenceAntisense Oligonucleotide 3cgagaggcgg
acgggaccg
19419RNAArtificial SequenceAntisense Oligonucleotide 4gcucuccgcc
ugcccuggc
19521DNAArtificial SequenceAntisense Oligonucleotide 5cgagaggcgg
acgggaccgt t
21621DNAArtificial SequenceAntisense Oligonucleotide 6ttgcucuccg
ccugcccugg c
21720DNAArtificial SequenceAntisense Oligonucleotide 7tccgtcatcg
ctcctcaggg
20820DNAArtificial SequenceAntisense Oligonucleotide 8gtgcgcgcga
gcccgaaatc
20920DNAArtificial SequenceAntisense Oligonucleotide 9atgcattctg
cccccaagga 201014121DNAH.
sapiens 10attcccaccg ggacctgcgg ggctgagtgc ccttctcggt tgctgccgct
gaggagcccg 60cccagccagc cagggccgcg aggccgaggc caggccgcag cccaggagcc
gccccaccgc 120agctggcgat ggacccgccg aggcccgcgc tgctggcgct gctggcgctg
cctgcgctgc 180tgctgctgct gctggcgggc gccagggccg aagaggaaat gctggaaaat
gtcagcctgg 240tctgtccaaa agatgcgacc cgattcaagc acctccggaa gtacacatac
aactatgagg 300ctgagagttc cagtggagtc cctgggactg ctgattcaag aagtgccacc
aggatcaact 360gcaaggttga gctggaggtt ccccagctct gcagcttcat cctgaagacc
agccagtgca 420ccctgaaaga ggtgtatggc ttcaaccctg agggcaaagc cttgctgaag
aaaaccaaga 480actctgagga gtttgctgca gccatgtcca ggtatgagct caagctggcc
attccagaag 540ggaagcaggt tttcctttac ccggagaaag atgaacctac ttacatcctg
aacatcaaga 600ggggcatcat ttctgccctc ctggttcccc cagagacaga agaagccaag
caagtgttgt 660ttctggatac cgtgtatgga aactgctcca ctcactttac cgtcaagacg
aggaagggca 720atgtggcaac agaaatatcc actgaaagag acctggggca gtgtgatcgc
ttcaagccca 780tccgcacagg catcagccca cttgctctca tcaaaggcat gacccgcccc
ttgtcaactc 840tgatcagcag cagccagtcc tgtcagtaca cactggacgc taagaggaag
catgtggcag 900aagccatctg caaggagcaa cacctcttcc tgcctttctc ctacaacaat
aagtatggga 960tggtagcaca agtgacacag actttgaaac ttgaagacac accaaagatc
aacagccgct 1020tctttggtga aggtactaag aagatgggcc tcgcatttga gagcaccaaa
tccacatcac 1080ctccaaagca ggccgaagct gttttgaaga ctctccagga actgaaaaaa
ctaaccatct 1140ctgagcaaaa tatccagaga gctaatctct tcaataagct ggttactgag
ctgagaggcc 1200tcagtgatga agcagtcaca tctctcttgc cacagctgat tgaggtgtcc
agccccatca 1260ctttacaagc cttggttcag tgtggacagc ctcagtgctc cactcacatc
ctccagtggc 1320tgaaacgtgt gcatgccaac ccccttctga tagatgtggt cacctacctg
gtggccctga 1380tccccgagcc ctcagcacag cagctgcgag agatcttcaa catggcgagg
gatcagcgca 1440gccgagccac cttgtatgcg ctgagccacg cggtcaacaa ctatcataag
acaaacccta 1500cagggaccca ggagctgctg gacattgcta attacctgat ggaacagatt
caagatgact 1560gcactgggga tgaagattac acctatttga ttctgcgggt cattggaaat
atgggccaaa 1620ccatggagca gttaactcca gaactcaagt cttcaatcct caaatgtgtc
caaagtacaa 1680agccatcact gatgatccag aaagctgcca tccaggctct gcggaaaatg
gagcctaaag 1740acaaggacca ggaggttctt cttcagactt tccttgatga tgcttctccg
