Method to predict response to pharmacological chaperone treatment of diseases

Abstract

To provide methods to determine whether a patient with a lysosomal storage disorder will benefit from treatment with a specific pharmacological chaperone.SOLUTION: The present invention exemplifies an in vitro method for determining α-galactosidase A responsiveness to a pharmacological chaperone such as 1-deoxygalactonojirimycin in a cell line expressing a mutant from of α-galactosidase A. The invention also provides a method for diagnosing Fabry disease in patients suspected of having Fabry disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 028,141, filed February 12, 2008, U.S. Provisional Patent Application No. 61 / 035,684, filed March 11, 2008, U.S. Provisional Patent Application No. 61 / 093,631, filed September 2, 2008, and U.S. Provisional Patent Application No. 61 / 113,496, filed November 11, 2008, the disclosures of which are incorporated herein by reference in their entireties.

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

[0003] Proteins are involved in nearly every aspect of cellular function within the human body. Proteins are linear strings of amino acids that fold and twist into specific three-dimensional shapes to function properly. Some human diseases result from mutations that cause changes in the amino acid sequence that can reduce protein stability and 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 amino acid within the protein. Because of this error, missense mutations often result in proteins with reduced levels of biological activity. In addition to missense mutations, there are other types of mutations that can result in proteins with reduced biological activity.

[0004] Proteins generally fold within a specific cellular region known as the endoplasmic reticulum, or ER. Cells have quality control mechanisms that ensure proteins fold into their correct three-dimensional shape before movement from the ER to their appropriate destination within the cell, a process commonly referred to as protein trafficking. Misfolded proteins are often initially retained within the ER and then removed by quality control mechanisms. In some cases, misfolded proteins may accumulate within the ER before being removed.

[0005] Retention of misfolded proteins in the ER disrupts their proper transport, and the resulting reduction in biological activity can lead to cellular dysfunction and ultimately disease. Furthermore, the accumulation of misfolded proteins in the ER may lead to various types of stress on the cell, which may also contribute to cellular dysfunction and disease.

[0006] Lysosomal storage diseases (LSDs) are characterized by lysosomal enzyme deficiencies due to mutations in the genes encoding these enzymes. This results in the pathological accumulation of substrates for these enzymes, including lipids, carbohydrates, and polysaccharides. Approximately 50 LSDs are known, including Gaucher disease, Fabry disease, Pompe disease, Tay-Sachs disease, and mucopolysaccharidoses (MPS). Most LSDs are inherited as autosomal recessive traits, but males with Fabry disease and MPS II are hemizygous because the disease gene is encoded on the X chromosome. For most LSDs, no treatment beyond symptomatic management is available. Enzyme replacement therapy (ERT) using recombinant enzymes is available for some LSDs, including Gaucher, Fabry, Pompe, and MPS I and II. Substrate reduction therapy (SRT) is also available for Gaucher disease in select circumstances. SRT utilizes small-molecule inhibitors of enzymes required for the synthesis of glucosylceramide (the GD substrate). The ultimate goal of SRT is to reduce substrate production and pathological accumulation.

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

[0008] Diagnosis of Fabry disease Fabry disease is a rare disease with multisystem involvement, a wide age range for onset, and heterogeneity, making proper diagnosis a challenge. Awareness is low among medical professionals, leading to frequent misdiagnosis. Diagnoses seriously considered in patients ultimately diagnosed with Fabry disease include amplifying valve prolapse, glomerulonephritis, idiopathic proteinuria, systemic lupus erythematosus, Whipple's disease, acute abdomen, ulcerative colitis, acute intermittent porphyria, and occult malignancy. Thus, even for men affected by the classic form, diagnosis typically takes approximately 5 to 7 years or longer. This is concerning because the longer an individual lives with Fabry disease, the greater the likelihood of damage occurring within the affected organs and tissues, potentially increasing the severity of the individual's condition. The diagnosis of Fabry disease is most often confirmed based on reduced α-Gal A activity in plasma or peripheral white blood cells (WBCs) once a patient becomes symptomatic, in combination with mutation analysis. In females, diagnosis is even more challenging due to the unreliable enzymatic identification of carrier females due to random X-chromosome inactivation in a proportion of carrier cells. For example, some obligate carriers (daughters of men affected with the classic form) have α-Gal A enzyme activity ranging from normal to very low activity. Because carriers may have normal α-Gal A enzyme activity in their white blood cells, only identification of the α-Gal A mutation by genetic testing provides accurate carrier identification and / or diagnosis.

[0009] Treatment of Fabry disease One approved therapy for treating Fabry disease is enzyme replacement therapy, which typically involves the intravenous infusion of a purified form of the corresponding wild-type protein (Fabrazyme®, Genzyme Corp.). One of the major challenges with protein replacement therapy is achieving and maintaining a therapeutically effective amount of protein due to the rapid degradation of the infused protein. The current approach to overcoming this problem is to administer numerous, costly high-dose infusions.

[0010] Protein replacement therapy has several additional problems, including the difficulty of large-scale production, purification, and storage of properly folded proteins; the acquisition of glycosylated native proteins; the development of anti-protein immune responses; and the inability of proteins to cross the blood-brain barrier (i.e., low bioavailability) to alleviate central nervous system pathology. Furthermore, replacement enzymes cannot penetrate the heart or kidney in sufficient quantities to reduce substrate accumulation in renal podocytes or cardiomyocytes, which is prominent in Fabry disease.

[0011] Gene therapy, using recombinant vectors containing nucleic acid sequences encoding functional proteins or genetically engineered human cells expressing functional proteins, 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 defective enzyme protein's natural substrate, thereby ameliorating the pathology. This "substrate reduction" approach has been specifically described for approximately 40 related enzyme disorders called lysosomal storage disorders, including glycosphingolipid storage disorders. The small-molecule inhibitors proposed for use as therapy are specific for inhibiting enzymes involved in glycolipid synthesis, thereby reducing the amount of cellular glycolipids that the defective enzyme needs to destroy.

[0013] It has previously been shown that binding of small molecule inhibitors of LSD-related enzymes can increase the stability of both mutant and corresponding wild-type enzymes (see U.S. Patent Nos. 5,629,997; 5,629,997; 5,629,997; 5,629,997; and 5,629,997, all of which are incorporated herein by reference). In particular, it has been discovered that administration of small molecule derivatives of glucose and galactose, which are specific, selective competitive inhibitors for several target lysosomal enzymes, effectively increases the stability of the enzymes in cells in vitro, increasing their transport to lysosomes. Therefore, increasing the amount of enzyme in lysosomes is expected to increase the hydrolysis of the enzyme substrate. The original theory behind this strategy was as follows: Because mutant enzyme proteins are unstable in the ER (Non-Patent Document 1), the enzyme proteins are delayed and prematurely degraded in the normal transport pathway (ER → Golgi apparatus → endosomes → lysosomes). Therefore, compounds that bind to and increase the stability of mutant enzymes may serve as "chaperones" for the enzymes, increasing the amount that can exit the ER and travel to lysosomes. Furthermore, because the folding and trafficking of some wild-type proteins is defective, and up to 70% of some wild-type proteins are degraded before reaching their final cellular location, it is possible to use chaperones to stabilize wild-type enzymes and increase the amount of enzyme that can exit the ER and be transported to lysosomes. This strategy has been shown in vitro and in vivo to increase the expression of several lysosomal enzymes, including β-glucocerebrosidase and α-glucosidase, deficiencies of which are associated with Gaucher disease and Pompe disease, respectively.

[0014] However, as mentioned above, promising candidates for SPC therapy must have mutations that result in the production of an enzyme that is stabilized and has the potential to fold into a conformation that allows export from the ER. Mutations that severely truncate the enzyme, such as nonsense mutations, or mutations in the catalytic domain that prevent chaperone binding, are unlikely to be "rescueable" or "enhanced," i.e., responsive to SPC therapy. While missense mutations outside the catalytic site are more likely to be rescuable with SPC, there is no guarantee, and screening for responsive mutations is necessary. This means that even if Fabry disease is diagnosed by detecting deficient α-Gal A activity in WBCs, without the benefit of the present invention, it is very difficult, if not impossible, to predict whether a particular Fabry disease patient will respond to treatment with SPC. Furthermore, because WBCs can only survive for a short time in culture (in vitro), screening for enhanced α-Gal A SPC is difficult and suboptimal for patients.

[0015] To effectively apply SPC therapy, it is necessary to employ a widely applicable, fast, and efficient method for screening patients for responsiveness to SPC therapy prior to initiation of treatment, and treatment can then be implemented based on the results of the screening. Thus, there remains a need in the art for a relatively non-invasive method for rapidly assessing the enzyme enhancement of potential therapies before making a treatment decision, to provide both cost and emotional benefits to patients.

[0016] [Patent Document 1] U.S. Patent No. 6,274,597 [Patent Document 2] U.S. Patent No. 6,583,158 [Patent Document 3] U.S. Patent No. 6,589,964 [Patent Document 4] U.S. Patent No. 6,599,919 [Patent Document 5] U.S. Patent No. 6,916,829 [Patent Document 6] U.S. Patent No. 7,141,582

[0017] [Non-Patent Document 1] Ishii et al., Biochem. Biophys. Res. Comm. 1996; 220: 812-815) Summary of the Invention [Means for solving the problem]

[0018] One embodiment of the present invention provides a method for determining whether a patient is a candidate for SPC therapy. Specifically, the present invention provides an in vitro assay for assessing 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 whether the mutant protein contacted with the SPC exhibits an increased level of activity (preferably a statistically significant increase) compared to a mutant protein expressed in a host cell that has not been contacted with the candidate SPC. If the candidate SPC increases the activity of the mutant protein according to the assay of the present invention, such candidate SPC can be used for SPC therapy to treat patients expressing the same mutant protein tested in the in vitro assay.

[0019] In one embodiment, the protein is an enzyme. In another embodiment, the protein is a lysosomal enzyme. In yet another embodiment, the protein is α-galactosidase A (α-Gal; α-Gal A). In other embodiments, the protein is alpha-glucosidase (acid α-glucosidase; α-glucosidase; GAA). In other embodiments, the protein is glucocerebrosidase (β-glucosidase; Gba; GCase).

[0020] The present invention also includes a basis for evaluating SPC as a treatment option for any number of other protein disorders and / or enzyme deficiencies and / or protein folding disorders.

[0021] The present invention further provides a documented list (e.g., a "treatment benchmark table") that lists protein mutations and the responsiveness of each mutation to SPC therapy. Such a list can be used to determine treatment options for a patient, thereby allowing the patient or the patient's physician or doctor to select an appropriate therapeutic approach, e.g., a therapeutic SPC, by identifying the patient's protein mutations and cross-referencing the mutations with the list to identify whether the SPC increases the activity of the patient's particular mutant enzyme.

[0022] In another embodiment, the "Criteria for Treatment" lists mutations for lysosomal enzymes, and the Criteria for Treatment is used to determine the best treatment regimen for treating a lysosomal storage disorder. In a further embodiment of the invention, the protein is α-Gal A and the disease is Fabry disease. In another embodiment of the invention, the protein is GAA and the disease is Pompe disease. In another embodiment of the invention, the protein is Gba and the disease is Gaucher disease.

[0023] In one embodiment, the treatment reference table describes mutant forms of enzymes such as lysosomal enzymes (e.g., α-Gal A, Gcase, and GAA) and identifies treatment options for lysosomal storage disorders (e.g., Fabry disease, Gaucher disease, and Pompe disease).

[0024] In one embodiment, the present invention also provides a method for generating a treatment reference table that may be directed to any protein folding disorder or disorder treatable with SPC, including 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 amyloidosis (transthyretin), among others.

[0025] In another embodiment, the present invention provides a method for treating a patient diagnosed as expressing a mutant protein (e.g., a lysosomal enzyme such as α-Gal A), in which the activity of the mutant protein (e.g., α-Gal A) when expressed in a host cell can be increased upon administration of an SPC directed to that protein (e.g., 1-deoxygalactonojirimycin, DGJ, as an SPC directed to mutant α-Gal A).

[0026] The present invention also provides diagnostic kits which contain the components needed to perform the assays. [Brief explanation of the drawings]

[0027] [Figure 1A] Figure 1 shows a list of Fabry mutations generated by site-directed mutagenesis. The text indicates whether HEK293 cells expressing each of the listed mutations responded to DGJ treatment in a transient transfection assay: italics = not tested; bold and underlined = no response to DGJ; plain text (not italics, bold, or underlined) = response to DGJ. [Figure 1B]Figure 1 shows a list of Fabry mutations generated by site-directed mutagenesis. The text indicates whether HEK293 cells expressing each of the listed mutations responded to DGJ treatment in a transient transfection assay: italics = not tested; bold and underlined = no response to DGJ; plain text (not italics, bold, or underlined) = response to DGJ. [Figure 1C] Figure 1 shows a list of Fabry mutations generated by site-directed mutagenesis. The text indicates whether HEK293 cells expressing each of the listed mutations responded to DGJ treatment in a transient transfection assay: italics = not tested; bold and underlined = no response to DGJ; plain text (not italics, bold, or underlined) = response to DGJ. [Figure 2A] The responsiveness of different α-Gal A mutants to DGJ treatment is shown. The magnitude of increase in α-Gal A activity levels after DGJ treatment and the EC50 values ​​are listed for all tested mutants in Figures 1A-D in response to DGJ treatment. The increase in enzyme activity is shown as a percentage of wild-type α-Gal A activity. [Figure 2B] The responsiveness of different α-Gal A mutants to DGJ treatment is shown. The magnitude of increase in α-Gal A activity levels after DGJ treatment and the EC50 values ​​are listed for all tested mutants in Figures 1A-D in response to DGJ treatment. The increase in enzyme activity is shown as a percentage of wild-type α-Gal A activity. [Figure 2C] The responsiveness of different α-Gal A mutants to DGJ treatment is shown. The magnitude of increase in α-Gal A activity levels after DGJ treatment and the EC50 values ​​are listed for all tested mutants in Figures 1A-D in response to DGJ treatment. The increase in enzyme activity is shown as a percentage of wild-type α-Gal A activity. [Figure 3]Representative examples of the response of wild-type and mutant α-Gal A to DGJ treatment are shown. α-Gal A activity (expressed as nmol / mg protein / h of 4-MU released) was measured in lysates prepared from transfected HEK293 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. The wild-type exhibits high baseline activity and therefore does not respond to DGJ in this assay. [Figure 4] This shows that the mutant response in HEK293 cells is comparable to that in patient-derived T cells, lymphoblasts, or leukocytes in vivo. α-Gal A levels measured in three different assays, reported as a percentage of wild-type, are compared for each mutation tested. α-Gal A levels in T cells, lymphoblasts, leukocytes, and HEK293 expressing mutant α-Gal A were measured before and after exposure to DGJ. Open bars represent basal levels (without DGJ treatment), and closed bars represent elevated levels after DGJ treatment. [Figure 5] This figure shows that DGJ-responsive α-Gal A mutations are widely distributed across the α-Gal-A protein sequence. The Fabry mutations tested are depicted on the α-Gal A secondary structure. No significant correlation was observed between the location of the mutation on the protein sequence and response, suggesting that responsive and non-responsive mutations are widely distributed throughout the protein. The text color indicates DGJ response: green = responsive; red = unresponsive; brown indicates that among multiple mutations at the same site, some responded to DGJ treatment but others did not. [Figure 6] 1 shows the oligonucleotide primer pairs used to generate point mutations in the α-Gal A gene through site-directed mutagenesis. [Figure 7] 1 shows the α-Gal A cDNA sequence mutated through site-directed mutagenesis. [Figure 8]Figure 1 shows the effect of isofagomine tartrate on patient-derived macrophages and lymphoblasts isolated from Gaucher disease patients with different mutations in the glucocerebrosidase (Gba; GCase) enzyme. [Figure 9] FIG. 1 shows the effect on GL-3 levels in 8-week-old male hR301Qα-Gal A Tg / KO mice treated daily or less frequently (4 days on / 3 days off) with 300 mg of DGJ per kg of body weight in drinking water for 4 weeks. [Figure 10] Figure 1 shows a list of Pompe mutations generated by site-directed mutagenesis. The text indicates whether COS-7 cells expressing each of the listed mutations responded to DNJ treatment in a transient transfection assay. [Figure 11] 1 shows the nucleic acid sequence of human lysosomal alpha-glucosidase (GAA) (GenBank accession number Y00839). [Figure 12] Figure 12 shows the response of four different GAA mutants to DNJ treatment at concentrations of 0 μM, 20 μM, 50 μM, and 100 μM. The increase in enzyme activity is shown as specific activity (nmol / mg protein / hr). Figure 12 also shows that DNJ promoted the processing of GAA to the 95 / 76 / 70 kDa forms. [Figure 13] Figure 1 shows the responsiveness of fibroblasts from Pompe disease patients to DNJ treatment. Fibroblasts were homozygous for either the P545L or R854X GAA mutation. [Figure 14] EC50 for DNJ-induced GAA activity in HEK-293 cells transiently transfected with the P545L GAA mutation is shown. [Figure 15] Figure 1 shows the responsiveness of lymphocytes from a Pompe disease patient to DNJ treatment. The lymphocytes were heterozygous for the (IVS1AS, T>G, -13) GAA splicing defect and GAA frameshift mutation. [Figure 16] 1 shows the amino acid sequence encoded by the human lysosomal alpha-glucosidase (GAA) nucleic acid (GenBank accession number Y00839). DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides an in vitro assay for accurately determining whether an SPC enhances the activity of a mutant protein.

