METHOD TO PREDICT RESPONSE TO PHARMACOLOGICAL CHAPERONE TREATMENT OF DISEASES

Abstract

The present invention provides methods to determine whether a patient with a lysosomal storage disorder will benefit from treatment with a specific pharmacological chaperone. The present invention exemplifies an in vitro method for determining .alpha.-galactosidase A responsiveness to a pharmacological chaperone such as 1-deoxygalactonojirimycin in a cell line expressing a mutant from of .alpha.-galactosidase A. The invention also provides a method for diagnosing Fabry disease in patients suspected of having Fabry disease.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is a continuation of U.S. patent application Ser. No. 14/731,603 filed Jun. 5, 2015, allowed, which is a continuation of U.S. patent application Ser. No. 14/054,369, filed Oct. 15, 2013, now U.S. Pat. No. 9,905,584, which is a divisional of U.S. patent application Ser. No. 12/855,468, filed Aug. 12, 2010, now U.S. Pat. No. 8,592,362, which claims the benefit of International Application No. PCT/US09/033963, filed Feb. 12, 2009, U.S. Provisional Patent Application No. 61/028,141 filed Feb. 12, 2008, U.S. Provisional Patent Application No. 61/035,684, filed Mar. 11, 2008, U.S. Provisional Patent Application No. 61/093,631, filed Sep. 2, 2008 and U.S. Provisional Patent Application No. 61/113,496, filed Nov. 11, 2008. Each of these applications are hereby incorporated by reference and their entirety.

FIELD OF THE INVENTION

[0002] The present invention provides methods to determine whether a patient with a lysosomal storage disorder will benefit from treatment with a specific pharmacological chaperone. The present invention also provides an in vitro method for determining enzyme (e.g., .alpha.-galactosidase A, .alpha.-glucosidase or glucocerebrosidase) responsiveness to a pharmacological chaperone (e.g., 1-deoxygalactonojirimycin, 1-deoxynojirimycin or isofagomine) in a cell line expressing a mutant form of the enzyme. The invention also provides a method for diagnosing a lysosomal storage disorder (e.g., Fabry disease, Pompe disease or Gaucher disease) in patients suspected of having a lysosomal storage disorder, and implementing the proper treatment based on the diagnosis (e.g., choosing a particular therapeutic agent to administer to the patient).

BACKGROUND

[0003] In the human body, proteins are involved in almost every aspect of cellular function. Proteins are linear strings of amino acids that fold and twist into specific three-dimensional shapes in order to function properly. Certain human diseases result from mutations that cause changes in the amino acid sequence of a protein which reduce its stability and may prevent it from folding properly. The majority of genetic mutations that lead to the production of less stable or misfolded proteins are called missense mutations. These mutations result in the substitution of a single amino acid for another in the protein. Because of this error, missense mutations often result in proteins that have a reduced level of biological activity. In addition to missense mutations, there are also other types of mutations that can result in proteins with reduced biological activity.

[0004] Proteins generally fold in a specific region of the cell known as the endoplasmic reticulum, or ER. The cell has quality control mechanisms that ensure that proteins are folded into their correct three-dimensional shape before they can move from the ER to the appropriate destination in the cell, a process generally referred to as protein trafficking. Misfolded proteins are often eliminated by the quality control mechanisms after initially being retained in the ER. In certain instances, misfolded proteins can accumulate in the ER before being eliminated.

[0005] The retention of misfolded proteins in the ER interrupts their proper trafficking, and the resulting reduced biological activity can lead to impaired cellular function and ultimately to disease. In addition, the accumulation of misfolded proteins in the ER may lead to various types of stress on cells, which may also contribute to cellular dysfunction and disease.

[0006] Lysosomal storage diseases (LSDs) are characterized by deficiencies of lysosomal enzymes due to mutations in the genes encoding the lysosomal enzymes. This results in the pathologic accumulation of substrates of those enzymes, which include lipids, carbohydrates, and polysaccharides. There are about fifty known LSDs to date, which include Gaucher disease, Fabry disease, Pompe disease, Tay Sachs disease and the mucopolysaccharidoses (MPS). Most LSDs are inherited as an autosomal recessive trait, although males with Fabry disease and MPS II are hemizygotes because the disease genes are encoded on the X chromosome. For most LSDs, there is no available treatment beyond symptomatic management. For several LSDs, including Gaucher, Fabry, Pompe, and MPS I and VI, enzyme replacement therapy (ERT) using recombinant enzymes is available. For Gaucher disease, substrate reduction therapy (SRT) also is available in limited situations. SRT employs a small molecule inhibitor of an enzyme required for the synthesis of glucosylceramide (the GD substrate). The goal of SRT is to reduce production of the substrate and reduce pathologic accumulation.

[0007] Although there are many different mutant genotypes associated with each LSD, some of the mutations, including some of the most prevalent mutations, are missense mutations which can lead to the production of a less stable enzyme. These less stable enzymes are sometimes prematurely degraded by the ER-associated degradation pathway. This results in the enzyme deficiency in the lysosome, and the pathologic accumulation of substrate. Such mutant enzymes are sometimes referred to in the pertinent art as "folding mutants" or "conformational mutants."

Diagnosis of Fabry Disease

[0008] Because Fabry disease is rare, involves multiple organs, has a wide age range of onset, and is heterogeneous, proper diagnosis is a challenge. Awareness is low among health care professionals and misdiagnoses are frequent. Some examples of diagnoses seriously considered in patients who were eventually diagnosed with Fabry's disease include: mitral valve prolapse, glomerulonephritis, idiopathic proteinuria, systemic lupus erythematosus, Whipple's disease, acute abdomen, ulcerative colitis, acute intermittent porphyrias, and occult malignancies. Thus, even for classically affected males, diagnosis typically takes from about 5-7 years or even longer. This is a concern because the longer a person has Fabry disease, the more damage is likely to occur in the affected organs and tissues and the more serious the person's condition may become. Diagnosis of Fabry disease is most often confirmed on the basis of decreased .alpha.-Gal A activity in plasma or peripheral leukocytes (WBCs) once a patient is symptomatic, coupled with mutational analysis. In females, diagnosis is even more challenging since the enzymatic identification of carrier females is less reliable due to random X-chromosomal inactivation in some cells of carriers. For example, some obligate carriers (daughters of classically affected males) have .alpha.-Gal A enzyme activities ranging from normal to very low activities. Since carriers can have normal .alpha.-Gal A enzyme activity in leukocytes, only the identification of an .alpha.-Gal A mutation by genetic testing provides precise carrier identification and/or diagnosis.

Treatment of Fabry Disease

[0009] One approved therapy for treating Fabry disease diseases is enzyme replacement therapy, which typically involves intravenous, infusion of a purified form of the corresponding wild-type protein (Fabrazyme.RTM., Genzyme Corp.). One of the main complications with protein replacement therapy is attainment and maintenance of therapeutically effective amounts of protein in vivo due to rapid degradation of the infused protein. The current approach to overcome this problem is to perform numerous costly high dose infusions.

[0010] Protein replacement therapy has several additional caveats, such as difficulties with large-scale generation, purification, and storage of properly folded protein; obtaining glycosylated native protein; generation of an anti-protein immune response; and inability of protein to cross the blood-brain barrier to mitigate central nervous system pathologies (i.e., low bioavailability). In addition, replacement enzyme cannot penetrate the heart or kidney in sufficient amounts to reduce substrate accumulation in the renal podocytes or cardiac myocytes, which figure prominently in Fabry pathology.

[0011] Gene therapy using recombinant vectors containing nucleic acid sequences that encode a functional protein, or using genetically modified human cells that express a functional protein, is also being developed to treat protein deficiencies and other disorders that benefit from protein replacement.

[0012] A third, relatively recent approach to treating some enzyme deficiencies involves the use of small molecule inhibitors to reduce production of the natural substrate of deficient enzyme proteins, thereby ameliorating the pathology. This "substrate reduction" approach has been specifically described for a class of about 40 related enzyme disorders called lysosomal storage disorders that include glycosphingolipid storage disorders. The small molecule inhibitors proposed for use as therapy are specific for inhibiting the enzymes involved in synthesis of glycolipids, reducing the amount of cellular glycolipid that needs to be broken down by the deficient enzyme.

[0013] It has previously been shown that the binding of small molecule inhibitors of enzymes associated with LSDs can increase the stability of both mutant enzyme and the corresponding wild-type enzyme (see U.S. Pat. Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; 6,916,829, and 7,141,582 all incorporated herein by reference). In particular, it was discovered that administration of small molecule derivatives of glucose and galactose, which are specific, selective competitive inhibitors for several target lysosomal enzymes, effectively increased the stability of the enzymes in cells in vitro and, thus, increased trafficking of the enzymes to the lysosome. Thus, by increasing the amount of enzyme in the lysosome, hydrolysis of the enzyme substrates is expected to increase. The original theory behind this strategy was as follows: since the mutant enzyme protein is unstable in the ER (Ishii et al., Biochem. Biophys. Res. Comm. 1996; 220: 812-815), the enzyme protein is retarded in the normal transport pathway (ER.fwdarw.Golgi apparatus.fwdarw.endosomes.fwdarw.lysosome) and prematurely degraded. Therefore, a compound which binds to and increases the stability of a mutant enzyme, may serve as a "chaperone" for the enzyme and increase the amount that can exit the ER and move to the lysosomes. In addition, because the folding and trafficking of some wild-type proteins is incomplete, with up to 70% of some wild-type proteins being degraded in some instances prior to reaching their final cellular location, the chaperones can be used to stabilize wild-type enzymes and increase the amount of enzyme which can exit the ER and be trafficked to lysosomes. This strategy has been shown to increase several lysosomal enzymes in vitro and in vivo, including .beta.-glucocerebrosidase and .alpha.-glucosidase, deficiencies of which are associated with Gaucher and Pompe disease, respectively.

[0014] However, as indicated above, successful candidates for SPC therapy should have a mutation which results in the production of an enzyme that has the potential to be stabilized and folded into a conformation that permits trafficking out of the ER. Mutations which severely truncate the enzyme, such as nonsense mutations, or mutations in the catalytic domain which prevent binding of the chaperone, will not be as likely to be "rescuable" or "enhanceable" using SPC therapy, i.e., to respond to SPC therapy. While missense mutations outside the catalytic site are more likely to be rescuable using SPCs, there is no guarantee, necessitating screening for responsive mutations. This means that, even when Fabry disease is diagnosed by detecting deficient .alpha.-Gal A activity in WBCs, it is very difficult, if not impossible, to predict whether a particular Fabry patient will respond to treatment with an SPC without benefit of the present invention. Moreover, since WBCs only survive for a short period of time in culture (in vitro), screening for SPC enhancement of .alpha.-Gal A is difficult and not optimal for the patient.

[0015] In order to apply SPC therapy effectively, a broadly applicable, fast and efficient method for screening patients for responsiveness to SPC therapy needs to be adopted prior to initiation of treatment. Treatment can then be implemented based on the results of the screening. Thus, there remains in the art a need for relatively non-invasive methods to rapidly assess enzyme enhancement with potential therapies prior to making treatment decisions, for both cost and emotional benefits to the patient.

SUMMARY OF THE INVENTION

[0016] One embodiment of the present invention provides a method for determining whether a patient will be a candidate for SPC therapy. Specifically, the present invention provides an in vitro assay to evaluate protein activity in the presence or absence of an SPC, wherein an SPC that increases the activity of the protein in the in vitro assay is an SPC that can be used for SPC therapy. In one embodiment, the in vitro assay comprises expressing a mutant protein in a host cell, contacting the mutant protein with a candidate SPC, and determining if the mutant protein contacted with the SPC exhibits an increased level of activity (preferably a statistically significant increase) when compared to a mutant protein expressed in a host cell that is not contacted with the candidate SPC. When a candidate SPC increases the activity of a mutant protein according to the assay of the invention, such a candidate SPC can be used for SPC therapy to treat a patient expressing the same mutant protein tested in the in vitro assay.

[0017] In one embodiment, the protein is an enzyme. In another embodiment, the protein is a lysosomal enzyme. In yet another embodiment, the protein is .alpha.-galactosidase A (.alpha.-GAL; .alpha.-GAL A). In other embodiments, the protein is alpha-glucosidase (Acid .alpha.-glucosidase; .alpha.-glucosidase; GAA). In other embodiments, the protein is glucocerebrosidase (.beta.-glucosidase; Gba; GCase).

[0018] The present invention also includes the basis for evaluation of SPC as a treatment option for any number of other protein abnormalities and/or enzyme deficiencies and/or a protein folding disorders.

[0019] The present invention further provides a written record (e.g., a "treatment reference table") listing protein mutations and the responsiveness of each of the mutations to SPC therapy. Such a list can be used in determining treatment options for a patient, whereby the patient, or the patient's physician or doctor, can select the proper therapeutic approach, for example, an SPC for treatment by identifying the patient's protein mutation, and cross-referencing the mutation with the list to identify whether an SPC will increase the activity of the patients particular mutant enzyme.

[0020] In another embodiment, the "treatment reference table" lists mutations for a lysosomal enzyme, and the treatment reference table is employed to determine the best therapeutic approach to treat a lysosomal storage disorder. In a further embodiment of the invention, the protein is .alpha.-Gal A, and the disease is Fabry disease. In other embodiments of the invention, the protein is GAA, and the disease is Pompe disease. In other embodiments of the invention, the protein is Gba, and the disease is Gaucher disease.

[0021] In one embodiment, the treatment reference table describes mutant forms of enzyme, such as a lysomal enzyme (e.g., .alpha.-Gal A, Gcase, and GAA) and treatment options are ascertained for lysosomal storage disorders (e.g., Fabry, Gacher and Pompe Disease).

[0022] In one embodiment, the invention also provides for methods of creating a treatment reference table, wherein the treatment reference table can be for any protein folding disorder or disorder treatable with an SPC. This class of disease includes the other lysosomal storage disorders, Cystic Fibrosis (CFTR) (respiratory or sweat gland epithelial cells), familial hypercholesterolemia (LDL receptor; LPL-adipocytes or vascular endothelial cells), cancer (p53; PTEN-tumor cells), and amyloidoses (transthyretin) among others.

[0023] In another embodiment, the present invention provides for methods of treating a patient diagnosed as expressing certain mutant proteins (e.g., lysosomal enzymes such as .alpha.-GAL A), wherein activity of the mutant protein (e.g., .alpha.-Gal A), when expressed in a host cell, can be increased upon administration of an SPC for that protein (for example, 1-deoxygalactonojirimycin, DGJ, as an SPC for mutant .alpha.-GAL A).

[0024] The present invention also provides for diagnostic kits containing the components required to perform the assay.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1A-C. Shows a listing of Fabry mutations generated by site-directed mutagenesis. The text indicates whether HEK-293 cells expressing each of the listed mutations responds to DGJ treatment in the transient transfection assay: italics=not yet tested; bold and underscored=no response to DGJ; plain text (not italicized, bold, or underscored)=response to DGJ.

[0026] FIG. 2A-C Shows the responsiveness of different .alpha.-Gal A mutations to DGJ treatment. The magnitude of increase in .alpha.-Gal A activity levels after DGJ treatment and EC50 values are listed for every tested mutation in FIG. 1A-D that responded to DGJ treatment. The increase in enzyme activity is shown as a percentage of wild type .alpha.-Gal A activity.

[0027] FIG. 3 Shows representative examples of wild type and mutant .alpha.-Gal A responses to DGJ treatment. .alpha.-Gal A activity (expressed as nmol/mg protein/hr of 4-MU released) was measured in lysates prepared from transfected HEK 293 cells incubated with increasing concentrations of DGJ. A typical concentration-dependent response is shown for L300P and a typical negative response to DGJ is shown for R227Q. Wild type exhibits high baseline activity and thus does not respond to DGJ in this assay.

[0028] FIG. 4 Shows that the mutation response in HEK 293 cells are comparable to patient-derived T-cells, lymphoblasts or white blood cells in vivo. .alpha.-Gal A levels measured in three different assays, reported as percentage of wild type, are compared for each mutation examined. .alpha.-Gal A levels were measured in T-Cells, lymphoblasts, white blood cells and HEK 293 cells expressing mutant .alpha.-Gal A before and after exposure to DGJ. Blank bars indicate basal level (without DGJ treatment) and filled bars indicate the elevated level after DGJ treatment.

[0029] FIG. 5 Shows that DGJ-responsive .alpha.-Gal A mutations are widely distributed on the .alpha.-Gal A protein sequence. Tested Fabry mutations are illustrated on the .alpha.-Gal A secondary structure. No significant correlation between response and location on the protein sequence of a mutation was observed, suggesting that responsive as well as non-responsive mutations are distributed widely across the entire protein. Text color indicates DGJ response: green=response; red=no response; brown indicates that of the multiple mutations on that same site some responded to DGJ treatment, while others did not.

[0030] FIG. 6 Shows the oligonucleotide primer pairs used to generate the point mutations in the .alpha.-Gal A gene through site-directed mutagenesis.

[0031] FIG. 7 Shows the .alpha.-Gal A cDNA sequence that was mutated through the site-directed mutagenesis.

[0032] FIG. 8 Shows the effect of isofagomine tartrate on patient-derived macrophages and lymphoblasts isolated from Gaucher disease patients with different mutations in their glucocerebrosidase (Gba; GCase) enzyme.

[0033] FIG. 9 Shows the effect on GL-3 levels of eight-week old male hR301Q .alpha.-Gal A Tg/KO mice which were treated for 4 weeks with 300 mg/kg DGJ in drinking water either daily or less frequently (4 days ON/3 days OFF).

[0034] FIG. 10 Shows a listing of Pompe mutations generated by site-directed mutagenesis. The text indicates whether COS-7 cells expressing each of the listed mutations responds to DNJ treatment in the transient transfection assay.

[0035] FIGS. 11A and 11B Show the nucleic acid sequence of human lysosomal alpha-glucosidase (GAA) (GenBank Accession No.: Y00839).

[0036] FIG. 12 Shows the responsiveness of four different GAA mutations to DNJ treatment at concentrations of 0 .mu.M, 20 .mu.M, 50 .mu.M and 100 .mu.M. The increase in enzyme activity is shown as specific activity (nmol/mg protein/hour). FIG. 12 also shows that DNJ promoted processing of GAA to the 95/76/70 kDa forms.

[0037] FIG. 13 Shows the responsiveness of Pompe patient-derived fibroblasts to DNJ treatment. The fibroblasts were homozygous for either the P545L or R854X GAA mutation.

[0038] FIG. 14 Shows the EC.sub.50 for DNJ induced GAA activity in HEK-293 cells transiently transfected with the P545L GAA mutation.

[0039] FIG. 15 Shows the responsiveness of Pompe patient-derived lymphocytes to DNJ treatment. The lymphocytes were heterozygous for the (IVS1AS, T>G, -13) GAA splicing defect and a GAA frameshift mutation.

[0040] FIGS. 16A, 16B, and 16C Show the amino acid sequence encoded by a human lysosomal alpha-glucosidase (GAA) nucleic acid (GenBank Accession No.: Y00839).

DETAILED DESCRIPTION

[0041] The present invention provides an in vitro assay to provide accurate determination of whether an SPC enhances activity of a mutant protein.

[0042] In one embodiment, the protein is a lysosomal enzyme, wherein the lysosomal enzyme, when mutated, causes a lysosomal storage disorder. The concepts of the present invention, however, can be globally applied to any disease or condition characterized by mutant proteins amenable to SPC-therapy, in which the proteins have one or more specific mutations that can be generated in vitro, for example, by site-directed mutagenesis.

[0043] In one specific embodiment, the invention provides methods for determining whether an SPC enhances enzyme activity of a mutant .alpha.-Gal A enzyme, and can therefore be utilized as an effective therapeutic treatment for a Fabry disease patient expressing the same .alpha.-Gal A mutation.