ggagataagc 1800gactggctgc ctatcttatg ttgatgagga gtccttcaca ggcagatatt
aacaaaattg 1860tccaaattct accatgggaa cagaatgagc aagtgaagaa ctttgtggct
tcccatattg 1920ccaatatctt gaactcagaa gaattggata tccaagatct gaaaaagtta
gtgaaagaag 1980ctctgaaaga atctcaactt ccaactgtca tggacttcag aaaattctct
cggaactatc 2040aactctacaa atctgtttct cttccatcac ttgacccagc ctcagccaaa
atagaaggga 2100atcttatatt tgatccaaat aactaccttc ctaaagaaag catgctgaaa
actaccctca 2160ctgcctttgg atttgcttca gctgacctca tcgagattgg cttggaagga
aaaggctttg 2220agccaacatt ggaagctctt tttgggaagc aaggattttt cccagacagt
gtcaacaaag 2280ctttgtactg ggttaatggt caagttcctg atggtgtctc taaggtctta
gtggaccact 2340ttggctatac caaagatgat aaacatgagc aggatatggt aaatggaata
atgctcagtg 2400ttgagaagct gattaaagat ttgaaatcca aagaagtccc ggaagccaga
gcctacctcc 2460gcatcttggg agaggagctt ggttttgcca gtctccatga cctccagctc
ctgggaaagc 2520tgcttctgat gggtgcccgc actctgcagg ggatccccca gatgattgga
gaggtcatca 2580ggaagggctc aaagaatgac ttttttcttc actacatctt catggagaat
gcctttgaac 2640tccccactgg agctggatta cagttgcaaa tatcttcatc tggagtcatt
gctcccggag 2700ccaaggctgg agtaaaactg gaagtagcca acatgcaggc tgaactggtg
gcaaaaccct 2760ccgtgtctgt ggagtttgtg acaaatatgg gcatcatcat tccggacttc
gctaggagtg 2820gggtccagat gaacaccaac ttcttccacg agtcgggtct ggaggctcat
gttgccctaa 2880aagctgggaa gctgaagttt atcattcctt ccccaaagag accagtcaag
ctgctcagtg 2940gaggcaacac attacatttg gtctctacca ccaaaacgga ggtgatccca
cctctcattg 3000agaacaggca gtcctggtca gtttgcaagc aagtctttcc tggcctgaat
tactgcacct 3060caggcgctta ctccaacgcc agctccacag actccgcctc ctactatccg
ctgaccgggg 3120acaccagatt agagctggaa ctgaggccta caggagagat tgagcagtat
tctgtcagcg 3180caacctatga gctccagaga gaggacagag ccttggtgga taccctgaag
tttgtaactc 3240aagcagaagg tgcgaagcag actgaggcta ccatgacatt caaatataat
cggcagagta 3300tgaccttgtc cagtgaagtc caaattccgg attttgatgt tgacctcgga
acaatcctca 3360gagttaatga tgaatctact gagggcaaaa cgtcttacag actcaccctg
gacattcaga 3420acaagaaaat tactgaggtc gccctcatgg gccacctaag ttgtgacaca
aaggaagaaa 3480gaaaaatcaa gggtgttatt tccatacccc gtttgcaagc agaagccaga
agtgagatcc 3540tcgcccactg gtcgcctgcc aaactgcttc tccaaatgga ctcatctgct
acagcttatg 3600gctccacagt ttccaagagg gtggcatggc attatgatga agagaagatt
gaatttgaat 3660ggaacacagg caccaatgta gataccaaaa aaatgacttc caatttccct
gtggatctct 3720ccgattatcc taagagcttg catatgtatg ctaatagact cctggatcac
agagtccctg 3780aaacagacat gactttccgg cacgtgggtt ccaaattaat agttgcaatg
agctcatggc 3840ttcagaaggc atctgggagt cttccttata cccagacttt gcaagaccac
ctcaatagcc 3900tgaaggagtt caacctccag aacatgggat tgccagactt ccacatccca
gaaaacctct 3960tcttaaaaag cgatggccgg gtcaaatata ccttgaacaa gaacagtttg