[0029] In one embodiment, the protein is a lysosomal enzyme, which when mutated causes a lysosomal storage disorder. However, the concepts of the present invention are generally applicable to any disease or condition characterized by a mutant protein suitable for SPC therapy that has one or more specific mutations that can be generated in vitro, for example, by site-directed mutagenesis.

[0030] In one specific embodiment, the present invention provides a method for determining whether an SPC enhances the enzymatic activity of a mutant α-Gal A enzyme and therefore can be used as an effective therapeutic treatment for Fabry disease patients expressing the same α-Gal A mutation.

[0031] In another specific embodiment, the present invention provides a method for determining whether an SPC enhances the enzymatic activity of a mutant GAA enzyme and therefore can be used as an effective therapeutic treatment for Pompe disease patients expressing the same GAA mutation.

[0032] In another specific embodiment, the present invention provides methods for determining whether an SPC enhances the enzymatic activity of a mutant Gba enzyme and therefore can be used as an effective therapeutic treatment for Gaucher disease patients expressing the same Gba mutation.

[0033] The methods of the present invention provide an assay that allows for determining whether a patient expressing a mutant lysosomal enzyme is a candidate for SPC treatment. The novel in vitro assay is highly sensitive and can be performed on host cells transfected with a nucleic acid construct encoding the mutant lysosomal enzyme. Specific candidate SPCs can then be assayed to determine whether they are capable of increasing the activity of the mutant enzyme expressed by the host cells. Thus, unlike assays that utilize cells derived from patients with lysosomal storage disorders, the assay of the present invention avoids the time-consuming steps of collecting a specimen from the patient, purifying cells from the specimen, and culturing the cells from the specimen in vitro.

[0034] The present invention also provides a method for determining whether a patient expressing a mutant protein (e.g., a lysosomal enzyme) is a candidate for SPC therapy, by which the patient's physician or other medical professional can determine whether the patient's mutation will respond to SPC therapy by examining the mutant protein (e.g., a lysosomal enzyme mutation) within a therapeutic reference table, which is generated from in vitro analysis of SPC responses in cell lines transformed with a nucleic acid vector encoding the mutant protein.

[0035] Furthermore, the present invention also provides a "treatment benchmark" that provides information describing whether 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 benchmark provides information indicating whether 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 therapies tailored to a patient's specific mutation.

[0036] In one non-limiting embodiment, the mutant protein is a mutant lysosomal enzyme, such as mutant α-Gal A, GAA, or Gba, and the cell line is transfected with a nucleic acid vector encoding the mutant lysosomal enzyme.

[0037] In another non-limiting embodiment, the present invention provides a method of treating a patient with Fabry disease, comprising administering a therapeutically effective dose of 1-deoxygalactonojirimycin (DGJ) to the patient, wherein the patient expresses a mutant α-Gal A whose activity when expressed in a host cell upon contact with an SPC (e.g., DGJ) can be increased. Such α-Gal A mutations treatable according to this method include A121T, A156V, A20P, A288D, A288P, A292P, and A316T. A348P, A73V, C52R, C94Y, D234E, D244H, D244N, D264Y, E338K, E341D, E358K, E39 8K, E48K, E59K, E66Q, F113L, G144V, G183D, G260A, G271S, G325D, G328A, G35R, G3 73D, G373S, H225R, I219N, I242N, I270T, I289F, I303N, I317T, I354K, I91T, L14P , L166V, L243F, L300F, L310F, L32P, L45R, M267I, M284T, M296I, M296V, M72V, M76 R, N224S, N263S, N298K, N298S, N320I, N320Y, N34K, P205R, P259L, P265L, P265R , P293A, P293S, P409S, P40L, P40S, Q279E, Q279H, Q279R, Q280H, Q280K, Q312H, Q3 These include, but are not limited to, 21E, Q321R, Q327E, R301P, R342Q, R363C, R363H, R49G, R49L, R49S, S201Y, S276N, S297C, S345P, T194I, V269M, V316E, W340R, W47L, and W95S mutations.

[0038] In one embodiment, the method of treating Fabry disease patients with a therapeutically effective dose of DGJ excludes the following α-Gal A mutations: D244N, E358K, E59K, E66Q, G183D, G325D, I289F, I91T, L45R, M296V, N263S, N320Y, P205R, P40S, Q279E, R342Q, R363C, R49L, V316E.

[0039] One advantage of the assay described by this invention is that it can be applied to female patients suffering from X-linked lysosomal storage disorders, such as Fabry disease. Due to X chromosome inactivation, specimens collected from female patients contain both normal, healthy cells and enzyme-deficient mutant cells. Assays for the effect of SPC on such specimens will show enhanced enzyme activity due to normal wild-type enzyme expression in healthy cells, even though abnormal cells with mutant enzymes may not be responsive to SPC. The present invention overcomes this obstacle because cell lines transfected with vectors encoding mutant proteins express only the mutant form of the protein, and therefore there is no wild-type protein expressed by the cell lines that would cause this spurious enhancement seen in assays using patient-derived cells.

[0040] In another non-limiting embodiment, the present invention provides a method of treating a Pompe disease patient, comprising administering a therapeutically effective dose of 1-deoxynojirimycin (DNJ) to the Pompe disease patient, wherein the patient expresses a mutant GAA whose activity when expressed in a host cell upon contact with an SPC (e.g., DNJ) can be increased. 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 ΔN470 mutations.

[0041] In another non-limiting embodiment, the present invention provides a method of treating a patient with Gaucher disease with a therapeutically effective dose of isofagomine (IFG), wherein the patient expresses a mutant Gba whose activity when expressed in a host cell upon contact with an SPC (e.g., IFG) can be increased.

[0042] definition The terms used herein generally have their ordinary meaning in the art, within the context of this invention and within the specific context in which each term is used. Some terms are discussed below or elsewhere herein to provide additional guidance to the practitioner in describing the compositions and methods of this invention and how to use them.

[0043] The term "Fabry disease" refers to an X-linked inborn error of glycosphingolipid catabolism caused by deficient activity of lysosomal α-galactosidase A. This defect leads to the accumulation of globotriaosylceramide (ceramide trihexoside) and related glycosphingolipids in vascular endothelial lysosomes in the heart, kidney, skin, and other tissues.

[0044] "Atypical Fabry disease" refers to patients with the predominantly cardiac manifestation of α-Gal A deficiency, namely, progressive globotriaosylceramide (GL-3) accumulation within cardiomyocytes leading to marked hypertrophy of the heart, particularly the left ventricle.

[0045] A "carrier" is a female who has one X chromosome with a defective α-Gal A gene and one X chromosome with a normal gene, and in which X chromosome inactivation of the normal allele is present in one or more cell types in the body. Carriers are often diagnosed with Fabry disease.

[0046] "Pompe disease" refers to an autosomal recessive LSD characterized by defective acid alpha-glucosidase (GAA) activity, which impairs lysosomal glycogen metabolism. The enzyme deficiency leads to lysosomal glycogen accumulation, resulting in progressive skeletal muscle weakness, cardiac decline, respiratory failure, and / or CNS dysfunction in the later stages of the disease. Genetic mutations within the GAA gene result in decreased expression or produce mutant forms of the enzyme with altered stability and / or biological activity that ultimately lead to disease (see generally Hirschhorn R, 1995, Glycogen Storage Disease Type II: Acid α-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 (pediatric, juvenile, and adult) are correlated with residual α-glucosidase activity levels (Reuser AJ et al., 1995, Glycogenosis Type II (Acid Maltase Deficiency) Muscle & Nerve Supplement 3, S61-S69). ASSCs (also known as "pharmacological chaperones") are a promising new therapeutic approach for the treatment of genetic diseases such as lysosomal storage disorders (e.g., Pompe disease).

[0047] Pediatric Pompe disease (type I or A) is the most common and most severe form, characterized by growth retardation, systemic hypotension, cardiac hypertrophy, and cardiopulmonary failure within the first two years of life. Juvenile Pompe disease (type II or B) is moderately severe and characterized by a predominance of muscular manifestations without cardiac hypertrophy. Individuals with juvenile Pompe disease usually die before reaching the age of 20 due to respiratory failure. Adult Pompe disease (type III or C) often presents as a slowly progressive myopathy in the teens or late 60s (Felice KJ et al., 1995, Clinical Variability in Adult-Onset Acid Maltase Deficiency: Report of Affected Sibs and Review of the Literature, Medicine 74, 131-135).

[0048] In Pompe disease, α-glucosidase has been shown to be extensively modified post-translationally by glycosylation, phosphorylation, and proteolytic processes. Conversion of a 110 kilodalton (kDa) precursor to mature forms of 76 and 70 kDa by proteolysis in lysosomes is required for optimal glycogen catalysis.

[0049] 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.

[0050] The term "Gaucher disease" refers to a deficiency of the lysosomal enzyme β-glucocerebrosidase (Gba), which breaks down the fatty acid glucocerebroside. Fat then accumulates primarily 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. Type I patients present in either childhood or adolescence and suffer from easy bruising, anemia, low platelets, enlarged liver and spleen, fatigue due to skeletal weakness, and, in some cases, pulmonary and renal dysfunction. There are no signs of brain involvement. In early-onset type II, enlarged liver and spleen develop by the age of three months, and widespread brain involvement is present. Mortality by age two years is high. Type III is characterized by enlarged liver and spleen and encephalopathy. The β-glucocerebrosidase gene is located on human chromosome 1q21. The protein precursor contains 536 amino acids, and the mature protein is 497 amino acids long.

[0051] "Patient" means a subject diagnosed with or suspected of having a particular disease. The patient may be a human or an animal.

[0052] "Fabry disease patient" means an individual who has been diagnosed with or is suspected of having Fabry disease and who has mutant α-Gal A as further defined below. The characteristic markers of Fabry disease can occur with equal prevalence in male hemizygotes and female carriers, although females are typically less severely affected.

[0053] "Pompe disease patient" means an individual diagnosed with or suspected of having Pompe disease and having mutant GAA as further defined below.

[0054] "Gaucher disease patient" means an individual who has been diagnosed with or is suspected of having Gaucher disease and who has a mutation Gba as further defined below.

[0055] Human α-galactosidase A (α-Gal A) refers to the enzyme encoded by the human GLA gene. The human α-Gal A enzyme consists of 429 amino acids and has GenBank accession number U78027.

[0056] In one non-limiting embodiment, human lysosomal alpha-glucosidase (acid α-glucosidase; GAA) is a lysosomal enzyme that hydrolyzes alpha-1,4- and alpha-1,6-linked D-glucose polymers present in glycogen, maltose, and isomaltose. Alternative names include glucoamylase; 1,4-α-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase; and exo-1,4-α-glucosidase. The human GAA gene is located on chromosome 17q25.2-25.3 and has the nucleotide and amino acid sequence set forth in GenBank accession number Y00839.

[0057] The term "human Gba gene" refers to the gene encoding acid β-glucosidase, also known as glucocerebrosidase or Gba. The Gba gene is located on chromosome 1q21 and involves 11 exons (GenBank accession number J03059). A homologous pseudogene of Gba also exists, located approximately 16 kb downstream from the Gba gene (GenBank accession number M16328).

[0058] "Human Gba" protein refers to the wild-type human Gba protein, which consists of 536 amino acids and has GenBank accession number J03059.

[0059] The term "mutant protein" includes proteins that have a mutation in the gene encoding the protein that results in the protein being unable to achieve a stable conformation under conditions in which the protein normally resides in the ER. This inability to achieve a stable conformation results in a significant amount of the enzyme being degraded rather than transported to the lysosome. Such mutations are sometimes referred to as "conformational mutants." Such mutations include, but are not limited to, missense mutations and in-frame small deletions and insertions.

[0060] As used herein, in one embodiment, the term "mutated α-Gal A" includes α-Gal A that has a mutation in the gene encoding α-Gal A that results in the enzyme being unable to achieve a stable conformation under conditions normally present in the ER. The inability to achieve a stable conformation results in a significant amount of the enzyme being degraded rather than being transported to lysosomes.

[0061] Non-limiting exemplary α-Gal A mutations associated with Fabry disease resulting in unstable α-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;I289F;M296I;M296V;L300P;R301Q;V316E;N320Y;G325D;G328A;R342Q;E358A;E358K;R363C;R363H;G370S; and P409A.

[0062] In one embodiment, the term "mutated GAA," as used herein, includes GAA that has a mutation in the gene encoding GAA that results in the enzyme being unable to achieve a stable conformation under conditions normally present in the ER. The inability to achieve a stable conformation results in a significant amount of the enzyme being degraded rather than being transported to the lysosome.

[0063] As used herein, in one embodiment, the term "mutant Gba" includes Gba that has a mutation in the gene encoding Gba that results in the enzyme being unable to achieve a stable conformation under conditions normally present in the ER. This inability to achieve a stable conformation results in a significant amount of the enzyme being degraded rather than being transported to lysosomes.

[0064] As used herein, the term "specific pharmacological chaperone" ("SPC") or "pharmacological chaperone" refers to any molecule, including small molecules, proteins, peptides, nucleic acids, carbohydrates, etc., that specifically binds to a protein and has one or more of the following effects: (i) enhancing the formation of a stable molecular conformation of that protein; (ii) inducing transport of the protein from the ER to another cellular site, preferably a native cellular site, i.e., preventing ER-associated degradation of the protein; (iii) preventing aggregation of misfolded proteins; and / or (iv) restoring or enhancing at least partial wild-type function and / or activity of the protein. For example, a compound that specifically binds to α-Gal A, GAA, or Gba means that it binds to and exerts a chaperone effect on that enzyme, rather than on a generic group of related or unrelated enzymes. More specifically, the term does not refer to endogenous chaperones such as BiP, or nonspecific agents, i.e., chemical chaperones, such as glycerol, DMSO, or heavy water, which have demonstrated nonspecific chaperone activity for a variety of proteins (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. Nos. 5,900,360; 6,270,954; and 6,541,195). In the present invention, the SPC may be a reversible competitive inhibitor.

[0065] A "competitive inhibitor" of an enzyme can refer to a compound whose chemical structure and molecular geometry are structurally similar to the enzyme's substrate, thereby binding the enzyme in approximately the same location as the substrate. Thus, the inhibitor competes for the same active site as the substrate molecules, increasing the Km. Competitive inhibition is usually reversible if enough substrate molecules are available to displace the inhibitor, i.e., if the competitive inhibitor can reversibly bind. Therefore, the amount of enzyme inhibition depends on the inhibitor concentration, the substrate concentration, and the relative affinities of the inhibitor and substrate for the active site.

[0066] Below is a description of some specific pharmacological chaperones (SPCs) contemplated by the present invention.

[0067] In one specific, non-limiting embodiment, the SPC is [ka] or a pharmaceutically acceptable salt, ester, or prodrug of 1-deoxygalactonojirimycin. The hydrochloride salt of DGJ is known as miglustat hydrochloride.