[0044] In another specific embodiment, the invention provides methods for determining whether an SPC enhances enzyme activity of a mutant GAA enzyme, and can therefore be utilized as an effective therapeutic treatment for a Pompe disease patient expressing the same GAA mutation.

[0045] In another specific embodiment, the invention provides methods for determining whether an SPC enhances enzyme activity of a mutant Gba enzyme, and can therefore be utilized as an effective therapeutic treatment for a Gaucher disease patient expressing the same Gba mutation.

[0046] According to the methods of the present invention, assays are provided that allow for the determination of whether a patient expressing a mutant lysosomal enzyme will be a candidate for SPC therapy. The new in vitro assay is extremely sensitive and can be performed on a host cell transfected with a nucleic acid construct encoding a mutant lysosomal enzyme. Specific candidate SPCs can then be assayed to determine if the candidate SPC is capable of increasing the activity of the mutant enzyme expressed by the host cell. Thus, unlike assays which utilize cells derived from a patient with a lysosomal storage disorder, the assay of the invention avoids time consuming steps such as collection of a sample from a patient, purification of cells from the sample, and culturing the cells from the sample in vitro.

[0047] The present invention also provides for a method of determining whether a patient expressing a mutant protein (e.g. a lysosomal enzyme) will be a candidate for SPC therapy, wherein a person, for example, a patient's physician or doctor, can look up the mutant protein (e.g. a lysosomal enzyme mutation) in a treatment reference table to determine if the patient's mutation will respond to SPC therapy. The reference table is generated from the results of in vitro analysis of SPC response in a cell line that has been transformed with a nucleic acid vector which encodes the mutant protein.

[0048] Furthermore, the invention also provides a "Treatment Reference Table" that provides information describing if a particular SPC will be a successful therapy for enhancing the activity of a specific lysosomal enzyme mutation. According to the present invention, the treatment reference table provides information indicating if a candidate SPC can increase the activity of a mutant lysosomal enzyme expressed by a host cell. Based on the response of different mutations to different SPC therapies, the present invention can provide SPC therapy tailored to the patient's specific mutation.

[0049] In one non-limiting embodiment, the mutant protein is a mutant lysosomal enzyme, such as, for example, a mutant .alpha.-Gal A, GAA or Gba, and the cell line is transfected with a nucleic acid vector which encodes the mutant lysosomal enzyme.

[0050] In another non-limiting embodiment, the present invention provides a method of treating a Fabry patient that includes the step of administering to the Fabry patient a therapeutically effective dose of 1-deoxygalactonojirimycin (DGJ), wherein the patient expresses a mutant .alpha.-Gal A, the activity of which, when expressed in a host cell, can be increased when contacted with an SPC (e.g. DGJ). Such .alpha.-Gal A mutations treatable according to this method include, but are not limited to A121T, A156V, A20P, A288D, A288P, A292P A348P, A73V, C52R, C94Y, D234E, D244H, D244N, D264Y, E338K, E341D, E358K, E398K, E48K, E59K, E66Q, F113L, G144V, G183D, G260A, G271S, G325D, G328A, G35R, G373D, G373S, H225R, I219N, I242N, I270T, I289F, I303N, I317T, I354K, I91T, L14P, L166V, L243F, L300F, L310F, L32P, L45R, M2671, M284T, M296I, M296V, M72V, M76R, N224S, N263S, N298K, N298S, N320I, N320Y, N34K, P205R, P259L, P265L, P265R, P293A, P293S, P409S, P40L, P40S, Q279E, Q279H, Q279R, Q280H, Q280K, Q312H, Q321E, Q321R, Q327E, R301P, R342Q, R363C, R363H, R49G, R49L, R49S, S201Y, S276N, S297C, S345P, T1941, V269M, V316E, W340R, W47L, and W95S mutations.

[0051] In one embodiment, the following .alpha.-Gal A mutations are excluded from the methods of treating a Fabry patient with a therapeutically effective dose of DGJ: D244N, E358K, E59K, E66Q, G183D, G325D, 1289F, I91T, L45R, M296V, N263S, N320Y, P205R, P40S, Q279E, R342Q, R363C, R49L, V316E.

[0052] One advantage of the assay described by the present invention is its applicability to female patients with an X-linked lysosomal storage disorder, such as Fabry disease. Because of X-chromosome inactivation, a sample taken from a female patient will comprise both normal healthy cells and enzyme deficient mutant cells. An assay for an SPC's effect on such a sample will show an enhancement in enzyme activity due to the normal wild type enzyme expression of the healthy cells even though the diseased cells with the mutant enzyme may not be responsive to the SPC. The present invention overcomes this obstacle because a cell line transfected with a vector encoding a mutant protein will only express the mutant form of the protein, and thus, there will be no wild type protein expressed by the cell line to cause such pseudo enhancement observed in assays with patient derived cells.

[0053] In another non-limiting embodiment, the present invention provides a method of treating a Pompe patient that includes the step of administering to the Pompe patient a therapeutically effective dose of 1-deoxynojirimycin (DNJ), wherein the patient expresses a mutant GAA, the activity of which, when expressed in a host cell, can be increased when contacted with an SPC (e.g. DNJ). Such GAA mutations treatable according to this method include, but are not limited to, E262K, P266S, P285R, P285S, L291F, L291H, L291P, M318K, G377R, A445P, Y455C, Y455F, P457L, G483R, G483V, M519V, S529V, P545L, G549R, L552P, Y575S, E579K, A610V, H612Q, A644P, and .DELTA.N470 mutations.

[0054] In another non-limiting embodiment, the present invention provides a method of treating a Gaucher patient with a therapeutically effective dose of isofagomine (IFG), wherein the patient expresses a mutant Gba, the activity of which, when expressed in a host cell, can be increased when contacted with an SPC (e.g. IFG).

DEFINITIONS

[0055] The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.

[0056] The term "Fabry disease" refers to an X-linked inborn error of glycosphingolipid catabolism due to deficient lysosomal .alpha.-galactosidase A activity. This defect causes accumulation of globotriaosylceramide (ceramide trihexoside) and related glycosphingolipids in vascular endothelial lysosomes of the heart, kidneys, skin, and other tissues.

[0057] The term "atypical Fabry disease" refers to patients with primarily cardiac manifestations of the .alpha.-Gal A deficiency, namely progressive globotriaosylceramide (GL-3) accumulation in myocardial cells that leads to significant enlargement of the heart, particularly the left ventricle.

[0058] A "carrier" is a female who has one X chromosome with a defective .alpha.-Gal A gene and one X chromosome with the normal gene and in whom X chromosome inactivation of the normal allele is present in one or more cell types. A carrier is often diagnosed with Fabry disease.

[0059] "Pompe disease" refers to an autosomal recessive LSD characterized by deficient acid alpha glucosidase (GAA) activity which impairs lysosomal glycogen metabolism. The enzyme deficiency leads to lysosomal glycogen accumulation and results in progressive skeletal muscle weakness, reduced cardiac function, respiratory insufficiency, and/or CNS impairment at late stages of disease. Genetic mutations in the GAA gene result in either lower expression or produce mutant forms of the enzyme with altered stability, and/or biological activity ultimately leading to disease. (see generally Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid .alpha.-Glucosidase (Acid Maltase) Deficiency, The Metabolic and Molecular Bases of Inherited Disease, Scriver et al., eds., McGraw-Hill, New York, 7th ed., pages 2443-2464). The three recognized clinical forms of Pompe disease (infantile, juvenile and adult) are correlated with the level of residual .alpha.-glucosidase activity (Reuser A J et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency), Muscle & Nerve Supplement 3, S61-S69). ASSC (also referred to elsewhere as "pharmacological chaperones") represent a promising new therapeutic approach for the treatment of genetic diseases, such as lysosomal storage disorders (e.g. Pompe Disease).

[0060] Infantile Pompe disease (type I or A) is most common and most severe, characterized by failure to thrive, generalized hypotonia, cardiac hypertrophy, and cardiorespiratory failure within the second year of life. Juvenile Pompe disease (type II or B) is intermediate in severity and is characterized by a predominance of muscular symptoms without cardiomegaly. Juvenile Pompe individuals usually die before reaching 20 years of age due to respiratory failure. Adult Pompe disease (type III or C) often presents as a slowly progressive myopathy in the teenage years or as late as the sixth decade (Felice K J et al., 1995, Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature, Medicine 74, 131-135).

[0061] In Pompe, it has been shown that .alpha.-glucosidase is extensively modified post-translationally by glycosylation, phosphorylation, and proteolytic processing. Conversion of the 110 kilodalton (kDa) precursor to 76 and 70 kDa mature forms by proteolysis in the lysosome is required for optimum glycogen catalysis.

[0062] As used herein, the term "Pompe Disease" refers to all types of Pompe Disease. The formulations and dosing regimens disclosed in this application may be used to treat, for example, Type I, Type II or Type III Pompe Disease.

[0063] The term "Gaucher disease" refers to a deficiency of the lysosomal enzyme .beta.-glucocerebrosidase (Gba) that breaks down fatty glucocerebrosides. The fat then accumulates, mostly in the liver, spleen and bone marrow. Gaucher disease can result in pain, fatigue, jaundice, bone damage, anemia and even death. There are three clinical phenotypes of Gaucher disease. Patients with, Type 1 manifest either early in life or in young adulthood, bruise easily and experience fatigue due to anemia, low blood platelets, enlargement of the liver and spleen, weakening of the skeleton, and in some instances have lung and kidney impairment. There are no signs of brain involvement. In Type II, early-onset, liver and spleen enlargement occurs by 3 months of age and there is extensive brain involvement. There is a high mortality rate by age 2. Type III is characterized by liver and spleen enlargement and brain seizures. The .beta.-glucocerebrosidase gene is located on the human 1q21 chromosome. Its protein precursor contains 536 amino acids and its mature protein is 497 amino acids long.

[0064] A "patient" refers to a subject who has been diagnosed with or is suspected of having a particular disease. The patient may be human or animal.

[0065] A "Fabry disease patient" refers to an individual who has been diagnosed with or suspected of having Fabry disease and has a mutated .alpha.-Gal A as defined further below. Characteristic markers of Fabry disease can occur in male hemizygotes and female carriers with the same prevalence, although females typically are less severely affected.

[0066] A "Pompe disease patient" refers to an individual who has been diagnosed with or suspected of having Pompe disease and has a mutated GAA as defined further below.

[0067] A "Gaucher disease patient" refers to an individual who has been diagnosed with or suspected of having Gaucher disease and has a mutated Gba as defined further below.

[0068] Human .alpha.-galactosidase A (.alpha.-Gal A) refers to an enzyme encoded by the human GLA gene. The human .alpha.-Gal A enzyme consists of 429 amino acids and is in GenBank Accession No. U78027.

[0069] In one non-limiting embodiment, human lysosomal alpha-glucosidase (Acid .alpha.-glucosidase; GAA) is a lysosomal enzyme which hydrolyzes alpha-1,4- and alpha-1,6-linked-D-glucose polymers present in glycogen, maltose, and isomaltose. Alternative names are as follows: glucoamylase; 1,4-.alpha.-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase; and exo-1,4-.alpha.-glucosidase. The human GAA gene has been mapped to chromosome 17q25.2-25.3 and has nucleotide and amino acid sequences depicted in GenBank Accession No. Y00839.

[0070] The term "human Gba gene" refers to the gene encoding acid .beta.-glucosidase, also referred to as glucocerebrosidase or Gba. The Gba gene is on chromosome 1q21 and involves 11 exons (GenBank Accession No. J03059). There is also a homologous pseudogene for Gba located about 16 kb downstream of the Gba gene (GenBank Accession No. M16328).

[0071] The "human Gba" protein refers to the wild-type human Gba protein. The Gba protein consists of 536 amino acids and is in GenBank Accession No. J03059.

[0072] The term "mutant protein" includes a protein which has a mutation in the gene encoding the protein which results in the inability of the protein to achieve a stable conformation under the conditions normally present in the ER. The failure to achieve a stable conformation results in a substantial amount of the enzyme being degraded, rather than being transported to the lysosome. Such a mutation is sometimes called a "conformational mutant." Such mutations include, but are not limited to, missense mutations, and in-frame small deletions and insertions.

[0073] As used herein in one embodiment, the term "mutant .alpha.-Gal A" includes an .alpha.-Gal A which has a mutation in the gene encoding .alpha.-Gal A which results in the inability of the enzyme to achieve a stable conformation under the conditions normally present in the ER. The failure to achieve a stable conformation results in a substantial amount of the enzyme being degraded, rather than being transported to the lysosome.

[0074] Non-limiting, exemplary .alpha.-Gal A mutations associated with Fabry disease which result in unstable .alpha.-Gal A include L32P; N34S; T41I; M51K; E59K; E66Q; I91T; A97V; R100K; R112C; R112H; F113L; T141L; A143T; G144V; S148N; A156V; L166V; D170V; C172Y; G183D; P205T; Y207C; Y207S; N215S; A228P; S235C; D244N; P259R; N263S; N264A; G272S; S276G; Q279E; Q279K; Q279H; M284T; W287C; 1289F; M296I; M296V; L300P; R301Q; V316E; N320Y; G325D; G328A; R342Q; E358A; E358K; R363C; R363H; G370S; and P409A.

[0075] As used herein in one embodiment, the term "mutant GAA" includes a GAA which has a mutation in the gene encoding GAA which results in the inability of the enzyme to achieve a stable conformation under the conditions normally present in the ER. The failure to achieve a stable conformation results in a substantial amount of the enzyme being degraded, rather than being transported to the lysosome.

[0076] As used herein in one embodiment, the term "mutant Gba" includes a Gba which has a mutation in the gene encoding Gba which results in the inability of the enzyme to achieve a stable conformation under the conditions normally present in the ER. The failure to achieve a stable conformation results in a substantial amount of the enzyme being degraded, rather than being transported to the lysosome.

[0077] As used herein, the term "specific pharmacological chaperone" ("SPC") or "pharmacological chaperone" refers to any molecule including a small molecule, protein, peptide, nucleic acid, carbohydrate, etc. that specifically binds to a protein and has one or more of the following effects: (i) enhances the formation of a stable molecular conformation of the protein; (ii) induces trafficking of the protein from the ER to another cellular location, preferably a native cellular location, i.e., prevents ER-associated degradation of the protein; (iii) prevents aggregation of misfolded proteins; and/or (iv) restores or enhances at least partial wild-type function and/or activity to the protein. A compound that specifically binds to e.g., .alpha.-Gal A, GAA or Gba, means that it binds to and exerts a chaperone effect on the enzyme and not a generic group of related or unrelated enzymes. More specifically, this term does not refer to endogenous chaperones, such as BiP, or to non-specific agents which have demonstrated non-specific chaperone activity against various proteins, such as glycerol, DMSO or deuterated water, i.e., chemical chaperones (see Welch et al., Cell Stress and Chaperones 1996; 1(2):109-115; Welch et al., Journal of Bioenergetics and Biomembranes 1997; 29(5):491-502; U.S. Pat. No. 5,900,360; U.S. Pat. No. 6,270,954; and U.S. Pat. No. 6,541,195). In the present invention, the SPC may be a reversible competitive inhibitor.

[0078] A "competitive inhibitor" of an enzyme can refer to a compound which structurally resembles the chemical structure and molecular geometry of the enzyme substrate to bind the enzyme in approximately the same location as the substrate. Thus, the inhibitor competes for the same active site as the substrate molecule, thus increasing the Km. Competitive inhibition is usually reversible if sufficient substrate molecules are available to displace the inhibitor, i.e., competitive inhibitors can bind reversibly. Therefore, the amount of enzyme inhibition depends upon the inhibitor concentration, substrate concentration, and the relative affinities of the inhibitor and substrate for the active site.

[0079] Following is a description of some specific pharmacological chaperones (SPCs) contemplated by this invention:

[0080] In one particular non-limiting embodiment, the SPC is 1-deoxygalactonorjirimycin which refers to a compound having the following structures:

##STR00001##

[0081] or a pharmaceutically acceptable salt, ester or prodrug of 1-deoxygalactonorjirimycin. The hydrochloride salt of DGJ is known as migalastat hydrochloride (Migalastat).

[0082] Still other SPCs for .alpha.-Gal A are described in U.S. Pat. Nos. 6,274,597, 6,774,135, and 6,599,919 to Fan et al., and include .alpha.-3,4-di-epi-homonojirimycin, 4-epi-fagomine, .alpha.-allo-homonojirimycin, N-methyl-deoxygalactonojirimycin, .beta.-1-C-butyl-deoxygalactonojirimycin, .alpha.-galacto-homonojirimycin, calystegine A.sub.3, calystegine B.sub.2, calystegine B.sub.3, N-methyl-calystegine A.sub.3, N-methyl-calystegine B.sub.2 and N-methyl-calystegine B.sub.3.

[0083] In one particular non-limiting embodiment, the SPC is isofagomine (IFG; (3R,4R,5R)-5-(hydroxymethyl)-3,4-piperidinediol) which is represented by the following formula:

##STR00002##

[0084] or a pharmaceutically acceptable salt, ester or prodrug of isofagomine, such as, for example, IFG tartrate (see, e.g., U.S. Patent Application Publication 20070281975.) IFG has a molecular formula of C.sub.6H.sub.13NO3 and a molecular weight of 147.17. This compound is further described in U.S. Pat. No. 5,844,102 to Sierks et al., and U.S. Pat. No. 5,863,903, to Lundgren et al.

[0085] Still other SPCs for Gba are described in U.S. Pat. No. 6,916,829 to Fan et al., and include C-benzyl isofagomine and derivatives, N-alkyl (C9-12)-DNJ, Glucoimidazole (and derivatives), C-alkyl-IFG (and derivatives), N-alkyl-.beta.-valeinamines, Fluphenozine, N-dodecyl-DNJ, calystegines A.sub.3, B.sub.1, B.sub.2 and C.sub.1

[0086] In one particular non-limiting embodiment, the SPC is 1-deoxynojirimycin (1-DNJ), which is represented by the following formula:

##STR00003##

[0087] or a pharmaceutically acceptable salt, ester or prodrug of 1-deoxynojirimycin. In one embodiment, the salt is hydrochloride salt (i.e. 1-deoxynojirimycin-HCl).

[0088] Still other SPCs for GAA are described in U.S. Pat. Nos. 6,274,597; 6,583,158; 6,599,919 and 6,916,829 to Fan et al., and U.S. Published Application No. 2006/0264467, and include N-methyl-DNJ, N-ethyl-DNJ, N-propyl-DNJ, N-butyl-DNJ, N-pentyl-DNJ, N-hexyl-DNJ, N-heptyl-DNJ, N-octyl-DNJ, N-nonyl-DNJ, N-methylcyclopropyl-DNJ, N-methylcyclopentyl-DNJ, N-2-hydroxyethyl-DNJ, 5-N-carboxypentyl DNJ, .alpha.-homonojirimycin, and castanospermine.

[0089] As used herein, the term "specifically binds" refers to the interaction of a pharmacological chaperone with a protein such as .alpha.-Gal A, Gba or GAA, specifically, an interaction with amino acid residues of the protein that directly participate in contacting the pharmacological chaperone. A pharmacological chaperone specifically binds a target protein, e.g., .alpha.-Gal A, Gba or GAA, to exert a chaperone effect on the protein and not a generic group of related or unrelated proteins. The amino acid residues of a protein that interact with any given pharmacological chaperone may or may not be within the protein's "active site." Specific binding can be evaluated through routine binding assays or through structural studies, e.g., co-crystallization, NMR, and the like. The active site for .alpha.-Gal A, Gba or GAA is the substrate binding site.

[0090] "Deficient .alpha.-Gal A activity" refers to .alpha.-Gal A activity in cells from a patient which is below the normal range as compared (using the same methods) to the activity in normal individuals not having or suspected of having Fabry or any other disease (especially a blood disease).

