Optimized gene therapy targeting muscle in muscle disease

Abstract

The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAV), optimized for delivery of transgenes to muscle. The optimized vectors contain: a constitutive or muscle-specific promoter for delivery of whole-body or skeletal muscle / cardiac muscle-specific transgene expression, respectively, and in combination with the transgene cDNA, replaces the gene mutation found in muscle disease with a normal gene copy; an internal ribosome entry site (IRES) to allow production of a second protein from the same transcript; and a muscle growth factor for establishment of new muscle growth and strength. For example, the present disclosure provides gene therapy vectors, such as recombinant adeno-associated viruses (rAAV), designed for treatment of GNE myopathy, where the rAAV expresses UDP-GlcNAc-epimerase / ManNAc-6 alone or in combination with a muscle growth factor or muscle transdifferentiation factor. The AAVs provided replace the mutated GNE gene expression, while also expressing a protein that stimulates muscle growth.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 951,564, filed December 20, 2019, the entire contents of which are incorporated herein by reference.

[0002] Materials submitted electronically are incorporated by reference.

[0003] This application contains a computer-readable list of sequences that is a separate part of this disclosure, which is incorporated herein by reference in its entirety and identified as a 233,379-byte file named “54649_Sqlisting.txt” created on December 21, 2020. Technical Field

[0004] This disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), optimized for delivering transgenes to muscle. The optimized vector contains: a constitutive or muscle-specific promoter for delivering systemic or skeletal / cardiac muscle-specific transgene expression, respectively, and binding to the transgene cDNA to replace gene mutations found in muscle diseases with normal gene copies; an internal ribosome entry site (IRES) to allow the production of a second protein from the same transcript; and muscle growth factor for building new muscle growth and strength. The transgene and muscle growth factor genes are expressed by the same mRNA, which expresses two proteins due to the presence of the internal ribosome entry site (or IRES) in the fibroblast growth factor 1A gene sequence, allowing the second protein to be produced from a single mRNA. Background Technology

[0005] GNE myopathy is an adult-onset autosomal recessive genetic disorder characterized by progressive muscle weakness that can lead to loss of walking ability and independent living. As the name suggests, GNE myopathy is caused by a loss-of-function variant or mutation in the GNE gene. This disease is also known as hereditary inclusion body myopathy, quadriceps-preserving myopathy, distal myopathy with rimmed vacuoles, and Nonaka myopathy. The GNE gene encodes a bifunctional UDP-GlcNAc-epimerase / ManNAc-6 kinase, whose enzymatic activity is essential in the sialic acid biosynthesis pathway.

[0006] Sialic acid is an acidic monosaccharide that modifies the non-reducing terminal carbohydrate chains on glycoproteins and glycolipids, playing a crucial role in various processes such as cell adhesion and cell-cell interactions. Sialic acid is associated with health and disease and is present in the terminal glycan chains of proteins that regulate their cellular functions. UDP-N-acetylglucosamine 2-epimerase / N-acetylmnotose kinase (GNE) is a key enzyme in sialic acid biosynthesis. Furthermore, GNE expression has been shown to be induced when myofibrils are damaged or regenerated, and GNE plays a role in muscle regeneration. Myoblasts carrying the mutant GNE gene exhibit reduced epimerase activity, and therefore cells carrying homozygous epimerase mutations also show only a significant reduction in total membrane-bound sialic acid (Pogoryleva et al., *Orphanet J Rare Dis.* 13:70, 2018).

[0007] GNE myopathy causes weakness and wasting of the leg and arm muscles. Initial symptoms typically occur in young adults (usually in their third decade of life), but later onset has been observed in some patients. Diagnosis of GNE myopathy should primarily consider patients presenting with distal weakness (foot drop) in early adulthood (other symptoms may also be present). The disease progresses slowly to other lower and upper limb muscles, usually with significant preservation of the quadriceps. Characteristic findings in biopsies of affected muscles include "rimmed" (autophagic) vacuoles, aggregation of various proteins, and changes in fiber size.

[0008] Although mutations in the GNE gene were shown to cause GNE myopathy in 2001, there is currently no effective treatment for the disease. Attempts to develop a sustained-release sialic acid therapy failed in a phase 3 clinical trial, and a ManNAc glycan therapy is currently under investigation. While developing gene therapy for GNE gene replacement may seem straightforward, it is actually complicated by many unresolved issues in GNE myopathy research: First, there is a lack of robust and reproducible models of the disease. Although Noguchi and Nishino published several papers on the transgenic GNED176VTg Gne- / - mouse model, which showed clear aspects of the disease pathology, other populations have failed to show the same phenotype in subsequent breeding, likely as a result of genetic drift in the founder transgenic lines (see Nishino et al., J. Neurol. Neurosurg. Psychiatry 86(4):385-392). M712TThe knock-in mouse model showed premature death from kidney disease in the first few weeks of life, a clinical phenotype absent in patients with GNE myopathy. Other strains bred with the same gene mutation did not exhibit any phenotype at all. Secondly, measurable natural history data from rare and geographically diverse patient populations are lacking. Thirdly, due to the late progression of the disease, trials of GNE myopathy with gene replacement alone are unlikely to demonstrate clinical efficacy, only slowing or halting disease progression.

[0009] Cells lacking GNE activity can be rescued by the addition of sialic acid (SA) or by the addition of ManNAc, the latter of which can be converted from the non-mutated GlcNAc-6 kinase activity in the disease to the final product of GNE activity, ManNAc-6 phosphate. Several glycan therapies have been shown in GNED176VTgGne- / - mice and Gne... M712T The treatment showed efficacy in knock-in mice. This has led to two clinical trials, one using sustained-release SA (completed in Phase 3) (Lochmuller et al., Neurology 92(18):e2109-e17, 2019) and the other using ManNAc (completed in Phase 1) (Xu et al., Molecular Genetics and Metabolism, 12291-2:126-34, 2017). Although SA and ManNAc showed significant therapeutic effects in mice, sustained-release SA therapy (ACE-ER) did not reach clinical milestones in a Phase 3 clinical trial in patients with GNE myopathy

[16] . There were no significant changes compared to placebo for any clinical measure.

[0010] The lack of efficacy of glycan therapy in patients with GNE myopathy makes gene therapy a very attractive alternative. However, a major problem remains that human disease progresses slowly and variably, and robust short-term clinical milestones are lacking. For example, the current phase 3 clinical trial lasted 48 weeks

[16] . During this time, no intensity measure in the patient population showed a significant decrease compared to pre-treatment baseline, although some measures did show a downward trend.

[0011] The goal of the GNE treatment proposed in this article is to create a tandem gene therapy—utilizing muscle-specific IRES to create a bicistronic gene therapy vector expressing both the normal GNE gene and a known muscle growth factor. Such an AAV vector can both correct the genetic defects of GNE myopathy and increase muscle strength, thereby reversing rather than merely preventing the decline in clinical indicators of muscle strength. Therapies that build new muscle and muscle strength while simultaneously preventing further disease progression by adding the normal GNE gene will offer greater benefits to patients with GNE myopathy and provide a more easily demonstrable method for clinical improvement.

[0012] Given the pathophysiology of this disease, recent clinical trials have evaluated the use of sialic acid or ManNAc (a precursor of sialic acid) in patients with GNE myopathy, as well as early gene therapy trials. For example, AAV8 viral vectors carrying wild-type human GNE cDNA have been shown to transduce cultured mouse muscle cells and human GNE myopathy-derived muscle cells, and to express transgenes in these cells (Mitrani-Rosenbaum et al., Neuromusculoskeletal Disorders, 22(11):1015-24, 2012). Existing gene therapies focus only on delivering wild-type GNE genes and do not utilize the bifunctional bicistronic technology disclosed herein.

[0013] This disclosure provides gene therapies to increase muscle strength, along with transgenic vectors for gene replacement to prevent further muscle damage or promote muscle growth. For example, gene therapy vectors providing GNE gene replacement may be one of the only ways to demonstrate clinical efficacy in GNE myopathy within less than 5 years, given the slow and constantly evolving natural history of disease progression. It will also be one of the only ways to demonstrate clinical efficacy in all patients with GNE myopathy, many of whom, despite losing the ability to walk shortly after diagnosis, still exhibit significant arm function, such as self-feeding, thus allowing this therapy to preserve or improve that function. Because this disease is a myopathy rather than malnutrition, once the muscles are repaired, they should be permanently retained in situ. Summary of the Invention

[0014] This disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), optimized for delivering transgenes to muscle. The optimized vectors comprise: a constitutive or muscle-specific promoter for delivering systemic or skeletal / cardiac muscle-specific transgene expression, respectively, and binding to the transgene cDNA, replacing gene mutations found in muscle diseases with a normal gene copy (or alternative gene substitution); an internal ribosome entry site (IRES) to allow the production of a second protein from the same transcript; and muscle growth factor for building new muscle growth and strength. The transgene and muscle growth factor genes are expressed by the same mRNA, which expresses two proteins due to the presence of the internal ribosome entry site (or IRES) in the fibroblast growth factor 1A gene sequence, allowing the second protein to be produced from a single mRNA. For example, this disclosure provides gene therapy vectors designed for treating GNE myopathy. AAVs express the GNE gene, which, alone or in combination with muscle growth factors such as follicle-stimulating hormone (FST), heparin-bound modified insulin-like growth factor 1 (HB-IGF), natural IGF1, or SMAD7, encodes a bifunctional UDP-GlcNAc-epimerase / ManNAc-6 kinase. In this context, the provided AAV replaces the mutated GNE gene expression in patients with GNE myopathy with the normal GNE gene, while simultaneously expressing proteins that stimulate muscle growth and strength, which can counteract or even reverse the progression of the disease. The unique aspect of tandem vectors is that they simultaneously provide two essential therapeutic elements—1) a gene replacement therapy to prevent the development of another disease when expressed in cells or tissues; and 2) a muscle growth therapy to reverse the disease by building new muscle growth and strength. In muscular dystrophy and myopathy, muscle tissue loss is caused by gene mutations that lead to the disease. The proposed therapy will not only halt the disease in such patients by reintroducing a non-mutated version of the disease gene, but also build and reverse persistent muscle loss through the co-expression of muscle growth factors. This growth factor may double the amount of muscle in the tissue, but it may also double the weakness caused by these diseases (thus reversing the effects).