[0068] Still other SPCs for α-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 α-3,4-di-epi-homonojirimycin, 4-epi-fagomine, α-allo-homonojirimycin, N-methyl-deoxygalactonojirimycin, β-1-C-butyl-deoxygalactonojirimycin, α-galacto-homonojirimycin, calystegine A3, calystegine B2, calystegine B3, N-methyl-calystegine A3, N-methyl-calystegine B2, and N-methyl-calystegine B3.

[0069] In one specific, non-limiting embodiment, the SPC is [ka] or a pharmaceutically acceptable salt, ester, or prodrug of isofagomine, such as IFG tartrate (see, e.g., U.S. Patent Application Publication No. 20070281975). IFG is CH 13 It has a molecular formula of NO3 and a molecular weight of 147.17. This compound is further described in U.S. Patent No. 5,844,102 to Sierks et al. and U.S. Patent No. 5,863,903 to Lundgren et al.

[0070] 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-β-bareinamines, flufenozine, N-dodecyl-DNJ, calystegines A3, B1, B2 and C1.

[0071] In one specific, non-limiting embodiment, the SPC is [ka] or a pharmaceutically acceptable salt, ester, or prodrug of 1-deoxynojirimycin (1-DNJ), which has the structural formula: In one embodiment, the salt is the hydrochloride salt (i.e., 1-deoxynojirimycin-HCl).

[0072] 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. Patent Application Publication 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, α-homonojirimycin, and castanospermine.

[0073] As used herein, the term "specifically binds" refers to the interaction of a protein, such as α-Gal A, Gba, or GAA, with a pharmacological chaperone, specifically with amino acid residues of the protein that directly participate in contacting the pharmacological chaperone. The pharmacological chaperone specifically binds a target protein, such as α-Gal A, Gba, or GAA, and exerts a chaperone effect on that protein, rather than on a general 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 "active site" of that protein. Specific binding can be assessed through routine binding assays or through structural studies, such as cocrystallization, NMR, and the like. The active site of α-Gal A, Gba, or GAA is the substrate binding site.

[0074] "Deficient α-Gal A activity" means α-Gal A activity in cells from a patient that is lower than the normal range compared (using the same method) to activity in normal individuals who do not have or are not suspected of having Fabry disease or any other disease (especially a blood disorder).

[0075] "Gba activity deficiency" means Gba activity in cells from a patient that is lower than the normal range compared (using the same method) to activity in normal individuals who do not have or are not suspected of having Gaucher disease or any other disease.

[0076] "GAA activity deficiency" means GAA activity in cells from a patient that is lower than the normal range compared (using the same method) to activity in normal individuals who do not have or are not suspected of having Pompe disease or any other disease.

[0077] As used herein, the terms "enhancing α-Gal A activity," "enhancing Gla activity," and "enhancing GAA activity" or "increasing α-Gal A activity," "increasing Gba activity," and "increasing GAA activity" refer to increasing the amount of α-Gal A, Gba, or GAA, respectively, that assumes a stable conformation in cells contacted with an α-Gal A, Gba, or GAA-specific pharmacological chaperone compared to the amount in the same cells (preferably, e.g., cells of the same cell type at an earlier stage) that have not been contacted with the α-Gal A, Gba, or GAA-specific pharmacological chaperone. The terms also refer to increasing the transport of α-Gal A, Gba, or GAA to lysosomes in cells contacted with an α-Gal A, Gba, or GAA-specific pharmacological chaperone compared to the transport of α-Gal A, Gba, or GAA that has not been contacted with the protein-specific pharmacological chaperone. These terms refer to both wild-type and mutant α-Gal A, Gba, or GAA. In one embodiment, an increase in the amount of α-Gal A, Gba, or GAA in cells is measured by measuring the hydrolysis of an artificial substrate in a lysate from cells treated with SPC. Increased hydrolysis indicates increased α-Gal A, Gba, or GAA activity.

[0078] The term "α-Gal A activity" refers to the normal physiological function of wild-type α-Gal A in a cell. For example, α-Gal A activity includes the hydrolysis of GL-3.

[0079] The term "Gba activity" refers to the normal physiological functions of wild-type αGba in a cell, such as the metabolism of lipid glucocerebrosidase.

[0080] The term "GAA activity" refers to the normal physiological functions of wild-type Gaa in a cell, such as lysosomal glycogen metabolism.

[0081] A "responder" is an individual diagnosed with or suspected of having a lysosomal storage disorder, such as, but not limited to, Fabry disease, Pompe disease, or Gaucher disease, whose cells respond to contact with SPC by exhibiting sufficient increased α-Gal A, GAA, or Gba activity and / or symptomatic improvement or improvement in surrogate markers, respectively. Non-limiting examples of improvements in surrogate markers for Fabry disease and Pompe disease are disclosed in U.S. Patent Application Nos. 60 / 909,185 and 61 / 035,869, respectively.

[0082] Non-limiting examples of improvements in surrogate markers of Fabry disease disclosed in U.S. Patent Application No. 60 / 909,185 include increased α-Gal A levels or activity in cells (e.g., fibroblasts) and tissues; decreased GL-3 accumulation; decreased plasma concentrations of homocysteine ​​and vascular cell adhesion molecule-1 (VCAM-1); decreased GL-3 accumulation in cardiomyocytes and valvular fibroblasts; reduced cardiac hypertrophy (particularly of the left ventricle), improved valvular dysfunction, and arrhythmias; improved proteinuria; decreased urinary concentrations of lipids such as CTH, lactosylceramide, and ceramide, and increased urinary concentrations of glucosylceramide and sphingomyelin (Fuller et al., Clinical Chemistry. 2005;51: 688-694); absence of laminated inclusion bodies (zebra bodies) in glomerular epithelial cells; improved renal function; alleviation of hypohidrosis; absence of angiokeratomas and improvement in hearing abnormalities such as high-frequency sensorineural hearing loss, progressive hearing loss, sudden hearing loss, or tinnitus. Improvement in neurological symptoms includes prevention of transient ischemic attack (TIA) or stroke; and improvement in neuropathic pain manifested as acroparesthesia (hot or tingling sensations in the extremities).

[0083] A dose that achieves one or more of the above responses is a "therapeutically effective dose."

[0084] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically do not produce undesirable reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle used when administering a compound. Such pharmaceutical carriers can be sterile liquids, such as water and oils. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin, 18th Edition.

[0085] As used herein, the term "isolated" refers to the removal of a reference material from the environment in which it is normally found. Thus, isolated biological material can be free of cellular components, i.e., components of the cell in which the material is found or produced. In the case of nucleic acid molecules, isolated nucleic acids include PCR products, mRNA bands on gels, cDNA, or restriction fragments. In another embodiment, isolated nucleic acids are preferably excised from the chromosome in which they are found and, more preferably, are no longer joined to non-regulatory, non-coding regions, or other genes located upstream or downstream of the gene that the isolated nucleic acid molecule contained when discovered in the chromosome. In yet another embodiment, isolated nucleic acids lack one or more introns. Isolated nucleic acids include sequences inserted into plasmids, cosmids, artificial chromosomes, etc. Thus, in specific embodiments, recombinant nucleic acids are isolated nucleic acids. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it is associated within the cell, or, if it is a membrane-bound protein, with the cell membrane. Isolated organelles, cells, or tissues are removed from the anatomical site in which they are found in vivo. An isolated material may, but need not be, purified.

[0086] The terms "about" and "approximately" generally refer to an acceptable degree of error for a measured quantity given the nature or precision of the measurement. Typically, 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, particularly in biological systems, the terms "about" and "approximately" can refer to an average value that is preferably within 10-fold or 5-fold, and more preferably 2-fold, of a given value. Numerical quantities given herein are approximations unless otherwise specified, meaning that the terms "about" or "approximately" can be implied when not explicitly stated.

[0087] How treatment options are determined To readily determine whether SPC therapy is a viable treatment for patients, including those with Fabry, Pompe, or Gaucher disease, and female carriers of X-linked lysosomal storage disorders such as Fabry disease, a simple, non-invasive SPC rescue assay of protein activity in cell lines expressing mutant forms of the protein was developed.

[0088] In vitro testing In one embodiment, the diagnostic method of the invention involves transforming a cell line with a nucleic acid vector encoding a mutant lysosomal enzyme, such as α-Gal A, GAA, or Gba. The cell line is then treated with or without an SPC, such as DGJ, DNJ, or IFG, for a time sufficient to demonstrate enhanced (i.e., increased) α-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 α-Gal A, GAA, or Gba activity in lysates from SPC-treated cells relative to activity in lysates from untreated cells indicates a high probability that a patient expressing α-Gal A, GAA, or Gba with the same mutation as the cell line will respond to SPC therapy (i.e., the patient is a "responder").

[0089] Transient transfection of cell lines and expression of mutant lysosomal enzymes In one embodiment, to identify SPC-responsive mutations, mutations in all known lysosomal enzymes (e.g., α-Gal A, GAA, or Gba), such as 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. The mutant enzyme constructs can then be transiently expressed in cell lines, such as mammalian COS-7, HEK-293, or GripTite293MSR (Invitrogen Corp., Carlsbad, CA, USA) cells. The transformed cells can then be incubated with increasing concentrations of SPC, and enzyme activity can be measured in cell lysates.

[0090] Mutagenesis: Nucleic acid vectors encoding mutant proteins (eg, mutant α-Gal A, GAA, or Gba) can be produced by conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of those in the art. (e.g. Sambrook, Fritsch & Maniatis, 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, ed., 1985, DNA Cloning: A Practical Approach, Volumes I and II, Second Edition; Gait, MJ, ed., 1984, Oligonucleotide Synthesis: A practical approach; Hames, BD & Higgins, SJ eds., 1985, Nucleic Acid Hybridization;Hames, BD & Higgins, SJ, eds., 1984, Transcription And Translation;Freshney, RI, 2000, Culture of Animal Cells: A Manual of Basic Technique;Woodward, J., 1986, Immobilized Cells And Enzymes: A practical approach, IRL Press;Perbal, BE, 1984, A Practical Guide To Molecular For example, a single α-Gal A, GAA, or Gba mutation can be introduced into a nucleic acid encoding a wild-type α-Gal A, GAA, or Gba gene through site-directed mutagenesis of a nucleic acid encoding the wild-type enzyme.

[0091] Transient transfection and expression: The coding sequence of the gene to be delivered, e.g., mutant α-Gal A, GAA, or Gba, is operably linked to expression control sequences, such as a promoter, that direct expression of the gene. As used herein, the term "operably linked" refers to the functional relationship of the polynucleotide / gene with regulatory and effector sequences, such as promoters, enhancers, transcription and translation stop sites, and other signal sequences. For example, operably linking 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 transcription of RNA from the polynucleotide. Expression of a mutant protein (e.g., mutant α-Gal A, GAA, or Gba) may be controlled by any promoter / enhancer element known in the art, provided that these regulatory elements are functional in the host selected for expression.

[0092] In a specific embodiment, a vector is used in which the coding sequence, and any other sequences, are flanked by regions that promote homologous recombination at the desired site within the genome, thereby providing for expression of the construct from a nucleic acid molecule integrated within 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.).

[0093] The term "host cell" refers to any cell of any organism that is selected, modified, transformed, grown, used, or otherwise manipulated for the purpose of cellular production of a substance, such as, for example, cellular expression of a gene, DNA or RNA sequence, protein, or enzyme. In one embodiment, host cells transfected with vectors encoding mutant α-Gal A, GAA, or Gba can be used to screen candidate SPCs, such as DGJ, DNJ, or IFG, to determine whether the candidate SPCs are compounds effective in increasing the activity of mutant α-Gal A, GAA, or Gba expressed by the host cell.

[0094] The term "expression system" refers to a host cell and a compatible vector under appropriate conditions for the expression of a protein encoded by foreign DNA, e.g., carried by the vector and introduced into the host cell. Expression systems include mammalian host cells and vectors. Suitable cells include PC12 cells, CHO cells, HeLa cells, GripTite 293MSR cells (Invitrogen Corp., Carlsbad, CA, USA), 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, and NIH3T3 cells.

[0095] Suitable vectors include viruses such as adenoviruses, adeno-associated viruses (AAV), vaccinia, herpes viruses, baculoviruses and retroviruses, parvoviruses, lentiviruses, bacteriophages, cosmids, plasmids, fungal vectors, naked DNA, DNA-lipid complexes and other recombinant vehicles typically used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts and may be used for gene therapy as well as simple protein expression.

[0096] In one non-limiting example, transient transfection can be performed in GripTite 293MSR cells (Invitrogen Corp., Carlsbad, CA, USA) using the Fugene HD reagent (Roche). Cells can be seeded in an appropriate assay vessel, such as a 96-well plate (Costar), at a density of, for example, 7.5-10k cells / well and incubated under appropriate conditions, for example, 37°C and 5% CO2, for 24 hours prior to transfection. After transfection with an expression construct containing a specific α-Gal A variant, the cells can be reincubated for, for example, 1 hour at 37°C and 5% CO2, after which DGJ can be added at 50nM-1mM. Cells can then be incubated for 4-5 days before digestion and assay.

[0097] Enzyme activity / enhancement assays: Typically, after incubation with SPC (e.g., DGJ, DNJ, or IFG), host cells are lysed by addition of lysis buffer (or deionized water) and physical disruption (pipetting, vortexing and / or stirring and / or sonication) at room temperature or on ice, after which the lysates are pooled on ice, and the pooled lysates are then divided into small aliquots and frozen.

[0098] Lysates can be thawed immediately prior to the assay, suspended and sonicated using a vortex mixer, and then added to the appropriate wells, e.g., in a microplate. For Fabry disease, N-acetylgalactosamine (GalNAc) is then added to each well (to inhibit α-galactosidase B), followed by a brief incubation. 4-Methylumbelliferyl-α-D-galactopyranoside (4-MU Gal) or other suitable labeled DGJ substrate is then added, and the plate is gently mixed briefly, covered, and incubated at 37°C for a time sufficient for substrate hydrolysis, usually about 1 hour. To stop the reaction, a pH 10.7 NaOH-glycine buffer is added to each well, and the plate is then read in a fluorescence plate reader (e.g., Wallac 1420 Victor3). TMThe enzyme activity is measured by a chromatographic method (or similar instrument). The excitation and emission wavelengths are routinely 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 specimen, at least three normal specimens may be tested simultaneously.

[0099] Those skilled in the art can readily ascertain various modifications of this assay. Examples of artificial substrates that can be used to detect α-Gal A activity include, but are not limited to, p-nitrophenyl-α-D-galactopyranoside and 4-MUGAL. Obviously, only substrates that are cleavable by human α-Gal A are suitable for use. While the use of fluorogenic substrates is preferred, other methods of determining enzyme activity, including the use of chromogenic substrates or immunoquantification techniques, are contemplated for use with this method.

[0100] In one specific example, after incubation with an SPC, e.g., DGJ, host cells can be washed twice with PBS and then incubated in 200 μL of fresh medium at 37°C with 5% CO for 2 hours, followed by two more washes with PBS. Cells can then be lysed in 60 μL of lysis buffer (27 mM sodium citrate / 46 mM dibasic sodium phosphate, 0.5% Triton-X-100, pH 4.6). At this time, 10 μL of the lysate can be added to 50 μL of assay buffer (lysis buffer without Triton-X-100, containing 6 mM 4-MU-α-D-galactopyranoside (4-MUG) and 117 mM N-acetyl-D-galactosamine (GalNac)) and incubated for 1 hour at 37°C. Seventy microliters of stop solution (0.4 M glycine, pH 10.8) can then be added, and fluorescence can be read on a Victor plate reader (Perkin Elmer) at excitation of 355 nm and emission of 460 nm. Raw fluorescence counts can be background-subtracted, as defined by counts from the substrate solution alone. Protein concentration was determined from 40 μL of cell lysate using the MicroBCA Protein Assay Kit (Pierce) according to the manufacturer's instructions. For calculation of absolute α-Gal A activity, expressed as nmoles / mg protein / h, a 4-methylumbelliferone (4-MU) standard curve ranging from 30 μM to 1.3 nM was generated in parallel or further normalized to the % of untreated wild-type enzyme activity.