[0091] "Deficient Gba activity" refers to Gba activity in cells from a patient which is below the normal range as compared (using the same methods) to the activity in normal individuals not having or suspected of having Gaucher or any other disease.

[0092] "Deficient GAA activity" refers to GAA activity in cells from a patient which is below the normal range as compared (using the same methods) to the activity in normal individuals not having or suspected of having Pompe or any other disease.

[0093] As used herein, the terms "enhance .alpha.-Gal A activity," "enhance Gba activity," and "enhance GAA activity" or "increase .alpha.-Gal A activity," "increase Gba activity," and "increase GAA activity" refer to increasing the amount of .alpha.-Gal A, Gba or GAA, respectively, that adopts a stable conformation in a cell contacted with a pharmacological chaperone specific for the .alpha.-Gal A, Gba or GAA, relative to the amount in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for the .alpha.-Gal A, Gba or GAA. This term also refers to increasing the trafficking of .alpha.-Gal A, Gba or GAA to the lysosome in a cell contacted with a pharmacological chaperone specific for the .alpha.-Gal A, Gba or GAA, relative to the trafficking of .alpha.-Gal A, Gba or GAA not contacted with the pharmacological chaperone specific for the protein. These terms refer to both wild-type and mutant .alpha.-Gal A, Gba or GAA. In one embodiment, the increase in the amount of .alpha.-Gal A, Gba or GAA in the cell is measured by measuring the hydrolysis of an artificial substrate in lysates from cells that have been treated with the SPC. An increase in hydrolysis is indicative of increased .alpha.-Gal A, Gba or GAA activity.

[0094] The term ".alpha.-Gal A activity" refers to the normal physiological function of a wild-type .alpha.-Gal A in a cell. For example, .alpha.-Gal A activity includes hydrolysis of GL-3.

[0095] The term "Gba activity" refers to the normal physiological function of a wild-type .alpha.Gba in a cell. For example, Gba activity includes metabolism of fatty glucocerebrosides.

[0096] The term "GAA activity" refers to the normal physiological function of a wild-type Gaa in a cell. For example, GAA activity includes lysosomal glycogen metabolism.

[0097] A "responder" is an individual diagnosed with or suspected of having a lysosomal storage disorder, such, for example, but not limited to, Fabry disease, Pompe disease or Gaucher disease, whose cells exhibit sufficiently increased .alpha.-Gal A, GAA or Gba activity, respectively, and/or amelioration of symptoms or improvement in surrogate markers, in response to contact with an SPC. Non-limiting examples of improvements in surrogate markers for Fabry and Pompe disease are disclosed in U.S. Ser. No. 60/909,185 and 61/035,869, respectively.

[0098] Non-limiting examples of improvements in surrogate markers for Fabry disease disclosed in U.S. Ser. No. 60/909,185 include increases in .alpha.-Gal A levels or activity in cells (e.g., fibroblasts) and tissue; reductions in of GL-3 accumulation; decreased plasma concentrations of homocysteine and vascular cell adhesion molecule-1 (VCAM-1); decreased GL-3 accumulation within myocardial cells and valvular fibrocytes; reduction in cardiac hypertrophy (especially of the left ventricle), amelioration of valvular insufficiency, and arrhythmias; amelioration of proteinuria; decreased urinary concentrations of lipids such as CTH, lactosylceramide, ceramide, and increased urinary concentrations of glucosylceramide and sphingomyelin (Fuller et al., Clinical Chemistry. 2005; 51: 688-694); the absence of laminated inclusion bodies (Zebra bodies) in glomerular epithelial cells; improvements in renal function; mitigation of hypohidrosis; the absence of angiokeratomas; and improvements hearing abnormalities such as high frequency sensorineural hearing loss progressive hearing loss, sudden deafness, or tinnitus. Improvements in neurological symptoms include prevention of transient ischemic attack (TIA) or stroke; and amelioration of neuropathic pain manifesting itself as acroparaesthesia (burning or tingling in extremities).

[0099] The dose that achieves one or more of the aforementioned responses is a "therapeutically effective dose."

[0100] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other editions.

[0101] As used herein, the term "isolated" means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an mRNA band on a gel, a cDNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome. In yet another embodiment, the isolated nucleic acid lacks one or more introns. Isolated nucleic acids include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism. An isolated material may be, but need not be, purified.

[0102] The terms "about" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 10- or 5-fold, and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

Method of Determining Treatment Options

[0103] To easily determine whether SPC therapy will be a viable treatment for patients, for example, Fabry, Pompe or Gaucher patients, and including female carriers of X-linked lysosomal storage disorders such as Fabry disease, a simple, non-invasive SPC rescue assay of protein activity in a cell line expressing a mutant form of the protein was developed.

In Vitro Assay

[0104] In one embodiment, the diagnostic method of the present invention involves transforming a cell line with a nucleic acid vector which encodes a mutant lysosomal enzyme, for example, .alpha.-Gal A, GAA or Gba. The cell line is then treated with or without an SPC, e.g., DGJ, DNJ or IFG, for a sufficient time period to demonstrate enhancement (i.e., increase) of .alpha.-Gal A, GAA or Gba activity. The transformed cells are then lysed, and the lysate is used in an assay to determine enzyme activity. A sufficient increase in .alpha.-Gal A, GAA or Gba activity in the lysates from cells treated with the SPC over the activity in the lysates from untreated cells indicates that a patient who expresses .alpha.-Gal A, GAA or Gba with the same mutation as the cell line will likely respond to SPC therapy (i.e., the patient will be a "responder").

[0105] Transient Transfection of a Cell Line and Expression of a Mutant Lysosmal Enzyme

[0106] In one embodiment, to identify SPC-responsive mutations, all known lysosomal enzyme (e.g., .alpha.-Gal A, GAA or Gba) mutations, for example, missense mutations and in-frame small deletions and insertions, can be generated according to techniques known in the art, for example, by site-directed mutagenesis. Mutant enzyme constructs can then be transiently expressed in a cell line, for example, mammalian COS-7, HEK-293 or GripTite 293 MSR (Invitrogen Corp., Carlsbad, Calif., U.S.A.) cells. Transformed cells can then be incubated with increasing concentrations of SPC and enzymatic activity can be measured in cell lysates.

[0107] Mutagenesis:

[0108] Nucleic acid vectors encoding a mutant protein (e.g. mutant .alpha.-Gal A, GAA or Gba) can be generated by conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. (See, e.g., Sambrook, Fritsch & Maniatis, 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover, ed., 1985, DNA Cloning: A Practical Approach, Volumes I and II, Second Edition; Gait, M. J., ed., 1984, Oligonucleotide Synthesis: A practical approach; Hames, B. D. & Higgins, S. J. eds., 1985, Nucleic Acid Hybridization; Hames, B. D. & Higgins, S. J., eds., 1984, Transcription And Translation; Freshney, R. I., 2000, Culture of Animal Cells: A Manual of Basic Technique; Woodward, J., 1986, Immobilized Cells And Enzymes: A practical approach, IRL Press; Perbal, B. E., 1984, A Practical Guide To Molecular Cloning). For example, a single .alpha.-Gal A, GAA or Gba mutation can be introduced into a nucleic acid encoding a wild type .alpha.-Gal A, GAA or Gba gene through site directed mutagenesis of a nucleic acid encoding the wild type enzyme.

[0109] Transient Transfection and Expression:

[0110] The coding sequences of the gene to be delivered, for example, a mutant .alpha.-Gal A, GAA or Gba, are operably linked to expression control sequences, e.g., a promoter that directs expression of the gene. As used herein, the phrase "operatively linked" refers to the functional relationship of a polynucleotide/gene with regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of a nucleic acid to a promoter refers to the physical and functional relationship between the polynucleotide and the promoter such that transcription of DNA is initiated from the promoter by an RNA polymerase that specifically recognizes and binds to the promoter. The promoter directs the transcription of RNA from the polynucleotide. Expression of a mutant protein (e.g. mutant .alpha.-Gal A, GAA or Gba) may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.

[0111] In one specific embodiment, a vector is used in which the coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for expression of the construct from a nucleic acid molecule that has integrated into the genome (See Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA, 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438; U.S. Pat. No. 6,244,113 to Zarling et al.; and U.S. Pat. No. 6,200,812 to Pati et al.).

[0112] The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. In one embodiment, a host cells that is transfected with a vector encoding a mutant .alpha.-Gal A, GAA or Gba can be used for screening a candidate SPC, for example, DGJ, DNJ or IFG, to determine if the candidate SPC is an effective compound for increasing the activity of the mutant .alpha.-Gal A, GAA or Gba expressed by the host cell.

[0113] The term "expression system" means a host cell and compatible vector under suitable conditions, e.g., for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Expression systems include mammalian host cells and vectors. Suitable cells include PC12 cells, CHO cells, HeLa cells, GripTite 293 MSR cells (Invitrogen Corp., Carlsbad, Calif., U.S.A.), HEK-293 (also known as 293 cells) and 293T cells (derived from human embryonic kidney cells), COS cells (e.g. COS-7 cells), mouse primary myoblasts, NIH 3T3 cells.

[0114] Suitable vectors include viruses, such as adenoviruses, adeno-associated virus (AAV), vaccinia, herpesviruses, baculoviruses and retroviruses, parvovirus, lentivirus, bacteriophages, cosmids, plasmids, fungal vectors, naked DNA, DNA lipid complexes, and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.

[0115] In one non-limiting example, transient transfection can be carried out in GripTite 293 MSR cells (Invitrogen Corp., Carlsbad, Calif., U.S.A.) using the reagent Fugene HD (Roche). The cells can be seeded in a suitable assay container, such as a 96-well plate (Costar) at a density of, for example, 7.5-10 k cells/well, and incubated under suitable conditions, such as, for example, 37.degree. C., 5% CO.sub.2 for 24 hours before transfection. After transfection with expression constructs containing a specific .alpha.-Gal A mutant, cells can be incubated again in, for example, 37.degree. C., 5% CO.sub.2 for one hour before adding DGJ at 50 nM to 1 mM. Cells can then be incubated for 4-5 days before lysis and assay.

[0116] Enzyme Activity/Enhancement Assay:

[0117] Typically, following incubation with an SPC (e.g. DGJ, DNJ or IFG), host cells are lysed by the addition of lysis buffer (or deionized water) and physical disruption (pipetting, vortexing and/or agitation, and/or sonication) at room temperature or on ice, followed by pooling of the lysates on ice, then splitting the pooled lysate into small aliquots and freezing.

[0118] The lysates can be thawed immediately prior to the assay and should be suspended by use of a vortex mixer and sonicated prior to addition to appropriate wells e.g., in a microplate. In the context of Fabry disease, N-acetylgalactosamine (GalNAc) is then added to each well (to inhibit .alpha.-galactosidase B), followed by a short incubation. 4-methylumbelliferyl-.alpha.-D-galactopyranoside (4-MU Gal), or other appropriate labeled DGJ substrate, is then added and the plate is gently mixed for a brief period of time, covered, and incubated at 37.degree. C. for a sufficient time for substrate hydrolysis, usually about 1 hour. To stop the reaction, NaOH-glycine buffer, pH 10.7, is added to each well and the plate is read on a fluorescent plate reader (e.g. Wallac 1420 Victor3.TM. or similar instrument). Excitation and emission wavelengths were customarily set at 355 nm and 460 nm, respectively. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 nmole of 4-methylumbelliferone per hour. For each patient sample at least three normal samples may be tested concurrently.

[0119] Various modifications of this assay will be readily ascertainable to one of ordinary skill in the art. Examples of artificial substrates that can be used to detect .alpha.-Gal A activity include but are not limited to p-nitrophenyl-.alpha.-D-galactopyranoside and 4-MU GAL. Obviously, only substrates that can be cleaved by human .alpha.-Gal A are suitable for use. It is noted that while use of a fluorogenic substrate is preferred, other methods of determining enzymatic activity are contemplated for use in the method, including using chromogenic substrates or immunoquantification techniques.

[0120] In one specific example, following incubation with an SPC, for example, DGJ, the host cells can be washed two times with PBS then incubated in 200 .mu.l fresh media at 37.degree. C., 5% CO.sub.2 for two hours followed by 2 additional PBS washes. After, cells can be lysed in 60 .mu.L Lysis Buffer (27 mM sodium citrate/46 mM sodium phosphate dibasic, 0.5% Triton X-100, pH 4.6). Ten .mu.L lysate can then be added to 50 .mu.l assay buffer (Lysis Buffer without Triton X-100, but containing 6 mM 4-MU-.alpha.-D-galactopyranoside (4-MUG) and 117 mM N-acetyl-D-galactosamine (GalNac)), and incubated at 37.degree. C. for 1 hr. Seventy .mu.L Stop Solution (0.4 M glycine, pH 10.8) can then be added and fluorescence read on a Victor plate reader (Perkin Elmer) at 355 nm excitation and 460 nm emission. Raw fluorescence counts can be background subtracted as defined by counts from substrate solution only. A MicroBCA Protein Assay Kit (Pierce) was used according to manufacturer's instructions to determine protein concentration from 40 .mu.L of cell lysate. A 4-methylumbelliferone (4-MU) standard curve ranging from 30 .mu.M to 1.3 nM was run in parallel for calculation of absolute .alpha.-Gal A activity expressed as nmoles/mg protein/hr or further normalized to % of untreated wild type enzyme activity.

Treatment Reference Table

[0121] In another embodiment, the methods described supra can be used to generate a "treatment reference table" or "treatment therapy table," wherein the treatment reference table comprises a list of protein mutations, and further wherein the table indicates the responsiveness of each mutation to an SPC, such as DGJ, DNJ or IFG. The treatment reference table can then be used to determine if a particular SPC, for example, DGJ, DNJ or IFG, would be an effective SPC for treating a patient with a particular .alpha.-Gal A, GAA or Gba mutation, respectively.

[0122] As used herein "treatment therapy table" or "treatment reference table" refers to any written record that conveys whether a particular mutation is responsive to SPC therapy, and is not necessarily limited to written records presented in tabular form.

[0123] In one embodiment, the treatment reference table can be used by a treating physician or clinical professional to select an SPC for treating a patient, for example, a Fabry, Pompe or Gaucher patient who expresses a specific mutant .alpha.-Gal A, GAA or Gba, respectively, wherein the SPC is selected because the treatment reference table identifies the SPC as a compound that can increase the activity of the patient's mutant .alpha.-Gal A, GAA or Gba when the mutant .alpha.-Gal A, GAA or Gba is expressed in a host cell.

Treatable Disorders

[0124] While the present application has been discussed largely in the context of Fabry, Pompe and Gaucher diseases, and the SPCs DGJ, DNJ and IFG, respectively, it should be understood that it is applicable to any SPC and disease. In one non-limiting embodiment, a treatment reference table can be generated for any candidate SPC and any lysosomal storage disorder, or any disorder involving protein misfolding. These diseases include other lysosomal storage disorders, for example, Cystic Fibrosis (CFTR) (respiratory or sweat gland epithelial cells), familial hypercholesterolemia (LDL receptor; LPL-adipocytes or vascular endothelial cells), cancer (p53; PTEN-tumor cells), Alzheimer's disease (.alpha.-secretase), Parkinson's disease (glucocerebrosidase), obesity (MC4R), and amyloidoses (transthyretin) among others.

Eligibility Determination Criteria

[0125] The criteria for determining eligibility for SPC therapy depends on the type of mutant GLA, GAA or Gba a patient expresses. In one embodiment, patients with Fabry, Pompe, or Gaucher disease could be categorized as eligible for SPC therapy if .alpha.-Gal A, GAA or Gba activity, respectively, in a host cell expressing the same mutation as the patient, in the presence of an SPC such as DGJ, DNJ or IFG, is at least about 1.5- to 20-fold (2% to 100%) activity of a host cell expressing a wild type .alpha.-Gal A, GAA or Gba.

[0126] This discovery provides a method for improving the diagnosis of and facilitating clinical treatment decisions for Fabry, Pompe and Gaucher diseases in particular, and lysosomal storage disease in general. Moreover, this method can be extended to a wide range of genetically defined diseases in appropriate cell types. This class of disease includes the other lysosomal storage disorders, Cystic Fibrosis (CFTR) (respiratory or sweat gland epithelial cells), familial hypercholesterolemia (LDL receptor; LPL-adipocytes or vascular endothelial cells), cancer (p53; PTEN-tumor cells), Alzheimer's disease (.alpha.-secretase), Parkinson's disease (glucocerebrosidase), obesity (MC4R), and amyloidoses (transthyretin) among others.

Kits

[0127] The present invention also provides for a commercial diagnostic test kit in order to make therapeutic treatment decisions. The kit provides all materials discussed above and more particularly in the Examples below, for preparing and running each assay in one convenient package, optionally including instructions and an analytic guide.

[0128] As one non-limiting example, a kit for evaluating .alpha.-Gal A activity may contain, at a minimum:

[0129] a. a panel of host cells, each expressing a mutant .alpha.-Gal A, or alternatively, a host cell, a vector encoding a mutant .alpha.-Gal A, and a means of transfecting the host cell such that the host cell expresses the mutant .alpha.-Gal A;

[0130] b. a specific pharmacological chaperone;

[0131] c. a chromogenic or fluorogenic substrate for the enzyme assay (including an appropriate standard); and

[0132] d. GalNAc. The kit may also contain instructions for optimally performing the protein enhancement assay. In another embodiment, the kit will contain the appropriate tubes, buffers (e.g., lysis buffer), and microplates.

[0133] In one embodiment, the SPC is supplied in dry form, and will be re-constituted prior to addition.

[0134] Patients who express a mutant .alpha.-Gal A, GAA or Gba that previously tested positive for enzyme enhancement with a candidate SPC in assays of the present invention can then be treated with that candidate SPC agent, whereas patients who express a mutant .alpha.-Gal A, GAA or Gba that does not display enzyme enhancement with a candidate SPC can avoid treatment which will save money and prevent the emotional toll of not responding to a treatment modality.

EXAMPLES

[0135] The present invention is further described by means of the examples, presented below. The use of such examples is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.

Example 1

Identification of Fabry Disease-Causing Mutations that are Responsive to the Pharmacological Chaperone DGJ

[0136] The present Example provides the in vitro diagnostic assay to determine a Fabry patient's responsiveness to a specific pharmacological chaperone.

Introduction

[0137] Fabry disease is a lysosomal storage disorder caused by mutations in the gene that encodes .alpha.-galactosidase A (.alpha.-GAL A). Over 600 Fabry mutations have been reported, and about 60% are missense. The iminosugar DGJ is currently being studied in Phase 2 clinical trials as a pharmacological chaperone for the treatment of Fabry disease. Previously, it has been shown that DGJ mediates selective and dose-dependent increases in .alpha.-Gal A levels in many Fabry patient-derived lymphoid cell lines. To identify additional DGJ-responsive mutations, GripTite 293 MSR, (Invitrogen Corp., Carlsbad, Calif., U.S.A.) cells were transiently transfected with expression vectors containing all known .alpha.-Gal A missense mutations and several in-frame small deletions and insertions generated by site-directed mutagenesis. Mutant .alpha.-Gal A constructs were transiently expressed in HEK-293 cells. Cells were incubated with increasing concentrations of DGJ and .alpha.-Gal A activity was measured in cell lysates. Assay validation has been carried out on more than 35 missense mutations and the results obtained in HEK-293 cells were similar to those obtained from both Fabry patient-derived lymphoid cells and primary T-cell cultures (see U.S. Ser. No. 11/749,512), as well as to the .alpha.-Gal A enzyme responses observed in the white blood cells of Fabry patients after oral administration of DGJ in Phase 2 clinical trials.