[0015] This disclosure also provides alternative gene therapy vectors for treating muscular dystrophy, such as Duchenne muscular dystrophy, limb-girdle muscular dystrophy 2L (LGMD2A), and congenital muscular dystrophy 1a (MDC1A). The AAV expresses the GALGT2 (B4GALNT2) gene, which, alone or in combination with muscle growth factors such as folliculostatin (FST), heparin-bound modified insulin-like growth factor 1 (HB-IGF), native IGF1, or SMAD7, encodes GalNAc transferase (β1,4–N-acetylgalactosamine galactosyltransferase). This is an alternative gene therapy because instead of replacing a mutated gene, it provides an enzyme that transfers complex sugar molecules to specific proteins, such as dystrophic glycans.

[0016] This article provides an AAV with a genome containing a constitutive or muscle-specific promoter that drives the expression of a nucleotide sequence encoding a target transgene in combination with a nucleotide sequence encoding a muscle growth factor, such as a protein that induces muscle growth and a muscle-specific IRES (e.g., FGF IRES), or a muscle transdifferentiation factor (e.g., myoD). This gene therapy can be used to treat any disease requiring gene replacement and the need to increase muscle growth or muscle strength, such as GNE myopathy, limb girdle muscular dystrophy, congenital muscular dystrophy 1A, and Duchenne muscular dystrophy.

[0017] The polynucleotide disclosed herein comprises: a) a promoter element, such as a constitutive or muscle-specific promoter; b) a transgene; c) an internal ribosome entry site (IRES); and d) a nucleotide sequence encoding muscle growth factor or muscle transdifferentiation factor (i.e., a second transgene). For example, a constitutive or muscle-specific promoter is operatively linked to the transgene and / or an IRES is operatively linked to a nucleotide sequence encoding muscle growth factor or muscle transdifferentiation factor. The fact that the element is linked to a single mRNA allows a gene therapy product mediated by a single AAV to provide two functions. Due to the high cost of AAV production and safety concerns regarding AAV administration, using a single AAV vector with two gene therapies would be significantly superior to achieving the same results by mixing two single-gene AAV gene therapies, where twice (or more) the amount of AAV must be manufactured and delivered to the patient to achieve the same outcome.

[0018] This disclosure also provides a polynucleotide comprising: a) one or more constitutive or muscle-specific promoter elements; and b) a GNE cDNA sequence or a GALGT2 cDNA sequence. For example, the polynucleotide comprises: a) additional constitutive or muscle-specific promoter elements; b) a GNE cDNA sequence; c) an internal ribosome entry site (IRES); and d) a polynucleotide sequence that induces muscle growth or differentiates cells into muscle cells. In some embodiments, the muscle-specific control element is operatively linked to the GNE cDNA sequence and / or the IRES is operatively linked to the polynucleotide that induces muscle growth. In another example, the polynucleotide comprises: a) additional constitutive or muscle-specific promoter elements; b) a GALGT2 cDNA sequence; c) an internal ribosome entry site (IRES); and d) a polynucleotide sequence that induces muscle growth or differentiates cells into muscle cells.

[0019] GNE myopathy is an adult-onset, slowly progressive muscle disease. To demonstrate therapeutic efficacy within a reasonable timeframe and provide maximum benefit to patients already affected by muscle weakness at diagnosis, a gene therapy is needed that not only corrects the genetic defects in GNE gene function but also builds new muscle mass. Follicle-stimulating hormone (FSH), IGF1, SMAD7, and HB-IGF are known to significantly stimulate muscle growth in mice, macaques, and / or humans. FSH does this partly by competitively inhibiting and suppressing Smad2 / 3 signaling, while IGF1 does it partly by activating muscle IGF1 receptors and activating Akt / mTOR signaling. This paper presents a bicistronic AAV expressed in GNE using an IRES sequence from FGF1A, which is known to be most potent in skeletal muscle tissue. The use of muscle-specific IRES is ideal for FSH because it promotes optimal muscle growth through localized expression, while the use of a CMV promoter for GNE expression would be ideal, as GNE is typically expressed in all tissues.

[0020] This document provides AAVs with genomes containing promoter elements, such as constitutive promoters or muscle-specific promoters, that drive the expression of GNE cDNA sequences or GALGT2 cDNA sequences. In particular, this disclosure provides rAAVs with genomes designed to promote GNE gene substitution. In these AAVs, the genome contains: a) one or more muscle-specific promoter elements and b) a GNE cDNA sequence. In another aspect, this disclosure provides rAAVs with genomes designed to promote GALGT2 substitution gene therapy (expression of the substitution gene).

[0021] For example, this disclosure provides an rAAV genome comprising a polynucleotide comprising: a nucleotide sequence encoding a wild-type human GNE gene, such as variant 2GNE wild-type human cDNA (SEQ ID NO:1); and a muscle-specific promoter such as a CMV promoter (SEQ ID NO:3), an MCK promoter (SEQ ID NO:4), an MHCK7 promoter (SEQ ID NO:5), or a mini CMV promoter (SEQ ID NO:7); or a human GNE promoter sequence (SEQ ID NO:6). In some embodiments, a human GNE promoter element is found between exons 1 and 2 to drive the expression of variant 2 (722 amino acids) GNE cDNA containing the nucleic acid sequence of SEQ ID NO:1 (thus allowing endogenous natural gene expression).

[0022] This disclosure also provides a polynucleotide comprising: a) one or more constitutive or muscle-specific promoter elements; and b) a GALGT2 cDNA sequence (SEQ ID NO:36). For example, the polynucleotide comprises: a) additional constitutive or muscle-specific promoter elements; b) a GALGT2 cDNA sequence; c) an internal ribosome entry site (IRES); and d) a polynucleotide sequence that induces muscle growth or differentiates cells into muscle cells. In some embodiments, the muscle-specific control element is operatively linked to the GALGT2 cDNA sequence and / or the IRES is operatively linked to the polynucleotide that induces muscle growth.

[0023] For example, this disclosure also provides an rAAV genome containing a polynucleotide comprising: a nucleotide sequence encoding the wild-type human GALGT2 gene (SEQ ID NO:36); and a muscle-specific promoter such as the MCK promoter (SEQ ID NO:4) or the MHCK7 promoter (SEQ ID NO:5).

[0024] This disclosure also provides rAAVs having a genome designed to contain a second transgene that will induce muscle growth or differentiate cells into muscle. For example, an rAAV has: a genome containing a GNE cDNA or GALGT2 cDNA sequence, an internal ribosome entry site (IRES) from a fibroblast growth factor 1A gene known to function in skeletal muscle, a 3' in the GNE cDNA or GALGT2 cDNA sequence, followed by a nucleotide sequence encoding a gene known to induce muscle growth, such as folliculorin (e.g., folliculorin 344 (FS344)) or a variant of IGF1 (e.g., HB-IGF1), and then a poly A sequence or SMAD7. An FGF IRES contains the nucleotide sequence of a fragment of SEQ ID NO:30. An exemplary fragment of an FGF IRES contains the nucleotide sequence of SEQ ID NO:8, which is also referred to herein as a “mini IRES”.

[0025] This disclosure relates to gene therapy vectors, such as AAVs, for expressing the wild-type human GNE gene into skeletal muscle to reduce or replace a defective GNE gene. The gene therapy vectors of this invention can also be AAVs that express the wild-type human GNE gene and muscle-inducing genes such as folliculorin, IGF1, or SMAD7 in a single rAAV genome.

[0026] This disclosure provides a polynucleotide comprising a) one or more promoter elements, such as constitutive or muscle-specific promoters, and b) a GNE cDNA sequence. The polynucleotide provided in this disclosure comprises: a) multiple promoter elements, such as constitutive muscle-specific promoters; b) a GNE cDNA sequence or a GALGT2 cDNA sequence; c) an internal ribosome entry site (IRES); and d) a nucleotide sequence encoding muscle growth factor or muscle transdifferentiation factor. The GNE cDNA is a nucleic acid sequence encoding UDP-GlcNAc-episomerase / ManNAc-6. In an exemplary embodiment, the GNE cDNA is a wild-type variant 2 GNE cDNA encoding UDP-GlcNAc-episomerase / ManNAc-6 kinase. The wild-type GNE cDNA sequence of variant 2 is shown as the nucleic acid sequence of SEQ ID NO:1. This disclosure also provides a polynucleotide comprising a GNE promoter element found between exons 1 and 2 to drive the expression of the same variant 2 (722 amino acids) GNE cDNA. The GNE promoter sequence is shown as SEQ ID NO:6. GALGT2 cDNA is the nucleic acid sequence encoding GalNAc transferase. The GALGT2 cDNA sequence is shown in SEQ ID NO:36. The GalNAc transferase amino acid sequence is shown in SEQ ID NO:37.

[0027] In some aspects, this disclosure provides a polynucleotide comprising a GNE cDNA sequence or a GALGT2 cDNA sequence and a nucleotide sequence encoding a protein that induces muscle growth, such as folliculitis, an insulin-like growth factor 1 (IGF1) variant, or SMAD7. For example, folliculitis 344 is encoded by the nucleotide sequence of SEQ ID NO:9. Another exemplary folliculitis is folliculitis 317, encoded by the nucleotide sequence of SEQ ID NO:28. Furthermore, the IGF1 variant is HB-IGF, encoded by the nucleotide sequence of SEQ ID NO:11. SMAD7 is encoded by the nucleotide sequence of SEQ ID NO:39.

[0028] In some aspects, this disclosure provides a polynucleotide comprising a GNE cDNA sequence or a GALGT2 cDNA sequence and a sequence encoding a protein (transdifferentiation factor) that induces cell differentiation into muscle, such as myoD (SEQ ID NO:31).

[0029] In some respects, the polynucleotide includes an internal ribosome entry site (IRES), such as the IRES from the fibroblast growth factor 1A gene (FGF IRES). The nucleotide sequence of FGF IRES is shown in SEQ ID NO:30 or a fragment thereof. FGF IRES can be miniaturized, for example, into mini FGF IRES as shown in SEQ ID NO:8.