[0101] Treatment standard table In another embodiment, the methods described above can be used to generate a "treatment reference table" or "treatment therapy table," where the treatment reference table includes a list of protein mutations and further indicates the responsiveness of each mutation to an SPC, e.g., DGJ, DNJ, or IFG. The treatment reference table can then be used to determine whether a particular SPC, e.g., DGJ, DNJ, or IFG, is likely to be an effective SPC for treating a patient with a particular α-Gal A, GAA, or Gba mutation, respectively.

[0102] As used herein, "treatment therapy table" or "treatment reference table" means any documentation that conveys whether a particular mutation will respond to an SPC therapy, and is not necessarily limited to documentation presented in the form of a table.

[0103] In one embodiment, the therapeutic reference table can be used by a treating physician or clinical professional to select an SPC for treatment of a patient with, e.g., Fabry disease, Pompe disease, or Gaucher disease, expressing a specific mutant α-Gal A, GAA, or Gba, respectively, where the SPC is selected because the therapeutic reference table identifies it as a compound that can increase the activity of the patient's mutant α-Gal A, GAA, or Gba when expressed in a host cell.

[0104] Treatable disorders While this application has been primarily discussed in relation to 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 enzyme, or for any disorder involving protein misfolding. These diseases include other lysosomal storage disorders, such as cystic fibrosis (CFTR) (respiratory or sweat gland epithelial cells), familial hypercholesterolemia (LDL receptor; LPL-containing adipocytes or vascular endothelial cells), cancer (p53; PTEN-tumor cells), Alzheimer's disease (α-secretase), Parkinson's disease (glucocerebrosidase), obesity (MC4R), and amyloidosis (transthyretin), among others.

[0105] Eligibility Criteria The criteria for determining eligibility for SPC therapy depend on the type of mutant GAL A, GAA, or Gba expressed by the patient. In one embodiment, a patient with Fabry, Pompe, or Gaucher disease may be classified as eligible for SPC therapy if, in the presence of an SPC such as DGJ, DNJ, or IFG, the α-Gal A, GAA, or Gba activity in host cells expressing the same mutation as the patient is at least 1.5-fold to 20-fold (2% to 100%) higher than the activity in host cells expressing wild-type α-Gal A, GAA, or Gba, respectively.

[0106] This discovery provides a method to improve the diagnosis of Fabry, Pompe, and Gaucher diseases in particular, and lysosomal storage disorders in general, and to facilitate clinical treatment decisions for these diseases. Furthermore, this method can be extended to a wide range of genetically defined diseases in the relevant cell types. This class of diseases includes other lysosomal storage disorders, among them cystic fibrosis (CFTR) (respiratory or sweat gland epithelial cells), familial hypercholesterolemia (LDL receptor; LPL-containing adipocytes or vascular endothelial cells), cancer (p53; PTEN-tumor cells), Alzheimer's disease (α-secretase), Parkinson's disease (glucocerebrosidase), obesity (MC4R), and amyloidosis (transthyretin).

[0107] kit The present invention also provides commercially available diagnostic test kits for making therapeutic treatment decisions, which kits provide all of the materials described above, and more particularly discussed in the Examples below, for preparing and performing each assay in one convenient package, optionally including instructions for use and analytical guidelines.

[0108] As one non-limiting example, a kit for assessing α-Gal A activity may include, at a minimum: a. a panel of host cells each expressing a mutant α-Gal A, or alternatively, a host cell, a vector encoding a mutant α-Gal A, and a means for transfecting the host cell such that the host cell expresses the mutant α-Gal A; b.Specific pharmacological chaperones; c. Chromogenic or fluorogenic substrates for enzyme assays (including appropriate standards); and d.GaINAc.

[0109] The kit may also include instructions for optimally performing the protein enhancement assay. In another embodiment, the kit includes appropriate test tubes, buffers (e.g., lysis buffer), and microplates.

[0110] In one embodiment, the SPC is supplied in dry form and is reconstituted prior to addition.

[0111] Patients expressing mutant α-Gal A, GAA, or Gba who test positive for enzyme enhancement at a candidate SPC in the assay of the present invention can then be treated with the candidate SPC agent, while patients expressing mutant α-Gal A, GAA, or Gba who do not show enzyme enhancement at a candidate SPC can avoid treatment, thus saving the cost and emotional pain of not responding to one therapy. [Example]

[0112] The present invention is further described using the examples provided below. The use of such examples is merely for illustrative purposes and is not intended to limit in any way the scope or meaning of the invention or any exemplified term. Likewise, the present invention is not limited to any particular preferred embodiment described herein. Indeed, many modifications and variations of the present invention will become apparent to those skilled in the art upon reading this specification. Therefore, the present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0113] Example 1: Identification of Fabry disease-causing mutations that are responsive to the pharmacological chaperone DGJ This Example 1 provides an in vitro diagnostic assay for determining the responsiveness of Fabry disease patients to specific pharmacological chaperones.

[0114] Preface Fabry disease is a lysosomal storage disorder caused by mutations in the gene encoding α-galactosidase A (α-Gal A). Over 600 Fabry mutations have been reported, with approximately 60% being missense. The iminosugar DGJ is currently being investigated in phase II clinical trials as a pharmacological chaperone for the treatment of Fabry disease. Previously, it was shown that DGJ mediates a selective and dose-dependent increase in α-Gal A levels in lymphoid cell lines derived from multiple Fabry disease patients. To identify additional DGJ-responsive mutations, GripTite293MSR (Invitrogen Corp., Carlsbad, CA, USA) cells were transiently transfected with expression vectors containing all known α-Gal A missense mutations and multiple in-frame small deletions and insertions generated by site-directed mutagenesis. Mutant α-Gal A constructs were transiently expressed in HEK-293 cells. Cells were incubated with increasing concentrations of DGJ, and α-Gal A activity in cell lysates was measured. Validation of the assay was performed for over 35 missense mutations, and the results obtained in HEK-293 cells were similar to those obtained from both lymphoid and primary T cell cultures from Fabry patients (see U.S. Patent Application No. 11 / 749,512), as well as to the α-Gal A enzyme responses seen in leukocytes from Fabry patients after oral administration of DGJ in a phase II clinical trial.

[0115] Methods and Materials Mutagenesis: All mutations were generated by site-directed mutagenesis according to standard molecular biology protocols. To generate point mutations, site-directed mutagenesis was used on the expression vector pcDNA3.1 (Invitrogen), which contains an in-frame human α-Gal A cDNA. Specific primer pairs containing the desired mutations were designed (Figure 6). Mutagenesis was performed via polymerase chain reaction using PfuUltra High Fidelity DNA polymerase (Stratagene) in a thermocycler. Each reaction mixture contained the following in a total volume of 50 μl: 1.6 μl dHO, 5.0 μl 10x PfuUltraHF reaction buffer, 0.5 μl forward 5'-primer (50 μM), 0.5 μl reverse 3'-primer, 1.0 μl dNTP mix (containing 25 mM each of dA, dT, dC, and dG), 0.9 μl human GLA in pcDNA3 (2 ng / μl DNA), and 0.5 μl PfuUltraHD DNA polymerase. The thermocycler parameters used were: i) 94°C for 30 s, ii) 94°C for 30 s, 50–60°C for 30 s, 68°C for 6 min, and iii) 16 cycles of ii). Then, 0.5 μl of DpnI (New England Biolabs) was added to each reaction, which was then incubated at 37°C for 2 h. A 7.5 μl volume of each mutagenesis reaction was used to transform DH5α cells (New England Biolabs). Cells were then plated onto LB agar plates with 75 μg / ml ampicillin and incubated overnight at 37°C. Bacterial colonies were picked and grown overnight in liquid LB with ampicillin at 37°C with shaking, and plasmid DNA was extracted using a QuickLyse Miniprep Kit (Qiagen). Mutants were confirmed by sequencing the full-length human GLA gene. For some mutants, the human GLA DNA was contained within the vector plasmid pCXN. Mutagenesis was performed in this vector using NEB fusion protein polymerase. After confirming mutations through sequencing, the plasmid was digested with EcoRI and subcloned into the expression vector pcDNA3.1. Correct orientation was confirmed by digestion with XhoI.

[0116] Transient transfection and expression: Transient transfections were performed in GripTite 293MSR cells (Invitrogen Corp., Carlsbad, CA, USA) using the Fugene HD reagent (Roche). Briefly, cells were seeded in 96-well plates (Costar) at a density of 7.5–10 k cells / well and incubated at 37°C and 5% CO2 for 24 h prior to transfection. Cells were transfected with 0.35 μL of Fugene HD reagent and 0.1 μg of DNA per well (DNA:reagent ratio 2:7). After transfection with expression constructs containing specific α-Gal A variants, cells were again incubated at 37°C and 5% CO2 for 1 h before adding DGJ at 20 nM–1 mM. Cells were then incubated for 4–5 days before lysis and assay.

[0117] α-Gal A activity measurement: Cells were washed twice with PBS and then incubated in 200 μL of fresh medium at 37°C with 5% CO for 2 hours, followed by two more washes with PBS. Cells were then lysed in 60 μL of lysis buffer (27 mM sodium citrate / 46 mM dibasic sodium phosphate, 0.5% Triton-X-100, pH 4.6). At this time, 10 μL of the lysate was added to 50 μL of assay buffer (lysis buffer without Triton-X-100, containing 6 mM 4-MU-α-D-galactopyranoside (4-MUG) and 117 mM N-acetyl-D-galactosamine (GalNac)) and incubated for 1 hour at 37°C. Next, 70 μL of stop solution (0.4 M glycine, pH 10.8) was added, and fluorescence was read on a Victor plate reader (Perkin Elmer) at excitation of 355 nm and emission of 460 nm. Raw fluorescence counts were background subtracted, as defined by counts from the substrate solution alone. To determine protein concentration from 40 μL of cell lysate, a MicroBCA Protein Assay Kit (Pierce) was used according to the manufacturer's instructions. For calculation of absolute α-Gal A activity, expressed as nmoles / mg protein / h, a 4-methylumbelliferone (4-MU) standard curve ranging from 30 μM to 1.3 nM was generated in parallel or further normalized to % of untreated wild-type enzyme activity.

[0118] Transient transfections and α-Gal A activity measurements were performed in quadruplicate, with each mutation repeated at least three times, and the average α-Gal A activity at each DGJ concentration was calculated. Significant responses to DGJ were determined by a two-tailed paired Student's t-test (p<0.05).

[0119] result All listed Fabry mutations were generated by site-directed mutagenesis (Figure 1). Mutations identified in italic text were not tested, whereas mutations identified in plain text are α-Gal A mutants that were responsive to DGJ treatment in transient transfection assays. Mutations identified in bold and underlined text were not responsive to DGJ treatment in transient transfection assays. The magnitude of increase in α-Gal A levels after DGJ treatment and the EC50 values ​​are listed for all tested mutations that responded to DGJ treatment (Figure 2).

[0120] α-Gal A activity (expressed as nmol / mg protein / h of 4-MU released) was measured in lysates prepared from transfected GripTite293 cells incubated with increasing concentrations of DGJ. L300P exhibited a typical concentration-dependent response, while R227Q exhibited a typical negative response to DGJ. The wild-type exhibited high baseline activity and was unresponsive to DGJ in this assay (Figure 3).

[0121] α-Gal A levels were measured in three different assays, reported as a percentage of wild-type, and compared for each mutation by side-by-side plotting. Three different assays examined α-Gal A levels in T cells and lymphoblasts isolated from Fabry disease patients (e.g., U.S. Patent Application No. 11 / 749,512) and white blood cells (WBCs) from the DGJ Phase II study.

[0122] White bars represent basal levels (no DGJ treatment), and black bars represent elevated levels after DGJ treatment (Figure 4).

[0123] The Fabry mutations tested are depicted on the α-Gal A secondary structure (Figure 5). No significant correlation was observed between the mutation's location on the protein sequence and response, suggesting a broad distribution of responsive and non-responsive mutations throughout the protein. Text color indicates DGJ response: green = responsive; red = unresponsive; brown indicates that among multiple mutations at that same site, some responded to DGJ treatment but others did not.

[0124] conclusion These described results are comparable to those obtained with lymphoid cells or T cells from patients with Fabry disease, as well as to the α-Gal A enzyme response observed in leukocytes from patients with Fabry disease after oral administration of DGJ in a phase II clinical trial.

[0125] Thus, the GripTite293MSR transient transfection assay is a reliable method for identifying DGJ-responsive mutations and characterizing the magnitude and potency of this response.

[0126] Among the identified responsive mutants, the increase in α-Gal A levels upon DGJ treatment ranged from 1.3- to 40-fold (2% to 100% of wild type), with EC 50 Values ​​were between 200 nM and over 100 mM.

[0127] The DGJ-responsive and -unresponsive mutant forms did not appear to map to specific regions or domains on the α-Gal A protein structure.

[0128] Example 2: In vitro method to assess the effect of SPC on glucocerebrosidase activity - prophetic example Gaucher disease (GD) is caused by a deficiency of lysosomal glucocerebrosidase (GCase). Deficiency in GCase activity leads to the accumulation of glucosylceramide (GlcCer) and the development of symptoms such as anemia, thrombocytopenia, hepatosplenomegaly, osteonecrosis, infarction, osteoporosis, and in some cases, neuralgia. The specific pharmacological chaperone isofagomine tartrate (IFG) binds to mutant (N370S / N370S) GCase in the ER and enhances its transport to lysosomes.

[0129] To assess the effect of IFG on different GCase variants, an in vitro diagnostic assay is set up using Cos7 cells to identify IFG-responsive mutations.

[0130] COS-7 cell lines expressing missense mutations and multiple in-frame small deletions and insertions are prepared by site-directed mutagenesis using the techniques described in Examples 1 and 4. Assays are prepared for all mutations listed on the X-axis of Figure 8. IFG activity responses are confirmed for each assay according to methods known in the art (see, e.g., U.S. Patent No. 6,916,829, incorporated herein by reference).

[0131] To determine the correlation of IFG responses measured in COS-7 cells with those from patients, we also measured IFG activity responses in macrophages and lymphoblasts from patients. Macrophages were successfully derived from 46 of 63 patients, and incubation with IFG (3, 10, 30, or 100 μM) for 5 days increased GCase levels in macrophages from 42 of 46 patients (mean = 2.3-fold; range: 1.1-6.5-fold). Residual activity levels and responses to IFG were more consistent for the same genotype when measured in lymphoblasts than in macrophages, due to variability in macrophage viability between different patients. Results are shown in Table 8.

[0132] The response to IFG for patient-derived cells will be compared to the results obtained in the Cos7 cell line.

[0133] Example 3: In vivo effects of SPC on α-Gal A activity in skin, heart, kidney, and plasma To determine whether increased mutant α-Gal A levels manifest as increased in situ α-Gal A activity, the effect of DGJ administration on tissue GL-3 levels was investigated in vivo in hR301Q α-Gal A Tg / KO mice.

[0134] Eight-week-old male hR301Q α-Gal A Tg / KO mice were treated daily (4 days on / 3 days off) with 300 mg of DGJ per kg body weight in their drinking water for 4 weeks. After dosing, lysates from skin, heart, kidney, and plasma were prepared by homogenizing approximately 50 mg of tissue in lysis buffer (see above). 20 μL of lysate was mixed with 50 μL of substrate (as detailed above). The reaction mixture was incubated for 1 hour at 37°C. 70 μL of stop solution was then 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 generated to convert fluorescence data to absolute α-Gal A activity expressed as nmol / mg protein / hr.

[0135] Tissue specimens were washed free of blood, weighed, and homogenized with a solvent system in a FastPrep® system. The homogenate was then extracted using solid-phase extraction on a C18 cartridge. The eluent was evaporated and reconstituted before injection onto the LC-MS / MS system. The 12 GL-3 isoforms were measured using positive ESI-MS / MS. LC separation was achieved on a 00829a Zorbax C18 column.

[0136] Daily and less frequent DGJ dosing resulted in significant reductions in GL-3 levels in the skin, heart, kidney, and plasma (Figure 9). A trend toward even greater reductions in GL-3 levels was observed 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.