aaaattgaga 4020ttcctttgcc ttttggtggc aaatcctcca gagatctaaa gatgttagag
actgttagga 4080caccagccct ccacttcaag tctgtgggat tccatctgcc atctcgagag
ttccaagtcc 4140ctacttttac cattcccaag ttgtatcaac tgcaagtgcc tctcctgggt
gttctagacc 4200tctccacgaa tgtctacagc aacttgtaca actggtccgc ctcctacagt
ggtggcaaca 4260ccagcacaga ccatttcagc cttcgggctc gttaccacat gaaggctgac
tctgtggttg 4320acctgctttc ctacaatgtg caaggatctg gagaaacaac atatgaccac
aagaatacgt 4380tcacactatc atgtgatggg tctctacgcc acaaatttct agattcgaat
atcaaattca 4440gtcatgtaga aaaacttgga aacaacccag tctcaaaagg tttactaata
ttcgatgcat 4500ctagttcctg gggaccacag atgtctgctt cagttcattt ggactccaaa
aagaaacagc 4560atttgtttgt caaagaagtc aagattgatg ggcagttcag agtctcttcg
ttctatgcta 4620aaggcacata tggcctgtct tgtcagaggg atcctaacac tggccggctc
aatggagagt 4680ccaacctgag gtttaactcc tcctacctcc aaggcaccaa ccagataaca
ggaagatatg 4740aagatggaac cctctccctc acctccacct ctgatctgca aagtggcatc
attaaaaata 4800ctgcttccct aaagtatgag aactacgagc tgactttaaa atctgacacc
aatgggaagt 4860ataagaactt tgccacttct aacaagatgg atatgacctt ctctaagcaa
aatgcactgc 4920tgcgttctga atatcaggct gattacgagt cattgaggtt cttcagcctg
ctttctggat 4980cactaaattc ccatggtctt gagttaaatg ctgacatctt aggcactgac
aaaattaata 5040gtggtgctca caaggcgaca ctaaggattg gccaagatgg aatatctacc
agtgcaacga 5100ccaacttgaa gtgtagtctc ctggtgctgg agaatgagct gaatgcagag
cttggcctct 5160ctggggcatc tatgaaatta acaacaaatg gccgcttcag ggaacacaat
gcaaaattca 5220gtctggatgg gaaagccgcc ctcacagagc tatcactggg aagtgcttat
caggccatga 5280ttctgggtgt cgacagcaaa aacattttca acttcaaggt cagtcaagaa
ggacttaagc 5340tctcaaatga catgatgggc tcatatgctg aaatgaaatt tgaccacaca
aacagtctga 5400acattgcagg cttatcactg gacttctctt caaaacttga caacatttac
agctctgaca 5460agttttataa gcaaactgtt aatttacagc tacagcccta ttctctggta
actactttaa 5520acagtgacct gaaatacaat gctctggatc tcaccaacaa tgggaaacta
cggctagaac 5580ccctgaagct gcatgtggct ggtaacctaa aaggagccta ccaaaataat
gaaataaaac 5640acatctatgc catctcttct gctgccttat cagcaagcta taaagcagac
actgttgcta 5700aggttcaggg tgtggagttt agccatcggc tcaacacaga catcgctggg
ctggcttcag 5760ccattgacat gagcacaaac tataattcag actcactgca tttcagcaat
gtcttccgtt 5820ctgtaatggc cccgtttacc atgaccatcg atgcacatac aaatggcaat
gggaaactcg 5880ctctctgggg agaacatact gggcagctgt atagcaaatt cctgttgaaa
gcagaacctc 5940tggcatttac tttctctcat gattacaaag gctccacaag tcatcatctc
gtgtctagga 6000aaagcatcag tgcagctctt gaacacaaag tcagtgccct gcttactcca
gctgagcaga 6060caggcacctg gaaactcaag acccaattta acaacaatga atacagccag
gacttggatg 6120cttacaacac taaagataaa attggcgtgg agcttactgg acgaactctg