[0030] Another aspect of this disclosure provides compositions comprising: a nucleic acid molecule comprising a genome within a nucleotide sequence of any one of SEQ ID NO:12-26 and 36; an rAAV having a genome within a nucleic acid sequence of SEQ ID NO:12-26 and 36; or rAAV particles comprising a genome within a nucleic acid sequence of any one of SEQ ID NO:12-26 and 36. Any method disclosed herein can be performed using these compositions.

[0031] The disclosed AAV contains: a genome including the CMV promoter and variant 2 wild-type human GNE cDNA, for example Figure 1A The genome provided or the genome listed in SEQ ID NO:12.

[0032] The disclosed AAV contains: a genome including the MCK promoter and variant 2 wild-type human GNE cDNA, for example Figure 1B The genome provided or the genome listed in SEQ ID NO:13.

[0033] The disclosed AAV contains: a genome including the MHCK promoter and variant 2 wild-type human GNE cDNA, for example Figure 1C The genome provided or the genome listed in SEQ ID NO:14.

[0034] The disclosed AAV contains: a genome including the GNE promoter and variant 2 wild-type human GNE cDNA, for example Figure 1D The genome provided or the genome listed in SEQ ID NO:15.

[0035] The disclosed AAV contains: a genome including the MHCK7 promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344, for example Figure 1E The genome provided in or listed in SEQ ID NO:16.

[0036] The disclosed AAV contains: a genome including the MHCK7 promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1IRES, and a nucleic acid sequence encoding HB-IGF1, for example Figure 1F The genome provided or the genome listed in SEQ ID NO:17.

[0037] The disclosed AAV comprises: a genome containing the CMV promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344, for example Figure 1G The genome provided or the genome listed in SEQ ID NO:18.

[0038] The disclosed AAV contains: a genome including a CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example Figure 1H The genome provided or the genome listed in SEQ ID NO:19.

[0039] The disclosed AAV comprises: a genome containing the MCK promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344, for example Figure 1I The genome provided in or listed in SEQ ID NO:20.

[0040] The disclosed AAV comprises: a genome containing the MCK promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example Figure 1J The genome provided or the genome listed in SEQ ID NO:21.

[0041] The disclosed AAV contains: a genome including the GNE promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344, for example Figure 1K The genome provided in or listed in SEQ ID NO:22.

[0042] The disclosed AAV contains: a genome including the GNE promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example Figure 1L The genome provided or the genome listed in SEQ ID NO:23.

[0043] The disclosed AAV contains: a genome including a mini CMV promoter and variant 2 wild-type GNE cDNA, for example Figure 1M The genome provided or the genome listed in SEQ ID NO:24.

[0044] The disclosed AAV comprises: a genome containing a mini CMV promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344, for example Figure 1N The genome provided in or listed in SEQ ID NO:25.

[0045] The disclosed AAV comprises: a genome containing a mini CMV promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example Figure 10 The genome provided or the genome listed in SEQ ID NO:26.

[0046] The disclosed AAV contains: a genome including the MHCK7 promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1P The genome provided in [the text].

[0047] The disclosed AAV contains: a genome including the MHCK7 promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1Q The genome provided in [the text].

[0048] The disclosed AAV comprises: a genome containing a CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1R The genome provided in [the text].

[0049] The disclosed AAV contains: a genome including a CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding a mini FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1S The genome provided in [the text].

[0050] The disclosed AAV comprises: a genome containing the MCK promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1T The genome provided in [the text].

[0051] The disclosed AAV contains: a genome including the MCK promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1U The genome provided in [the text].

[0052] The disclosed AAV comprises: a genome containing the GNE promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1V The genome provided in [the text].

[0053] The disclosed AAV contains: a genome including the GNE promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding mini FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1W The genome provided in [the text].

[0054] The disclosed AAV comprises: a genome containing a mini CMV promoter, variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1X The genome provided in [the text].

[0055] The disclosed AAV contains: a genome including a mini CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding a mini FGF1IRES, and a nucleic acid sequence encoding SMAD7, for example Figure 1Y The genome provided in [the text].

[0056] The disclosed AAV includes: a genome containing the MCK promoter, GALGT2 cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344, for example... Figure 1Z The genome provided or the genome listed in SEQ ID NO:38.

[0057] The disclosed AAV comprises: a genome containing the MCK promoter, GALGT2 cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example... Figure 1AA The genome provided in [the text].

[0058] The disclosed AAV comprises: a genome containing the MCK promoter, GALGT2 cDNA, a nucleic acid sequence encoding FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example... Figure 1BB The genome provided in [the text].

[0059] This disclosure provides a method for treating GNE myopathy in human subjects in need, comprising the steps of administering the recombinant adenovirus-associated (rAAV) or AAV disclosed herein. The method of treating GNE myopathy includes methods for reducing, inhibiting, or slowing the progression of muscle weakness symptoms and muscle atrophy in GNE, and / or increasing muscle strength in the subject in need. The subject in need may experience a reduction in muscle weakness symptoms of GNE myopathy. The subject in need may have a mutation in the GNE gene.

[0060] This disclosure provides methods for treating muscular dystrophy, including Duchenne muscular dystrophy, LGMD2A, and MDC1A, in human subjects in need, comprising the steps of administering a recombinant adenovirus-associated (rAAV) or AAV disclosed herein. Methods for treating muscular dystrophy include reducing, inhibiting, or slowing the progression of muscle weakness symptoms, muscle atrophy, and / or increasing muscle strength in subjects in need. Subjects in need may experience a reduction in muscle weakness symptoms of GNE myopathy. Subjects in need may have mutations in the GNE gene.

[0061] In any of the methods disclosed herein, a dose of rAAV may be administered via intramuscular, intraperitoneal, intravenous, intraarterial, oral, oral, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal routes of administration. For example, the route of administration may be systemic, such as by injection, infusion, or implantation. For instance, a dose of rAAV may be administered by infusion over approximately one hour. Alternatively, a dose of rAAV may be administered via an intravenous route through peripheral veins of the extremities, such as peripheral veins of the arm or leg. Or, it may be administered by injection over approximately 30 minutes, or approximately 1.5 hours, or approximately 2 hours, or approximately 2.5 hours, or approximately 3 hours.

[0062] In any method of this disclosure, the administered rAAV is serotype AAVrh7.4. The rAAV vector of this disclosure can be any AAV serotype, such as serotypes AAVrh.74, Anc80, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, AAV12, AAV13, AAVTT, AAV7m8, and derivatives thereof.

[0063] In one aspect, this disclosure provides an rAAV comprising a muscle-specific control element nucleotide sequence and a nucleotide sequence encoding UDP-N-acetylglucosamine 2-epomerase / N-acetylmnosamine kinase. For example, the nucleotide sequence encodes a functional UDP-N-acetylglucosamine 2-epomerase / N-acetylmnosamine kinase, wherein the nucleotide has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% sequence identity with SEQ ID NO:1, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100%, wherein the encoded protein retains kinase activity. In addition, the nucleotide sequence encodes a functional protein comprising an amino acid sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually at least 90%, 91%, 92%, 93%, or 94%, and even more usually at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2, and retains kinase activity.

[0064] In another aspect, this disclosure provides an rAAV comprising a muscle-specific control element nucleotide sequence and a nucleotide sequence encoding a GalNAc transferase. For example, the nucleotide sequence encodes a functional GalNAc transferase, wherein the nucleotide has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89% sequence identity with SEQ ID NO:36, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100%, wherein the encoded protein retains transferase activity. In addition, the nucleotide sequence encodes a functional protein containing an amino acid sequence, wherein the amino acid sequence has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually at least 90%, 91%, 92%, 93%, or 94%, and even more usually at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ IDNO:37, and retains transferase activity.

[0065] In another aspect, this disclosure provides an rAAV comprising a muscle-specific control element nucleotide sequence and a nucleotide sequence encoding a follistatin, such as follistatin 344 or follistatin 317. For example, the nucleotide sequence encodes a functional follistatin, wherein the nucleotide has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity with SEQ ID NO:9 or 28, wherein the encoded protein retains follistatin activity. In addition, the nucleotide sequence encodes a functional protein comprising an amino acid sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually at least 90%, 91%, 92%, 93%, or 94%, and even more usually at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:10 or 29, and retains folliculorine activity, which refers to folliculorine binding to activin, thereby antagonizing activin activity. Folliculorine exerts its effect by competitively inhibiting and suppressing Smad2 / 3 signaling by utilizing myostatin to inhibit repressive growth signaling.

[0066] In one embodiment, this disclosure provides an rAAV comprising a muscle-specific promoter element nucleotide sequence and a nucleotide sequence encoding an IGF variant, such as HB-IGF. For example, the nucleotide sequence encodes an IGF variant wherein the nucleotide has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:11, wherein the encoded protein retains IGF activity. Additionally, the nucleotide sequence encodes a functional protein comprising an amino acid sequence having, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:27, and retains IGF-1 activity. IGF-1 activity refers to IGF-1 binding to and activating the IGF receptor (IGFR) and / or insulin receptor IGF-1 by activating muscle IGFR and Akt / mTOR signaling. IGF-1 activity includes stimulating cell growth and proliferation (e.g., muscle cell growth) and inhibiting programmed cell death. This disclosure also provides an rAAV wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO:11 or its complementary sequence and encodes a functional IGF variant.

[0067] This disclosure also provides an rAAV wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 9 or 28 or its complementary sequence and encodes a functional follicle-suppressing hormone.

[0068] This disclosure also provides an rAAV, wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO:11 or its complementary sequence and encodes a functional IGF.

[0069] The term "strict" is used to refer to conditions that are generally understood in the art as stringent. The stringency of hybridization is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions used for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65°C to 68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, New York, 1989). Even more stringent conditions (e.g., higher temperatures, lower ionic strengths, higher concentrations of formamide or other denaturing agents) can be used; however, the hybridization rate will be affected. In cases involving deoxyoligonucleotide hybridization, additional exemplary stringent hybridization conditions include washing in 6×SSC 0.05% sodium pyrophosphate at 37°C (for 14-base oligomers), 48°C (for 17-base oligomers), 55°C (for 20-base oligomers), and 60°C (for 23-base oligomers).