[0137] Example 4: Identification of Pompe disease-causing mutations that are responsive to the pharmacological chaperone DNJ Pompe disease is caused by a deficiency in acid alpha-glucosidase (GAA) activity, which impairs lysosomal glycogen metabolism. The enzyme deficiency leads to lysosomal glycogen accumulation, resulting in progressive skeletal muscle weakness, cardiac decline, respiratory failure, and CNS dysfunction in the later stages of the disease. Genetic mutations within the GAA gene result in lower expression or generate mutant forms of the enzyme with altered stability and / or biological activity, ultimately leading to disease. Pharmacological chaperones are a promising new therapeutic approach for the treatment of genetic diseases.

[0138] To assess the DNJ effect on different GAA variants, in vitro diagnostic assays were set up using COS-7 and HEK-293 cells to identify DNJ-responsive mutations (Figs. 10, 12, and 14).

[0139] A site-directed mutagenesis approach was used to introduce specific mutations into the complementary DNA (cDNA) encoding wild-type human acid α-glucosidase (GAA). An initial wild-type GAA DNA construct was generated by subcloning the GAA coding region from cDNA clone 5739991 (Invitrogen) into the pcDNA6 / V5-HisA mammalian expression vector (Invitrogen). The resulting DNA construct (referred to as wild-type GAA DNA) was used as a DNA template for subsequent mutagenesis. These missense, small insertion, or deletion mutations are cited in the Erasmus database and are known to be associated with glycogen storage disorder type 2 (GSDII), also known as Pompe disease. Briefly, wild-type GAA DNA was PCR amplified using mutagenic primers to obtain plasmid DNA bearing the desired mutations. These mutations were confirmed by DNA sequencing prior to protein expression in cells.

[0140] COS-7 cells (derived from green monkey embryonic kidney cells) were cultured at approximately 1.4 × 10 cells per well in 3 ml of Dulbecco's modified essential medium (DMEM) containing 10% (v / v) fetal bovine serum. 5Cells were aseptically seeded into 12-well tissue culture plates at a density of 1 / 3000 cells per well and grown overnight at 37°C in a humidified 5% CO2 atmosphere. The next day, cells (typically 60-80% confluent) were transfected with 0.75 μg of each DNA construct via a lipid transfection reagent such as FUGENE HD (Roche) according to the manufacturer's instructions. Two wells were transfected with each DNA construct, with one well incubated with DNJ (typically 0 μM, 20 μM, 50 μM, or 100 μM), while an equal volume of PBS was added to the other well. Two additional wells were transfected with an empty vector (no GAA cDNA) and incubated with or without DNJ to serve as background controls for endogenous monkey GAA expression. Similarly, two additional wells were transfected with wild-type human GAA cDNA and incubated with or without DNJ to serve as positive controls. All specimens were incubated at 37°C in a humidified 5% CO atmosphere for approximately 48 hours.

[0141] After a 48-hour incubation period, the spent medium was removed, and the cells were washed with PBS and then incubated with 1–2 ml of fresh DMEM medium for 3 hours at 37°C in a humidified 5% CO2 atmosphere. The medium was then removed, and the cells were immediately washed with PBS and lysed in 200 μl 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 plates were then gently swirled on an orbital shaker at room temperature for 10 minutes to ensure complete cell lysis. The resulting cell lysates were transferred to clean 1.5 ml microcentrifuge tubes and spun at 20,000 x g for 10 minutes to pellet cell debris. Approximately 175 μl of each supernatant sample was then transferred to a new 1.5 ml microcentrifuge tube. This cell lysate was used for all subsequent assays, including GAA enzyme activity, total protein concentration determination and Western blotting.

[0142] Residual GAA enzyme activity was determined for each transiently expressed GAA using fluorescent 4-methylumbelliferyl-α-glucopyranoside (4-MU-α-glucose) substrate (Sigma). Briefly, 10 μl of each cell lysate was assayed (in triplicate) in 100 μl reactions in 96-well, clear-bottom, black plates using 3 mM A4-MU-α-glucose and 50 mM KOAc (pH 4.0). Transiently expressed wild-type GAA preparations were diluted 20-fold with lysis buffer to ensure the enzymatic reaction remained within the linear range of the instrument. The enzymatic reaction was carried out for 1 h at 37°C and terminated by the addition of 50 μl of 500 mM Na2CO3 (pH 10.5). The assay was then read in a fluorescence plate reader (using excitation at 355 nm / emission at 460 nm) to quantify the amount of GAA-dependent 4-MU fluorescence released. GAA enzyme activity was then extrapolated from a free 4-MU standard curve after subtracting background fluorescence (i.e., empty vector control).

[0143] Twenty-five microliters of each cell lysate was used in parallel assays to determine total cellular protein concentration using the bicinchoninic acid (BCA) protein assay (Pierce) according to the manufacturer's protocol, and total cellular protein concentration was extrapolated from a bovine serum albumin (BSA) standard curve.

[0144] GAA enzyme activity for each sample was normalized to total cellular protein concentration and expressed as 4-MU nmoles released per mg of total protein per hour to define specific GAA activity. The resulting specific GAA activity after DNJ treatment was compared with the GAA enzyme activity of the corresponding untreated sample to determine whether specific GAA mutants responded to DNJ.

[0145] For the GAA mutation, transfected single HEK-293 cell line P545L, DNJEC 50 was also determined in the same way (Figure 14).

[0146] To determine the correlation of the DNJ response measured in COS-7 cells to patient-derived cells, the DNJ-active response was measured in vitro in macrophages and lymphoblasts from the patient as well.

[0147] Fibroblast and lymphoid cell lines derived from Pompe patients were also generated as previously described (see U.S. Patent Application No. 11 / 794,512). Fibroblast cell lines were derived from patients homozygous for the P545L or R854X GAA mutation (FIG. 13). Lymphoid cell lines were derived from patients heterozygous for the (IVS1AS, T>G, -13) GAA splicing defect and GAA frameshift mutation (FIG. 15).

[0148] GAA activity was measured in lymphocyte cell lines after incubation in 0 μM, 30 μM, 100 μM, or 300 μM DNJ (FIG. 15). GAA activity was also measured in fibroblast cell lines after DNJ incubation (FIG. 13).

[0149] In this study, the pharmacological chaperone 1-deoxygalactonojirimycin-HCl (DNJ) was shown to bind to and enhance the activity of mutant GAA. DNJ significantly increased GAA levels in fibroblasts (Fig. 13) and lymphocytes (Fig. 15) from Pompe disease patients, as well as in transiently transfected COS-7 (Figs. 10 and 12) or HEK-293 (Fig. 14) cells expressing some GAA missense mutations.

[0150] DNJ increased GAA activity for 26 of the 131 mutants tested (data not shown) (Figure 10). In addition to increasing the activity of these mutant GAA, DNJ also promoted the processing of GAA to the 95 / 76 / 70 kDa forms.

[0151] Furthermore, a dose-dependent increase in GAA activity was observed in lymphocytes from a patient containing the common IVS1AS, T>G, −13 splicing in one allele and a frameshift mutation in the second allele.