Methods and Materials

[0138] Mutagenesis:

[0139] All mutations were generated by site-directed mutagenesis following standard molecular biology protocols. To generate point mutations, site-directed mutagenesis was used on the expression vector pcDNA3.1 (Invitrogen) containing human .alpha.-GAL A cDNA in-frame. Specific primer pairs were designed containing the desired mutation (FIG. 6). The mutagenesis was performed through the polymerase chain reaction using PfuUltra high-fidelity DNA polymerase (Stratagene) in a thermocycler. Each reaction mixture contained a total volume of 50 .mu.l with the following: 41.6 .mu.l dH.sub.2O, 5.0 .mu.l 10.times. PfuUltra HF reaction buffer, 0.5 .mu.L Forward-5'-primer (50 uM), 0.5 .mu.l Reverse-3'-primer, 1.0 .mu.l dNTP mix (containing 25 mM each dA, dT, dC, dG), 0.9 .mu.l human GLA in pcDNA3 (2 ng/.mu.l DNA), 0.5 .mu.l PfuUltra HD DNA polymerase. Thermocycler parameter used was the following: i) 94.degree. C. for 30 seconds, ii) 94.degree. C. for 30 seconds, 55-60.degree. C. for 30 seconds, 68.degree. C. for 6 minutes, iii) Repeat (ii) 16 times. Afterwards, 0.5 .mu.l Dpn I (New England Biolabs) was added to each reaction and incubated at 37.degree. C. for 2 hours. A volume of 7.5 .mu.l for each mutagenesis reaction was used to transform DH5.alpha. cells (New England Biolabs). Cells were then plated on LB-agar plates with 75 ug/ml ampicillin, and incubated at 37.degree. C. overnight. Bacterial colonies were picked, grown in liquid LB with ampicillin overnight, shaking, at 37.degree. C., and plasmid DNA extracted using QuickLyse Miniprep Kit (Qiagen). Mutants were confirmed by sequencing the full-length human GLA gene. For some of the mutants, human GLA cDNA was contained in the vector plasmid pCXN. Mutagenesis was performed in this vector with the NEB Fusion DNA polymerase. After confirming the mutation through sequencing, the plasmid was digested with EcoRI and subcloned into expression vector pcDNA3.1. Correct orientation was confirmed by digestion with Xho I.

[0140] Transient Transfection and Expression:

[0141] Transient transfection was carried out in GripTite 293 MSR cells (Invitrogen Corp., Carlsbad, Calif., U.S.A.) using the reagent Fugene HD (Roche). Briefly, cells were seeded in 96-well plates (Costar) at a density of 7.5-10 k cells/well and incubated at 37.degree. C., 5% CO.sub.2 for 24 hours before transfection. Cells were transfected with 0.1 .mu.g DNA and 0.35 .mu.L of Fugene HD reagent per well (DNA:Reagent ratio of 2:7). After transfection with expression constructs containing the specific .alpha.-Gal A mutants, cells were incubated again in 37.degree. C., 5% CO.sub.2 for one hour before adding DGJ at 20 nM to 1 mM. Cells were then incubated for 4-5 days before lysis and assay.

[0142] .alpha.-GAL A Activity Measurement:

[0143] Cells were washed two times with PBS then incubated in 200 .mu.l fresh media at 37.degree. C., 5% CO.sub.2 for two hours followed by 2 additional PBS washes. After, cells were lysed in 60 .mu.L Lysis Buffer (27 mM sodium citrate/46 mM sodium phosphate dibasic, 0.5% Triton X-100, pH 4.6). Ten .mu.L lysate were added to 50 .mu.l assay buffer (Lysis Buffer without Triton X-100, but containing 6 mM 4-MU-.alpha.-D-galactopyranoside (4-MUG) and 117 mM N-acetyl-D-galactosamine (GalNac)), and incubated at 37.degree. C. for 1 hr. Seventy pt Stop Solution (0.4 M glycine, pH 10.8) were then added and fluorescence read on a Victor plate reader (Perkin Elmer) at 355 nm excitation and 460 nm emission. Raw fluorescence counts were background subtracted as defined by counts from substrate solution only. A MicroBCA Protein Assay Kit (Pierce) was used according to manufacturer's instructions to determine protein concentration from 404 of cell lysate. A 4-methylumbelliferone (4-MU) standard curve ranging from 30 .mu.M to 1.3 nM was run in parallel for calculation of absolute .alpha.-Gal A activity expressed as nmoles/mg protein/hr or further normalized to % of untreated wild type enzyme activity.

[0144] Transient transfection and .alpha.-Gal A activity measurements were performed in quadruplicates and repeated at least 3 times for each mutation to calculate the average .alpha.-Gal A activity at each DGJ concentration. Significant response to DGJ was determined by a two-tailed, paired Student's T-test (p<0.05).

Results

[0145] All listed Fabry mutations were generated by site-directed mutagenesis (FIG. 1). Mutations identified in italicized text were not tested, while those identified in plain text were .alpha.-Gal A mutants that were responsive to DGJ treatment in the transient transfection assay, and those identified in bold and underscored text were not responsive to DGJ treatment in the transient transfection assay. The magnitude of increase in .alpha.-Gal A levels after DGJ treatment and EC50 values are listed for every tested mutation that responded to DGJ treatment (FIG. 2).

[0146] .alpha.-Gal A activity (expressed as nmol/mg protein/hr of 4-MU released) was measured in lysates prepared from transfected GripTite 293 cells incubated with increasing concentrations of DGJ. A typical concentration-dependent response is shown for L300P and a typical negative response to DGJ is shown for R227Q. Wild type exhibits high baseline activity and does not respond to DGJ in this assay (FIG. 3).

[0147] .alpha.-Gal A levels were measured in three different assays, reported as percentage of wild type, are compared for each mutation by plotting side by side. The three different assays examined .alpha.-Gal A levels in T-cells and lymphoblasts isolated from Fabry patients (for example, see U.S. Ser. No. 11/749,512), as well as in white blood cell (WBC) from DGJ Phase 2 studies

[0148] Blank bars indicate basal level (without DGJ treatment) and filled bars indicate the elevated level after DGJ treatment (FIG. 4).

[0149] Tested Fabry mutations were illustrated on the .alpha.-Gal A secondary structure (FIG. 5). No significant correlation between response and location on the protein sequence of a mutation was observed, suggesting that responsive as well as non-responsive mutations are distributed widely across the entire protein. Text color indicates DGJ response: green=response; red=no response; brown indicates that of the multiple mutations on that same site some responded to DGJ treatment, while others did not.

Conclusion

[0150] These described results are comparable to those obtained from Fabry patient-derived lymphoid or T cells, as well as to the .alpha.-Gal A enzyme responses observed in the white blood cells of Fabry patients after oral administration of DGJ in Phase 2 clinical trials.

[0151] Thus, the GripTite 293 MSR transient transfection assay is a reliable method for identifying DGJ-responsive mutations and characterizing the magnitude and potency of this response.

[0152] Among the responsive mutations identified, the increases in .alpha.-Gal A levels by DGJ treatment ranged from 1.3- to 40-fold (2% to 100% wild type), with EC.sub.50 values between 200 nM and >100 mM.

[0153] DGJ-responsive and non-responsive mutant forms did not appear to be located to particular regions or domains on the .alpha.-Gal A protein structure.

Example 2

Ex Vivo Method for Evaluating Effects of an SPC on Glucocerebrosidase Activity--Prophetic Example

[0154] Gaucher disease (GD) is caused by a deficiency of lysosomal glucocerebrosidase (GCase). Deficient GCase activity leads to an accumulation of glucosylceramide (GlcCer) and the development of symptoms such as anemia, thrombocytopenia, hepatosplenomegaly, bone necrosis, infarcts and osteoporosis, and in some cases, neuropathic disease. The specific pharmacological chaperone isofagomine tartrate (IFG) selectively binds and stabilizes mutant (N370S/N370S) GCase in the ER and increases its trafficking to the lysosome.

[0155] To evaluate the effects of IFG on different GCase variants, an ex vivo diagnostic assay will be prepared using Cos7 cells in order to ascertain IFG-responsive mutations.

[0156] Using the techniques described in Examples 1 and 4, COS-7 cell lines will be prepared that express missense mutations and several in-frame small deletions and insertions by site-directed mutagenesis. Assays will be prepared for all of the mutations listed in the x-axis of FIG. 8. IFG-activity response will be ascertained for each assay according to methods known in the art (see, e.g., U.S. Pat. No. 6,916,829, which is hereby incorporated by reference).

[0157] To determine the correlation of the IFG-response measured in the COS-7 cells to patient-derived cells, IFG-activity response was also measured in Patient-Derived Macrophages and Lymphoblasts. Macrophages were successfully derived from 46 of 63 patients and incubation with IFG (3, 10, 30 or 100 .mu.M) for 5 days increased GCase levels in macrophages from 42 of 46 patients (mean=2.3-fold; range: 1.1- to 6.5-fold). Residual activity levels and response to IFG was more consistent for the same genotypes when measured in lymphoblasts compared to macrophages, potentially due to the variability in macrophage viability between different patients. The results are shown in FIG. 8.

[0158] The response to IFG for the patient-derived cells will be compared to the results obtained in the Cos7 cell line.

Example 3

In Vivo Effect of an SPC on .alpha.-GAL a Activity in Skin, Heart, Kidney and Plasma

[0159] To determine if increased mutant .alpha.-Gal A levels translate to increased .alpha.-Gal A activity in situ, the effect of DGJ administration on tissue GL-3 levels was investigated in vivo in hR301Q .alpha.-Gal A Tg/KO mice.

[0160] Eight-week old male hR301Q .alpha.-Gal A Tg/KO mice were treated for 4 weeks with 300 mg/kg DGJ in drinking water either daily or less frequently (4 days ON/3 days OFF). After dosing, lysates were prepared from skin, heart, kidney, and plasma by homogenizing .about.50 mg tissue in Lysis Buffer (see above). 20 .mu.L lysate were mixed with 50 .mu.L of substrate (as detailed above). Reaction mixtures were incubated at 37.degree. C. for 1 hr. After, 70 .mu.L Stop Solution were added and fluorescence was read on a Victor plate reader as described above. Enzyme activity in the lysates was background subtracted, and normalized for protein concentration. A 4-MU standard curve was run for conversion of fluorescence data to absolute .alpha.-Gal A activity expressed as nmol/mg protein/hr.

[0161] Tissue samples were washed free of blood, weighed and homogenized with a solvent system in a FastPrep.RTM. system. Homogenate was then extracted using Solid Phase Extraction on a C18 cartridge. The eluent was evaporated and reconstituted prior to injection onto a LC-MS/MS system. Twelve GL-3 isoforms were measured using positive ESI-MS/MS. LC separation was achieved on 00839a Zorbax C18 column.

[0162] Significant decreases in GL-3 levels were seen with daily and less frequent DGJ dosing in skin, heart, kidney, and plasma (FIG. 9). A trend of greater reduction in GL-3 levels was seen in multiple tissues and plasma with less frequent DGJ dosing. Collectively, these results indicate that DGJ merits further evaluation for the treatment of patients with Fabry disease.

Example 4

Identification of Pompe Disease-Causing Mutations that are Responsive to the Pharmacological Chaperone DNJ

[0163] Pompe disease is caused by deficient acid alpha glucosidase (GAA) activity which impairs lysosomal glycogen metabolism. The enzyme deficiency leads to lysosomal glycogen accumulation and results in progressive skeletal muscle weakness, reduced cardiac function, respiratory insufficiency, and CNS impairment at late stages of disease. Genetic mutations in the GAA gene result in either lower expression or produce mutant faints of the enzyme with altered stability, and/or biological activity ultimately leading to disease. Pharmacological chaperones represent a promising new therapeutic approach for the treatment of genetic diseases.

[0164] To evaluate the effects of DNJ on different GAA variants, an in vitro diagnostic assay was prepared using COS-7 and HEK-293 cells in order to ascertain DNJ-responsive mutations (FIGS. 10, 12 and 14)

[0165] A site-directed mutagenesis approach was employed to introduce specific mutations into the complementary DNA (cDNA) encoding wild-type human acid .alpha.-glucosidase (GAA). The initial wild-type GAA DNA construct was generated by subcloning the GAA coding region from cDNA clone 5739991 (Invitrogen) into the pcDNA6N5-HisA mammalian expression vector (Initrogen). The resultant DNA construct (designated as wild-type GAA cDNA) was used as the DNA template for subsequent mutagenesis. These missense, small insertion or deletion mutations are cited in the Erasmus database and known to be associated with type 2 glycogen storage disorder (GSD II), also known as Pompe disease. Briefly, wild-type GAA cDNA was PCR-amplified using mutagenic primers to obtain plasmid DNA with the desired mutation These mutations were confirmed by DNA sequencing prior to protein expression in cells.

[0166] COS-7 cells (derived from green monkey embryonic kidney cells) were aseptically seeded in 12-well tissue culture plates at a cell density of .about.1.4.times.10.sup.5 cells per well in 3 ml of Dulbecco's Modified Essential Medium (DMEM) containing 10% (v/v) fetal bovine serum and grown overnight at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere. On the following day, the cells (typically 60-80% confluent) were transfected with 0.75 .mu.g of the individual DNA construct via a lipid transfection reagent such as FUGENE HD (Roche) according the manufacturer's instructions. Two wells were transfected with each DNA construct such that one well was incubated with DNJ (typically 0 .mu.M, 20 .mu.M, 50 .mu.M or 100 .mu.M) while an equivalent volume of PBS was added to the other well. Two additional wells were transfected with the empty vector (no GAA cDNA) and incubated with or without DNJ to serve as the background control for endogenous monkey GAA expression. Similarly, 2 additional wells were transfected with the wild-type human GAA cDNA and incubated with or without DNJ to serve as the positive control. All samples were incubated for .about.48 hrs at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere.

[0167] After the 48-hour incubation period, the spent media was removed and the cells were washed with PBS and then incubated with fresh 1-2 ml DMEM medium for 3 hours at 37.degree. C. in a humidified 5% CO.sub.2 atmosphere. The medium was subsequently removed and cells were immediately washed with PBS and lysed with 200 .mu.l of Lysis Buffer (25 mM Bis-Tris (pH 6.5), 150 mM NaCl, 1% (v/v) Triton X-100) containing a cocktail of protease inhibitors. The cell culture plate were then gently swirled on a rotating orbital shaker apparatus for 10 min at room temperature for complete cell lysis. The resultant cell lysates were transferred to clean 1.5 ml microcentrifuge tubes and spun at 20,000.times.g for 10 mM to pellet cellular debris. Approximately 175 .mu.l of each supernatant sample was then transferred to a 1.5 ml fresh microcentrifuge tube. This cell lysate was used for all subsequent assays including GAA enzyme activity, total protein concentration determination, and Western blotting.

[0168] Residual GAA enzyme activity was determined for each transiently-expressed GAA using a fluorogenic 4-methylumbelliferyl-.alpha.-glucopyranoside (4-MU-.alpha.-glucose) substrate (Sigma). Briefly, 10 .mu.l of each cell lysate was assayed (in triplicate) in a 100 .mu.l reaction in 96-well clear bottom black plates using 3 mM 4-MU-.alpha.-glucose and 50 mM KOAc (pH 4.0). The transiently-expressed wild-type GAA sample was diluted 20-fold with Lysis Buffer to ensure that the enzymatic reaction is maintained within the linear range of the instrument. The enzyme reactions were performed at 37.degree. C. for 1 hour and terminated by the addition of 50 .mu.l of 500 mM Na.sub.2CO.sub.3 (pH 10.5). The assay was then read in a fluorescence plate reader (using 355 nm excitation/460 nm emission) to quantitate the amount of GAA-dependent 4-MU fluorescence liberated. The GAA enzyme activity was then extrapolated from a free 4-MU standard curve after subtracting the background fluorescence (i.e., empty vector control).

[0169] Twenty five microliters of each cell lysate was used in a parallel assay to determine the total cellular protein concentration using the bicinchoninic acid (BCA) protein assay (Pierce) according to the manufacturer's protocol. The total cellular protein concentration was extrapolated from a bovine serum albumin (BSA) standard curve.

[0170] The GAA enzyme activity for each sample was normalized to the total cellular protein concentration and expressed as the nmoles of 4-MU released/mg total protein/hr to define the GAA specific activity. The resultant GAA specific activity after DNJ treatment was compared to GAA enzyme activity of the corresponding untreated sample to determine whether a specific GAA mutant responds to DNJ.

[0171] For a single HEK-293 cell line transfected with the GAA mutation, P545L, the DNJ EC.sub.50 was also determined (FIG. 14).

[0172] To determine the correlation of the DNJ-response measured in the COS-7 cells to patient-derived cells, DNJ-activity response was also measured ex vivo in Patient-Derived Macrophages and Lymphoblasts.

[0173] Fibroblast and lymphocyte cell lines derived from Pompe patients were also generated as previously described (see U.S. Ser. No. 11/749,512). Fibroblast cell lines were derived from patients homozygous for the P545L or R854X GAA mutations (FIG. 13). Lymphocyte cell lines were derived from patients heterozygous for the (IVS1AS, T>G, -13) GAA splicing defect and GAA frameshift mutation (FIG. 15).

[0174] GAA activity was measured in the lymphocyte cell lines following incubation in 0 .mu.M, 30 .mu.M, 100 .mu.M, or 300 .mu.M DNJ (FIG. 15). GAA activity was also measured in the fibroblast cell lines following DNJ incubation (FIG. 13).

[0175] In this study, the pharmacological chaperone 1-deoxynojirimycin-HCl (DNJ) is shown to bind mutant GAA and increase its activity. In Pompe patient-derived fibroblasts (FIG. 13) and lymphocytes (FIG. 15), as well as in transiently transfected COS-7 (FIGS. 10 and 12) or HEK-293 (FIG. 14) cells expressing certain GAA missense mutations, DNJ significantly increases GAA levels.

[0176] DNJ increased GAA activity for 26 mutations (FIG. 10) out of 131 mutants tested (data not shown). In addition to increasing the activity of these mutant GAA's, DNJ also promoted processing of GAA to the 95/76/70 kDa forms.

[0177] Furthermore, dose-dependent increases in GAA activity was observed in patient-derived lymphocytes containing the common IVS1AS, T>G, -13 splicing defect in one allele and a frameshift mutation in the second allele (FIG. 15).

[0178] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0179] Patents, patent applications, publications, product descriptions, GenBank Accession Numbers, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purpose.