[0070] To reduce nonspecific and / or background hybridization, additional reagents can be included in the hybridization and wash buffers. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4, (SDS), Ficoll, Denhardt solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable reagents may also be used. The concentration and type of these additives can be varied without significantly affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8–7.4; however, under typical ionic strength conditions, the hybridization rate is almost pH-independent. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Chapter 4, IRL Press (Oxford, UK). Those skilled in the art can adjust hybridization conditions to accommodate these variables and allow hybrids of DNAs with different sequence correlations.

[0071] The term "muscle-specific promoter element" refers to a nucleotide sequence that regulates the expression of a coding sequence specifically expressed in muscle tissue. These control elements include enhancers and promoters. This disclosure provides a polynucleotide or AAV containing a genome comprising one or more muscle-specific control elements, such as the MCKH7 promoter, MCK promoter, or MCK enhancer. The GNE promoter may be the promoter of the human wild-type GNE gene. Other promoter elements, such as CMV, mini-CMV, and GNE promoters, allow expression in virtually all tissues and will be referred to as "constitutive promoters."

[0072] The term "constitutive promoter element" refers to an unregulated promoter that allows its associated gene to be transcribed continuously. Examples of constitutive promoter elements include hACTB, hEF-1α, CAG, CMV, herpes simplex virus thymidine kinase (HSV-TK), SP1, C-FOS, or C-MYC promoters.

[0073] The term "operably linked" refers to the positioning of a regulatory element nucleotide sequence (e.g., a promoter nucleotide sequence) to confer expression of the nucleotide sequence through the regulatory element.

[0074] For example, the muscle-specific promoter element is the MHCK7 promoter nucleotide sequence shown in SEQ ID NO:5, or the CMV promoter nucleic acid sequence shown in SEQ ID NO:3, or the MCK nucleotide sequence shown in SEQ ID NO:4, or the GNE promoter nucleotide sequence shown in SEQ ID NO:6, or the mini-CMV nucleotide sequence shown in SEQ ID NO:7. Furthermore, in any rAAV vector of this disclosure, the muscle-specific promoter element nucleotide sequence is operatively linked to the GNE cDNA sequence (SEQ ID NO:1).

[0075] On the other hand, this disclosure provides an rAAV construct contained in a plasmid, comprising a nucleotide sequence of any one of SEQ ID NO:12-26 and 38 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence of any one of SEQ ID NO:12-26.

[0076] This disclosure also provides pharmaceutical compositions (or sometimes simply referred to herein as "compositions") comprising any of the rAAV carriers of the present invention.

[0077] In another embodiment, this disclosure provides a method for generating rAAV particles, comprising culturing cells transfected with any of the rAAV vectors of the present invention and recovering rAAV particles from the supernatant of the transfected cells. This disclosure also provides viral particles comprising any disclosed recombinant AAV vector.

[0078] In any treatment for GNE myopathy, the expression level of the GNE gene in the subject's cells increases after administration of rAAV. Cellular GNE gene expression is detected by measuring the levels of UDP-N-acetylglucosamine 2-epimerase / N-acetylmnosamine kinase in various tissues (e.g., muscle, heart, liver, kidney, brain, colon) before and after rAAV administration via Western blotting, immunohistochemistry, or enzymatic assays. Attached Figure Description

[0079] Figure 1A-1BB A schematic diagram of the AAV genome presented in this article is provided.

[0080] Figure 2 provides a representation of... Figure 1A The plasmid sequence of the rAAVrh74.CMV.GNE (variant 2) (SEQ ID NO:12) genome is shown.

[0081] Figure 3 provides a representation of... Figure 1B The plasmid sequence of the rAAVrh74.MCK.GNE (variant 2) (SEQ ID NO:13) genome is shown.

[0082] Figure 4 provides a summary of the data. Figure 1C The plasmid sequence of the rAAVrh74.MHCK7.GNE (variant 2) (SEQ ID NO:14) genome is shown.

[0083] Figure 5 provides a summary of the data. Figure 1D The plasmid sequence of the rAAVrh74.GNE promoter.GNE (variant 2) (SEQ ID NO:15) genome is shown.

[0084] Figure 6 provides a summary of the data. Figure 1E The plasmid sequence of the rAAVrh74.MHCK7.GNE(variant 2).FGF1IRES.FS344(SEQ ID NO:16) genome is shown.

[0085] Figure 7 provides a representation of... Figure 1F The plasmid sequence of the rAAVrh74.MHCK7.GNE(variant 2).FGF1IRES.HB-IGF1(SEQ ID NO:17) genome is shown.

[0086] Figure 8 provides a representation of... Figure 1GThe plasmid sequence of the rAAVrh74.CVM.GNE(variant 2).FGF1IRES.FS344(SEQ ID NO:18) genome is shown.

[0087] Figure 9 provides a representation of... Figure 1H The plasmid sequence of the rAAVrh74.CMV.GNE(variant 2).FGF1IRES.HB-IGF1(SEQ ID NO:19) genome is shown.

[0088] Figure 10 provides a representation of... Figure 1I The plasmid sequence of the genome of rAAVrh74.MCK.GNE (variant 2).FGF1IRES.FS344 (SEQ ID NO:20) is shown.

[0089] Figure 11 provides a representation of... Figure 1J The plasmid sequence of the rAAVrh74.MCK.GNE(variant 2).FGF1IRES.HB-IGF1(SEQ ID NO:21) genome is shown.

[0090] Figure 12 provides a summary of the data. Figure 1K The plasmid sequence of the rAAVrh74.GNE promoter.GNE(variant 2).FGF1IRES.FS344(SEQ ID NO:22) genome is shown.

[0091] Figure 13 provides a summary of the data. Figure 1L The plasmid sequence of the rAAVrh74.GNE promoter.GNE (variant 2).FGF1IRES.HB-IGFI (SEQ ID NO:23) genome is shown.

[0092] Figure 14 provides a summary of the data. Figure 1M The plasmid sequence of the genome of rAAVrh74. mini CMV.GNE (SEQ ID NO:24) is shown.

[0093] Figure 15 provides a summary of the data. Figure 1N The plasmid sequence of the rAAVrh74.miniCMV.GNE(variant 2).FGF1IRES.FS344(SEQ ID NO:25) genome is shown.

[0094] Figure 16 provides a summary of the data. Figure 10 The plasmid sequence of the rAAVrh74.mini CMV.GNE(variant 2).FGF1IRES.HB-IGF1(SEQ ID NO:26) genome is shown.

[0095] Figure 17 provides a summary of the data. Figure 1ZThe plasmid sequence of the rAAVrh74.MCK.GALGT2.FGF1IRES.FS344 genome is shown (SEQ ID NO:38).

[0096] Figure 18 Sialic acid staining of liver and muscle in GNED176V TgGne- / - mice after intramuscular injection of rAAVrh74.MCK.GNE or IP injection of rAAVrh74.LSP.GNE is shown. The bars are 100 μm in size.

[0097] Figure 19 Genotyping data from generative mice with Cas9-CRISPR Gne exon 3 deletion / loxP recombination experiments are provided. Generative CR10646-8 and -9 contain genomic deletions in GNE exon 3.

[0098] Figure 20 Staining of Gne-deficient Lec3 CHO cells with rAAV.CMV.GNE.mini-IRES.GFP was provided to demonstrate the expression of a second protein using the mini-IRES sequence. GFP showed endogenous fluorescence, while Gne showed immunostaining, and DAPI was used as a staining agent for the cell nucleus in triple exposure.

[0099] Figure 21 Staining of Gne-deficient Lec3 CHO cells transfected with rAAV.miniCMV.GNE is provided. Full-length (FL)IRES.GFP is used to visualize the expression of the second protein using the full-length IRES sequence. GFP shows endogenous fluorescence, while Gne shows immunostaining. DAPI is used as a staining agent for the cell nucleus in triple exposure.

[0100] Figure 22 Muscle growth following intramuscular injection of IGF1, HB-IGF1, or FST344 using rAAVrh74 was demonstrated. The tibialis anterior (TA, left) was injected with 1 x 10¹¹ vg (vector genome), and the gastrocnemius (Gastroc, right) was injected with 5 x 10¹¹ vg of AAV expressing insulin-like growth factor 1 (IGF1, muscle form Ea), HB-IGF1, or follicle-stressor (FST) form 344. Muscles were dissected and weighed 2 months post-injection. Significant increases in HB-IGF1 and FST344 were observed in TA and in FST344 in Gastroc compared to buffer injection alone. Error is represented by SEM images of n = 12 muscles per group. *p < 0.05, ***p < 0.001.

[0101] Figure 23This indicates that CMV.GNE.IRES.GFP allows the induction of sialic acid expression on the membrane of Lec3 Gne-deficient CHO cells, while IRES allows the expression of a second protein, in this case, GFP. Endogenous GFP expression is shown in the green channel, while MAA staining of sialic acid appears red. Normal CHO cells exhibit MAA staining because they have normal Gne function, while Lec3 cells typically do not express MAA because they lack functional Gne. The introduction of CMV.GNE.IRES.GFP, due to the presence of IRES, allows for the expression of functional GNE and the second protein, GFP, in Lec3 cells. DAPI, as shown in the triple exposure, reveals the cell nuclei stained blue.

[0102] Figure 24 This indicates that, due to the presence of a bicistronic vector in the IRES sequence, muscle cells (C2C12 cells) transfected with MCK.GALGT2.IRES.FS344 (or FST) can express both GALGT2 (green) and FST (red) in the same cells. Using time-matched images, C2 cells transfected with the mimic in the absence of bicistronic DNA showed low or no expression of either protein.

[0103] Figure 25 The changes in MAA signaling in Lec3 cells after infection with rAAVrh74.CMV.GNE were demonstrated. Maackia amurensis agglutinin (MAA), conjugated with horseradish peroxidase (HRP), was used to measure sialic acid expression in CHO or Lec3 cells in a 96-well ELISA plate assay, using a colorimetric assay of HRP activity as the output. Lec3 cells grown in Opti-MEM for 3 days showed reduced MAA binding compared to CHO cells, and this binding was likely partially rescued by the addition of rAAVrh74.CMV.GNE for two days. For each group n=2, the error is SD. MOI, multiplicity of infection, OD, optical density, **p<0.01.