[0152] 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 accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0153] Patents, patent applications, publications, product literature, GenBank accession numbers, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0154] [Sequence table] SEQUENCE LISTING <110> AMICUS THERAPEUTICS, INC. BENJAMIN, ELFRIDA DO, HUNG WU, XIAOYANG FLANAGAN, JOHN WUSTMAN, BRANDON <120> METHOD TO PREDICT RESPONSE TO PHARMACOLOGICAL CHAPERONE TREATMENT OF DISEASES <130> 077376.0520 <140> PCT / US09 / 033963 <141> 2009-02-12 <150> US 61 / 028,141 <151> 2008-02-12 <150> US 61 / 035,684 <151> 2008-03-11 <150> US 61 / 093,631 <151> 2008-09-02 <150> US 61 / 113,496 <151> 2008-11-11 <160> 566 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 398 <212> PRT <213> Homo sapiens <400> 1 Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp 1 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 Gln 65 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 Leu 145 150 155 160 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 Gly 225 230 235 240 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 Val 305 310 315 320 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 Leu 385 390 395 <210> 2 <211> 1312 <212> DNA <213> Homo sapiens <400> 2 gaattctccg gtcaccgtga caatgcagct gaggaccca gaactacatc tgggctgcgc gcttgcgctt cgcttcctgg ccctcgtttc ctgggacatc cctggggcta gagcactgga 120 caatggattg gcaaggacgc ctaccatggg ctggctgcac tgggagcgct tcatgtgcaa ccttgactgc caggaagagc cagattcctg catcagtgag aagctcttca tggagatggc agagctcatg gtctcagaag gctggaagga tgcaggttat gagtacctct gcattgatga ctgttggatg gctccccaaa gagattcaga aggcagactt caggcagacc ctcagcgctt tcctcatggg attcgccagc tagctaatta tgttcacagc aaaggactga agctagggat 420 ttatgcagat gttggaaata aaacctgcgc aggcttccct gggagttttg gatactacga cattgatgcc cagacctttg ctgactgggg agtagtctg ctaaaatttg atggttgtta 540 ctgtgacagt ttggaaaatt tggcagatgg ttataagcac atgtccttgg ccctgaatag gactggcaga agcattgtgt actcctgtga gtggcctctt tatatgtggc cctttcaaaa 660 gcccaattat acagaaatcc gacagtactg caatcactgg cgaaattttg ctgacattga 720 tgattcctgg aaaagtataa agagtatctt ggactggaca tcttttaacc aggagagaat 780 tgttgatgtt gctggaccag ggggttggaa tgacccagat atgttagtga ttggcaactt 840 tggcctcagc tggaatcagc aagtaactca gatggccctc tgggctatca tggctgctcc 900 tttattcatg tctaatgacc tccgacacat cagccctcaa gccaaagctc tccttcagga 960 taaggacgta attgccatca atcaggaccc cttgggcaag caagggtacc agcttagaca 1020 gggagacaac tttgaagtgt gggaacgacc tctctcaggc ttagcctggg ctgtagctat 1080 gataaaccgg caggagattg gtggacctcg ctcttatacc atcgcagttg cttccctggg 1140 taaaggagtg gcctgtaatc ctgcctgctt catcacacag ctcctccctg tgaaaaggaa 1200 gctagggttc tatgaatgga cttcaaggtt aagaagtcac ataaatccca caggcactgt 1260 tttgcttcag ctagaaaata caatgcagat gtcattaaaa gacttacttt aa 1312 <210> 3 <211> 3624 <212> DNA <213> Homo sapiens <400> 3 cagttgggaa agctgaggtt gtcgccgggg ccgcgggtgg aggtcgggga tgaggcagca 60 ggtaggacag tgacctcggt gacgcgaagg accccggcca cctctaggtt ctcctcgtcc 120 gcccgttgtt cagcgaggga ggctctgggc ctgccgcagc tgacggggaa actgaggcac 180 ggagcgggcc tgtaggagct gtccaggcca tctccaacca tgggagtgag gcacccgccc 240 tgctcccacc ggctcctggc cgtctgcgcc ctcgtgtcct tggcaaccgc tgcactcctg 300 gggcacatcc tactccatga tttcctgctg gttccccgag agctgagtgg ctcctcccca 360 gtcctggagg agactcaccc agctcaccag cagggagcca gcagaccagg gccccgggat 420 gcccaggcac accccggccg tcccagagca gtgcccacac agtgcgacgt cccccccaac 480 agccgcttcg attgcgcccc tgacaaggcc atcacccagg aacagtgcga ggcccgcggc 540 tgctgctaca tccctgcaaa gcaggggctg cagggagccc agatggggca gccctggtgc 600 ttcttcccac ccagctaccc cagctacaag ctggagaacc tgagctcctc tgaaatgggc 660 tacacggcca ccctgacccg taccaccccc accttcttcc ccaaggacat cctgaccctg 720 cggctggacg tgatgatgga gactgagaac cgcctccact tcacgatcaa agatccagct 780 aacaggcgct acgaggtgcc cttggagacc ccgcgtgtcc acagccgggc accgtcccca 840 ctctacagcg tggagttctc cgaggagccc ttcggggtga tcgtgcaccg gcagctggac 900 ggccgcgtgc tgctgaacac gacggtggcg cccctgttct ttgcggacca gttccttcag 960 ctgtccacct cgctgccctc gcagtatatc acaggcctcg ccgagcacct cagtcccctg 1020 atgctcagca ccagctggac caggatcacc ctgtggaacc gggaccttgc gcccacgccc 1080 ggtgcgaacc tctacgggtc tcaccctttc tacctggcgc tggaggacgg cgggtcggca 1140 cacggggtgt tcctgctaaa cagcaatgcc atggatgtgg tcctgcagcc gagccctgcc 1200 cttagctgga ggtcgacagg tgggatcctg gatgtctaca tcttcctggg cccagagccc 1260 aagagcgtgg tgcagcagta cctggacgtt gtgggatacc cgttcatgcc gccatactgg 1320 ggcctgggct tccacctgtg ccgctggggc tactcctcca ccgctatcac ccgccaggtg 1380 gtggagaaca tgaccagggc ccacttcccc ctggacgtcc aatggaacga cctggactac 1440 atggactccc ggagggactt cacgttcaac aaggatggct tccgggactt cccggccatg 1500 gtgcaggagc tgcaccaggg cggccggcgc tacatgatga tcgtggatcc tgccatcagc 1560 agctcgggcc ctgccgggag ctacaggccc tacgacgagg gtctgcggag gggggttttc 1620 atcaccaacg agaccggcca gccgctgatt gggaaggtat ggcccgggtc cactgccttc 1680 cccgacttca ccaaccccac agccctggcc tggtgggagg acatggtggc tgagttccat 1740 gaccaggtgc ccttcgacgg catgtggatt gacatgaacg agccttccaa cttcatcaga 1800 ggctctgagg acggctgccc caacaatgag ctggagaacc caccctacgt gcctggggtg 1860 gttgggggga ccctccaggc ggccaccatc tgtgcctcca gccaccagtt tctctccaca 1920 cactacaacc tgcacaacct ctacggcctg accgaagcca tcgcctccca cagggcgctg 1980 gtgaaggctc gggggacacg cccatttgtg atctcccgct cgacctttgc tggccacggc 2040 cgatacgccg gccactggac gggggacgtg tggagctcct gggagcagct cgcctcctcc 2100 gtgccagaaa tcctgcagtt taacctgctg ggggtgcctc tggtcggggc cgacgtctgc 2160 ggcttcctgg gcaacacctc agaggagctg tgtgtgcgct ggacccagct gggggccttc 2220 taccccttca tgcggaacca caacagcctg ctcagtctgc cccaggagcc gtacagcttc 2280 agcgagccgg cccagcaggc catgaggaag gccctcaccc tgcgctacgc actcctcccc 2340 cacctctaca cactgttcca ccaggcccac gtcgcggggg agaccgtggc ccggcccctc 2400 ttcctggagt tccccaagga ctctagcacc tggactgtgg accaccagct cctgtggggg 2460 gaggccctgc tcatcacccc agtgctccag gccgggaagg ccgaagtgac tggctacttc 2520 cccttgggca catggtacga cctgcagacg gtgccaatag aggcccttgg cagcctccca 2580 cccccacctg cagctccccg tgagccagcc atccacagcg aggggcagtg ggtgacgctg 2640 ccggcccccc tggacaccat caacgtccac ctccgggctg ggtacatcat ccccctgcag 2700 ggccctggcc tcacaaccac agagtcccgc cagcagccca tggccctggc tgtggccctg 2760 accaagggtg gagaggcccg aggggagctg ttctgggacg atggagagag cctggaagtg 2820 ctggagcgag gggcctacac acaggtcatc ttcctggcca ggaataacac gatcgtgaat 2880 gagctggtac gtgtgaccag tgagggagct ggcctgcagc tgcagaaggt gactgtcctg 2940 ggcgtggcca cggcgcccca gcaggtcctc tccaacggtg tccctgtctc caacttcacc 3000 tacagccccg acaccaaggt cctggacatc tgtgtctcgc tgttgatggg agagcagttt 3060 ctcgtcagct ggtgttagcc gggcggagtg tgttagtctc tccagaggga ggctggttcc 3120 ccagggaagc agagcctgtg tgcgggcagc agctgtgtgc gggcctgggg gttgcatgtg 3180 tcacctggag ctgggcacta accattccaa gccgccgcat cgcttgtttc cacctcctgg 3240 gccggggctc tggcccccaa cgtgtctagg agagctttct ccctagatcg cactgtgggc 3300 cggggcctgg agggctgctc tgtgttaata agattgtaag gtttgccctc ctcacctgtt 3360 gccggcatgc gggtagtatt agccaccccc ctccatctgt tcccagcacc ggagaagggg 3420 gtgctcaggt ggaggtgtgg ggtatgcacc tgagctcctg cttcgcgcct gctgctctgc 3480 cccaacgcga ccgcttcccg gctgcccaga gggctggatg cctgccggtc cccgagcaag 3540 cctgggaact caggaaaatt cacaggactt gggagattct aaatcttaag tgcaattatt 3600 ttataaag gggcattgg atc 3624 <210> 4 <211> 952 <212> PRT <213> Homo sapiens <400> 4 Met Gly Val Arg His Pro Pro Cys Ser His Arg Leu Leu Ala Val Cys 1 5 10 15 Leu Wing Val Ser Leu Wing Thr Wing 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 Thr 65 70 75 80 Gln Cys Asp Val Pro Asn Ser Arg Phe Asp Cys Ala Pro Asp Lys 85 90 95 Only Thr Gln Glue Gln Cys Glue Only 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 Phe 145 150 155 160 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 Phe 225 230 235 240 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 Val 305 310 315 320 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 Val 385 390 395 400 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 Val 465 470 475 480 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 Ser 545 550 555 560 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 Pro 625 630 635 640 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 Gly 705 710 715 720 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 Ser 785 790 795 800 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 Ala 865 870 875 880 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 Cys 945 950 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 5 cgtgacaata cagctgag 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 6 ctcagctgta ttgtcacg 18 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 7 caccgtgaca acgcagctga gg 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 8 cctcagctgc gttgtcacgg tg 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 9 ggctgcgcgc ctgcgcttcg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 10 cgaagcgcag gcgcgcagcc 20 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 11 gcgcttgcgc mtcgcttcct gg 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 12 ccaggaagcg akgcgcaagc gc 22 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 13 gcttcgcttc ccggccctcg tttc 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 14 gaaacgaggg ccgggaagcg aagc 24 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 15 ggggctagag tactggacaa tgg 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 16 ccattgtcca gtactctagc ccc 23 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 17 gctagagcac cggacaatgg a 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 18 tccattgtcc ggtgctctag c 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 19 gctagagcac cggacaatgg a 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 20 tccattgtcc ggtgctctag c 21 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 21 ctagagcact gtacaatgga ttg 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 22 caatccattg tacagtgctc tag 23 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 23 gcactggaca aaggattggc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 24 gccaatcctt tgtccagtgc 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 25 gcactggaca gtggattggc 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 26 gccaatccac tgtccagtgc 20 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 27 ctggacaata gattggcaag g 21 <210> 28 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 28 gtcagcaaaa ttttgccagt gattgc 26 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 29 gcgaaatttt actgacattg atg 23 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 30 catcaatgtc agtaaaattt cgc 23 <210> 31 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 31 ggcgaaattt tgctaacatt gatgattcct g 31 <210> 32 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 32 caggaatcat caatgttagc aaaatttcgc c 31 <210> 33 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 33 cgaaattttg ctggcattga tgatattc 28 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 34 gaatatcatc aatgccagca aaatttcg 28 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 35 tgacattgat gagtcctgga aaag 24 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 36 cttttccagg actcatcaat gtca 24 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 37 gctgacattg attattcctg gaaaag 26 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 38 cttttccagg aataatcaat gtcagc 26 <210> 39 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 39 ctgacattga tgattgctgg aaaagtataa agag 34 <210> 40 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 40 ctctttatac ttttccagca atcatcaatg tcag 34 <210> 41 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 41 gctgacattg atgattcccg gaaaagtata aagagtatc 39 <210> 42 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 42 gatactcttt atacttttcc gggaatcatc aatgtcagc 39 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 43 tgatgattcc ttgaaaagta taa 23 <210> 44 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 44 ttatactttt caaggaatca tca 23 <210> 45 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 45 ctgacattga tgattccttg aaaagtataa agag 34 <210> 46 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 46 ctctttatac ttttcaagga atcatcaatg tcag 34 <210> 47 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 47 gctgacattg atgattcctg taaaagtata aagagtatct tgg 43 <210> 48 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 48 ccaagatact ctttatactt ttacaggaat catcaatgtc agc 43 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 49 tggaaaagta caaagagtat c 21 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 50 gatactcttt gtacttttcc a 21 <210> 51 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 51 ccttgccaat ctattgtcca g 21 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 52 aatggattgg taaggacgcc 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 53 ggcgtcctta ccaatccatt 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 54 gcaaggacgc ttaccatggg 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 55 cccatggtaa gcgtccttgc 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 56 gcaaggacgt ctaccatggg 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 57 cccatggtag acgtccttgc 20 <210> 58 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 58 aggacgccta ccacgggctg gctgcac 27 <210> 59 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 59 gtgcagccag cccgtggtag gcgtcct 27 <210> 60 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 60 aggacgccta ccttgggctg gctgcac 27 <210> 61 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 61 gtgcagccag cccaaggtag gcgtcct 27 <210> 62 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 62 aggacgccta ccgtgggctg gctgc 25 <210> 63 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 63 gcagccagcc cacggtaggc gtcct 25 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 64 ctaccatggw ctggctgcac 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 65 gtgcagccag wccatggtag 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 66 ctaccatgcg ctggctgcac 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 67 gtgcagccag cgcatggtag 20 <210> 68 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 68 catgggctgt ctgcactgg 19 <210> 69 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 69 ccagtgcaga cagcccatg 19 <210> 70 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 70 atgggctggc ggcactggga g 21 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 71 ctcccagtgc cgccagccca t 21 <210> 72 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 72 ctggctgcgc tgggagc 17 <210> 73 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 73 gctcccagcg cagccag 17 <210> 74 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 74 ctggctgtac tgggagc 17 <210> 75 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 75 gctcccagta cagccag 17 <210> 76 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 76 gattcctgga aaagtacaaa gagtatcttg gactg 35 <210> 77 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 77 cagtccaaga tactctttgt acttttccag gaatc 35 <210> 78 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 78 agtataaaga gtttcttgga ctggac 26 <210> 79 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 79 gtccagtcca agaaactctt tatact 26 <210> 80 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 80 gtataaagag taacttggac tgg 23 <210> 81 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 81 ccagtccaag ttactcttta tac 23 <210> 82 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 82 agagtatctt cgactggaca tc 22 <210> 83 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 83 gatgtccagt cgaagatact ct 22 <210> 84 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 84 gagtatcttg cactggacat c 21 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 85 gatgtccagt cgaagatact ct 22 <210> 86 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 86 cttggactgg acatgtttta accaggagag 30 <210> 87 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 87 ctctcctggt taaaacatgt ccagtccaag 30 <210> 88 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 88 ggactggaca ccttttaacc a 21 <210> 89 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 89 tggttaaaag gtgtccagtc c 21 <210> 90 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 90 gttgatgttc ctggaccag 19 <210> 91 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 91 ctggtccagg aacatcaac 19 <210> 92 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 92 gatgttgctc gaccagggg 19 <210> 93 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 93 cccctggtcg agcaacatc 19 <210> 94 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 94 gatgttgctg gacgaggggg ttgga 25 <210> 95 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 95 tccaaccccc tcgtccagca acatc 25 <210> 96 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 96 gttgctggac tagggggttg g 21 <210> 97 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 97 ccaaccccct agtccagcaa c 21 <210> 98 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 98 gctggaccag cgggttggaa tg 22 <210> 99 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 99 cattccaacc cgctggtcca gc 22 <210> 100 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 100 ggaccagggg attggaatga c 21 <210> 101 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 101 ctggctgcac ggggagcgct tc 22 <210> 102 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 102 gaagcgctcc ccgtgcagcc ag 22 <210> 103 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 103 ctggctgcac ttggagcgct tc 22 <210> 104 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 104 gaagcgctcc aagtgcagcc ag 22 <210> 105 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 105 gctgcactgg aagcgcttca tg 22 <210> 106 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 106 catgaagcgc ttccagtgca gc 22 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 107 actgggagcy cttcatgtgc 20 <210> 108 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 108 gcacatgaag rgctcccagt 20 <210> 109 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 109 cactgggagr gcttcatgt 19 <210> 110 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 110 acatgaagcy ctcccagtg 19 <210> 111 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 111 ctgggagcgc tgcatgtgca ac 22 <210> 112 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 112 gttgcacatg cagcgctccc ag 22 <210> 113 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 113 gagcgcttca agtgcaacct tg 22 <210> 114 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 114 caaggttgca cttgaagcgc tc 22 <210> 115 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 115 gcgcttcata tgcaacc 17 <210> 116 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 116 ggttgcatat gaagcgc 17 <210> 117 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 117 gagcgcttca tgtccaacct tgactg 26 <210> 118 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 118 cagtcaaggt tggacatgaa gcgctc 26 <210> 119 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 119 cgcttcatgs gcaaccttga c 21 <210> 120 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 120 gtcaaggttg cscatgaagc g 21 <210> 121 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 121 caaccttgac ggccaggaag 20 <210> 122 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 122 cttcctggcc gtcaaggttg 20 <210> 123 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 123 caaccttgac twccaggaag ag 22 <210> 124 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 124 ctcttcctgg wagtcaaggt tg 22 <210> 125 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 125 gtgcaacctt gactaccagg aagagccag 29 <210> 126 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 126 gtcattccaa tcccctggtc c 21 <210> 127 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 127 cagggggttg caatgaccca g 21 <210> 128 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 128 ctgggtcatt gcaaccccct g 21 <210> 129 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 129 ggggttggag tgacccaga 19 <210> 130 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 130 tctgggtcac tccaacccc 19 <210> 131 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 131 ggttggaatg tcccagatat g 21 <210> 132 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 132 catatctggg acattccaac c 21 <210> 133 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 133 gggttggaat tacccagata tg 22 <210> 134 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 134 catatctggg taattccaac cc 22 <210> 135 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 135 tggaatgacc gagatatgtt a 21 <210> 136 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 136 taacatatct cggtcattcc a 21 <210> 137 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 137 ttggaatgac ctagatatgt tag 23 <210> 138 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 138 ctaacatatc taggtcattc caa 23 <210> 139 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 139 ggaatgaccc amatatgtta gtg 23 <210> 140 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 140 cactaacata ttgggtcatt cc 22 <210> 141 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 141 ggttggaatg acccacatat gttagtgatt gg 32 <210> 142 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 142 ccaatcacta acatatgtgg gtcattccaa cc 32 <210> 143 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 143 gaatgaccca gttatgttag tg 22 <210> 144 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 144 cactaacata actgggtcat tc 22 <210> 145 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 145 atgacccaga aatgttagtg a 21 <210> 146 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 146 tcactaacat ttctgggtca t 21 <210> 147 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 147 gacccagata ggttagtgat tg 22 <210> 148 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 148 caatcactaa cctatctggg tc 22 <210> 149 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 149 gacccagata tattagtgat tgg 23 <210> 150 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 150 ccaatcacta atatatctgg gtc 23 <210> 151 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 151 ctggctcttc ctggtagtca aggttgcac 29 <210> 152 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 152 gagccagatt cctacatcag tgagaagc 28 <210> 153 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 153 gcttctcact gatgtaggaa tctggctc 28 <210> 154 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 154 tcctgcatca ctgagaagct c 21 <210> 155 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 155 gagcttctca gtgatgcagg a 21 <210> 156 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 156 ctgcatcagt aagaagctct tc 22 <210> 157 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 157 gaagagcttc ttactgatgc ag 22 <210> 158 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 158 ctgcatcagt gggaagctct tc 22 <210> 159 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 159 gaagagcttc ccactgatgc ag 22 <210> 160 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 160 cagtgagaag ttcttcatgg 20 <210> 161 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 161 ccaggaagaa cttctcactg 20 <210> 162 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 162 gcatcagtga gaagttcttc atggagatg 29 <210> 163 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 163 catctccatg aagaacttct cactgatgc 29 <210> 164 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 164 cttcatggag agggcagagc tc 22 <210> 165 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 165 gagctctgcc ctctccatga ag 22 <210> 166 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 166 cttcatggag atagcagagc tc 22 <210> 167 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 167 gagctctgct atctccatga ag 22 <210> 168 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 168 catggagatg gtagagctca tg 22 <210> 169 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 169 catgagctct accatctcca tg 22 <210> 170 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 170 gcagagctca gggtctcaga ag 22 <210> 171 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 171 cttctgagac cctgagctct gc 22 <210> 172 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 172 ctcagaaggc tgtaaggatg caggt 25 <210> 173 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 173 acctgcatcc ttacagcctt ctgag 25 <210> 174 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 174 ctcagaaggc tcgaaggatg ca 22 <210> 175 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 175 tgcatccttc gagccttctg ag 22 <210> 176 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 176 ccagatatgt cagtgattgg c 21 <210> 177 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 177 gccaatcact gacatatctg g 21 <210> 178 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 178 gatatgttag cgattggcaa c 21 <210> 179 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 179 gttgccaatc gctaacatat c 21 <210> 180 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 180 cagatatgtt aatgattggc aac 23 <210> 181 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 181 gttgccaatc attaacatat ctg 23 <210> 182 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 182 tatgttagtg actggcaact ttg 23 <210> 183 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 183 caaagttgcc agtcactaac ata 23 <210> 184 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 184 ttagtgattg tcaactttg 19 <210> 185 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 185 caaagttgac aatcactaa 19 <210> 186 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 186 gttagtgatt tgcaactttg g 21 <210> 187 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 187 ccaaagttgc aaatcactaa c 21 <210> 188 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 188 tgttagtgat tagcaacttt ggc 23 <210> 189 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 189 gccaaagttg ctaatcacta aca 23 <210> 190 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 190 gtgattggca aatttggcct cag 23 <210> 191 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 191 ctgaggccaa atttgccaat cac 23 <210> 192 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 192 gtgattggca gctttggcct c 21 <210> 193 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 193 gaggccaaag ctgccaatca c 21 <210> 194 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 194 caactttggc ctcggctgga atcag 25 <210> 195 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 195 ctgattccag ccgaggccaa agttg 25 <210> 196 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 196 ctttggcctc aactggaatc agc 23 <210> 197 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 197 gctgattcca gttgaggcca aag 23 <210> 198 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 198 agctggaatc ggcaagtaac tc 22 <210> 199 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 199 gagttacttg ccgattccag ct 22 <210> 200 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 200 cagctggaat caccaagtaa ctc 23 <210> 201 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 201 gaaggatgca gattatgagt ac 22 <210> 202 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 202 gtactcataa tctgcatcct tc 22 <210> 203 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 203 ggatgcaggt tgtgagtacc tctgc 25 <210> 204 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 204 gcagaggtac tcacaacctg catcc 25 <210> 205 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 205 ggttatgagg acctctgcat tg 22 <210> 206 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 206 caatgcagag gtcctcataa cc 22 <210> 207 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 207 gttatgagta ccsctgcatt gatg 24 <210> 208 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 208 catcaatgca gsggtactca taac 24 <210> 209 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 209 cctctgcatt hatgactgtt g 21 <210> 210 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 210 caacagtcat daatgcagag g 21 <210> 211 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 211 tacctctgca ttaatgactg ttggatg 27 <210> 212 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 212 catccaacag tcattaatgc agaggta 27 <210> 213 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 213 tacctctgca ttcatgactg ttggatg 27 <210> 214 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 214 catccaacag tcatgaatgc agaggta 27 <210> 215 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 215 gagtacctct gcatttatga ctgttggatg gctc 34 <210> 216 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 216 gagccatcca acagtcataa atgcagaggt actc 34 <210> 217 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 217 ctgcattgat ggctgttgga tg 22 <210> 218 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 218 catccaacag ccatcaatgc ag 22 <210> 219 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 219 ctgcattgat gtctgttgga tg 22 <210> 220 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 220 catccaacag acatcaatgc ag 22 <210> 221 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 221 ctgcattgat aactgttgga tg 22 <210> 222 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 222 catccaacag ttatcaatgc ag 22 <210> 223 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 223 gcattgatga ctcttggatg gctc 24 <210> 224 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 224 gagccatcca agagtcatca atgc 24 <210> 225 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 225 gagttacttg gtgattccag ctg 23 <210> 226 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 226 cagctggaat aagcaagtaa c 21 <210> 227 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 227 gttacttgct tattccagct g 21 <210> 228 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 228 ggaatcagca tgtaactcag a 21 <210> 229 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 229 tctgagttac atgctgattc c 21 <210> 230 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 230 ctggaatcag aaagtaactc ag 22 <210> 231 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 231 ctgagttact ttctgattcc a 21 <210> 232 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 232 cagcaagtaa atcagatggc c 21 <210> 233 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 233 ggccatctga tttacttgct g 21 <210> 234 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 234 caagtaactc cgatggccct c 21 <210> 235 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 235 gagggccatc ggagttactt g 21 <210> 236 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 236 gtaactcaga cggccctctg 20 <210> 237 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 237 cagagggccg tctgagttac 20 <210> 238 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 238 taactcagat gcccctctgg gct 23 <210> 239 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 239 agcccagagg ggcatctgag tta 23 <210> 240 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 240 aactcagatg gacctctggg ct 22 <210> 241 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 241 agcccagagg tccatctgag tt 22 <210> 242 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 242 gatggccctc ggggctatca t 21 <210> 243 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 243 atgatagccc cgagggccat c 21 <210> 244 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 244 atggccctct gtgctatcat g 21 <210> 245 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 245 catgatagca cagagggcca t 21 <210> 246 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 246 gccctctggg atatcatggc tg 22 <210> 247 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 247 cagccatgat atcccagagg