gctgacctaa 6180ctctactaga ctccccaatt aaagtgccac ttttactcag tgagcccatc
aatatcattg 6240atgctttaga gatgagagat gccgttgaga agccccaaga atttacaatt
gttgcttttg 6300taaagtatga taaaaaccaa gatgttcact ccattaacct cccatttttt
gagaccttgc 6360aagaatattt tgagaggaat cgacaaacca ttatagttgt agtggaaaac
gtacagagaa 6420acctgaagca catcaatatt gatcaatttg taagaaaata cagagcagcc
ctgggaaaac 6480tcccacagca agctaatgat tatctgaatt cattcaattg ggagagacaa
gtttcacatg 6540ccaaggagaa actgactgct ctcacaaaaa agtatagaat tacagaaaat
gatatacaaa 6600ttgcattaga tgatgccaaa atcaacttta atgaaaaact atctcaactg
cagacatata 6660tgatacaatt tgatcagtat attaaagata gttatgattt acatgatttg
aaaatagcta 6720ttgctaatat tattgatgaa atcattgaaa aattaaaaag tcttgatgag
cactatcata 6780tccgtgtaaa tttagtaaaa acaatccatg atctacattt gtttattgaa
aatattgatt 6840ttaacaaaag tggaagtagt actgcatcct ggattcaaaa tgtggatact
aagtaccaaa 6900tcagaatcca gatacaagaa aaactgcagc agcttaagag acacatacag
aatatagaca 6960tccagcacct agctggaaag ttaaaacaac acattgaggc tattgatgtt
agagtgcttt 7020tagatcaatt gggaactaca atttcatttg aaagaataaa tgatgttctt
gagcatgtca 7080aacactttgt tataaatctt attggggatt ttgaagtagc tgagaaaatc
aatgccttca 7140gagccaaagt ccatgagtta atcgagaggt atgaagtaga ccaacaaatc
caggttttaa 7200tggataaatt agtagagttg acccaccaat acaagttgaa ggagactatt
cagaagctaa 7260gcaatgtcct acaacaagtt aagataaaag attactttga gaaattggtt
ggatttattg 7320atgatgctgt gaagaagctt aatgaattat cttttaaaac attcattgaa
gatgttaaca 7380aattccttga catgttgata aagaaattaa agtcatttga ttaccaccag
tttgtagatg 7440aaaccaatga caaaatccgt gaggtgactc agagactcaa tggtgaaatt
caggctctgg 7500aactaccaca aaaagctgaa gcattaaaac tgtttttaga ggaaaccaag
gccacagttg 7560cagtgtatct ggaaagccta caggacacca aaataacctt aatcatcaat
tggttacagg 7620aggctttaag ttcagcatct ttggctcaca tgaaggccaa attccgagag
actctagaag 7680atacacgaga ccgaatgtat caaatggaca ttcagcagga acttcaacga
tacctgtctc 7740tggtaggcca ggtttatagc acacttgtca cctacatttc tgattggtgg
actcttgctg 7800ctaagaacct tactgacttt gcagagcaat attctatcca agattgggct
aaacgtatga 7860aagcattggt agagcaaggg ttcactgttc ctgaaatcaa gaccatcctt
gggaccatgc 7920ctgcctttga agtcagtctt caggctcttc agaaagctac cttccagaca
cctgatttta 7980tagtccccct aacagatttg aggattccat cagttcagat aaacttcaaa
gacttaaaaa 8040atataaaaat cccatccagg ttttccacac cagaatttac catccttaac
accttccaca 8100ttccttcctt tacaattgac tttgtcgaaa tgaaagtaaa gatcatcaga
accattgacc 8160agatgcagaa cagtgagctg cagtggcccg ttccagatat atatctcagg
gatctgaagg 8220tggaggacat tcctctagcg agaatcaccc tgccagactt ccgtttacca
gaaatcgcaa 8280ttccagaatt cataatccca actctcaacc ttaatgattt tcaagttcct
gaccttcaca 8340taccagaatt ccagcttccc cacatctcac acacaattga agtacctact