[0104] Figure 26This study demonstrates GNE enzyme activity in CHO, Lec3, and Lec3 cells transfected with pAAV.CMV.GNE. Cells were lysed, and UDP-GlcNAc epimerase activity was measured using 0.3 mg of total protein per sample. ManNAc was measured using a colorimetric assay, and samples were compared to a ManNAc standard curve. CHO cells showed significantly higher UDP-GlcNAc epimerase activity compared to Lec3 cells lacking functional GNE enzymes. Lec3 cells transfected with pAAV.CMV.GNE showed GNE enzyme activity exceeding that of CHO cells. For each group n=2, the error is SD. **p<0.01, ***p<0.001.

[0105] Figure 27 The efficacy of bistronic GALGT2 and follicle-stabilizing hormone 344 (FST) gene therapy in MDX mice was demonstrated. Figure 27 A demonstrates that intramuscular injection of TA at the same dose, using either 1x10¹¹ vg rAAVrh74.MCK.GALGT2.IRES.FST or the single-gene vector rAAVrh74.MCK.FST, resulted in an increase in muscle size, measured as muscle weight (mg / g) relative to the total body mass. For each group n=4, the error is SD. *p<0.05, **p<0.01. Figure 27 B provides images of TA muscle stained with antibodies against FST and WFA (to identify GalNAc produced by GALGT2) after injection. Detailed Implementation

[0106] This disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), optimized for delivering transgenes to muscle. The optimized vectors comprise: a constitutive or muscle-specific promoter for delivering systemic or skeletal / cardiac muscle-specific transgene expression, respectively, and binding to the transgene cDNA, replacing gene mutations found in muscle diseases with normal gene copies; an internal ribosome entry site (IRES) to allow the production of a second protein from the same transcript; and muscle growth factor for building new muscle growth and strength. The transgene and muscle growth factor genes are expressed by the same mRNA, which expresses two proteins due to the presence of the internal ribosome entry site (or IRES) in the fibroblast growth factor 1A gene sequence, allowing the second protein to be produced from a single mRNA.

[0107] This disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), designed for the treatment of GNE myopathy. AAV expresses UDP-GlcNAc-epimerase / ManNAc-6, alone or in combination with follistatin or IGF1. The provided AAV replaces the expression of the mutated GNE gene while simultaneously expressing proteins that stimulate muscle growth. A strategy combining gene substitution functions that prevent the development of further disease (direct gene substitution or substitution with alternative gene functions) with muscle growth or muscle transdifferentiation therapies that build new muscle mass and strength has the potential not only to halt disease progression but also to reverse it by stimulating new muscle growth and strength while simultaneously inhibiting the disease's pathogenesis.

[0108] The gene therapy vectors provided in this article consist of: 1) transgenes for gene replacement or as alternative gene therapy; and 2) genes encoding growth factors that induce muscle growth or increase muscle strength. This gene therapy is encoded by a single gene therapy genome, such as a single AAV genome. This combined therapy has the potential not only to halt disease progression but also to reverse it by stimulating new muscle growth and strength while simultaneously inhibiting the disease's pathogenesis.

[0109] The gene therapy offered can be used to treat GNE myopathy, Duchenne muscular dystrophies (DMD), Becker muscular dystrophies (BMD), and limb girdle muscular dystrophies. dystrophies, LGMD), such as LGMD2A (CAPN3, LGMD2C (SGCG), LGMD2D (SGCA), LGMD2E (SGCB), LGMD2F (SGCD), LGMD2G (TCAP), LGMD2H (TRIM32), LGMD2I (FKRP), LGMD2K (POMT1), LGMD2L (ANO5), LGM D2M(FKTN), LGMD2O(POMT2), LGMD2P(DAG1), LGMD2R(DES), LGMD2T(GMPPB), LGMD2U(ISPD), LGMD2X(BVES), LGMD2Y(TOR1AIP1), LGMD2Z(POGLUT1), LGMD1A(TTID, MYOT), LGMD1B(LMNA), LGMD 1C (Cav3), LGMD1D (DES), LGMD1F (TNPO3), LGMD1G (HNRPDL), and MDC1A. In different cases, the first transgene (e.g., GALGT2 or B4GALNT2) can be used for gene substitution or replacement of a missing gene in the disease, while the second transgene is muscle growth factor (e.g., FS344, HB-IGF1, IGF1, or SMAD7) to reverse disease symptoms by building new muscle growth and strength. The gene therapy disclosed herein can also be used to treat diseases by using a replacement gene instead of gene substitution as the first transgene for disease prevention, and is applicable to therapies where muscle growth from the second transgene placement is derived from muscle transdifferentiation factor (e.g., MyoD) rather than muscle growth factor, where muscle is built up by converting adipocytes or fibroblasts into muscle rather than through a muscle growth factor carrier.

[0110] In some implementations, a construct with available space is provided in which the AAV genome contains a second IRES and a third transgene to simultaneously deliver three gene therapies.

[0111] AAV has a genome containing a muscle-specific promoter that drives the expression of a combination of nucleotide sequences encoding the target transgene and nucleotide sequences encoding muscle growth factors, such as proteins that induce muscle growth and muscle-specific IRES (e.g., FGF IRES). This gene therapy can be used to treat any disease requiring gene replacement and the need to increase muscle growth or muscle strength, such as GNE myopathy, limb girdle muscular dystrophy, and Duchenne muscular dystrophy.

[0112] Growth factors and transdifferentiation factors

[0113] Growth factors that induce muscle growth or increase muscle strength include: IGF, HB-IGF, Pax7, HGF (hepatocyte growth factor), HGH (human growth hormone), FGF19 (fibroblast growth factor 19), FGF21 (fibroblast growth factor 21), VEGF (vascular endothelial growth factor), IL6 (interleukin-6), IL15 (interleukin-15), and SMAD7 (mothers against decapentaplegic homolog 7, MADH7).

[0114] Growth factors that induce muscle growth or increase muscle strength also include follistatin. Folliculin is a secreted protein that inhibits the activity of TGF-β family members such as GDF-11 / BMP-11. Folliculin 344 is a follistatin precursor that, through peptide cleavage, forms a circulating follistatin 315 isoform containing an acidic C-terminal region. It circulates in a complex with myostatin propeptide, which contains two other proteins: the follistatin-associated gene (FLRG) and GDF-associated serum protein (GASP-1). Folliculin 317 is another follistatin precursor that, through peptide cleavage, forms a membrane-bound follistatin 288 isoform.

[0115] The DNA and amino acid sequences of the follistatin 344 precursor are listed in SEQ ID NO:9 and 10, respectively. The follistatin 288 isoform lacks the C-terminal acidic region and exhibits a strong affinity for heparin-sulfate-proteoglycan, making it a potent inhibitor of pituitary follicle-stimulating hormone. It is present in the follicular fluid of the ovary and has a high affinity for ovarian granulosa cells. Folllistatin 288 is also produced by the testes. The DNA and amino acid sequences of the follistatin 317 precursor are listed in SEQ ID NO:28 and 29, respectively. Deficiency of follistatin leads to reduced muscle mass at birth.

[0116] Examples of follicle-stimulating hormone are provided in U.S. Patent No. 5,041,538 by Shimasaki et al.; other follicle-stimulating hormone-like proteins are provided in U.S. Patent Nos. 5,942,420, 6,410,232, 6,537,966 and 6,953,662; FLRG (SEQ ID NO:33, corresponding nucleotide sequence is SEQ ID NO:32) was proposed by Hill et al., Journal of Biochemistry, 277(43):40735-40741 (2002); GASP-1 (SEQ ID NO:35, corresponding nucleotide sequence is SEQ ID NO:34) was proposed by Hill et al., Molecular Endocrinology, 17:1144-1154 (2003).

[0117] SMAD7 is known to inhibit TGF-β-activated signaling responses by binding to the active TGF-β complex, thereby reducing TGF-β signaling. Myostatin and TGF-β signaling induce SMAD7 expression, establishing a negative feedback loop to inhibit TGF-β signaling. Specifically, SMAD7 is known to utilize this negative feedback loop to regulate myocyte production (Kollias et al., Molecular Cell Biology, 26(16):6248-6260, 2006). The nucleotide sequence encoding the SMAD7 protein is shown in SEQ ID NO:39 (Genbank accession number NM_005904.4), and the amino acid sequence is shown in SEQ ID NO:40 (Genbank accession number NP_005895).

[0118] Transdifferentiation factors are agents that convert or induce non-muscle cells to differentiate into muscle. For example, MyoD is known to convert various cell types into muscle, including dermal fibroblasts, chondrocytes, smooth muscle, retinal pigment epithelial cells, adipocytes, as well as melanoma, neuroblastoma, osteosarcoma, and liver cancer cells (Abraham & Tapscott, Progress in Gene Lines (Curr. Opin. Genet. Dev. 23(5): 568-573(2013)). Examples of transdifferentiation factors: Myocd (cardioxin), Mef2C (myocytic enhancer 2C), Mef2B (myocytic enhancer 2B), Mkl1 (MKL [megakaryocytic leukemia] Myocd-like 1), Gata4 (GATA-binding protein 4), Gata5 (GATA-binding protein 5), Gata6 (GATA-binding protein 6), Ets1 (E26 avian leukemia oncogene 1, 5' domain).

[0119] GNE myopathy

[0120] GNE myopathy is characterized by progressive muscle atrophy and weakness. It typically begins in the third decade of life, starting with weakness in the tibialis anterior (TA) and hamstring muscles, and by the second decade after diagnosis, patients often require a wheelchair. Eventually, patients may require assistance with daily living functions, such as eating. Muscle biopsies often reveal bordered vacuoles and inclusion bodies. GNE myopathy is caused by mutations in the GNE gene, which encodes a bifunctional UDP-GlcNAc epimerase / ManNAc-6 kinase. GNE function is required for the synthesis of all sialic acid (SA). The SA biosynthetic pathway ultimately produces CMP-SA, which sialyltransferases use to transfer SA to glycoproteins and glycolipids in all mammalian cells.

[0121] The incidence of GNE myopathy is recently estimated to be between 1 and 6 per million, making it a rare disease. However, some founder effector mutations lead to a higher incidence of GNE myopathy in certain populations, such as patients of Japanese descent (D176V, D207V in the new nomenclature) and patients of Middle Eastern descent (M712T, M743T in the new nomenclature). In a study of 1,000 Iranian Jews, the frequency of disease mutation carriers was found to be one in eleven. A partial reduction in GNE activity in patients results in decreased but not absent SA expression.