Sequence CWU 1

1

5661398PRTHomo sapiens 1Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp1 5 10 15 Glu Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys 20 25 30 Ile Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu 35 40 45 Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp 50 55 60 Met Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln65 70 75 80 Arg Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys 85 90 95 Gly Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala 100 105 110 Gly Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe 115 120 125 Ala Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp 130 135 140 Ser Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu145 150 155 160 Asn Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr 165 170 175 Met Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys 180 185 190 Asn His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile 195 200 205 Lys Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp 210 215 220 Val Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly225 230 235 240 Asn Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp 245 250 255 Ala Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile 260 265 270 Ser Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile 275 280 285 Asn Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp 290 295 300 Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val305 310 315 320 Ala Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile 325 330 335 Ala Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe 340 345 350 Ile Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp 355 360 365 Thr Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu 370 375 380 Gln Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu385 390 395 21312DNAHomo sapiens 2gaattctccg gtcaccgtga caatgcagct gaggaaccca gaactacatc tgggctgcgc 60gcttgcgctt cgcttcctgg ccctcgtttc ctgggacatc cctggggcta gagcactgga 120caatggattg gcaaggacgc ctaccatggg ctggctgcac tgggagcgct tcatgtgcaa 180ccttgactgc caggaagagc cagattcctg catcagtgag aagctcttca tggagatggc 240agagctcatg gtctcagaag gctggaagga tgcaggttat gagtacctct gcattgatga 300ctgttggatg gctccccaaa gagattcaga aggcagactt caggcagacc ctcagcgctt 360tcctcatggg attcgccagc tagctaatta tgttcacagc aaaggactga agctagggat 420ttatgcagat gttggaaata aaacctgcgc aggcttccct gggagttttg gatactacga 480cattgatgcc cagacctttg ctgactgggg agtagatctg ctaaaatttg atggttgtta 540ctgtgacagt ttggaaaatt tggcagatgg ttataagcac atgtccttgg ccctgaatag 600gactggcaga agcattgtgt actcctgtga gtggcctctt tatatgtggc cctttcaaaa 660gcccaattat acagaaatcc gacagtactg caatcactgg cgaaattttg ctgacattga 720tgattcctgg aaaagtataa agagtatctt ggactggaca tcttttaacc aggagagaat 780tgttgatgtt gctggaccag ggggttggaa tgacccagat atgttagtga ttggcaactt 840tggcctcagc tggaatcagc aagtaactca gatggccctc tgggctatca tggctgctcc 900tttattcatg tctaatgacc tccgacacat cagccctcaa gccaaagctc tccttcagga 960taaggacgta attgccatca atcaggaccc cttgggcaag caagggtacc agcttagaca 1020gggagacaac tttgaagtgt gggaacgacc tctctcaggc ttagcctggg ctgtagctat 1080gataaaccgg caggagattg gtggacctcg ctcttatacc atcgcagttg cttccctggg 1140taaaggagtg gcctgtaatc ctgcctgctt catcacacag ctcctccctg tgaaaaggaa 1200gctagggttc tatgaatgga cttcaaggtt aagaagtcac ataaatccca caggcactgt 1260tttgcttcag ctagaaaata caatgcagat gtcattaaaa gacttacttt aa 131233624DNAHomo sapiens 3cagttgggaa agctgaggtt gtcgccgggg ccgcgggtgg aggtcgggga tgaggcagca 60ggtaggacag tgacctcggt gacgcgaagg accccggcca cctctaggtt ctcctcgtcc 120gcccgttgtt cagcgaggga ggctctgggc ctgccgcagc tgacggggaa actgaggcac 180ggagcgggcc tgtaggagct gtccaggcca tctccaacca tgggagtgag gcacccgccc 240tgctcccacc ggctcctggc cgtctgcgcc ctcgtgtcct tggcaaccgc tgcactcctg 300gggcacatcc tactccatga tttcctgctg gttccccgag agctgagtgg ctcctcccca 360gtcctggagg agactcaccc agctcaccag cagggagcca gcagaccagg gccccgggat 420gcccaggcac accccggccg tcccagagca gtgcccacac agtgcgacgt cccccccaac 480agccgcttcg attgcgcccc tgacaaggcc atcacccagg aacagtgcga ggcccgcggc 540tgctgctaca tccctgcaaa gcaggggctg cagggagccc agatggggca gccctggtgc 600ttcttcccac ccagctaccc cagctacaag ctggagaacc tgagctcctc tgaaatgggc 660tacacggcca ccctgacccg taccaccccc accttcttcc ccaaggacat cctgaccctg 720cggctggacg tgatgatgga gactgagaac cgcctccact tcacgatcaa agatccagct 780aacaggcgct acgaggtgcc cttggagacc ccgcgtgtcc acagccgggc accgtcccca 840ctctacagcg tggagttctc cgaggagccc ttcggggtga tcgtgcaccg gcagctggac 900ggccgcgtgc tgctgaacac gacggtggcg cccctgttct ttgcggacca gttccttcag 960ctgtccacct cgctgccctc gcagtatatc acaggcctcg ccgagcacct cagtcccctg 1020atgctcagca ccagctggac caggatcacc ctgtggaacc gggaccttgc gcccacgccc 1080ggtgcgaacc tctacgggtc tcaccctttc tacctggcgc tggaggacgg cgggtcggca 1140cacggggtgt tcctgctaaa cagcaatgcc atggatgtgg tcctgcagcc gagccctgcc 1200cttagctgga ggtcgacagg tgggatcctg gatgtctaca tcttcctggg cccagagccc 1260aagagcgtgg tgcagcagta cctggacgtt gtgggatacc cgttcatgcc gccatactgg 1320ggcctgggct tccacctgtg ccgctggggc tactcctcca ccgctatcac ccgccaggtg 1380gtggagaaca tgaccagggc ccacttcccc ctggacgtcc aatggaacga cctggactac 1440atggactccc ggagggactt cacgttcaac aaggatggct tccgggactt cccggccatg 1500gtgcaggagc tgcaccaggg cggccggcgc tacatgatga tcgtggatcc tgccatcagc 1560agctcgggcc ctgccgggag ctacaggccc tacgacgagg gtctgcggag gggggttttc 1620atcaccaacg agaccggcca gccgctgatt gggaaggtat ggcccgggtc cactgccttc 1680cccgacttca ccaaccccac agccctggcc tggtgggagg acatggtggc tgagttccat 1740gaccaggtgc ccttcgacgg catgtggatt gacatgaacg agccttccaa cttcatcaga 1800ggctctgagg acggctgccc caacaatgag ctggagaacc caccctacgt gcctggggtg 1860gttgggggga ccctccaggc ggccaccatc tgtgcctcca gccaccagtt tctctccaca 1920cactacaacc tgcacaacct ctacggcctg accgaagcca tcgcctccca cagggcgctg 1980gtgaaggctc gggggacacg cccatttgtg atctcccgct cgacctttgc tggccacggc 2040cgatacgccg gccactggac gggggacgtg tggagctcct gggagcagct cgcctcctcc 2100gtgccagaaa tcctgcagtt taacctgctg ggggtgcctc tggtcggggc cgacgtctgc 2160ggcttcctgg gcaacacctc agaggagctg tgtgtgcgct ggacccagct gggggccttc 2220taccccttca tgcggaacca caacagcctg ctcagtctgc cccaggagcc gtacagcttc 2280agcgagccgg cccagcaggc catgaggaag gccctcaccc tgcgctacgc actcctcccc 2340cacctctaca cactgttcca ccaggcccac gtcgcggggg agaccgtggc ccggcccctc 2400ttcctggagt tccccaagga ctctagcacc tggactgtgg accaccagct cctgtggggg 2460gaggccctgc tcatcacccc agtgctccag gccgggaagg ccgaagtgac tggctacttc 2520cccttgggca catggtacga cctgcagacg gtgccaatag aggcccttgg cagcctccca 2580cccccacctg cagctccccg tgagccagcc atccacagcg aggggcagtg ggtgacgctg 2640ccggcccccc tggacaccat caacgtccac ctccgggctg ggtacatcat ccccctgcag 2700ggccctggcc tcacaaccac agagtcccgc cagcagccca tggccctggc tgtggccctg 2760accaagggtg gagaggcccg aggggagctg ttctgggacg atggagagag cctggaagtg 2820ctggagcgag gggcctacac acaggtcatc ttcctggcca ggaataacac gatcgtgaat 2880gagctggtac gtgtgaccag tgagggagct ggcctgcagc tgcagaaggt gactgtcctg 2940ggcgtggcca cggcgcccca gcaggtcctc tccaacggtg tccctgtctc caacttcacc 3000tacagccccg acaccaaggt cctggacatc tgtgtctcgc tgttgatggg agagcagttt 3060ctcgtcagct ggtgttagcc gggcggagtg tgttagtctc tccagaggga ggctggttcc 3120ccagggaagc agagcctgtg tgcgggcagc agctgtgtgc gggcctgggg gttgcatgtg 3180tcacctggag ctgggcacta accattccaa gccgccgcat cgcttgtttc cacctcctgg 3240gccggggctc tggcccccaa cgtgtctagg agagctttct ccctagatcg cactgtgggc 3300cggggcctgg agggctgctc tgtgttaata agattgtaag gtttgccctc ctcacctgtt 3360gccggcatgc gggtagtatt agccaccccc ctccatctgt tcccagcacc ggagaagggg 3420gtgctcaggt ggaggtgtgg ggtatgcacc tgagctcctg cttcgcgcct gctgctctgc 3480cccaacgcga ccgcttcccg gctgcccaga gggctggatg cctgccggtc cccgagcaag 3540cctgggaact caggaaaatt cacaggactt gggagattct aaatcttaag tgcaattatt 3600ttaataaaag gggcatttgg aatc 36244952PRTHomo sapiens 4Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys1 5 10 15 Ala Leu Val Ser Leu Ala Thr Ala Ala Leu Leu Gly His Ile Leu Leu 20 25 30 His Asp Phe Leu Leu Val Pro Arg Glu Leu Ser Gly Ser Ser Pro Val 35 40 45 Leu Glu Glu Thr His Pro Ala His Gln Gln Gly Ala Ser Arg Pro Gly 50 55 60 Pro Arg Asp Ala Gln Ala His Pro Gly Arg Pro Arg Ala Val Pro Thr65 70 75 80 Gln Cys Asp Val Pro Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 85 90 95 Ala Ile Thr Gln Glu Gln Cys Glu Ala Arg Gly Cys Cys Tyr Ile Pro 100 105 110 Ala Lys Gln Gly Leu Gln Gly Ala Gln Met Gly Gln Pro Trp Cys Phe 115 120 125 Phe Pro Pro Ser Tyr Pro Ser Tyr Lys Leu Glu Asn Leu Ser Ser Ser 130 135 140 Glu Met Gly Tyr Thr Ala Thr Leu Thr Arg Thr Thr Pro Thr Phe Phe145 150 155 160 Pro Lys Asp Ile Leu Thr Leu Arg Leu Asp Val Met Met Glu Thr Glu 165 170 175 Asn Arg Leu His Phe Thr Ile Lys Asp Pro Ala Asn Arg Arg Tyr Glu 180 185 190 Val Pro Leu Glu Thr Pro Arg Val His Ser Arg Ala Pro Ser Pro Leu 195 200 205 Tyr Ser Val Glu Phe Ser Glu Glu Pro Phe Gly Val Ile Val His Arg 210 215 220 Gln Leu Asp Gly Arg Val Leu Leu Asn Thr Thr Val Ala Pro Leu Phe225 230 235 240 Phe Ala Asp Gln Phe Leu Gln Leu Ser Thr Ser Leu Pro Ser Gln Tyr 245 250 255 Ile Thr Gly Leu Ala Glu His Leu Ser Pro Leu Met Leu Ser Thr Ser 260 265 270 Trp Thr Arg Ile Thr Leu Trp Asn Arg Asp Leu Ala Pro Thr Pro Gly 275 280 285 Ala Asn Leu Tyr Gly Ser His Pro Phe Tyr Leu Ala Leu Glu Asp Gly 290 295 300 Gly Ser Ala His Gly Val Phe Leu Leu Asn Ser Asn Ala Met Asp Val305 310 315 320 Val Leu Gln Pro Ser Pro Ala Leu Ser Trp Arg Ser Thr Gly Gly Ile 325 330 335 Leu Asp Val Tyr Ile Phe Leu Gly Pro Glu Pro Lys Ser Val Val Gln 340 345 350 Gln Tyr Leu Asp Val Val Gly Tyr Pro Phe Met Pro Pro Tyr Trp Gly 355 360 365 Leu Gly Phe His Leu Cys Arg Trp Gly Tyr Ser Ser Thr Ala Ile Thr 370 375 380 Arg Gln Val Val Glu Asn Met Thr Arg Ala His Phe Pro Leu Asp Val385 390 395 400 Gln Trp Asn Asp Leu Asp Tyr Met Asp Ser Arg Arg Asp Phe Thr Phe 405 410 415 Asn Lys Asp Gly Phe Arg Asp Phe Pro Ala Met Val Gln Glu Leu His 420 425 430 Gln Gly Gly Arg Arg Tyr Met Met Ile Val Asp Pro Ala Ile Ser Ser 435 440 445 Ser Gly Pro Ala Gly Ser Tyr Arg Pro Tyr Asp Glu Gly Leu Arg Arg 450 455 460 Gly Val Phe Ile Thr Asn Glu Thr Gly Gln Pro Leu Ile Gly Lys Val465 470 475 480 Trp Pro Gly Ser Thr Ala Phe Pro Asp Phe Thr Asn Pro Thr Ala Leu 485 490 495 Ala Trp Trp Glu Asp Met Val Ala Glu Phe His Asp Gln Val Pro Phe 500 505 510 Asp Gly Met Trp Ile Asp Met Asn Glu Pro Ser Asn Phe Ile Arg Gly 515 520 525 Ser Glu Asp Gly Cys Pro Asn Asn Glu Leu Glu Asn Pro Pro Tyr Val 530 535 540 Pro Gly Val Val Gly Gly Thr Leu Gln Ala Ala Thr Ile Cys Ala Ser545 550 555 560 Ser His Gln Phe Leu Ser Thr His Tyr Asn Leu His Asn Leu Tyr Gly 565 570 575 Leu Thr Glu Ala Ile Ala Ser His Arg Ala Leu Val Lys Ala Arg Gly 580 585 590 Thr Arg Pro Phe Val Ile Ser Arg Ser Thr Phe Ala Gly His Gly Arg 595 600 605 Tyr Ala Gly His Trp Thr Gly Asp Val Trp Ser Ser Trp Glu Gln Leu 610 615 620 Ala Ser Ser Val Pro Glu Ile Leu Gln Phe Asn Leu Leu Gly Val Pro625 630 635 640 Leu Val Gly Ala Asp Val Cys Gly Phe Leu Gly Asn Thr Ser Glu Glu 645 650 655 Leu Cys Val Arg Trp Thr Gln Leu Gly Ala Phe Tyr Pro Phe Met Arg 660 665 670 Asn His Asn Ser Leu Leu Ser Leu Pro Gln Glu Pro Tyr Ser Phe Ser 675 680 685 Glu Pro Ala Gln Gln Ala Met Arg Lys Ala Leu Thr Leu Arg Tyr Ala 690 695 700 Leu Leu Pro His Leu Tyr Thr Leu Phe His Gln Ala His Val Ala Gly705 710 715 720 Glu Thr Val Ala Arg Pro Leu Phe Leu Glu Phe Pro Lys Asp Ser Ser 725 730 735 Thr Trp Thr Val Asp His Gln Leu Leu Trp Gly Glu Ala Leu Leu Ile 740 745 750 Thr Pro Val Leu Gln Ala Gly Lys Ala Glu Val Thr Gly Tyr Phe Pro 755 760 765 Leu Gly Thr Trp Tyr Asp Leu Gln Thr Val Pro Ile Glu Ala Leu Gly 770 775 780 Ser Leu Pro Pro Pro Pro Ala Ala Pro Arg Glu Pro Ala Ile His Ser785 790 795 800 Glu Gly Gln Trp Val Thr Leu Pro Ala Pro Leu Asp Thr Ile Asn Val 805 810 815 His Leu Arg Ala Gly Tyr Ile Ile Pro Leu Gln Gly Pro Gly Leu Thr 820 825 830 Thr Thr Glu Ser Arg Gln Gln Pro Met Ala Leu Ala Val Ala Leu Thr 835 840 845 Lys Gly Gly Glu Ala Arg Gly Glu Leu Phe Trp Asp Asp Gly Glu Ser 850 855 860 Leu Glu Val Leu Glu Arg Gly Ala Tyr Thr Gln Val Ile Phe Leu Ala865 870 875 880 Arg Asn Asn Thr Ile Val Asn Glu Leu Val Arg Val Thr Ser Glu Gly 885 890 895 Ala Gly Leu Gln Leu Gln Lys Val Thr Val Leu Gly Val Ala Thr Ala 900 905 910 Pro Gln Gln Val Leu Ser Asn Gly Val Pro Val Ser Asn Phe Thr Tyr 915 920 925 Ser Pro Asp Thr Lys Val Leu Asp Ile Cys Val Ser Leu Leu Met Gly 930 935 940 Glu Gln Phe Leu Val Ser Trp Cys945 950 518DNAArtificial SequenceSynthetic oligonucleotide 5cgtgacaata cagctgag 18618DNAArtificial SequenceSynthetic oligonucleotide 6ctcagctgta ttgtcacg 18722DNAArtificial SequenceSynthetic oligonucleotide 7caccgtgaca acgcagctga gg 22822DNAArtificial SequenceSynthetic oligonucleotide 8cctcagctgc gttgtcacgg tg 22920DNAArtificial SequenceSynthetic oligonucleotide 9ggctgcgcgc ctgcgcttcg 201020DNAArtificial SequenceSynthetic oligonucleotide 10cgaagcgcag gcgcgcagcc 201122DNAArtificial SequenceSynthetic oligonucleotide 11gcgcttgcgc mtcgcttcct gg 221222DNAArtificial SequenceSynthetic oligonucleotide 12ccaggaagcg akgcgcaagc gc 221324DNAArtificial SequenceSynthetic oligonucleotide 13gcttcgcttc ccggccctcg tttc 241424DNAArtificial SequenceSynthetic oligonucleotide 14gaaacgaggg ccgggaagcg aagc 241523DNAArtificial SequenceSynthetic oligonucleotide 15ggggctagag