tttggcaagc 8400tatacagtat tctgaaaatc caatctcctc ttttcacatt agatgcaaat
gctgacatag 8460ggaatggaac cacctcagca aacgaagcag gtatcgcagc ttccatcact
gccaaaggag 8520agtccaaatt agaagttctc aattttgatt ttcaagcaaa tgcacaactc
tcaaacccta 8580agattaatcc gctggctctg aaggagtcag tgaagttctc cagcaagtac
ctgagaacgg 8640agcatgggag tgaaatgctg ttttttggaa atgctattga gggaaaatca
aacacagtgg 8700caagtttaca cacagaaaaa aatacactgg agcttagtaa tggagtgatt
gtcaagataa 8760acaatcagct taccctggat agcaacacta aatacttcca caaattgaac
atccccaaac 8820tggacttctc tagtcaggct gacctgcgca acgagatcaa gacactgttg
aaagctggcc 8880acatagcatg gacttcttct ggaaaagggt catggaaatg ggcctgcccc
agattctcag 8940atgagggaac acatgaatca caaattagtt tcaccataga aggacccctc
acttcctttg 9000gactgtccaa taagatcaat agcaaacacc taagagtaaa ccaaaacttg
gtttatgaat 9060ctggctccct caacttttct aaacttgaaa ttcaatcaca agtcgattcc
cagcatgtgg 9120gccacagtgt tctaactgct aaaggcatgg cactgtttgg agaagggaag
gcagagttta 9180ctgggaggca tgatgctcat ttaaatggaa aggttattgg aactttgaaa
aattctcttt 9240tcttttcagc ccagccattt gagatcacgg catccacaaa caatgaaggg
aatttgaaag 9300ttcgttttcc attaaggtta acagggaaga tagacttcct gaataactat
gcactgtttc 9360tgagtcccag tgcccagcaa gcaagttggc aagtaagtgc taggttcaat
cagtataagt 9420acaaccaaaa tttctctgct ggaaacaacg agaacattat ggaggcccat
gtaggaataa 9480atggagaagc aaatctggat ttcttaaaca ttcctttaac aattcctgaa
atgcgtctac 9540cttacacaat aatcacaact cctccactga aagatttctc tctatgggaa
aaaacaggct 9600tgaaggaatt cttgaaaacg acaaagcaat catttgattt aagtgtaaaa
gctcagtata 9660agaaaaacaa acacaggcat tccatcacaa atcctttggc tgtgctttgt
gagtttatca 9720gtcagagcat caaatccttt gacaggcatt ttgaaaaaaa cagaaacaat
gcattagatt 9780ttgtcaccaa atcctataat gaaacaaaaa ttaagtttga taagtacaaa
gctgaaaaat 9840ctcacgacga gctccccagg acctttcaaa ttcctggata cactgttcca
gttgtcaatg 9900ttgaagtgtc tccattcacc atagagatgt cggcattcgg ctatgtgttc
ccaaaagcag 9960tcagcatgcc tagtttctcc atcctaggtt ctgacgtccg tgtgccttca
tacacattaa 10020tcctgccatc attagagctg ccagtccttc atgtccctag aaatctcaag
ctttctcttc 10080cacatttcaa ggaattgtgt accataagcc atatttttat tcctgccatg
ggcaatatta 10140cctatgattt ctcctttaaa tcaagtgtca tcacactgaa taccaatgct
gaacttttta 10200accagtcaga tattgttgct catctccttt cttcatcttc atctgtcatt
gatgcactgc 10260agtacaaatt agagggcacc acaagattga caagaaaaag gggattgaag
ttagccacag 10320ctctgtctct gagcaacaaa tttgtggagg gtagtcataa cagtactgtg
agcttaacca 10380cgaaaaatat ggaagtgtca gtggcaaaaa ccacaaaagc cgaaattcca
attttgagaa 10440tgaatttcaa gcaagaactt aatggaaata ccaagtcaaa acctactgtc
tcttcctcca 10500tggaatttaa gtatgatttc aattcttcaa tgctgtactc taccgctaaa
ggagcagttg 10560accacaagct tagcttggaa agcctcacct cttacttttc cattgagtca