[0122] Attenuated IGF1R signaling has been shown to be the basis for muscle stem cell death in GNE myopathy models, making IGF1 a potentially ideal growth factor element for gene therapy design. These tandem gene vectors are expected to not only inhibit disease progression (the function of GNE gene substitution) but also induce new muscle growth (thus increasing muscle strength) and potentially prevent stem cell death. These vectors are quite unique because GNE myopathy patients lose muscle and strength over decades, and the provided AAVs are expected to not only slow this progression but actually reverse it. The provided bifunctional AAVs will be able to demonstrate clinical efficacy because this disease exhibits high clinical variability (between disease mutations in patients, and even among patients with the same disease mutation) and because it progresses slowly (significant clinical changes occur over decades).

[0123] GNE myopathy mutation

[0124] In any of the methods provided, the subject has GNE myopathy. For example, the subject's GNE gene is mutated, resulting in decreased expression of UDP-N-acetylglucosamine 2-epimerase / N-acetylmnosamine kinase. The diagnosis of GNE myopathy in the subject is confirmed by determining the presence of pathogenic (primarily missense) mutations in the two alleles of the GNE gene. Table 1 lists known mutations in the GNE gene associated with GNE myopathy, as shown below. Objects of the claimed methods may include the mutations listed in this table.

[0125] In Table 1, bold text indicates truncated cDNA or protein variants. Italics and dark gray highlight “mild” variants. Question marks (?) indicate terms that could not be accurately extracted from the references. The DNA numbering system is based on the cDNA sequence. Nucleotide numbering uses +1 as the reference sequence for the ATG translation start codon A, which is codon 1.

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] 1 Amino acid substitutions were provided in the previously used hGNE1 (NP_005467.1) and the preferred new hGNE2 (NP_001121699.1) [Huizing et al., 2014b]. For certain variants, updated nomenclature extracted from the references is provided.

[0138] 2 The nucleotide variant (NM_001128227.2; the longest spliced ​​mRNA; encoding the hGNE2 protein) is provided in the mRNA variant 1 nomenclature.

[0139] 3 Based on the exon number (NC_000009.12) of the genome sequence, as shown in Figure 1. in = intron.

[0140] 4 For detailed information on GNE protein domains, please refer to the text; ep = UDP-GlcNAc 2-episomerase domain; ep-NES = nuclear output signal; ep-AR: allosteric region; UF = unknown function; kin = ManNAc kinase domain; UF epimerase.

[0141] 5 Based on the pathogenicity score, intron variants that predict splicing effects are classified as "splicing," while intron variants that do not predict splicing effects are classified as "splicing?".

[0142] 6 Excerpted from references.

[0143] GNE mouse model

[0144] Gne is an essential gene in mice; deletion of it leads to embryonic lethality (E) between day 8.5 and day 9.5. The most well-known GNE myopathy model was created by Malicdan et al. (Human Molecular Genetics 16(22):2669-82, 2007). This model was developed in mice with Gne - / - The background constitutively expresses mutated human GNE D207V Genetically modified (Tg). It has been reported that by week 30, GNE... D207V Tg Gne - / - Mice exhibited significantly shortened lifespans, reduced scores on pole climbing and constant-speed treadmill walking, and moderately elevated serum CK activity and Aβ1-42 peptide muscle production. By week 42, muscles displayed bordered vacuoles with Congo red inclusions, as well as respiratory and cardiac pathologies not observed in human GNE myopathy patients. Unfortunately, as these mice reproduced, most of these phenotypes were lost from the production line, thus we and others were unable to find evidence of muscle pathology or muscle defects at week 64.

[0145] The second model is a knock-in of the M712T (now called M743T) Iranian founder's GNE mutation, showing perinatal mortality due to kidney disease (via P3) (Galeno et al., Clin. Invest. 117(6): 1585-94, 2007). Similarly, others have found that such homozygous knock-in lines can be bred to produce phenotypically atypical animal subpopulations (Sela et al., Neuromuscular Med. 15(1): 180-91, 2013). Therefore, the robustness of all preclinical data for this disease is questionable due to the high phenotypic variability of the models used.

[0146] Because all current GNE myopathy mouse models exhibit complex and variable phenotypes, all preclinical data are highly complex. M743T The knock-in model showed early death due to kidney complications, which could be offset by ManNAc. Other strains with the same knock-in did not show the phenotype. In earlier studies, GNE D207V Tg Gne - / - The mouse model showed a clear disease phenotype at one year of age, and these phenotypes could not be replicated in mice currently in existence. Since Gne deficiency causes embryonic death in mice between E8.5 and E9.5, pure gene deletion mice are useless, although several groups, including ours, are working to create more precise gene deletion mice.

[0147] The mouse model is described in Example 3 of this paper. This mouse model was generated using Cas9-CRISPR, ultimately allowing the creation of a fluxed allele to enter exon 3 of the mouse Gne gene, and the introduction of this allele is sufficient to allow Cre-mediated deletion, thereby producing a GNE myopathy-like phenotype. Since Gne is essential in mice, contributing to lethality between E8.5 and E9, creating a fluxed allele in adult mice to delete this gene should allow for the creation of robust systemic or muscle-specific phenotypes using Cre-mediated deletion. This, in turn, allows for more reproducible evidence of therapeutic efficacy.

[0148] Muscular dystrophy

[0149] Muscular dystrophy (MD) is a group of genetic disorders. This group is characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD develop in infancy or childhood, while others may not appear until middle age or later. The disease varies in the distribution and severity of muscle weakness (some forms of MD also affect the cardiac muscle), age of onset, rate of progression, and inheritance patterns.

[0150] One form of MD is Duchenne muscular dystrophy (DMD). It is the most common and severe form of muscular dystrophy in children, affecting 1 in 5,000 newborn males. The genetic trait follows an X-linked recessive pattern. DMD is caused by mutations in the DMD gene that result in the absence of dystrophin (427 kDa) in skeletal and cardiac muscle, as well as in the gastrointestinal tract and retina. Dystrophin not only protects the sarcomere from eccentric contractions but also anchors many signaling proteins located near the sarcomere. Clinical symptoms of DMD typically first appear between 3 and 5 years of age, with gait changes and decreased motor skills usually determining the diagnostic assessment. DMD progresses uncontrollably, leading to loss of walking ability by age 12. Historically, patients die from respiratory complications late in the second decade, but improved supportive care—particularly the judicious use of nocturnal ventilation—has extended life expectancy by nearly a decade. This increased life expectancy is accompanied by a near-universal decline in cardiac function, accompanied by complications of dilated cardiomyopathy. This presents further clinical challenges and requires previously unmet diagnostic and medical intervention approaches. Non-progressive cognitive impairment may also be present in DMD. Despite hundreds of clinical trials conducted in DMD, corticosteroid therapy remains the only treatment consistently proven effective. Current standard of care for DMD involves the use of prednisone or deflazacort, which can extend walking ability for years at the cost of significant side effects, with limited evidence of any impact on survival.

[0151] Another type of MD is congenital muscular dystrophy 1A (MCD1A). MCD1A belongs to a group of neuromuscular disorders that occur at birth or in infancy and are characterized by hypotonia, muscle weakness, and muscle atrophy. MCD1A represents 30-40% of congenital muscular dystrophy, with some regional variations. The prevalence is estimated at 1 / 30,000. The disease presents with hypotonia and muscle weakness in the limbs and trunk at birth or in the first few months of life. Breathing and feeding difficulties may also occur. Motor development is delayed and restricted (sitting or standing can only be achieved with assistance). Early infancy presents with ankylosing spondylitis, scoliosis, and respiratory failure. The typical elongated myopathyal facies is characterized by facial involvement, and oculomotor palsy may occur. Seizures are also possible, although they occur in less than one-third of cases. Intellectual development is normal. MCD1A is caused by mutations in the LAMA2 gene, which encodes the α-2 laminin chain. Transmission is autosomal recessive. Current treatment is symptomatic. It comprises a multidisciplinary approach, including physical therapists, occupational therapists, and speech therapists, with the goal of optimizing each participant's abilities. Seizures or other neurological complications require specific treatment. The prognosis for MDC1A is very serious because most affected children do not reach puberty. Currently, the prognosis can only be improved through careful multidisciplinary management, particularly by orthopedic and respiratory specialists.

[0152] Another type of MD is limb-girdle muscular dystrophy (LGMD). LGMD is a rare condition, and individuals vary in age of onset, area of ​​muscle weakness, involvement of the heart and respiratory system, rate of progression, and severity. LGMD may begin in childhood, adolescence, young adulthood, or even later. Both sexes are affected equally. LGMD causes weakness in the shoulder and pelvic girdle, and muscles near the thighs and arms may sometimes become weak over time. Leg weakness usually precedes arm weakness. Facial muscles are usually unaffected. As the condition progresses, people may experience walking problems and may need to use a wheelchair over time. Involvement of the shoulder and arm muscles can make it difficult to raise the arms overhead and lift objects. In some types of LGMD, the heart and respiratory muscles may be involved.

[0153] There are at least 19 forms of LGMD, which are classified according to their associated genetic defects.

[0154] LGMD1A Autosomal dominant inheritance Myoconstrictor protein gene LGMD1B Autosomal dominant inheritance Lamin A / C gene LGMD1C Autosomal dominant inheritance Nestin gene LGMD1D Autosomal dominant inheritance chromosome 7 LGMD1E Autosomal dominant inheritance Desmin gene LGMD1F Autosomal dominant inheritance chromosome 7 LGMD1G Autosomal dominant inheritance chromosome 4 LGMD1H Autosomal dominant inheritance chromosome 3 LGMD2A Autosomal recessive inheritance Caloplasmin 3 gene LGMD2B Autosomal recessive inheritance dysferlin gene LGMD2C Autosomal recessive inheritance γ-sarcosin LGMD2D Autosomal recessive inheritance α-caryoga gene LGMD2E Autosomal recessive inheritance β-caryoga gene LGMD2F Autosomal recessive inheritance δ-sarcosin gene LGMD2G Autosomal recessive inheritance Telethonin gene LGMD2H Autosomal recessive inheritance TRIM32 LGMD2I Autosomal recessive inheritance FKRP gene LGMD2J Autosomal recessive inheritance Myoma giant protein gene LGMD2K Autosomal recessive inheritance POMT1 gene LGMD2L Autosomal recessive inheritance Anoctamin 5 gene LGMD2M Autosomal recessive inheritance Fukutin gene LGMD2N Autosomal recessive inheritance POMT2 gene LGMD2O Autosomal recessive inheritance POMGnT1 gene LGMD2Q Autosomal recessive inheritance lectin gene

[0155] Specialized testing for LGMD is now available through the National Diagnostic Program developed by the National Commissioning Group (NCG).