tactggacaa tgg 231623DNAArtificial SequenceSynthetic oligonucleotide 16ccattgtcca gtactctagc ccc 231721DNAArtificial SequenceSynthetic oligonucleotide 17gctagagcac cggacaatgg a 211821DNAArtificial SequenceSynthetic oligonucleotide 18tccattgtcc ggtgctctag c 211921DNAArtificial SequenceSynthetic oligonucleotide 19gctagagcac cggacaatgg a 212021DNAArtificial SequenceSynthetic oligonucleotide 20tccattgtcc ggtgctctag c 212123DNAArtificial SequenceSynthetic oligonucleotide 21ctagagcact gtacaatgga ttg 232223DNAArtificial SequenceSynthetic oligonucleotide 22caatccattg tacagtgctc tag 232320DNAArtificial SequenceSynthetic oligonucleotide 23gcactggaca aaggattggc 202420DNAArtificial SequenceSynthetic oligonucleotide 24gccaatcctt tgtccagtgc 202520DNAArtificial SequenceSynthetic oligonucleotide 25gcactggaca gtggattggc 202620DNAArtificial SequenceSynthetic oligonucleotide 26gccaatccac tgtccagtgc 202721DNAArtificial SequenceSynthetic oligonucleotide 27ctggacaata gattggcaag g 212826DNAArtificial SequenceSynthetic oligonucleotide 28gtcagcaaaa ttttgccagt gattgc 262923DNAArtificial SequenceSynthetic oligonucleotide 29gcgaaatttt actgacattg atg 233023DNAArtificial SequenceSynthetic oligonucleotide 30catcaatgtc agtaaaattt cgc 233131DNAArtificial SequenceSynthetic oligonucleotide 31ggcgaaattt tgctaacatt gatgattcct g 313231DNAArtificial SequenceSynthetic oligonucleotide 32caggaatcat caatgttagc aaaatttcgc c 313328DNAArtificial SequenceSynthetic oligonucleotide 33cgaaattttg ctggcattga tgatattc 283428DNAArtificial SequenceSynthetic oligonucleotide 34gaatatcatc aatgccagca aaatttcg 283524DNAArtificial SequenceSynthetic oligonucleotide 35tgacattgat gagtcctgga aaag 243624DNAArtificial SequenceSynthetic oligonucleotide 36cttttccagg actcatcaat gtca 243726DNAArtificial SequenceSynthetic oligonucleotide 37gctgacattg attattcctg gaaaag 263826DNAArtificial SequenceSynthetic oligonucleotide 38cttttccagg aataatcaat gtcagc 263934DNAArtificial SequenceSynthetic oligonucleotide 39ctgacattga tgattgctgg aaaagtataa agag 344034DNAArtificial SequenceSynthetic oligonucleotide 40ctctttatac ttttccagca atcatcaatg tcag 344139DNAArtificial SequenceSynthetic oligonucleotide 41gctgacattg atgattcccg gaaaagtata aagagtatc 394239DNAArtificial SequenceSynthetic oligonucleotide 42gatactcttt atacttttcc gggaatcatc aatgtcagc 394323DNAArtificial SequenceSynthetic oligonucleotide 43tgatgattcc ttgaaaagta taa 234423DNAArtificial SequenceSynthetic oligonucleotide 44ttatactttt caaggaatca tca 234534DNAArtificial SequenceSynthetic oligonucleotide 45ctgacattga tgattccttg aaaagtataa agag 344634DNAArtificial SequenceSynthetic oligonucleotide 46ctctttatac ttttcaagga atcatcaatg tcag 344743DNAArtificial SequenceSynthetic oligonucleotide 47gctgacattg atgattcctg taaaagtata aagagtatct tgg 434843DNAArtificial SequenceSynthetic oligonucleotide 48ccaagatact ctttatactt ttacaggaat catcaatgtc agc 434921DNAArtificial SequenceSynthetic oligonucleotide 49tggaaaagta caaagagtat c 215021DNAArtificial SequenceSynthetic oligonucleotide 50gatactcttt gtacttttcc a 215121DNAArtificial SequenceSynthetic oligonucleotide 51ccttgccaat ctattgtcca g 215220DNAArtificial SequenceSynthetic oligonucleotide 52aatggattgg taaggacgcc 205320DNAArtificial SequenceSynthetic oligonucleotide 53ggcgtcctta ccaatccatt 205420DNAArtificial SequenceSynthetic oligonucleotide 54gcaaggacgc ttaccatggg 205520DNAArtificial SequenceSynthetic oligonucleotide 55cccatggtaa gcgtccttgc 205620DNAArtificial SequenceSynthetic oligonucleotide 56gcaaggacgt ctaccatggg 205720DNAArtificial SequenceSynthetic oligonucleotide 57cccatggtag acgtccttgc 205827DNAArtificial SequenceSynthetic oligonucleotide 58aggacgccta ccacgggctg gctgcac 275927DNAArtificial SequenceSynthetic oligonucleotide 59gtgcagccag cccgtggtag gcgtcct 276027DNAArtificial SequenceSynthetic oligonucleotide 60aggacgccta ccttgggctg gctgcac 276127DNAArtificial SequenceSynthetic oligonucleotide 61gtgcagccag cccaaggtag gcgtcct 276225DNAArtificial SequenceSynthetic oligonucleotide 62aggacgccta ccgtgggctg gctgc 256325DNAArtificial SequenceSynthetic oligonucleotide 63gcagccagcc cacggtaggc gtcct 256420DNAArtificial SequenceSynthetic oligonucleotide 64ctaccatggw ctggctgcac 206520DNAArtificial SequenceSynthetic oligonucleotide 65gtgcagccag wccatggtag 206620DNAArtificial SequenceSynthetic oligonucleotide 66ctaccatgcg ctggctgcac 206720DNAArtificial SequenceSynthetic oligonucleotide 67gtgcagccag cgcatggtag 206819DNAArtificial SequenceSynthetic oligonucleotide 68catgggctgt ctgcactgg 196919DNAArtificial SequenceSynthetic oligonucleotide 69ccagtgcaga cagcccatg 197021DNAArtificial SequenceSynthetic oligonucleotide 70atgggctggc ggcactggga g 217121DNAArtificial SequenceSynthetic oligonucleotide 71ctcccagtgc cgccagccca t 217217DNAArtificial SequenceSynthetic oligonucleotide 72ctggctgcgc tgggagc 177317DNAArtificial SequenceSynthetic oligonucleotide 73gctcccagcg cagccag 177417DNAArtificial SequenceSynthetic oligonucleotide 74ctggctgtac tgggagc 177517DNAArtificial SequenceSynthetic oligonucleotide 75gctcccagta cagccag 177635DNAArtificial SequenceSynthetic oligonucleotide 76gattcctgga aaagtacaaa gagtatcttg gactg 357735DNAArtificial SequenceSynthetic oligonucleotide 77cagtccaaga tactctttgt acttttccag gaatc 357826DNAArtificial SequenceSynthetic oligonucleotide 78agtataaaga gtttcttgga ctggac 267926DNAArtificial SequenceSynthetic oligonucleotide 79gtccagtcca agaaactctt tatact 268023DNAArtificial SequenceSynthetic oligonucleotide 80gtataaagag taacttggac tgg 238123DNAArtificial SequenceSynthetic oligonucleotide 81ccagtccaag ttactcttta tac 238222DNAArtificial SequenceSynthetic oligonucleotide 82agagtatctt cgactggaca tc 228322DNAArtificial SequenceSynthetic oligonucleotide 83gatgtccagt cgaagatact ct 228421DNAArtificial SequenceSynthetic oligonucleotide 84gagtatcttg cactggacat c 218522DNAArtificial SequenceSynthetic oligonucleotide 85gatgtccagt cgaagatact ct 228630DNAArtificial SequenceSynthetic oligonucleotide 86cttggactgg acatgtttta accaggagag 308730DNAArtificial SequenceSynthetic oligonucleotide 87ctctcctggt taaaacatgt ccagtccaag 308821DNAArtificial SequenceSynthetic oligonucleotide 88ggactggaca ccttttaacc a 218921DNAArtificial SequenceSynthetic oligonucleotide 89tggttaaaag gtgtccagtc c 219019DNAArtificial SequenceSynthetic oligonucleotide 90gttgatgttc ctggaccag 199119DNAArtificial SequenceSynthetic oligonucleotide 91ctggtccagg aacatcaac 199219DNAArtificial SequenceSynthetic oligonucleotide 92gatgttgctc gaccagggg 199319DNAArtificial SequenceSynthetic oligonucleotide 93cccctggtcg agcaacatc 199425DNAArtificial SequenceSynthetic oligonucleotide 94gatgttgctg gacgaggggg ttgga 259525DNAArtificial SequenceSynthetic oligonucleotide 95tccaaccccc tcgtccagca acatc 259621DNAArtificial SequenceSynthetic oligonucleotide 96gttgctggac tagggggttg g 219721DNAArtificial SequenceSynthetic oligonucleotide 97ccaaccccct agtccagcaa c 219822DNAArtificial SequenceSynthetic oligonucleotide 98gctggaccag cgggttggaa tg 229922DNAArtificial SequenceSynthetic oligonucleotide 99cattccaacc cgctggtcca gc 2210021DNAArtificial SequenceSynthetic oligonucleotide 100ggaccagggg attggaatga c 2110122DNAArtificial SequenceSynthetic oligonucleotide 101ctggctgcac ggggagcgct tc 2210222DNAArtificial SequenceSynthetic oligonucleotide 102gaagcgctcc ccgtgcagcc ag 2210322DNAArtificial SequenceSynthetic oligonucleotide 103ctggctgcac ttggagcgct tc 2210422DNAArtificial SequenceSynthetic oligonucleotide 104gaagcgctcc aagtgcagcc ag 2210522DNAArtificial SequenceSynthetic oligonucleotide 105gctgcactgg aagcgcttca tg 2210622DNAArtificial SequenceSynthetic oligonucleotide 106catgaagcgc ttccagtgca gc 2210720DNAArtificial SequenceSynthetic oligonucleotide 107actgggagcy cttcatgtgc 2010820DNAArtificial SequenceSynthetic oligonucleotide 108gcacatgaag rgctcccagt 2010919DNAArtificial SequenceSynthetic oligonucleotide 109cactgggagr gcttcatgt 1911019DNAArtificial SequenceSynthetic oligonucleotide 110acatgaagcy ctcccagtg 1911122DNAArtificial SequenceSynthetic oligonucleotide 111ctgggagcgc tgcatgtgca ac 2211222DNAArtificial SequenceSynthetic oligonucleotide 112gttgcacatg cagcgctccc ag 2211322DNAArtificial SequenceSynthetic oligonucleotide 113gagcgcttca agtgcaacct tg 2211422DNAArtificial SequenceSynthetic oligonucleotide 114caaggttgca cttgaagcgc tc 2211517DNAArtificial SequenceSynthetic oligonucleotide 115gcgcttcata tgcaacc 1711617DNAArtificial SequenceSynthetic oligonucleotide 116ggttgcatat gaagcgc 1711726DNAArtificial SequenceSynthetic oligonucleotide 117gagcgcttca tgtccaacct tgactg 2611826DNAArtificial SequenceSynthetic oligonucleotide 118cagtcaaggt tggacatgaa gcgctc 2611921DNAArtificial SequenceSynthetic oligonucleotide 119cgcttcatgs gcaaccttga c 2112021DNAArtificial SequenceSynthetic oligonucleotide 120gtcaaggttg cscatgaagc g 2112120DNAArtificial SequenceSynthetic oligonucleotide 121caaccttgac ggccaggaag 2012220DNAArtificial SequenceSynthetic oligonucleotide 122cttcctggcc gtcaaggttg 2012322DNAArtificial SequenceSynthetic oligonucleotide 123caaccttgac twccaggaag ag 2212422DNAArtificial SequenceSynthetic oligonucleotide 124ctcttcctgg wagtcaaggt tg 2212529DNAArtificial SequenceSynthetic oligonucleotide 125gtgcaacctt gactaccagg aagagccag 2912621DNAArtificial SequenceSynthetic oligonucleotide 126gtcattccaa tcccctggtc c 2112721DNAArtificial SequenceSynthetic oligonucleotide 127cagggggttg caatgaccca g 2112821DNAArtificial SequenceSynthetic oligonucleotide 128ctgggtcatt gcaaccccct g 2112919DNAArtificial SequenceSynthetic oligonucleotide 129ggggttggag tgacccaga 1913019DNAArtificial SequenceSynthetic oligonucleotide 130tctgggtcac tccaacccc 1913121DNAArtificial SequenceSynthetic oligonucleotide 131ggttggaatg tcccagatat g 2113221DNAArtificial SequenceSynthetic oligonucleotide 132catatctggg acattccaac c 2113322DNAArtificial SequenceSynthetic oligonucleotide 133gggttggaat tacccagata tg 2213422DNAArtificial SequenceSynthetic oligonucleotide 134catatctggg taattccaac cc 2213521DNAArtificial SequenceSynthetic oligonucleotide 135tggaatgacc gagatatgtt a 2113621DNAArtificial SequenceSynthetic oligonucleotide 136taacatatct cggtcattcc a 2113723DNAArtificial SequenceSynthetic oligonucleotide 137ttggaatgac ctagatatgt tag 2313823DNAArtificial SequenceSynthetic oligonucleotide 138ctaacatatc taggtcattc caa 2313923DNAArtificial SequenceSynthetic oligonucleotide 139ggaatgaccc amatatgtta gtg 2314022DNAArtificial SequenceSynthetic oligonucleotide 140cactaacata ttgggtcatt cc

2214132DNAArtificial SequenceSynthetic oligonucleotide 141ggttggaatg acccacatat gttagtgatt gg 3214232DNAArtificial SequenceSynthetic oligonucleotide 142ccaatcacta acatatgtgg gtcattccaa cc 3214322DNAArtificial SequenceSynthetic oligonucleotide 143gaatgaccca gttatgttag tg 2214422DNAArtificial SequenceSynthetic oligonucleotide 144cactaacata actgggtcat tc 2214521DNAArtificial SequenceSynthetic oligonucleotide 145atgacccaga aatgttagtg a 2114621DNAArtificial SequenceSynthetic oligonucleotide 146tcactaacat ttctgggtca t 2114722DNAArtificial SequenceSynthetic oligonucleotide 147gacccagata ggttagtgat tg 2214822DNAArtificial SequenceSynthetic oligonucleotide 148caatcactaa cctatctggg tc 2214923DNAArtificial SequenceSynthetic oligonucleotide 149gacccagata tattagtgat tgg 2315023DNAArtificial SequenceSynthetic oligonucleotide 150ccaatcacta atatatctgg gtc 2315129DNAArtificial SequenceSynthetic oligonucleotide 151ctggctcttc ctggtagtca aggttgcac 2915228DNAArtificial SequenceSynthetic oligonucleotide 152gagccagatt cctacatcag tgagaagc 2815328DNAArtificial SequenceSynthetic oligonucleotide 153gcttctcact gatgtaggaa tctggctc 2815421DNAArtificial SequenceSynthetic oligonucleotide 154tcctgcatca ctgagaagct c 2115521DNAArtificial SequenceSynthetic oligonucleotide 155gagcttctca gtgatgcagg a 2115622DNAArtificial SequenceSynthetic oligonucleotide 156ctgcatcagt aagaagctct tc 2215722DNAArtificial SequenceSynthetic oligonucleotide 157gaagagcttc ttactgatgc ag 2215822DNAArtificial SequenceSynthetic oligonucleotide 158ctgcatcagt gggaagctct tc 2215922DNAArtificial SequenceSynthetic oligonucleotide 159gaagagcttc ccactgatgc ag 2216020DNAArtificial SequenceSynthetic oligonucleotide 160cagtgagaag ttcttcatgg 2016120DNAArtificial SequenceSynthetic oligonucleotide 161ccaggaagaa cttctcactg 2016229DNAArtificial SequenceSynthetic oligonucleotide 162gcatcagtga gaagttcttc atggagatg 2916329DNAArtificial SequenceSynthetic oligonucleotide 163catctccatg aagaacttct cactgatgc 2916422DNAArtificial SequenceSynthetic oligonucleotide 164cttcatggag agggcagagc tc 2216522DNAArtificial SequenceSynthetic oligonucleotide 165gagctctgcc ctctccatga ag 2216622DNAArtificial SequenceSynthetic oligonucleotide 166cttcatggag atagcagagc tc 2216722DNAArtificial SequenceSynthetic oligonucleotide 167gagctctgct atctccatga ag 2216822DNAArtificial SequenceSynthetic oligonucleotide 168catggagatg gtagagctca tg 2216922DNAArtificial SequenceSynthetic oligonucleotide 169catgagctct accatctcca tg 2217022DNAArtificial SequenceSynthetic oligonucleotide 170gcagagctca gggtctcaga ag 2217122DNAArtificial SequenceSynthetic oligonucleotide 171cttctgagac cctgagctct gc 2217225DNAArtificial SequenceSynthetic oligonucleotide 172ctcagaaggc tgtaaggatg caggt 2517325DNAArtificial SequenceSynthetic oligonucleotide 173acctgcatcc ttacagcctt ctgag 2517422DNAArtificial SequenceSynthetic oligonucleotide 174ctcagaaggc tcgaaggatg ca 2217522DNAArtificial SequenceSynthetic oligonucleotide 175tgcatccttc gagccttctg ag 2217621DNAArtificial SequenceSynthetic oligonucleotide 176ccagatatgt cagtgattgg c 2117721DNAArtificial SequenceSynthetic oligonucleotide 177gccaatcact gacatatctg g 2117821DNAArtificial SequenceSynthetic oligonucleotide 178gatatgttag cgattggcaa c 2117921DNAArtificial SequenceSynthetic oligonucleotide 179gttgccaatc gctaacatat c 2118023DNAArtificial SequenceSynthetic oligonucleotide 180cagatatgtt aatgattggc aac 2318123DNAArtificial SequenceSynthetic oligonucleotide 181gttgccaatc attaacatat ctg 2318223DNAArtificial SequenceSynthetic oligonucleotide 182tatgttagtg actggcaact ttg 2318323DNAArtificial SequenceSynthetic oligonucleotide 183caaagttgcc agtcactaac ata 2318419DNAArtificial SequenceSynthetic oligonucleotide 184ttagtgattg tcaactttg 1918519DNAArtificial SequenceSynthetic oligonucleotide 185caaagttgac aatcactaa 1918621DNAArtificial SequenceSynthetic oligonucleotide 186gttagtgatt tgcaactttg g 2118721DNAArtificial SequenceSynthetic oligonucleotide 187ccaaagttgc aaatcactaa c 2118823DNAArtificial SequenceSynthetic oligonucleotide 188tgttagtgat tagcaacttt ggc 2318923DNAArtificial SequenceSynthetic oligonucleotide 189gccaaagttg ctaatcacta aca 2319023DNAArtificial SequenceSynthetic oligonucleotide 190gtgattggca aatttggcct cag 2319123DNAArtificial SequenceSynthetic oligonucleotide 191ctgaggccaa atttgccaat cac 2319221DNAArtificial SequenceSynthetic oligonucleotide 192gtgattggca gctttggcct c 2119321DNAArtificial SequenceSynthetic oligonucleotide 193gaggccaaag ctgccaatca c 2119425DNAArtificial SequenceSynthetic oligonucleotide 194caactttggc ctcggctgga atcag 2519525DNAArtificial SequenceSynthetic oligonucleotide 195ctgattccag ccgaggccaa agttg 2519623DNAArtificial SequenceSynthetic oligonucleotide 196ctttggcctc aactggaatc agc 2319723DNAArtificial SequenceSynthetic oligonucleotide 197gctgattcca gttgaggcca aag 2319822DNAArtificial SequenceSynthetic oligonucleotide 198agctggaatc ggcaagtaac tc 2219922DNAArtificial SequenceSynthetic oligonucleotide 199gagttacttg ccgattccag ct 2220023DNAArtificial SequenceSynthetic oligonucleotide 200cagctggaat caccaagtaa ctc 2320122DNAArtificial SequenceSynthetic oligonucleotide 201gaaggatgca gattatgagt ac 2220222DNAArtificial SequenceSynthetic oligonucleotide 202gtactcataa tctgcatcct tc 2220325DNAArtificial SequenceSynthetic oligonucleotide 203ggatgcaggt tgtgagtacc tctgc 2520425DNAArtificial SequenceSynthetic oligonucleotide 204gcagaggtac tcacaacctg catcc 2520522DNAArtificial SequenceSynthetic oligonucleotide 205ggttatgagg acctctgcat tg 2220622DNAArtificial SequenceSynthetic oligonucleotide 206caatgcagag gtcctcataa cc 2220724DNAArtificial SequenceSynthetic oligonucleotide 207gttatgagta ccsctgcatt gatg 2420824DNAArtificial SequenceSynthetic oligonucleotide 208catcaatgca gsggtactca taac 2420921DNAArtificial SequenceSynthetic oligonucleotide 209cctctgcatt hatgactgtt g 2121021DNAArtificial SequenceSynthetic oligonucleotide 210caacagtcat daatgcagag g 2121127DNAArtificial SequenceSynthetic oligonucleotide 211tacctctgca ttaatgactg ttggatg 2721227DNAArtificial SequenceSynthetic oligonucleotide 212catccaacag tcattaatgc agaggta 2721327DNAArtificial SequenceSynthetic oligonucleotide 213tacctctgca ttcatgactg ttggatg 2721427DNAArtificial SequenceSynthetic oligonucleotide 214catccaacag tcatgaatgc agaggta 2721534DNAArtificial SequenceSynthetic oligonucleotide 215gagtacctct gcatttatga ctgttggatg gctc 3421634DNAArtificial SequenceSynthetic oligonucleotide 216gagccatcca acagtcataa atgcagaggt actc 3421722DNAArtificial SequenceSynthetic oligonucleotide 217ctgcattgat ggctgttgga tg 2221822DNAArtificial SequenceSynthetic oligonucleotide 218catccaacag ccatcaatgc ag 2221922DNAArtificial SequenceSynthetic oligonucleotide 219ctgcattgat gtctgttgga tg 2222022DNAArtificial SequenceSynthetic oligonucleotide 220catccaacag acatcaatgc ag 2222122DNAArtificial SequenceSynthetic oligonucleotide 221ctgcattgat aactgttgga tg 2222222DNAArtificial SequenceSynthetic oligonucleotide 222catccaacag ttatcaatgc ag 2222324DNAArtificial SequenceSynthetic oligonucleotide 223gcattgatga ctcttggatg gctc 2422424DNAArtificial SequenceSynthetic oligonucleotide 224gagccatcca agagtcatca atgc 2422523DNAArtificial SequenceSynthetic oligonucleotide 225gagttacttg gtgattccag ctg 2322621DNAArtificial SequenceSynthetic oligonucleotide 226cagctggaat aagcaagtaa c 2122721DNAArtificial SequenceSynthetic oligonucleotide 227gttacttgct tattccagct g 2122821DNAArtificial SequenceSynthetic oligonucleotide 228ggaatcagca tgtaactcag a 2122921DNAArtificial SequenceSynthetic oligonucleotide 229tctgagttac atgctgattc c 2123022DNAArtificial SequenceSynthetic oligonucleotide 230ctggaatcag aaagtaactc ag 2223121DNAArtificial SequenceSynthetic oligonucleotide 231ctgagttact ttctgattcc a 2123221DNAArtificial SequenceSynthetic oligonucleotide 232cagcaagtaa atcagatggc c 2123321DNAArtificial SequenceSynthetic oligonucleotide 233ggccatctga tttacttgct g 2123421DNAArtificial SequenceSynthetic oligonucleotide 234caagtaactc cgatggccct c 2123521DNAArtificial SequenceSynthetic oligonucleotide 235gagggccatc ggagttactt g 2123620DNAArtificial SequenceSynthetic oligonucleotide 236gtaactcaga cggccctctg 2023720DNAArtificial SequenceSynthetic oligonucleotide 237cagagggccg tctgagttac 2023823DNAArtificial SequenceSynthetic oligonucleotide 238taactcagat gcccctctgg gct 2323923DNAArtificial SequenceSynthetic oligonucleotide 239agcccagagg ggcatctgag tta 2324022DNAArtificial SequenceSynthetic oligonucleotide 240aactcagatg gacctctggg ct 2224122DNAArtificial SequenceSynthetic oligonucleotide 241agcccagagg tccatctgag tt 2224221DNAArtificial SequenceSynthetic oligonucleotide 242gatggccctc ggggctatca t 2124321DNAArtificial SequenceSynthetic oligonucleotide 243atgatagccc cgagggccat c 2124421DNAArtificial SequenceSynthetic oligonucleotide 244atggccctct gtgctatcat g 2124521DNAArtificial SequenceSynthetic oligonucleotide 245catgatagca cagagggcca t 2124622DNAArtificial SequenceSynthetic oligonucleotide 246gccctctggg atatcatggc tg 2224722DNAArtificial SequenceSynthetic oligonucleotide 247cagccatgat atcccagagg gc 2224819DNAArtificial SequenceSynthetic oligonucleotide 248gccctctggc ctatcatgg 1924924DNAArtificial SequenceSynthetic oligonucleotide 249gcattgatga ctattggatg gctc 2425024DNAArtificial SequenceSynthetic oligonucleotide 250gagccatcca atagtcatca atgc 2425121DNAArtificial SequenceSynthetic oligonucleotide 251gatgactgtt cgatggctcc c 2125221DNAArtificial SequenceSynthetic oligonucleotide 252gggagccatc gaacagtcat c 2125323DNAArtificial SequenceSynthetic oligonucleotide 253ctgttggatg cctccccaaa gag 2325423DNAArtificial SequenceSynthetic oligonucleotide 254ctctttgggg aggcatccaa cag 2325522DNAArtificial SequenceSynthetic oligonucleotide 255gctccccaaa magattcaga ag 2225622DNAArtificial SequenceSynthetic oligonucleotide 256cttctgaatc tktttgggga gc 2225724DNAArtificial SequenceSynthetic oligonucleotide 257ggctccccaa acagattcag aagg 2425824DNAArtificial SequenceSynthetic oligonucleotide 258ccttctgaat ctgtttgggg agcc 2425925DNAArtificial SequenceSynthetic oligonucleotide 259caaagagatt cacaaggcag acttc 2526025DNAArtificial SequenceSynthetic oligonucleotide 260gaagtctgcc ttgtgaatct ctttg 2526125DNAArtificial SequenceSynthetic oligonucleotide 261gcagaccctc agagctttcc tcatg 2526225DNAArtificial SequenceSynthetic oligonucleotide 262catgaggaaa gctctgaggg tctgc 2526324DNAArtificial SequenceSynthetic oligonucleotide 263cagaccctca gtgctttcct catg 2426424DNAArtificial SequenceSynthetic oligonucleotide 264catgaggaaa gcactgaggg tctg 2426520DNAArtificial SequenceSynthetic oligonucleotide 265ccctcagcgc tctcctcatg 2026620DNAArtificial SequenceSynthetic oligonucleotide 266catgaggaga