tctaccaaag 10620gagatgtcaa gggttcggtt ctttctcggg aatattcagg aactattgct
agtgaggcca 10680acacttactt gaattccaag agcacacggt cttcagtgaa gctgcagggc
acttccaaaa 10740ttgatgatat ctggaacctt gaagtaaaag aaaattttgc tggagaagcc
acactccaac 10800gcatatattc cctctgggag cacagtacga aaaaccactt acagctagag
ggcctctttt 10860tcaccaacgg agaacataca agcaaagcca ccctggaact ctctccatgg
caaatgtcag 10920ctcttgttca ggtccatgca agtcagccca gttccttcca tgatttccct
gaccttggcc 10980aggaagtggc cctgaatgct aacactaaga accagaagat cagatggaaa
aatgaagtcc 11040ggattcattc tgggtctttc cagagccagg tcgagctttc caatgaccaa
gaaaaggcac 11100accttgacat tgcaggatcc ttagaaggac acctaaggtt cctcaaaaat
atcatcctac 11160cagtctatga caagagctta tgggatttcc taaagctgga tgtaaccacc
agcattggta 11220ggagacagca tcttcgtgtt tcaactgcct ttgtgtacac caaaaacccc
aatggctatt 11280cattctccat ccctgtaaaa gttttggctg ataaattcat tactcctggg
ctgaaactaa 11340atgatctaaa ttcagttctt gtcatgccta cgttccatgt cccatttaca
gatcttcagg 11400ttccatcgtg caaacttgac ttcagagaaa tacaaatcta taagaagctg
agaacttcat 11460catttgccct caacctacca acactccccg aggtaaaatt ccctgaagtt
gatgtgttaa 11520caaaatattc tcaaccagaa gactccttga ttcccttttt tgagataacc
gtgcctgaat 11580ctcagttaac tgtgtcccag ttcacgcttc caaaaagtgt ttcagatggc
attgctgctt 11640tggatctaaa tgcagtagcc aacaagatcg cagactttga gttgcccacc
atcatcgtgc 11700ctgagcagac cattgagatt ccctccatta agttctctgt acctgctgga
attgtcattc 11760cttcctttca agcactgact gcacgctttg aggtagactc tcccgtgtat
aatgccactt 11820ggagtgccag tttgaaaaac aaagcagatt atgttgaaac agtcctggat
tccacatgca 11880gctcaaccgt acagttccta gaatatgaac taaatgtttt gggaacacac
aaaatcgaag 11940atggtacgtt agcctctaag actaaaggaa cacttgcaca ccgtgacttc
agtgcagaat 12000atgaagaaga tggcaaattt gaaggacttc aggaatggga aggaaaagcg
cacctcaata 12060tcaaaagccc agcgttcacc gatctccatc tgcgctacca gaaagacaag
aaaggcatct 12120ccacctcagc agcctcccca gccgtaggca ccgtgggcat ggatatggat
gaagatgacg 12180acttttctaa atggaacttc tactacagcc ctcagtcctc tccagataaa
aaactcacca 12240tattcaaaac tgagttgagg gtccgggaat ctgatgagga aactcagatc
aaagttaatt 12300gggaagaaga ggcagcttct ggcttgctaa cctctctgaa agacaacgtg
cccaaggcca 12360caggggtcct ttatgattat gtcaacaagt accactggga acacacaggg
ctcaccctga 12420gagaagtgtc ttcaaagctg agaagaaatc tgcagaacaa tgctgagtgg
gtttatcaag 12480gggccattag gcaaattgat gatatcgacg tgaggttcca gaaagcagcc
agtggcacca 12540ctgggaccta ccaagagtgg aaggacaagg cccagaatct gtaccaggaa
ctgttgactc 12600aggaaggcca agccagtttc cagggactca aggataacgt gtttgatggc
ttggtacgag 12660ttactcaaaa attccatatg aaagtcaagc atctgattga ctcactcatt
gattttctga 12720acttccccag attccagttt ccggggaaac ctgggatata cactagggag