gc 22 <210> 248 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 248 gccctctggc ctatcatgg 19 <210> 249 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 249 gcattgatga ctattggatg gctc 24 <210> 250 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 250 gagccatcca atagtcatca atgc 24 <210> 251 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 251 gatgactgtt cgatggctcc c 21 <210> 252 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 252 gggagccatc gaacagtcat c 21 <210> 253 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 253 ctgttggatg cctccccaaa gag 23 <210> 254 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 254 ctctttgggg aggcatccaa cag 23 <210> 255 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 255 gctccccaaa magattcaga ag 22 <210> 256 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 256 cttctgaatc tktttgggga gc 22 <210> 257 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 257 ggctccccaa acagattcag aagg 24 <210> 258 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 258 ccttctgaat ctgtttgggg agcc 24 <210> 259 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 259 caaagagatt cacaaggcag acttc 25 <210> 260 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 260 gaagtctgcc ttgtgaatct ctttg 25 <210> 261 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 261 gcagaccctc agagctttcc tcatg 25 <210> 262 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 262 catgaggaaa gctctgaggg tctgc 25 <210> 263 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 263 cagaccctca gtgctttcct catg 24 <210> 264 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 264 catgaggaaa gcactgaggg tctg 24 <210> 265 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 265 ccctcagcgc tctcctcatg 20 <210> 266 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 266 catgaggaga gcgctgaggg 20 <210> 267 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 267 ctcatgggat ttgccagcta gc 22 <210> 268 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 268 gctagctggc aaatcccatg ag 22 <210> 269 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 269 gattcgccag ccagctaatt atg 23 <210> 270 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 270 cataattagc tggctggcga atc 23 <210> 271 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 271 tcgccagcta mctaattatg ttcacagc 28 <210> 272 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 272 gctgtgaaca taattagkta gctggcga 28 <210> 273 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 273 gctagctaat tatgatcaca gcaaaggac 29 <210> 274 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 274 ccatgatagg ccagagggc 19 <210> 275 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 275 ctctgggctt tcatggctgc tcctt 25 <210> 276 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 276 aaggagcagc catgaaagcc cagag 25 <210> 277 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 277 gggctatcat cgctgctcct t 21 <210> 278 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 278 aaggagcagc gatgatagcc c 21 <210> 279 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 279 ctatcatggc tcctccttta ttc 23 <210> 280 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 280 gaataaagga ggagccatga tag 23 <210> 281 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 281 catggctgct gctttattca tg 22 <210> 282 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 282 catgaataaa gcagcagcca tg 22 <210> 283 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 283 catggctgct wctttattca tg 22 <210> 284 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 284 catgaataaa gwagcagcca tg 22 <210> 285 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 285 gctgctcctt tatgcatgtc taatgacc 28 <210> 286 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 286 ggtcattaga catgcataaa ggagcagc 28 <210> 287 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 287 ctttattcat gtktaatgac ctccg 25 <210> 288 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 288 cggaggtcat tamacatgaa taaag 25 <210> 289 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 289 attcatgtct agtgacctcc gac 23 <210> 290 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 290 gtcggaggtc actagacatg aat 23 <210> 291 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 291 tattcatgtc taaggacctc cgac 24 <210> 292 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 292 gtcggaggtc cttagacatg aata 24 <210> 293 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 293 tattcatgtc tcatgacctc cgac 24 <210> 294 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 294 gtcggaggtc atgagacatg aata 24 <210> 295 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 295 tcatgtctaa tggcctccga cacatc 26 <210> 296 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 296 gatgtgtcgg aggccattag acatga 26 <210> 297 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 297 gtctaatgac ccccgacaca tcag 24 <210> 298 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 298 ctgatgtgtc gggggtcatt agac 24 <210> 299 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 299 gtcctttgct gtgatcataa ttagctagc 29 <210> 300 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 300 cacagcaaag aactgaagct ag 22 <210> 301 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 301 ctagcttcag ttctttgctg tg 22 <210> 302 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 302 cagcaaagga ccgaagctag g 21 <210> 303 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 303 atccctagct tcggtccttt gctg 24 <210> 304 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 304 aggactgaag ccagggattt atgc 24 <210> 305 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 305 gcataaatcc ctggcttcag tcct 24 <210> 306 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 306 ggactgaagc tagagattta tgcagatg 28 <210> 307 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 307 catctgcata aatctctagc ttcagtcc 28 <210> 308 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 308 gactgaagct aaggatttat gcagatg 27 <210> 309 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 309 catctgcata aatccttagc ttcagtc 27 <210> 310 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 310 gctagggatt tctgcagatg ttgg 24 <210> 311 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 311 ccaacatctg cagaaatccc tagc 24 <210> 312 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 312 gctagggatt tatgtagatg ttgga 25 <210> 313 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 313 tccaacatct acataaatcc ctagc 25 <210> 314 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 314 ggatttatgc acatgttgga a 21 <210> 315 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 315 ttccaacatg tgcataaatc c 21 <210> 316 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 316 gggatttatg cacatgttgg aaataaaacc 30 <210> 317 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 317 ggttttattt ccaacatgtg cataaatccc 30 <210> 318 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 318 tgcagatgtt gaaaataaaa cctg 24 <210> 319 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 319 caggttttat tttcaacatc tgca 24 <210> 320 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 320 tgcagatgtt agaaataaaa cctg 24 <210> 321 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 321 caggttttat ttctaacatc tgca 24 <210> 322 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 322 ggatttatgc agatgttgaa aataaaacct gcgcagc 37 <210> 323 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 323 gctgcgcagg ttttattttc aacatctgca taaatcc 37 <210> 324 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 324 gtctaatgac ttccgacaca tc 22 <210> 325 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 325 gatgtgtcgg aagtcattag ac 22 <210> 326 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 326 gtctaatgac caccgacaca tc 22 <210> 327 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 327 gatgtgtcgg tggtcattag ac 22 <210> 328 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 328 ctaatgacct cggacacatc agc 23 <210> 329 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 329 gctgatgtgt ccgaggtcat tag 23 <210> 330 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 330 ctaatgacct cccacacatc agc 23 <210> 331 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 331 gctgatgtgt gggaggtcat tag 23 <210> 332 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 332 ctccgacaca acagccctca agc 23 <210> 333 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 333 gcttgagggc tgttgtgtcg gag 23 <210> 334 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 334 gccaaagctt tccttcagga 20 <210> 335 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 335 tcctgaagga aagctttggc 20 <210> 336 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 336 gctctccttc acgataagga cg 22 <210> 337 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 337 cgtccttatc gtgaaggaga gc 22 <210> 338 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 338 ctctccttca gtataaggac g 21 <210> 339 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 339 cgtccttata ctgaaggaga g 21 <210> 340 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 340 gataaggacg aaattgccat c 21 <210> 341 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 341 gatggcaatt tcgtccttat c 21 <210> 342 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 342 aaggacgtaa mtgccatcaa tc 22 <210> 343 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 343 gattgatggc akttacgtcc tt 22 <210> 344 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 344 aattgccatc attcaggacc cc 22 <210> 345 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 345 ggggtcctga atgatggcaa tt 22 <210> 346 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 346 aattgccatc aagcaggacc cc 22 <210> 347 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 347 ggggtcctgc ttgatggcaa tt 22 <210> 348 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 348 aattgccatc tatcaggacc cc 22 <210> 349 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 349 ggatttatgc agatgttcga aataaaacct gcgcagc 37 <210> 350 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 350 ggatttatgc agatgttcga aataaaacct gcgcagc 37 <210> 351 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 351 ggaaataaaa tctgcgcagg ct 22 <210> 352 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 352 agcctgcgca gattttattt cc 22 <210> 353 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 353 ggaaataaaa cccgcgcagg cttc 24 <210> 354 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 354 gaagcctgcg cgggttttat ttcc 24 <210> 355 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 355 gaaataaaac ctrcgcaggc ttcc 24 <210> 356 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 356 ggaagcctgc gyaggtttta tttc 24 <210> 357 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 357 ggaaataaaa cctgggcagg cttccctg 28 <210> 358 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 358 cagggaagcc tgcccaggtt ttatttcc 28 <210> 359 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 359 gaaataaaac ctgcacaggc ttccc 25 <210> 360 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 360 gggaagcctg tgcaggtttt atttc 25 <210> 361 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 361 ataaaacctg cccaggcttc cc 22 <210> 362 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 362 gggaagcctg ggcaggtttt at 22 <210> 363 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 363 cctgcgcagt cttccctgg 19 <210> 364 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 364 ccagggaaga ctgcgcagg 19 <210> 365 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 365 ggcttcccta ggagttttgg 20 <210> 366 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 366 ccaaaactcc tagggaagcc 20 <210> 367 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 367 ttccctggga attttggata c 21 <210> 368 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 368 gtatccaaaa ttcccaggga a 21 <210> 369 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 369 ccctgggagg tttggatact 20 <210> 370 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 370 agtatccaaa cctcccaggg 20 <210> 371 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 371 gttttggata ctgcgacatt gatg 24 <210> 372 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 372 catcaatgtc gcagtatcca aaac 24 <210> 373 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 373 ggggtcctga tagatggcaa tt 22 <210> 374 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 374 tgccatcaat gaggacccct tg 22 <210> 375 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 375 caaggggtcc tcattgatgg ca 22 <210> 376 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 376 tgccatcaat cgggacccct tg 22 <210> 377 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 377 caaggggtcc cgattgatgg ca 22 <210> 378 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 378 ggaccccttg gacaagcaag 20 <210> 379 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 379 cttgcttgtc caaggggtcc 20 <210> 380 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 380 cttgggcaag raagggtacc ag 22 <210> 381 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 381 ctggtaccct tycttgccca ag 22 <210> 382 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 382 ggcaagcaaa ggtaccagc 19 <210> 383 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 383 gctggtacct ttgcttgcc 19 <210> 384 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 384 ggcaagcaag ygtaccagc 19 <210> 385 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 385 gctggtacrc ttgcttgcc 19 <210> 386 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 386 tgggcaagca agagtaccag cttag 25 <210> 387 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 387 ctaagctggt actcttgctt gccca 25 <210> 388 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 388 gagacaactt taaagtgtgg g 21 <210> 389 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 389 cccacacttt aaagttgtct c 21 <210> 390 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 390 ctttgaagtg cgggaacgac 20 <210> 391 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 391 gtcgttcccg cacttcaaag 20 <210> 392 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 392 gaagtgtggg accgacctct ctc 23 <210> 393 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 393 gagagaggtc ggtcccacac ttc 23 <210> 394 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 394 gaagtgtgga aacgacctct ctc 23 <210> 395 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 395 gagagaggtc gtttccacac ttc 23 <210> 396 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 396 gtgtgggaac aacctctctc ag 22 <210> 397 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 397 ctacgacatt catgcccaga c 21 <210> 398 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 398 gtctgggcat gaatgtcgta g 21 <210> 399 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 399 gacattgata cccagacctt tg 22 <210> 400 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 400 caaaggtctg ggtatcaatg tc 22 <210> 401 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 401 ctttgctgac cggggagtag atc 23 <210> 402 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 402 gatctactcc ccggtcagca aag 23 <210> 403 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 403 ctttgctgac tgcggagtag atc 23 <210> 404 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 404 gatctactcc gcagtcagca aag 23 <210> 405 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 405 gctgactggg tagtagatct g 21 <210> 406 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 406 cagatctact acccagtcag c 21 <210> 407 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 407 ctggggagta gttctgctaa aatttg 26 <210> 408 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 408 caaattttag cagaactact ccccag 26 <210> 409 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 409 gagtagatct gccaaaattt gatgg 25 <210> 410 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 410 ccatcaaatt ttggcagatc tactc 25 <210> 411 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 411 gtagatctgc taagatttga tggtttg 27 <210> 412 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 412 caaaccatca aatcttagca gatctac 27 <210> 413 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 413 gtagatctgc taaaatctga tggttgttac tg 32 <210> 414 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 414 cagtaacaac catcagattt tagcagatct ac 32 <210> 415 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 415 gctaaaattt gttggttgtt actg 24 <210> 416 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 416 cagtaacaac caacaaattt tagc 24 <210> 417 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 417 gctaaaattt catggttgtt actg 24 <210> 418 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 418 cagtaacaac catgaaattt tagc 24 <210> 419 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 419 ctaaaatttg atgattgtta ctgtgac 27 <210> 420 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 420 gtcacagtaa caatcatcaa attttag 27 <210> 421 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 421 ctaaaatttg atcgttgtta ctgtgac 27 <210> 422 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 422 ctgagagagg ttgttcccac ac 22 <210> 423 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 423 gaacgacctc cctcaggctt ag 22 <210> 424 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 424 ctaagcctga gggaggtcgt tc 22 <210> 425 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 425 cgacctctcc caggcttagc c 21 <210> 426 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 426 ggctaagcct gggagaggtc g 21 <210> 427 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 427 ctcaggctta ccctgggctg tag 23 <210> 428 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 428 ctacagccca gggtaagcct gag 23 <210> 429 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 429 cttagcctgg cctgtagcta tg 22 <210> 430 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 430 catagctaca ggccaggcta ag 22 <210> 431 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 431 ctgggctgta gatatgataa ac 22 <210> 432 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 432 gtttatcata tctacagccc ag 22 <210> 433 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 433 gtagctatga aaaaccggca gg 22 <210> 434 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 434 cctgccggtt tttcatagct ac 22 <210> 435 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 435 gctatgataa aacggcagga g 21 <210> 436 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 436 ctcctgccgt tttatcatag c 21 <210> 437 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 437 gctatgataa actggcagga gatt 24 <210> 438 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 438 aatctcctgc cagtttatca tagc 24 <210> 439 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 439 accggcaggs gattggtgga c 21 <210> 440 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 440 gtccaccaat cscctgccgg t 21 <210> 441 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 441 gataaaccgg cagaagattg gtgg 24 <210> 442 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 442 ccaccaatct tctgccggtt tatc 24 <210> 443 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 443 gataaaccgg caggcgattg gtggacctc 29 <210> 444 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 444 gaggtccacc aatcgcctgc cggtttatc 29 <210> 445 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 445 ccggcaggag actggtggac ctc 23 <210> 446 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 446 gaggtccacc agtctcctgc cgg 23 <210> 447 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 447 gtcacagtaa caacgatcaa attttag 27 <210> 448 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 448 ctaaaatttg atggttwtta ctgtgacagt 30 <210> 449 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 449 actgtcacag taawaaccat caaattttag 30 <210> 450 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 450 ctaaaatttg atggttggta ctgtgacagt 30 <210> 451 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 451 actgtcacag taccaaccat caaattttag 30 <210> 452 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 452 ctaaaatttg atggtcgtta ctgtgacagt 30 <210> 453 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 453 actgtcacag taacgaccat caaattttag 30 <210> 454 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 454 ctaaaatttg atggtggtta ctgtgacagt 30 <210> 455 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 455 actgtcacag taaccaccat caaattttag 30 <210> 456 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 456 ttggcagatg attataagca c 21 <210> 457 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 457 gtgcttataa tcatctgcca a 21 <210> 458 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 458 ttggcagata gttataagca c 21 <210> 459 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 459 gtgcttataa ctatctgcca a 21 <210> 460 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 460 gttataagca cacgtccttg gccct 25 <210> 461 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 461 agggccaagg acgtgtgctt ataac 25 <210> 462 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 462 gttataagca cgtgtccttg gcc 23 <210> 463 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 463 ggccaaggac acgtgcttat aac 23 <210> 464 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 464 gtccttggcc cagaatagga ctg 23 <210> 465 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 465 cagtcctatt ctgggccaag gac 23 <210> 466 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 466 gtccttggcc ccgaatagga ctg 23 <210> 467 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 467 cagtcctatt cggggccaag gac 23 <210> 468 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 468 ctgaatagga ttggcagaag c 21 <210> 469 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 469 gcttctgcca atcctattca g 21 <210> 470 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 470 cagaagcatt atgtactcct g 21 <210> 471 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 471 caggagtaca taatgcttct g 21 <210> 472 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 472 caggagatta gtggacctcg c 21 <210> 473 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 473 gcgaggtcca ctaatctcct g 21 <210> 474 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 474 ggagattggt agacctcgct c 21 <210> 475 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 475 gagcgaggtc taccaatctc c 21 <210> 476 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 476 gattggtgga cttcgctctt atac 24 <210> 477 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 477 gtataagagc gaagtccacc aatc 24 <210> 478 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 478 ggtggacctc actcttatac 20 <210> 479 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 479 gtataagagt gaggtccacc 20 <210> 480 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 480 ggtggacctt gctcttatac 20 <210> 481 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 481 gtataagagc aaggtccacc 20 <210> 482 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 482 tgcttccctg cgtaaaggag tgg 23 <210> 483 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 483 ccactccttt acgcagggaa gca 23 <210> 484 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 484 gtaaaggagt ggactgtaat cctg 24 <210> 485 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 485 caggattaca gtccactcct ttac 24 <210> 486 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 486 aaaggagtgg cctataatcc tgcc 24 <210> 487 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 487 ggcaggatta taggccactc cttt 24 <210> 488 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 488 aaaggagtgg cccgtaatcc tgcc 24 <210> 489 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 489 ggcaggatta cgggccactc cttt 24 <210> 490 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 490 gtaatcctgc ctacttcatc acac 24 <210> 491 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 491 gtgtgatgaa gtaggcagga ttac 24 <210> 492 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 492 ctgcctgctt caacacacag ctcctc 26 <210> 493 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 493 gaggagctgt gtgttgaagc aggcag 26 <210> 494 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 494 cctgcttcat cccacagctc ctcc 24 <210> 495 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 495 ggaggagctg tgggatgaag cagg 24 <210> 496 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 496 cttcatcaca ccgctcctcc ctgt 24 <210> 497 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 497 cattgtgtac ttctgtgagt gg 22 <210> 498 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 498 ccactcacag aagtacacaa tg 22 <210> 499 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 499 cattgtgtac tactgtgagt ggc 23 <210> 500 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 500 gccactcaca gtagtacaca atg 23 <210> 501 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 501 gtgtactcct atgagtggcc tc 22 <210> 502 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 502 gaggccactc ataggagtac ac 22 <210> 503 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 503 gtgtactcct gggagtggcc tct 23 <210> 504 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 504 agaggccact cccaggagta cac 23 <210> 505 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 505 ctgtgagtgg actctttata tg 22 <210> 506 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 506 catataaaga gtccactcac ag 22 <210> 507 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 507 ctgtgagtgg cktctttata tg 22 <210> 508 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 508 catataaaga mgccactcac ag 22 <210> 509 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 509 tggcctcttt ctatgtggcc c 21 <210> 510 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 510 gggccacata gaaagaggcc a 21 <210> 511 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 511 agtggcctct ttgtatgtgg ccctt 25 <210> 512 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 512 aagggccaca tacaaagagg ccact 25 <210> 513 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 513 cctttcaaaa gcccaatgat acagaaatcc gacag 35 <210> 514 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 514 ctgtcggatt tctgtatcat tgggcttttg aaagg 35 <210> 515 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 515 caattataca gaaaaccgac agtactgc 28 <210> 516 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 516 gcagtactgt cggttttctg tataattg 28 <210> 517 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 517 cgacagtacs gcaatcactg g 21 <210> 518 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 518 ccagtgattg csgtactgtc g 21 <210> 519 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 519 cgacagtact acaatcactg g 21 <210> 520 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 520 acagggagga gcggtgtgat gaag 24 <210> 521 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 521 cacagctcct ccgtgtgaaa aggaagct 28 <210> 522 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 522 agcttccttt tcacacggag gagctgtg 28 <210> 523 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 523 ggaagctagg gtactatgaa tgg 23 <210> 524 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 524 ccattcatag taccctagct tcc 23 <210> 525 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 525 agggttctat aaatggactt ca 22 <210> 526 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 526 tgaagtccat ttatagaacc ct 22 <210> 527 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 527 gacttcaagg tcaagaagtc ac 22 <210> 528 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 528 gtgacttctt gaccttgaag tc 22 <210> 529 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 529 gaagtcacaa aaatcccaca g 21 <210> 530 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 530 ctgtgggatt tttgtgactt c 21 <210> 531 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 531 gtcacataaa tdccacaggc actg 24 <210> 532 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 532 cagtgcctgt gghatttatg tgac 24 <210> 533 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 533 cacataaatc ccaaaggcac tg 22 <210> 534 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 534 cagtgccttt gggatttatg tg 22 <210> 535 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 535 cacataaatc ccgcaggcac tg 22 <210> 536 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 536 cagtgcctgc gggatttatg tg 22 <210> 537 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 537 atcccacaga cactgttttg c 21 <210> 538 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 538 gcaaaacagt gtctgtggga t 21 <210> 539 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 539 ggcactgttt cgcttcagct ag 22 <210> 540 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 540 ctagctgaag cgaaacagtg cc 22 <210> 541 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 541 gcgcttcgct tcctggacat ccctggggc 29 <210> 542 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 542 gccccaggga tgtccaggaa gcgaagcgc 29 <210> 543 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 543 ccagtgattg tagtactgtc g 21 <210> 544 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 544 cagtactgca gtcactggcg a 21 <210> 545 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 545 tcgccagtga ctgcagtact g 21 <210> 546 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 546 cagtactgcg atcactggc 19 <210> 547 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 547 gccagtgatc gcagtactg 19 <210> 548 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 548 tactgcaatc gctggcgaaa t 21 <210> 549 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 549 atttcgccag cgattgcagt a 21 <210> 550 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 550 gcaatcaccg gcgaaat 17 <210> 551 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 551 atttcgccgg tgattgc 17 <210> 552 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 552 gcaatcactg tcgaaatttt gc 22 <210> 553 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 553 gcaaaatttc gacagtgatt gc 22 <210> 554 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 554 gcaatcactg gcaaaatttt gctgac 26 <210> 555 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 555 gcagaccctc agcgccagct agctaattat g 31 <210> 556 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 556 cataattagc tagctggcgc tgagggtctg c 31 <210> 557 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 557 gttttggata ctacattgat gcccag 26 <210> 558 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 558 ctgggcatca atgtagtatc caaaac 26 <210> 559 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 559 ctcctgtgag tggatgtggc cctt 24 <210> 560 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 560 aagggccaca tccactcaca ggag 24 <210> 561 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 561 caggagagaa ttgatgttgc tgg 23 <210> 562 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 562 ccagcaacat caattctctc ctg 23 <210> 563 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 563 gataaaccgg cagattggtg gacct 25 <210> 564 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 564 aggtccacca atctgccggt ttatc 25 <210> 565 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 565 gactggacat cttggacatc ttttaaccag gagag 35 <210> 566 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 566 ctctcctggt taaaagatgt ccaagatgtc cagtc 35