gcgctgaggg 2026722DNAArtificial SequenceSynthetic oligonucleotide 267ctcatgggat ttgccagcta gc 2226822DNAArtificial SequenceSynthetic oligonucleotide 268gctagctggc aaatcccatg ag 2226923DNAArtificial SequenceSynthetic oligonucleotide 269gattcgccag ccagctaatt atg 2327023DNAArtificial SequenceSynthetic oligonucleotide 270cataattagc tggctggcga atc 2327128DNAArtificial SequenceSynthetic oligonucleotide 271tcgccagcta mctaattatg ttcacagc 2827228DNAArtificial SequenceSynthetic oligonucleotide 272gctgtgaaca taattagkta gctggcga 2827329DNAArtificial SequenceSynthetic oligonucleotide 273gctagctaat tatgatcaca gcaaaggac 2927419DNAArtificial SequenceSynthetic oligonucleotide 274ccatgatagg ccagagggc 1927525DNAArtificial SequenceSynthetic oligonucleotide 275ctctgggctt tcatggctgc tcctt 2527625DNAArtificial SequenceSynthetic oligonucleotide 276aaggagcagc catgaaagcc cagag 2527721DNAArtificial SequenceSynthetic oligonucleotide 277gggctatcat cgctgctcct t 2127821DNAArtificial SequenceSynthetic oligonucleotide 278aaggagcagc gatgatagcc c 2127923DNAArtificial SequenceSynthetic oligonucleotide 279ctatcatggc tcctccttta ttc 2328023DNAArtificial SequenceSynthetic oligonucleotide 280gaataaagga ggagccatga tag 2328122DNAArtificial SequenceSynthetic oligonucleotide 281catggctgct gctttattca tg 2228222DNAArtificial SequenceSynthetic oligonucleotide 282catgaataaa gcagcagcca tg 2228322DNAArtificial SequenceSynthetic oligonucleotide 283catggctgct wctttattca tg 2228422DNAArtificial SequenceSynthetic oligonucleotide 284catgaataaa gwagcagcca tg 2228528DNAArtificial SequenceSynthetic oligonucleotide 285gctgctcctt tatgcatgtc taatgacc 2828628DNAArtificial SequenceSynthetic oligonucleotide 286ggtcattaga catgcataaa ggagcagc 2828725DNAArtificial SequenceSynthetic oligonucleotide 287ctttattcat gtktaatgac ctccg 2528825DNAArtificial SequenceSynthetic oligonucleotide 288cggaggtcat tamacatgaa taaag 2528923DNAArtificial SequenceSynthetic oligonucleotide 289attcatgtct agtgacctcc gac 2329023DNAArtificial SequenceSynthetic oligonucleotide 290gtcggaggtc actagacatg aat 2329124DNAArtificial SequenceSynthetic oligonucleotide 291tattcatgtc taaggacctc cgac 2429224DNAArtificial SequenceSynthetic oligonucleotide 292gtcggaggtc cttagacatg aata 2429324DNAArtificial SequenceSynthetic oligonucleotide 293tattcatgtc tcatgacctc cgac 2429424DNAArtificial SequenceSynthetic oligonucleotide 294gtcggaggtc atgagacatg aata 2429526DNAArtificial SequenceSynthetic oligonucleotide 295tcatgtctaa tggcctccga cacatc 2629626DNAArtificial SequenceSynthetic oligonucleotide 296gatgtgtcgg aggccattag acatga 2629724DNAArtificial SequenceSynthetic oligonucleotide 297gtctaatgac ccccgacaca tcag 2429824DNAArtificial SequenceSynthetic oligonucleotide 298ctgatgtgtc gggggtcatt agac 2429929DNAArtificial SequenceSynthetic oligonucleotide 299gtcctttgct gtgatcataa ttagctagc 2930022DNAArtificial SequenceSynthetic oligonucleotide 300cacagcaaag aactgaagct ag 2230122DNAArtificial SequenceSynthetic oligonucleotide 301ctagcttcag ttctttgctg tg 2230221DNAArtificial SequenceSynthetic oligonucleotide 302cagcaaagga ccgaagctag g 2130324DNAArtificial SequenceSynthetic oligonucleotide 303atccctagct tcggtccttt gctg 2430424DNAArtificial SequenceSynthetic oligonucleotide 304aggactgaag ccagggattt atgc 2430524DNAArtificial SequenceSynthetic oligonucleotide 305gcataaatcc ctggcttcag tcct 2430628DNAArtificial SequenceSynthetic oligonucleotide 306ggactgaagc tagagattta tgcagatg 2830728DNAArtificial SequenceSynthetic oligonucleotide 307catctgcata aatctctagc ttcagtcc 2830827DNAArtificial SequenceSynthetic oligonucleotide 308gactgaagct aaggatttat gcagatg 2730927DNAArtificial SequenceSynthetic oligonucleotide 309catctgcata aatccttagc ttcagtc 2731024DNAArtificial SequenceSynthetic oligonucleotide 310gctagggatt tctgcagatg ttgg 2431124DNAArtificial SequenceSynthetic oligonucleotide 311ccaacatctg cagaaatccc tagc 2431225DNAArtificial SequenceSynthetic oligonucleotide 312gctagggatt tatgtagatg ttgga 2531325DNAArtificial SequenceSynthetic oligonucleotide 313tccaacatct acataaatcc ctagc 2531421DNAArtificial SequenceSynthetic oligonucleotide 314ggatttatgc acatgttgga a 2131521DNAArtificial SequenceSynthetic oligonucleotide 315ttccaacatg tgcataaatc c 2131630DNAArtificial SequenceSynthetic oligonucleotide 316gggatttatg cacatgttgg aaataaaacc 3031730DNAArtificial SequenceSynthetic oligonucleotide 317ggttttattt ccaacatgtg cataaatccc 3031824DNAArtificial SequenceSynthetic oligonucleotide 318tgcagatgtt gaaaataaaa cctg 2431924DNAArtificial SequenceSynthetic oligonucleotide 319caggttttat tttcaacatc tgca 2432024DNAArtificial SequenceSynthetic oligonucleotide 320tgcagatgtt agaaataaaa cctg 2432124DNAArtificial SequenceSynthetic oligonucleotide 321caggttttat ttctaacatc tgca 2432237DNAArtificial SequenceSynthetic oligonucleotide 322ggatttatgc agatgttgaa aataaaacct gcgcagc 3732337DNAArtificial SequenceSynthetic oligonucleotide 323gctgcgcagg ttttattttc aacatctgca taaatcc 3732422DNAArtificial SequenceSynthetic oligonucleotide 324gtctaatgac ttccgacaca tc 2232522DNAArtificial SequenceSynthetic oligonucleotide 325gatgtgtcgg aagtcattag ac 2232622DNAArtificial SequenceSynthetic oligonucleotide 326gtctaatgac caccgacaca tc 2232722DNAArtificial SequenceSynthetic oligonucleotide 327gatgtgtcgg tggtcattag ac 2232823DNAArtificial SequenceSynthetic oligonucleotide 328ctaatgacct cggacacatc agc 2332923DNAArtificial SequenceSynthetic oligonucleotide 329gctgatgtgt ccgaggtcat tag 2333023DNAArtificial SequenceSynthetic oligonucleotide 330ctaatgacct cccacacatc agc 2333123DNAArtificial SequenceSynthetic oligonucleotide 331gctgatgtgt gggaggtcat tag 2333223DNAArtificial SequenceSynthetic oligonucleotide 332ctccgacaca acagccctca agc 2333323DNAArtificial SequenceSynthetic oligonucleotide 333gcttgagggc tgttgtgtcg gag 2333420DNAArtificial SequenceSynthetic oligonucleotide 334gccaaagctt tccttcagga 2033520DNAArtificial SequenceSynthetic oligonucleotide 335tcctgaagga aagctttggc 2033622DNAArtificial SequenceSynthetic oligonucleotide 336gctctccttc acgataagga cg 2233722DNAArtificial SequenceSynthetic oligonucleotide 337cgtccttatc gtgaaggaga gc 2233821DNAArtificial SequenceSynthetic oligonucleotide 338ctctccttca gtataaggac g 2133921DNAArtificial SequenceSynthetic oligonucleotide 339cgtccttata ctgaaggaga g 2134021DNAArtificial SequenceSynthetic oligonucleotide 340gataaggacg aaattgccat c 2134121DNAArtificial SequenceSynthetic oligonucleotide 341gatggcaatt tcgtccttat c 2134222DNAArtificial SequenceSynthetic oligonucleotide 342aaggacgtaa mtgccatcaa tc 2234322DNAArtificial SequenceSynthetic oligonucleotide 343gattgatggc akttacgtcc tt 2234422DNAArtificial SequenceSynthetic oligonucleotide 344aattgccatc attcaggacc cc 2234522DNAArtificial SequenceSynthetic oligonucleotide 345ggggtcctga atgatggcaa tt 2234622DNAArtificial SequenceSynthetic oligonucleotide 346aattgccatc aagcaggacc cc 2234722DNAArtificial SequenceSynthetic oligonucleotide 347ggggtcctgc ttgatggcaa tt 2234822DNAArtificial SequenceSynthetic oligonucleotide 348aattgccatc tatcaggacc cc 2234937DNAArtificial SequenceSynthetic oligonucleotide 349ggatttatgc agatgttcga aataaaacct gcgcagc 3735037DNAArtificial SequenceSynthetic oligonucleotide 350ggatttatgc agatgttcga aataaaacct gcgcagc 3735122DNAArtificial SequenceSynthetic oligonucleotide 351ggaaataaaa tctgcgcagg ct 2235222DNAArtificial SequenceSynthetic oligonucleotide 352agcctgcgca gattttattt cc 2235324DNAArtificial SequenceSynthetic oligonucleotide 353ggaaataaaa cccgcgcagg cttc 2435424DNAArtificial SequenceSynthetic oligonucleotide 354gaagcctgcg cgggttttat ttcc 2435524DNAArtificial SequenceSynthetic oligonucleotide 355gaaataaaac ctrcgcaggc ttcc 2435624DNAArtificial SequenceSynthetic oligonucleotide 356ggaagcctgc gyaggtttta tttc 2435728DNAArtificial SequenceSynthetic oligonucleotide 357ggaaataaaa cctgggcagg cttccctg 2835828DNAArtificial SequenceSynthetic oligonucleotide 358cagggaagcc tgcccaggtt ttatttcc 2835925DNAArtificial SequenceSynthetic oligonucleotide 359gaaataaaac ctgcacaggc ttccc 2536025DNAArtificial SequenceSynthetic oligonucleotide 360gggaagcctg tgcaggtttt atttc 2536122DNAArtificial SequenceSynthetic oligonucleotide 361ataaaacctg cccaggcttc cc 2236222DNAArtificial SequenceSynthetic oligonucleotide 362gggaagcctg ggcaggtttt at 2236319DNAArtificial SequenceSynthetic oligonucleotide 363cctgcgcagt cttccctgg 1936419DNAArtificial SequenceSynthetic oligonucleotide 364ccagggaaga ctgcgcagg 1936520DNAArtificial SequenceSynthetic oligonucleotide 365ggcttcccta ggagttttgg 2036620DNAArtificial SequenceSynthetic oligonucleotide 366ccaaaactcc tagggaagcc 2036721DNAArtificial SequenceSynthetic oligonucleotide 367ttccctggga attttggata c 2136821DNAArtificial SequenceSynthetic oligonucleotide 368gtatccaaaa ttcccaggga a 2136920DNAArtificial SequenceSynthetic oligonucleotide 369ccctgggagg tttggatact 2037020DNAArtificial SequenceSynthetic oligonucleotide 370agtatccaaa cctcccaggg 2037124DNAArtificial SequenceSynthetic oligonucleotide 371gttttggata ctgcgacatt gatg 2437224DNAArtificial SequenceSynthetic oligonucleotide 372catcaatgtc gcagtatcca aaac 2437322DNAArtificial SequenceSynthetic oligonucleotide 373ggggtcctga tagatggcaa tt 2237422DNAArtificial SequenceSynthetic oligonucleotide 374tgccatcaat gaggacccct tg 2237522DNAArtificial SequenceSynthetic oligonucleotide 375caaggggtcc tcattgatgg ca 2237622DNAArtificial SequenceSynthetic oligonucleotide 376tgccatcaat cgggacccct tg 2237722DNAArtificial SequenceSynthetic oligonucleotide 377caaggggtcc cgattgatgg ca 2237820DNAArtificial SequenceSynthetic oligonucleotide 378ggaccccttg gacaagcaag 2037920DNAArtificial SequenceSynthetic oligonucleotide 379cttgcttgtc caaggggtcc 2038022DNAArtificial SequenceSynthetic oligonucleotide 380cttgggcaag raagggtacc ag 2238122DNAArtificial SequenceSynthetic oligonucleotide 381ctggtaccct tycttgccca ag 2238219DNAArtificial SequenceSynthetic oligonucleotide 382ggcaagcaaa ggtaccagc 1938319DNAArtificial SequenceSynthetic oligonucleotide 383gctggtacct ttgcttgcc 1938419DNAArtificial SequenceSynthetic oligonucleotide 384ggcaagcaag ygtaccagc 1938519DNAArtificial SequenceSynthetic oligonucleotide 385gctggtacrc ttgcttgcc 1938625DNAArtificial SequenceSynthetic oligonucleotide 386tgggcaagca agagtaccag cttag 2538725DNAArtificial SequenceSynthetic oligonucleotide 387ctaagctggt actcttgctt gccca 2538821DNAArtificial SequenceSynthetic oligonucleotide 388gagacaactt taaagtgtgg g 2138921DNAArtificial SequenceSynthetic oligonucleotide 389cccacacttt aaagttgtct c 2139020DNAArtificial SequenceSynthetic oligonucleotide 390ctttgaagtg cgggaacgac 2039120DNAArtificial SequenceSynthetic oligonucleotide 391gtcgttcccg cacttcaaag