gaactttgca 12780ctatgttcat aagggaggta gggacggtac tgtcccaggt atattcgaaa
gtccataatg 12840gttcagaaat actgttttcc tatttccaag acctagtgat tacacttcct
ttcgagttaa 12900ggaaacataa actaatagat gtaatctcga tgtataggga actgttgaaa
gatttatcaa 12960aagaagccca agaggtattt aaagccattc agtctctcaa gaccacagag
gtgctacgta 13020atcttcagga ccttttacaa ttcattttcc aactaataga agataacatt
aaacagctga 13080aagagatgaa atttacttat cttattaatt atatccaaga tgagatcaac
acaatcttca 13140atgattatat cccatatgtt tttaaattgt tgaaagaaaa cctatgcctt
aatcttcata 13200agttcaatga atttattcaa aacgagcttc aggaagcttc tcaagagtta
cagcagatcc 13260atcaatacat tatggccctt cgtgaagaat attttgatcc aagtatagtt
ggctggacag 13320tgaaatatta tgaacttgaa gaaaagatag tcagtctgat caagaacctg
ttagttgctc 13380ttaaggactt ccattctgaa tatattgtca gtgcctctaa ctttacttcc
caactctcaa 13440gtcaagttga gcaatttctg cacagaaata ttcaggaata tcttagcatc
cttaccgatc 13500cagatggaaa agggaaagag aagattgcag agctttctgc cactgctcag
gaaataatta 13560aaagccaggc cattgcgacg aagaaaataa tttctgatta ccaccagcag
tttagatata 13620aactgcaaga tttttcagac caactctctg attactatga aaaatttatt
gctgaatcca 13680aaagattgat tgacctgtcc attcaaaact accacacatt tctgatatac
atcacggagt 13740tactgaaaaa gctgcaatca accacagtca tgaaccccta catgaagctt
gctccaggag 13800aacttactat catcctctaa ttttttaaaa gaaatcttca tttattcttc
ttttccaatt 13860gaactttcac atagcacaga aaaaattcaa actgcctata ttgataaaac
catacagtga 13920gccagccttg cagtaggcag tagactataa gcagaagcac atatgaactg
gacctgcacc 13980aaagctggca ccagggctcg gaaggtctct gaactcagaa ggatggcatt
ttttgcaagt 14040taaagaaaat caggatctga gttattttgc taaacttggg ggaggaggaa
caaataaatg 14100gagtctttat tgtgtatcat a
141211121DNAArtificial SequencePCR Primer 11tgctaaaggc
acatatggcc t
211223DNAArtificial SequencePCR Primer 12ctcaggttgg actctccatt gag
231328DNAArtificial SequencePCR
Probe 13cttgtcagag ggatcctaac actggccg
281419DNAArtificial SequencePCR Primer 14gaaggtgaag gtcggagtc
191520DNAArtificial SequencePCR
Primer 15gaagatggtg atgggatttc
201620DNAArtificial SequencePCR Probe 16caagcttccc gttctcagcc
20172354DNAM. musculus
17gaattccaac ttcctcacct ctcacataca attgaaatac ctgcttttgg caaactgcat
60agcatcctta agatccaatc tcctctcttt atattagatg ctaatgccaa catacagaat
120gtaacaactt cagggaacaa agcagagatt gtggcttctg tcactgctaa aggagagtcc
180caatttgaag ctctcaattt tgattttcaa gcacaagctc aattcctgga gttaaatcct
240catcctccag tcctgaagga atccatgaac ttctccagta agcatgtgag aatggagcat
300gagggtgaga tagtatttga tggaaaggcc attgagggga aatcagacac agtcgcaagt
360ttacacacag agaaaaatga agtagagttt aataatggta tgactgtcaa agtaaacaat
420cagctcaccc ttgacagtca cacaaagtac ttccacaagt tgagtgttcc taggctggac
480ttctccagta aggcttctct taataatgaa atcaagacac tattagaagc tggacatgtg