Claims

1. 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 that protein, comprising: a. contacting a first host cell with a pharmacological chaperone specific for the protein, wherein the first host cell expresses a mutant form of the protein; b. comparing the protein activity in the second host cell that has not been contacted with the specific pharmacological chaperone to the protein activity in the first host cell that has been 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 a second host cell not contacted with the specific pharmacological chaperone indicates that the patient will respond to treatment with the specific pharmacological chaperone.

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

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

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

7. α-galactosidase A is α-galactosidase A mutations 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, G 373S, H225R, I219N, I242N, I270T, I289F, I303N, I317T, I354K, I91T, L14P, L166V, L243F, L300F, L310F, L32P, L45R, M267I, M284T, M296I, M296V, M72V, M76R, N224S, N2 63S, N298K, N298S, N320I, N320Y, N34K, P205R, P259L, P265L, P265R, P293A, P293S, P409S, P40L, P40S, Q279E, Q279H, Q279R, Q280H, Q280K, Q312H, Q321E, Q321R, Q327E, 7. The method of claim 6, wherein the mutant α-galactosidase A is selected from the group consisting of R301P, R342Q, R363C, R363H, R49G, R49L, R49S, S201Y, S276N, S297C, S345P, T194I, V269M, V316E, W340R, W47L and W95S.

8. 8. The method of claim 7, wherein the mutant α-galactosidase A is selected from the group consisting of α-galactosidase A mutations G144V, H225R, S276G, R301P and N320I.

9. The method of any one of claims 1 to 8, wherein the specific pharmacological chaperone is 1-deoxygalactonojirimycin.

10. 6. The method of claim 5, wherein the lysosomal enzyme is α-glucosidase and the lysosomal storage disorder is Pompe disease.

11. 11. The method of claim 10, wherein the α-glucosidase is a mutant α-glucosidase selected from the group consisting of α-glucosidase mutations 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 ΔN470.

12. The method of claim 10 or 11, wherein the specific pharmacological chaperone is 1-deoxynojirimycin.

13. 6. The method of claim 5, wherein the lysosomal enzyme is glucocerebrosidase and the lysosomal storage disorder is Gaucher disease.

14. 14. The method of claim 13, wherein the specific pharmacological chaperone is isofagomine.

15. 2. The method of claim 1, wherein the host cells are selected from the group consisting of CHO cells, HeLa cells, HEK-293 cells, GripTite 293MSR cells, 293T cells, COS cells, COS-7 cells, mouse primary myoblasts, and NIH3T3 cells.

16. The method of claim 15, wherein the host cell is a HEK-293 cell.

17. The method of claim 15, wherein the host cell is a COS-7 cell.

18. 16. The method of claim 15, wherein protein activity is determined using a fluorescent assay that quantifies hydrolysis of a substrate in a lysate from the host cell.

19. 1. A method for generating a therapeutic regimen table indicating whether a specific pharmacological chaperone is an effective compound for increasing the activity of a mutant protein, comprising: a. contacting a first host cell with a protein-specific pharmacological chaperone, wherein the first host cell expresses a mutant protein; b. comparing the protein activity in a first host cell contacted with the specific pharmacological chaperone with the protein activity in a second host cell, the second host cell expressing the mutant protein and not contacted with the specific pharmacological chaperone; c. Recording the results of step (b) in a treatment regimen chart; wherein a specific pharmacological chaperone recorded in the therapeutic therapy table that increases the activity of the mutant protein in the first host cell relative 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 expressing the mutant protein.

20. 20. The method of claim 19, wherein the protein is an α-galactosidase A enzyme.

21. 21. The method of claim 20, wherein the specific pharmacological chaperone is 1-deoxygalactonojirimycin.

22. 20. The method of claim 19, wherein the protein is an α-glucosidase enzyme.

23. 23. The method of claim 22, wherein the specific pharmacological chaperone is 1-deoxynojirimycin.

24. 20. The method of claim 19, wherein the protein is a glucocerebrosidase enzyme.

25. 25. The method of claim 24, wherein the specific pharmacological chaperone is isofagomine.

26. 1. A method for selecting a specific pharmacological chaperone for treating a patient expressing a mutant form of a protein, comprising: - the specific pharmacological chaperone is selected from a therapeutic criteria table that includes a list of mutations in the protein and the effect of the specific pharmacological chaperone on the activity of the protein mutation; and - the specific pharmacological chaperone selected to treat the patient increases the activity of the mutant protein expressed by the patient; method.

27. 27. The method of claim 26, wherein the patient has been diagnosed with a lysosomal storage disorder.

28. 28. The method of any one of claims 26 or 27, wherein the protein is an α-galactosidase A enzyme.

29. 29. The method of claim 28, wherein the specific pharmacological chaperone is 1-deoxygalactonojirimycin.

30. 28. The method of claim 26 or 27, wherein the protein is an α-glucosidase enzyme.

31. 31. The method of claim 30, wherein the specific pharmacological chaperone is 1-deoxynojirimycin.

32. 28. The method of any one of claims 26 or 27, wherein the protein is a glucocerebrosidase enzyme.

33. 33. The method of claim 32, wherein the specific pharmacological chaperone is isofagomine.

34. 1. A method of treating a patient diagnosed with Fabry disease, comprising administering to the patient a therapeutically effective dose of 1-deoxygalactonojirimycin, wherein the patient has α-galactosidase A mutations A121T, A156V, A20P, A288D, A288P, A292P A348P, A73V, C52R, C94Y, D234E, D244H, D264Y, E338K, E341D, E398K, E48K, F113L, G144V, G260A, G271S, G328A, G35R, G373D, G373S, H 225R, I219N, I242N, I270T, I303N, I317T, I354K, L14P, L166V, L243F, L300F, L310F, L32P, M267I, M284T, M296I, M72V, M76R, N224S, N 298K, N298S, N320I, N34K, P259L, P265L, P265R, P293A, P293S, P409S, P40L, Q279H, Q279R, Q280H, Q280K, Q312H, Q321E, Q321R, Q327E, R301P, R363H, R49G, R49S, S201Y, S276N, S297C, S345P, T194I, V269M, W340R, W47L and W95S.

35. 1. A method of treating a patient diagnosed with Pompe disease, comprising administering to the patient a therapeutically effective dose of 1-deoxynojirimycin, wherein the patient expresses a mutant α-glucosidase selected from the group consisting of α-glucosidase mutations 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 ΔN470.

36. 1. A method for treating a patient expressing a mutant protein, the method comprising the steps of identifying the mutant protein, determining whether the patient will respond to a specific pharmacological chaperone using a treatment regimen, and administering the specific pharmacological chaperone to the patient if the patient is determined to respond to the specific pharmacological chaperone.

37. 37. The method of claim 36, wherein the mutant protein is a mutant lysosomal enzyme and the patient has been diagnosed with a lysosomal storage disorder.

38. 38. The method of claim 36 or 37, wherein the lysosomal enzyme is alpha-galactosidase A and the lysosomal storage disorder is Fabry disease.

39. 38. The method of claim 37, wherein the specific pharmacological chaperone is 1-deoxygalactonojirimycin.

40. 38. The method of claim 36 or 37, wherein the lysosomal enzyme is α-glucosidase and the lysosomal storage disorder is Pompe disease.

41. 41. The method of claim 40, wherein the specific pharmacological chaperone is 1-deoxynojirimycin.

42. 38. The method of claim 36 or 37, wherein the lysosomal enzyme is glucocerebrosidase and the lysosomal storage disorder is Gaucher disease.

43. 43. The method of claim 42, wherein the specific pharmacological chaperone is isofagomine.

44. The method of claim 34, wherein the treatment regimen table is generated according to the method of any one of claims 19 to 25.

Images