[0156] The GALGT2 gene (also known as B4GALNT2) encodes β1-4-N-acetyl-D-galactosamine (βGalNAc) glycosyltransferase. GALGT2 overexpression has been investigated in three different muscular dystrophy models: DMD, LGMD2D, and MDC1A [Xu et al., *American Journal of Pathology*, 175:235-247 (2009); Xu et al., *American Journal of Pathology*, 171:181-199 (2007); Xu et al., *Neuromuscular Disorders*, 171:181-199 (2007); Xu et al., *Neuromuscular Disorders*, 171:181-199 (2007); Xu et al., *Neuromuscular Disorders*, 171:181-199 (2007).] [sorrd.)》, 17:209-220 (2007); Martin et al., American Journal of Physiology: Cell Physiology (Am.J.Physiol.Cell.Physiol.), 296:C476-488 (2009); and Nguyen et al., Proceedings of the National Academy of Sciences (Proc.Natl.Acad.Sci.USA), 99:5616-5621 (2002)]. It has been reported that GALGT2 overexpression in skeletal muscle can induce glycosylation of α-sarcodystrophyglycans with β1-4-N-acetyl-D-galactosamine (GalNAc) carbohydrates, thereby generating the CT carbohydrate antigen (Neu5Ac / Gcα2-3[GalNAcβ1-4]Gal3Galβα1-4GlcNAcβ-). GALGT2 glycosyltransferase and the CT carbohydrates it produces are generally confined to the neuromuscular and tendon junctions in the skeletal muscle of adults, non-human primates, rodents, and all other unstudied mammals [Martin et al., J. Neurocytol., 32:915-929 (2003)]. It has been reported that overexpression of GALGT2 in skeletal muscle stimulates ectopic glycosylation of the synaptic membrane, thereby stimulating ectopic overexpression of the scaffold of normal synaptic proteins that are orthologs or homologs of proteins missing in various forms of muscular dystrophy, including dystrophin substitutes (e.g., utrophin, Plectin1) and laminin α2 substitutes (laminin α5 and aggregatein) [Xu et al., 2009, ibid.; Xu et al., J. Path., 2007, ibid.; Xu et al., Neuromuscul. Disord., 2007, ibid.; Nguyen et al., ibid.; Chicoine et al., Molecular Therapy 22:713-724, (2014).As a group, it has been reported that induction of GALGT2 by such substitutes enhances myofibril membrane integrity and prevents muscle damage in dystrophin-deficient and wild-type muscles [Martin et al., ibid.]. GALGT2 overexpression in skeletal muscle has been reported to prevent muscle damage and inhibit muscle disease. This was confirmed in mdx mouse models of DMD [Xu et al., Neuromuscular Disorders, 2007, ibid.; Martin et al. (2009), ibid.; Nguyen et al., ibid.], where improvements comparable to microdystrophin gene transfer were noted, even though only half the number of fibers were transduced [Martin et al. (2009), ibid.]. Notably, GALGT2 gene transfer has been reported in dystrophin 1A. W Model [Xu et al., American Journal of Pathology (Am.J.Path.), 2007, ibid.] and Sgca 2D type limb girdle muscular dystrophy - / - It has a preventive effect in mouse models [Xu et al., 2009, ibid.].

[0157] AAV gene therapy

[0158] This disclosure provides gene therapy vectors expressing the GNE gene, such as rAAV vectors, and methods for treating GNE myopathy.

[0159] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that is generated exclusively in cells, with some functions provided by co-infected helper viruses. Thirteen AAV serotypes have been characterized. Basic information and reviews of AAVs can be found, for example, Carter, 1989, *Handbook of Parvoviruses*, Vol. 1, pp. 169–228, and Berns, 1990, *Virology*, pp. 1743–1764, Raven Press, (New York). However, it is entirely reasonable to expect that the same principles will be applicable to additional AAV serotypes, given that it is well known that the various serotypes are closely related in both structure and function, even at the genetic level. (See, for example, Blacklowe, 1988, Parvoviruses and Human Disease, pp. 165–174, ed. JRPattison; and Rose, Comprehensive Virology 3:1–61 (1974)). For instance, all AAV serotypes exhibit remarkably similar replication characteristics mediated by homologous rep genes; and all possess three associated capsid proteins, such as those expressed in AAV2. The degree of correlation is further demonstrated by heteroduplex analysis, which reveals extensive cross-hybridization along the genome length between serotypes; and the presence of similar self-annealing fragments at the ends, corresponding to “inverted terminal repeats” (ITRs). Similar patterns of infectivity also suggest that replication function is under similar regulatory control for each serotype.

[0160] As used herein, “AAV vector” refers to a vector containing one or more target polynucleotides (or transgenes) flanked by AAV terminal repeat sequences (ITRs). When present in host cells transfected with a vector that encodes and expresses the rep and cap gene products, such an AAV vector can be replicated and packaged into infectious viral particles.

[0161] "AAV virion," "AAV virus particle," or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and a capsidated polynucleotide AAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to mammalian cells), it is generally referred to as an "AAV vector particle" or simply an "AAV vector." Therefore, the production of AAV vector particles necessarily involves the production of AAV vectors, thus the AAV vector particle contains a vector.

[0162] AAV

[0163] Adeno-associated virus (AAV) is a replication-defective parvovirus with a single-stranded DNA genome approximately 4.7 kb long, consisting of two 145-nucleotide inverted terminal repeats (ITRs). Multiple serotypes of AAV exist. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the genome of AAV serotype 2 (AAV2) was proposed in *Journal of Virology*, 45:555-564 (1983), as corrected by Ruffing et al. in *Journal of General Virology*, 75:3385-3392 (1994). As another example, the complete genome of AAV-1 is available in GenBank accession number NC_002077; the complete genome of AAV-3 is available in GenBank accession number NC_1829; the complete genome of AAV-4 is available in GenBank accession number NC_001829; the genome of AAV-5 is available in GenBank accession number AF085716; and the complete genome of AAV-6 is available in GenBank accession number NC_002077. The AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively (see also U.S. Patents 7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., Journal of Virology, 78:6381-6388 (2004); the AAV-10 genome is provided in Molecular Therapy, 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). A clone of serotype AAVrh.74 is described in Rodino-Klapac et al., Journal of Translational Medicine, 45 (2007). The cis-acting sequences that guide viral DNA replication (rep), capsidation / packaging, and host cell chromosome integration are contained in the ITR. Three AAV promoters (named p5, p19, and p40 at their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Differential splicing of the two rep promoters (p5 and p19) with a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227) results in the production of four rep proteins (rep 78, rep 68, rep 52, and rep40) from the rep gene. These rep proteins possess various enzymatic properties ultimately responsible for replicating the viral genome. The cap gene is expressed by the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3.Alternative splicing and non-shared translation initiation sites are responsible for the production of three related capsid proteins. A single shared polyadenylation site is located at position 95 on the AAV genome map. The AAV life cycle and genetics are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0164] AAV possesses unique characteristics that make it attractive as a vector for delivering exogenous DNA into cells, for example, in gene therapy. AAV infection of cultured cells is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells, allowing for the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells and can essentially prolong the lifespan of these cells as a transcriptionally active nuclear appendage (exochromosomal element). The AAV proviral genome is infectious as clonal DNA in a plasmid, enabling the construction of recombinant genomes. Furthermore, since the signals guiding AAV replication, genome capsidation, and integration are contained in the ITR of the AAV genome, approximately 4.3 kb of the internal genome (encoding replication and structural capsid proteins, rep-cap) can be partially or entirely replaced with exogenous DNA, such as gene cassettes containing promoters, DNA of interest, and polyadenylation signals. The rep and cap proteins can be provided in trans form. Another remarkable characteristic of AAV is its extremely stable and robust nature. It readily withstands the conditions used for inactivating adenovirus (56°C to 65°C for several hours), making cold storage of AAV less critical. AAV can even be freeze-dried. Finally, AAV-infected cells are intolerant of repeated infection.

[0165] Multiple studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Human Gene Therapy, 8:659-669 (1997); Kessler et al., Proceedings of the National Academy of Sciences of the United States of America, 93:14082-14087 (1996); and Xiao et al., Journal of Virology, 70:8098-8108 (1996). See also Chao et al., Molecular Therapy, 2:619-623 (2000) and Chao et al., Molecular Therapy, 4:217-222 (2001). Furthermore, due to the high vascularization of muscle, recombinant AAV transduction leads to the appearance of transgenic products in systemic circulation after intramuscular injection, as described in Herzog et al., Proc. Natl. Acad. Sci. USA, 94:5804-5809 (1997) and Murphy et al., Proc. Natl. Acad. Sci. USA, 94:13921-13926 (1997). In addition, Lewis et al., Journal of Virology, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cytokines for proper antibody glycosylation, folding, and secretion, indicating that muscle can stably express secreted protein therapeutic agents.

[0166] The recombinant AAV genome disclosed herein comprises the nucleic acid molecule of the present invention and one or more AAV ITRs of the side-attached nucleic acid molecule. The AAV DNA in the rAAV genome can be derived from any AAV serotype from which recombinant viruses can be derived, including but not limited to AAV serotypes AAVrh.74, AAVrh.10, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also considered. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To activate skeletal muscle-specific expression, AAV1, AAV6, AAV8, AAV9, AAVrh10, or AAVrh.74 can be used.