540gcattgacat cttcagggac agggtcatgg aactgggcct gtcccaactt ctcggatgaa
600ggcatacatt cgtcccaaat tagctttact gtggatggtc ccattgcttt tgttggacta
660tccaataaca taaatggcaa acacttacgg gtcatccaaa aactgactta tgaatctggc
720ttcctcaact attctaagtt tgaagttgag tcaaaagttg aatctcagca cgtgggctcc
780agcattctaa cagccaatgg tcgggcactg ctcaaggacg caaaggcaga aatgactggt
840gagcacaatg ccaacttaaa tggaaaagtt attggaactt tgaaaaattc tctcttcttt
900tcagcacaac catttgagat tactgcatcc acaaataatg aaggaaattt gaaagtgggt
960tttccactaa agctgactgg gaaaatagac ttcctgaata actatgcatt gtttctgagt
1020ccccgtgccc aacaagcaag ctggcaagcg agtaccagat tcaatcagta caaatacaat
1080caaaactttt ctgctataaa caatgaacac aacatagaag ccagtatagg aatgaatgga
1140gatgccaacc tggatttctt aaacatacct ttaacaattc ctgaaattaa cttgccttac
1200acggagttca aaactccctt actgaaggat ttctccatat gggaagaaac aggcttgaaa
1260gaatttttga agacaacaaa gcaatcattt gatttgagtg taaaggctca atataaaaag
1320aacagtgaca agcattccat tgttgtccct ctgggtatgt tttatgaatt tattctcaac
1380aatgtcaatt cgtgggacag aaaatttgag aaagtcagaa acaatgcttt acattttctt
1440accacctcct ataatgaagc aaaaattaag gttgataagt acaaaactga aaattccctt
1500aatcagccct ctgggacctt tcaaaatcat ggctacacta tcccagttgt caacattgaa
1560gtatctccat ttgctgtaga gacactggct tccaggcatg tgatccccac agcaataagc
1620accccaagtg tcacaatccc tggtcctaac atcatggtgc cttcatacaa gttagtgctg
1680ccacccctgg agttgccagt tttccatggt cctgggaatc tattcaagtt tttcctccca
1740gatttcaagg gattcaacac tattgacaat atttatattc cagccatggg caactttacc
1800tatgactttt cttttaaatc aagtgtcatc acactgaata ccaatgctgg actttataac
1860caatcagata tcgttgccca tttcctttct tcctcttcat ttgtcactga cgccctgcag
1920tacaaattag agggaacatc acgtctgatg cgaaaaaggg gattgaaact agccacagct
1980gtctctctaa ctaacaaatt tgtaaagggc agtcatgaca gcaccattag tttaaccaag
2040aaaaacatgg aagcatcagt gagaacaact gccaacctcc atgctcccat attctcaatg
2100aacttcaagc aggaacttaa tggaaatacc aagtcaaaac ccactgtttc atcatccatt
2160gaactaaact atgacttcaa ttcctcaaag ctgcactcta ctgcaacagg aggcattgat
2220cacaagttca gcttagaaag tctcacttcc tacttttcca ttgagtcatt caccaaagga
2280aatatcaaga gttccttcct ttctcaggaa tattcaggaa gtgttgccaa tgaagccaat
2340gtatatctga attc
23541819DNAArtificial SequencePCR Primer 18cgtgggctcc agcattcta
191921DNAArtificial SequencePCR
Primer 19agtcatttct gcctttgcgt c
212022DNAArtificial SequencePCR Probe 20ccaatggtcg ggcactgctc aa
222120DNAArtificial SequencePCR
Primer 21ggcaaattca acggcacagt
202220DNAArtificial SequencePCR Primer 22gggtctcgct cctggaagat
202327DNAArtificial SequencePCR
Probe 23aaggccgaga atgggaagct tgtcatc
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