2039223DNAArtificial SequenceSynthetic oligonucleotide 392gaagtgtggg accgacctct ctc 2339323DNAArtificial SequenceSynthetic oligonucleotide 393gagagaggtc ggtcccacac ttc 2339423DNAArtificial SequenceSynthetic oligonucleotide 394gaagtgtgga aacgacctct ctc 2339523DNAArtificial SequenceSynthetic oligonucleotide 395gagagaggtc gtttccacac ttc 2339622DNAArtificial SequenceSynthetic oligonucleotide 396gtgtgggaac aacctctctc ag 2239721DNAArtificial SequenceSynthetic oligonucleotide 397ctacgacatt catgcccaga c 2139821DNAArtificial SequenceSynthetic oligonucleotide 398gtctgggcat gaatgtcgta g 2139922DNAArtificial SequenceSynthetic oligonucleotide 399gacattgata cccagacctt tg 2240022DNAArtificial SequenceSynthetic oligonucleotide 400caaaggtctg ggtatcaatg tc 2240123DNAArtificial SequenceSynthetic oligonucleotide 401ctttgctgac cggggagtag atc 2340223DNAArtificial SequenceSynthetic oligonucleotide 402gatctactcc ccggtcagca aag 2340323DNAArtificial SequenceSynthetic oligonucleotide 403ctttgctgac tgcggagtag atc 2340423DNAArtificial SequenceSynthetic oligonucleotide 404gatctactcc gcagtcagca aag 2340521DNAArtificial SequenceSynthetic oligonucleotide 405gctgactggg tagtagatct g 2140621DNAArtificial SequenceSynthetic oligonucleotide 406cagatctact acccagtcag c 2140726DNAArtificial SequenceSynthetic oligonucleotide 407ctggggagta gttctgctaa aatttg 2640826DNAArtificial SequenceSynthetic oligonucleotide 408caaattttag cagaactact ccccag 2640925DNAArtificial SequenceSynthetic oligonucleotide 409gagtagatct gccaaaattt gatgg 2541025DNAArtificial SequenceSynthetic oligonucleotide 410ccatcaaatt ttggcagatc tactc 2541127DNAArtificial SequenceSynthetic oligonucleotide 411gtagatctgc taagatttga tggtttg 2741227DNAArtificial SequenceSynthetic oligonucleotide 412caaaccatca aatcttagca gatctac 2741332DNAArtificial SequenceSynthetic oligonucleotide 413gtagatctgc taaaatctga tggttgttac tg 3241432DNAArtificial SequenceSynthetic oligonucleotide 414cagtaacaac catcagattt tagcagatct ac 3241524DNAArtificial SequenceSynthetic oligonucleotide 415gctaaaattt gttggttgtt actg 2441624DNAArtificial SequenceSynthetic oligonucleotide 416cagtaacaac caacaaattt tagc 2441724DNAArtificial SequenceSynthetic oligonucleotide 417gctaaaattt catggttgtt actg 2441824DNAArtificial SequenceSynthetic oligonucleotide 418cagtaacaac catgaaattt tagc 2441927DNAArtificial SequenceSynthetic oligonucleotide 419ctaaaatttg atgattgtta ctgtgac 2742027DNAArtificial SequenceSynthetic oligonucleotide 420gtcacagtaa caatcatcaa attttag 2742127DNAArtificial SequenceSynthetic oligonucleotide 421ctaaaatttg atcgttgtta ctgtgac 2742222DNAArtificial SequenceSynthetic oligonucleotide 422ctgagagagg ttgttcccac ac 2242322DNAArtificial SequenceSynthetic oligonucleotide 423gaacgacctc cctcaggctt ag 2242422DNAArtificial SequenceSynthetic oligonucleotide 424ctaagcctga gggaggtcgt tc 2242521DNAArtificial SequenceSynthetic oligonucleotide 425cgacctctcc caggcttagc c 2142621DNAArtificial SequenceSynthetic oligonucleotide 426ggctaagcct gggagaggtc g 2142723DNAArtificial SequenceSynthetic oligonucleotide 427ctcaggctta ccctgggctg tag 2342823DNAArtificial SequenceSynthetic oligonucleotide 428ctacagccca gggtaagcct gag 2342922DNAArtificial SequenceSynthetic oligonucleotide 429cttagcctgg cctgtagcta tg 2243022DNAArtificial SequenceSynthetic oligonucleotide 430catagctaca ggccaggcta ag 2243122DNAArtificial SequenceSynthetic oligonucleotide 431ctgggctgta gatatgataa ac 2243222DNAArtificial SequenceSynthetic oligonucleotide 432gtttatcata tctacagccc ag 2243322DNAArtificial SequenceSynthetic oligonucleotide 433gtagctatga aaaaccggca gg 2243422DNAArtificial SequenceSynthetic oligonucleotide 434cctgccggtt tttcatagct ac 2243521DNAArtificial SequenceSynthetic oligonucleotide 435gctatgataa aacggcagga g 2143621DNAArtificial SequenceSynthetic oligonucleotide 436ctcctgccgt tttatcatag c 2143724DNAArtificial SequenceSynthetic oligonucleotide 437gctatgataa actggcagga gatt 2443824DNAArtificial SequenceSynthetic oligonucleotide 438aatctcctgc cagtttatca tagc 2443921DNAArtificial SequenceSynthetic oligonucleotide 439accggcaggs gattggtgga c 2144021DNAArtificial SequenceSynthetic oligonucleotide 440gtccaccaat cscctgccgg t 2144124DNAArtificial SequenceSynthetic oligonucleotide 441gataaaccgg cagaagattg gtgg 2444224DNAArtificial SequenceSynthetic oligonucleotide 442ccaccaatct tctgccggtt tatc 2444329DNAArtificial SequenceSynthetic oligonucleotide 443gataaaccgg caggcgattg gtggacctc 2944429DNAArtificial SequenceSynthetic oligonucleotide 444gaggtccacc aatcgcctgc cggtttatc 2944523DNAArtificial SequenceSynthetic oligonucleotide 445ccggcaggag actggtggac ctc 2344623DNAArtificial SequenceSynthetic oligonucleotide 446gaggtccacc agtctcctgc cgg 2344727DNAArtificial SequenceSynthetic oligonucleotide 447gtcacagtaa caacgatcaa attttag 2744830DNAArtificial SequenceSynthetic oligonucleotide 448ctaaaatttg atggttwtta ctgtgacagt 3044930DNAArtificial SequenceSynthetic oligonucleotide 449actgtcacag taawaaccat caaattttag 3045030DNAArtificial SequenceSynthetic oligonucleotide 450ctaaaatttg atggttggta ctgtgacagt 3045130DNAArtificial SequenceSynthetic oligonucleotide 451actgtcacag taccaaccat caaattttag 3045230DNAArtificial SequenceSynthetic oligonucleotide 452ctaaaatttg atggtcgtta ctgtgacagt 3045330DNAArtificial SequenceSynthetic oligonucleotide 453actgtcacag taacgaccat caaattttag 3045430DNAArtificial SequenceSynthetic oligonucleotide 454ctaaaatttg atggtggtta ctgtgacagt 3045530DNAArtificial SequenceSynthetic oligonucleotide 455actgtcacag taaccaccat caaattttag 3045621DNAArtificial SequenceSynthetic oligonucleotide 456ttggcagatg attataagca c 2145721DNAArtificial SequenceSynthetic oligonucleotide 457gtgcttataa tcatctgcca a 2145821DNAArtificial SequenceSynthetic oligonucleotide 458ttggcagata gttataagca c 2145921DNAArtificial SequenceSynthetic oligonucleotide 459gtgcttataa ctatctgcca a 2146025DNAArtificial SequenceSynthetic oligonucleotide 460gttataagca cacgtccttg gccct 2546125DNAArtificial SequenceSynthetic oligonucleotide 461agggccaagg acgtgtgctt ataac 2546223DNAArtificial SequenceSynthetic oligonucleotide 462gttataagca cgtgtccttg gcc 2346323DNAArtificial SequenceSynthetic oligonucleotide 463ggccaaggac acgtgcttat aac 2346423DNAArtificial SequenceSynthetic oligonucleotide 464gtccttggcc cagaatagga ctg 2346523DNAArtificial SequenceSynthetic oligonucleotide 465cagtcctatt ctgggccaag gac 2346623DNAArtificial SequenceSynthetic oligonucleotide 466gtccttggcc ccgaatagga ctg 2346723DNAArtificial SequenceSynthetic oligonucleotide 467cagtcctatt cggggccaag gac 2346821DNAArtificial SequenceSynthetic oligonucleotide 468ctgaatagga ttggcagaag c 2146921DNAArtificial SequenceSynthetic oligonucleotide 469gcttctgcca atcctattca g 2147021DNAArtificial SequenceSynthetic oligonucleotide 470cagaagcatt atgtactcct g 2147121DNAArtificial SequenceSynthetic oligonucleotide 471caggagtaca taatgcttct g 2147221DNAArtificial SequenceSynthetic oligonucleotide 472caggagatta gtggacctcg c 2147321DNAArtificial SequenceSynthetic oligonucleotide 473gcgaggtcca ctaatctcct g 2147421DNAArtificial SequenceSynthetic oligonucleotide 474ggagattggt agacctcgct c 2147521DNAArtificial SequenceSynthetic oligonucleotide 475gagcgaggtc taccaatctc c 2147624DNAArtificial SequenceSynthetic oligonucleotide 476gattggtgga cttcgctctt atac 2447724DNAArtificial SequenceSynthetic oligonucleotide 477gtataagagc gaagtccacc aatc 2447820DNAArtificial SequenceSynthetic oligonucleotide 478ggtggacctc actcttatac 2047920DNAArtificial SequenceSynthetic oligonucleotide 479gtataagagt gaggtccacc 2048020DNAArtificial SequenceSynthetic oligonucleotide 480ggtggacctt gctcttatac 2048120DNAArtificial SequenceSynthetic oligonucleotide 481gtataagagc aaggtccacc 2048223DNAArtificial SequenceSynthetic oligonucleotide 482tgcttccctg cgtaaaggag tgg 2348323DNAArtificial SequenceSynthetic oligonucleotide 483ccactccttt acgcagggaa gca 2348424DNAArtificial SequenceSynthetic oligonucleotide 484gtaaaggagt ggactgtaat cctg 2448524DNAArtificial SequenceSynthetic oligonucleotide 485caggattaca gtccactcct ttac 2448624DNAArtificial SequenceSynthetic oligonucleotide 486aaaggagtgg cctataatcc tgcc 2448724DNAArtificial SequenceSynthetic oligonucleotide 487ggcaggatta taggccactc cttt 2448824DNAArtificial SequenceSynthetic oligonucleotide 488aaaggagtgg cccgtaatcc tgcc 2448924DNAArtificial SequenceSynthetic oligonucleotide 489ggcaggatta cgggccactc cttt 2449024DNAArtificial SequenceSynthetic oligonucleotide 490gtaatcctgc ctacttcatc acac 2449124DNAArtificial SequenceSynthetic oligonucleotide 491gtgtgatgaa gtaggcagga ttac 2449226DNAArtificial SequenceSynthetic oligonucleotide 492ctgcctgctt caacacacag ctcctc 2649326DNAArtificial SequenceSynthetic oligonucleotide 493gaggagctgt gtgttgaagc aggcag 2649424DNAArtificial SequenceSynthetic oligonucleotide 494cctgcttcat cccacagctc ctcc 2449524DNAArtificial SequenceSynthetic oligonucleotide 495ggaggagctg tgggatgaag cagg 2449624DNAArtificial SequenceSynthetic oligonucleotide 496cttcatcaca ccgctcctcc ctgt 2449722DNAArtificial SequenceSynthetic oligonucleotide 497cattgtgtac ttctgtgagt gg 2249822DNAArtificial SequenceSynthetic oligonucleotide 498ccactcacag aagtacacaa tg 2249923DNAArtificial SequenceSynthetic oligonucleotide 499cattgtgtac tactgtgagt ggc 2350023DNAArtificial SequenceSynthetic oligonucleotide 500gccactcaca gtagtacaca atg 2350122DNAArtificial SequenceSynthetic oligonucleotide 501gtgtactcct atgagtggcc tc 2250222DNAArtificial SequenceSynthetic oligonucleotide 502gaggccactc ataggagtac ac 2250323DNAArtificial SequenceSynthetic oligonucleotide 503gtgtactcct gggagtggcc tct 2350423DNAArtificial SequenceSynthetic oligonucleotide 504agaggccact cccaggagta cac 2350522DNAArtificial SequenceSynthetic oligonucleotide 505ctgtgagtgg actctttata tg 2250622DNAArtificial SequenceSynthetic oligonucleotide 506catataaaga gtccactcac ag 2250722DNAArtificial SequenceSynthetic oligonucleotide 507ctgtgagtgg cktctttata tg 2250822DNAArtificial SequenceSynthetic oligonucleotide 508catataaaga mgccactcac ag 2250921DNAArtificial SequenceSynthetic oligonucleotide 509tggcctcttt ctatgtggcc c 2151021DNAArtificial SequenceSynthetic oligonucleotide 510gggccacata gaaagaggcc a 2151125DNAArtificial SequenceSynthetic oligonucleotide 511agtggcctct ttgtatgtgg ccctt 2551225DNAArtificial SequenceSynthetic oligonucleotide 512aagggccaca tacaaagagg ccact 2551335DNAArtificial SequenceSynthetic oligonucleotide 513cctttcaaaa gcccaatgat acagaaatcc gacag 3551435DNAArtificial SequenceSynthetic oligonucleotide 514ctgtcggatt tctgtatcat tgggcttttg aaagg 3551528DNAArtificial SequenceSynthetic oligonucleotide 515caattataca gaaaaccgac agtactgc 2851628DNAArtificial SequenceSynthetic oligonucleotide 516gcagtactgt cggttttctg tataattg 2851721DNAArtificial SequenceSynthetic oligonucleotide 517cgacagtacs

gcaatcactg g 2151821DNAArtificial SequenceSynthetic oligonucleotide 518ccagtgattg csgtactgtc g 2151921DNAArtificial SequenceSynthetic oligonucleotide 519cgacagtact acaatcactg g 2152024DNAArtificial SequenceSynthetic oligonucleotide 520acagggagga gcggtgtgat gaag 2452128DNAArtificial SequenceSynthetic oligonucleotide 521cacagctcct ccgtgtgaaa aggaagct 2852228DNAArtificial SequenceSynthetic oligonucleotide 522agcttccttt tcacacggag gagctgtg 2852323DNAArtificial SequenceSynthetic oligonucleotide 523ggaagctagg gtactatgaa tgg 2352423DNAArtificial SequenceSynthetic oligonucleotide 524ccattcatag taccctagct tcc 2352522DNAArtificial SequenceSynthetic oligonucleotide 525agggttctat aaatggactt ca 2252622DNAArtificial SequenceSynthetic oligonucleotide 526tgaagtccat ttatagaacc ct 2252722DNAArtificial SequenceSynthetic oligonucleotide 527gacttcaagg tcaagaagtc ac 2252822DNAArtificial SequenceSynthetic oligonucleotide 528gtgacttctt gaccttgaag tc 2252921DNAArtificial SequenceSynthetic oligonucleotide 529gaagtcacaa aaatcccaca g 2153021DNAArtificial SequenceSynthetic oligonucleotide 530ctgtgggatt tttgtgactt c 2153124DNAArtificial SequenceSynthetic oligonucleotide 531gtcacataaa tdccacaggc actg 2453224DNAArtificial SequenceSynthetic oligonucleotide 532cagtgcctgt gghatttatg tgac 2453322DNAArtificial SequenceSynthetic oligonucleotide 533cacataaatc ccaaaggcac tg 2253422DNAArtificial SequenceSynthetic oligonucleotide 534cagtgccttt gggatttatg tg 2253522DNAArtificial SequenceSynthetic oligonucleotide 535cacataaatc ccgcaggcac tg 2253622DNAArtificial SequenceSynthetic oligonucleotide 536cagtgcctgc gggatttatg tg 2253721DNAArtificial SequenceSynthetic oligonucleotide 537atcccacaga cactgttttg c 2153821DNAArtificial SequenceSynthetic oligonucleotide 538gcaaaacagt gtctgtggga t 2153922DNAArtificial SequenceSynthetic oligonucleotide 539ggcactgttt cgcttcagct ag 2254022DNAArtificial SequenceSynthetic oligonucleotide 540ctagctgaag cgaaacagtg cc 2254129DNAArtificial SequenceSynthetic oligonucleotide 541gcgcttcgct tcctggacat ccctggggc 2954229DNAArtificial SequenceSynthetic oligonucleotide 542gccccaggga tgtccaggaa gcgaagcgc 2954321DNAArtificial SequenceSynthetic oligonucleotide 543ccagtgattg tagtactgtc g 2154421DNAArtificial SequenceSynthetic oligonucleotide 544cagtactgca gtcactggcg a 2154521DNAArtificial SequenceSynthetic oligonucleotide 545tcgccagtga ctgcagtact g 2154619DNAArtificial SequenceSynthetic oligonucleotide 546cagtactgcg atcactggc 1954719DNAArtificial SequenceSynthetic oligonucleotide 547gccagtgatc gcagtactg 1954821DNAArtificial SequenceSynthetic oligonucleotide 548tactgcaatc gctggcgaaa t 2154921DNAArtificial SequenceSynthetic oligonucleotide 549atttcgccag cgattgcagt a 2155017DNAArtificial SequenceSynthetic oligonucleotide 550gcaatcaccg gcgaaat 1755117DNAArtificial SequenceSynthetic oligonucleotide 551atttcgccgg tgattgc 1755222DNAArtificial SequenceSynthetic oligonucleotide 552gcaatcactg tcgaaatttt gc 2255322DNAArtificial SequenceSynthetic oligonucleotide 553gcaaaatttc gacagtgatt gc 2255426DNAArtificial SequenceSynthetic oligonucleotide 554gcaatcactg gcaaaatttt gctgac 2655531DNAArtificial SequenceSynthetic oligonucleotide 555gcagaccctc agcgccagct agctaattat g 3155631DNAArtificial SequenceSynthetic oligonucleotide 556cataattagc tagctggcgc tgagggtctg c 3155726DNAArtificial SequenceSynthetic oligonucleotide 557gttttggata ctacattgat gcccag 2655826DNAArtificial SequenceSynthetic oligonucleotide 558ctgggcatca atgtagtatc caaaac 2655924DNAArtificial SequenceSynthetic oligonucleotide 559ctcctgtgag tggatgtggc cctt 2456024DNAArtificial SequenceSynthetic oligonucleotide 560aagggccaca tccactcaca ggag 2456123DNAArtificial SequenceSynthetic oligonucleotide 561caggagagaa ttgatgttgc tgg 2356223DNAArtificial SequenceSynthetic oligonucleotide 562ccagcaacat caattctctc ctg 2356325DNAArtificial SequenceSynthetic oligonucleotide 563gataaaccgg cagattggtg gacct 2556425DNAArtificial SequenceSynthetic oligonucleotide 564aggtccacca atctgccggt ttatc 2556535DNAArtificial SequenceSynthetic oligonucleotide 565gactggacat cttggacatc ttttaaccag gagag 3556635DNAArtificial SequenceSynthetic oligonucleotide 566ctctcctggt taaaagatgt ccaagatgtc cagtc 35

Claims

1. A method for determining whether a patient expressing a mutant form of a protein will respond to treatment with a specific pharmacological chaperone for the protein, which method comprises a. contacting a first host cell with a pharmacological chaperone specific for the protein, wherein the first host cell expresses the mutant form of the protein; and b. comparing protein activity in a second host cell not contacted with the specific pharmacological chaperone, with protein activity in the first host cell contacted with the specific pharmacological chaperone, wherein an increase in protein activity in the first host cell contacted with the specific pharmacological chaperone compared to the activity of the protein expressed by the second host cell not contacted with the specific pharmacological chaperone indicates that the patient will respond to treatment with the specific pharmacological chaperone.

2. The method of claim 1, wherein the mutant form of the protein is caused by a missense mutation in a gene encoding the protein.

3. The method of claim 1, wherein the protein is an enzyme.

4. The method of claim 3, wherein the enzyme is a lysosomal enzyme.

5. The method of claim 4, wherein the patient has been diagnosed with a lysosomal storage disorder.

6. The method of claim 5, wherein the lysosomal enzyme is .alpha.-galactosidase A and the lysosomal storage disorder is Fabry disease.

7. The method of claim 1, wherein the specific pharmacological chaperone is 1-deoxygalactonojirimycin.

8. A method for producing a treatment therapy table, wherein the treatment therapy table indicates if a specific pharmacological chaperone is an effective compound for increasing the activity of a mutant protein, which method comprises: a. contacting a first host cell with a pharmacological chaperone specific for the protein, wherein the first host cell expresses the mutant protein; b. comparing protein activity in the first host cell contacted with the specific pharmacological chaperone with protein activity in a second host cell, wherein the second host cell expresses the mutant protein and is not contacted with the specific pharmacological chaperone; and c. recording the result of step (b) in the treatment therapy table, wherein a specific pharmacological chaperone recorded in the treatment therapy table that increases the activity of the mutant protein in the first host cell compared to the activity of the mutant protein in the second cell is a specific pharmacological chaperone that can be used as a therapy for a patient who expresses the mutant protein.

Images