[0167] The DNA plasmid disclosed herein contains the rAAV genome of this disclosure. The DNA plasmid is transferred into cells that are permissible for infection with AAV helper viruses (e.g., adenovirus, E1-deficient adenovirus, or herpesvirus) to assemble the rAAV genome into infectious viral particles. Techniques for producing rAAV particles are standard in the art, wherein the AAV genome to be packaged, the rep and cap genes, and helper viral functions are provided to the cell. The production of rAAV requires the following components to be present within a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separate from (i.e., not present in) the rAAV genome, and the helper viral functions. The AAV rep and cap genes can originate from any AAV serotype from which recombinant viruses can be derived, and can originate from AAV serotypes that differ from the rAAV genomic ITR, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAVrh.10, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAVs is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0168] The method for generating packaging cells involves creating cell lines that stably express all the essential components for AAV particle production. This can be achieved by integrating plasmids (or multiple plasmids) containing the rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and optional markers such as neomycin resistance genes into the cell's genome. The AAV genome has been introduced into bacterial plasmids via procedures such as GC tailing (Samulski et al., 1982, Proceedings of the National Academy of Sciences, 79:2077-2081), by adding synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or by direct blunt-end ligation (Senapathy and Carter, 1984, Journal of Biochemistry, 259:4661-4666). The packaging cell lines are then infected with helper viruses such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0169] The general principles of rAAV generation are commented on, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial and Immunol., 158:97-129. Various methods are described in Ratschin et al., Molecular and Cellular Biology, 4:2072 (1984); Hermonat et al., Proceedings of the National Academy of Sciences, 81:6466 (1984); Tratschin et al., Molecular and Cellular Biology, 5:3251 (1985); McLaughlin et al., Journal of Virology, 62:1963 (1988); and Lebkowski et al., Molecular and Cellular Biology, 7:349 (1988). Samulski et al., *Journal of Virology*, 63:3822-3828 (1989); US Patent No. 5,173,414; WO 95 / 13365 and corresponding US Patent Nos. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO The references cited are: 99 / 11764; Perrin et al., Vaccine, 13:1244-1250 (1995); Paul et al., Human Gene Therapy, 4:609-615 (1993); Clark et al., Gene Therapy, 3:1124-1132 (1996); U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595. These references are incorporated herein by reference in their entirety, with particular emphasis on those relating to the generation of rAAV.

[0170] Therefore, this disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (homogeneous 293 lineage). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passaged 293 cells (human embryonic kidney cells transformed with adenovirus E1), MRC-5 cells (human embryonic fibroblasts), WI-38 cells (human embryonic fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus monkey embryonic lung cells).

[0171] The provided recombinant AAV (i.e., infectious capsidated rAAV particles) contains an rAAV genome. In an exemplary embodiment, the genomes of both rAAVs lack AAV rep and cap DNA; that is, there is no AAV rep or cap DNA between the ITRs of the genome.

[0172] In one exemplary embodiment, the recombinant AAV is generated via a triple transfection method (Xiao et al., Journal of Virology 72, 2224-2232 (1998)) using an AAV vector plasmid containing the GNE gene and the muscle-specific promoter elements pNLRep2-Caprh74 and pHelp. The rAAV contains a GNE gene expression cassette flanked by inverted terminal repeats (ITRs) of AAV2. It is this sequence that is capsidred into the AAVrh74 viral particle. The plasmid contains the GNE sequence and the muscle-specific promoter elements and core promoter elements of the muscle-specific promoter to drive gene expression. The expression cassette also contains SV40 introns (SD / SA) to promote high levels of gene expression, and bovine growth hormone polyadenylation signaling for efficient transcription termination.

[0173] pNLREP2-Caprh74 is an AAV helper plasmid that encodes four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins derived from serotype rh74.

[0174] The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. The plasmid contains adenovirus genomic regions important for AAV replication, namely E2A, E4ORF6, and VA RNA (adenovirus E1 function was provided by 293 cells). The adenovirus sequence present in this plasmid represents only about 40% of the adenovirus genome and does not contain cis-elements crucial for replication, such as adenovirus terminal repeat sequences. Therefore, infectious adenovirus is not expected to be generated from this production system.

[0175] rAAV can be purified using standard methods in the art, such as column chromatography or cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art and include, for example, Clark et al., Human Gene Therapy, 10(6):1031-1039 (1999); Schenpp and Clark, Methods in Molecular Medicine, 69 427-443 (2002); U.S. Patent Nos. 6,566,118 and WO 98 / 09657.

[0176] In another embodiment, this disclosure contemplates compositions comprising the rAAV of this disclosure. The compositions of this disclosure comprise rAAV and a pharmaceutically acceptable carrier. The compositions may also comprise other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to the recipient and are preferably inert at the doses and concentrations used, including buffers and surfactants such as pluronics.

[0177] In the methods disclosed herein, the titer of rAAV to be administered will vary depending on, for example, the specific rAAV, the route of administration, the treatment target, the individual, and one or more targeted cell types, and can be determined using methods standard in the art. The rAAV titer range can be approximately 1 × 10⁻⁶ per milliliter. 6 Approximately 1×10 7 Approximately 1×10 8 Approximately 1×10 9 Approximately 1×10 10 Approximately 1×10 11 Approximately 1×10 12 Approximately 1×10 13 To approximately 1×10 14 More or more DNase-resistant particles (DRPs). The dose can also be expressed in units of viral genome (vg). An exemplary method for determining the capsid vector genome titer is to use quantitative PCR, for example, the method described in (Pozsgai et al., Molecular Therapy 25(4):855-869, 2017).

[0178] This disclosure contemplates methods for transducing target cells with rAAV in vivo or in vitro. In vivo methods include the step of administering an effective dose or multiple effective doses of a composition comprising the rAAV of this disclosure to an animal (including a human) in need. If the dose is administered before the onset of a symptom / disease, the administration is preventative. If the dose is administered after the onset of a symptom / disease, the administration is therapeutic. In embodiments of this disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with a treated symptom / disease state, slows or prevents progression to a symptom / disease state, slows or prevents the progression of a symptom / disease state, reduces the severity of the disease, causes remission of the disease (partial or complete), and / or prolongs survival. An example of a disease contemplated for prevention or treatment using the methods of this disclosure is GNE myopathy.

[0179] This disclosure also considers combination therapies. Combinations, as used herein, include both simultaneous and sequential treatment. Particular consideration is given to combinations of the methods disclosed herein with standard medical treatments (e.g., corticosteroids), as well as combinations with novel therapies.

[0180] The composition can be administered at effective doses via conventional routes in the art, including but not limited to intramuscular, parenteral, intravenous, intra-arterial, oral, oral, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal routes. Considering the infection and / or the disease state being treated, and the presence of one or more target cells / tissues expressing UDP-GlcNAc-epimerase / ManNAc-6 kinase protein and follicle-suppressing hormone 344, follicle-suppressing hormone 317, or insulin-like growth factor 1, those skilled in the art can select and / or match one or more routes of administration and one or more serotypes of the AAV components (particularly AAV ITR and capsid protein) of the rAAV disclosed herein.

[0181] This disclosure provides effective doses of rAAV and topical and systemic administration of the compositions of this disclosure. For example, systemic administration is administration to the circulatory system, so that the whole body is affected. Systemic administration includes enteral administration, such as through gastrointestinal absorption, and parenteral administration via injection, infusion, or implantation.

[0182] In particular, the actual administration of the rAAV of this disclosure can be achieved by any physical method of delivering the recombinant rAAV vector to the target tissue of an animal. Administration according to this disclosure includes, but is not limited to, injection into muscle and injection into the bloodstream. Simply resuspending rAAV in a phosphate-buffered saline solution has been shown to be sufficient to provide a vehicle for expression in muscle tissue, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should generally be avoided when used with rAAV). The capsid protein of rAAV can be modified to target specific target tissues of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be formulated as injectable or topical formulations that can be delivered to muscle via transdermal transport. Many formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practices of this disclosure. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0183] In the methods disclosed herein, the dose of rAAV to be administered will vary depending on, for example, the specific rAAV, the route of administration, the therapeutic target, the individual, and one or more targeted cell types, and can be determined using methods standard in the art. The titer range for each rAAV administered can be from approximately 1 × 10⁻⁶ per milliliter. 6 Approximately 1×10 7 Approximately 1×10 8 Approximately 1×10 9 Approximately 1×10 10 Approximately 1×10 11 Approximately 1×10 12 Approximately 1×10 13 Approximately 1×10 14 Approximately 2×10 14 Or approximately 1×10 15 More or more DNase-resistant particles (DRP). Dosage can also be expressed in units of viral genome (vg) (i.e., 1 × 10⁻⁶, respectively). 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 vg, 2×10 14 vg, 1×10 15 The dosage can also be expressed in units of viral genome (vg) per kilogram (kg) of body weight (i.e., 1 × 10⁻⁶). 10 vg / kg, 1×10 11vg / kg, 1×10 12 vg / kg, 1×10 13 vg / kg, 1×10 14 vg / kg, 1.25×10 14 vg / kg, 1.5×10 14 vg / kg, 1.75×10 14 vg / kg, 2.0×10 14 vg / kg, 2.25×10 14 vg / kg, 2.5×10 14 vg / kg, 2.75×10 14 vg / kg, 3.0×10 14 vg / kg, 3.25×10 14 vg / kg, 3.5×10 14 vg / kg, 3.75×10 14 vg / kg, 4.0×10 14 vg / kg, 1×10 15 The method for titrating AAV is described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0184] For intramuscular injection, adjuvants such as sesame oil or peanut oil or aqueous propylene glycol, as well as sterile aqueous solutions, can be used. If desired, such aqueous solutions can be buffered, and the liquid diluent should first be isotonic with saline or glucose. rAAV solutions as free acids (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared in water appropriately mixed with a surfactant (e.g., hydroxypropyl cellulose). Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and oils. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In this regard, the sterile aqueous media used are readily available using standard techniques well known to those skilled in the art.

[0185] Suitable drug carriers, diluents, or excipients for injectable use include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and its flowability must be sufficient to allow for smooth injection. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. For example, suitable flowability can be maintained by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, will be preferred. Extended absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0186] As needed, a sterile injectable solution is prepared by incorporating rAAV in the desired amount into a suitable solvent containing the various other components listed above, followed by filtration sterilization. Typically, a dispersion is prepared by incorporating the sterilized active ingredient into a sterile carrier containing a base dispersion medium and any other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient plus any additional desired components from their previously sterile filtered solutions.

[0187] rAAV transduction can also be performed in vitro. In one implementation, desired target muscle cells are removed from the subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or allogeneic muscle cells can be used, where these cells will not elicit an inappropriate immune response in the subject.

[0188] Suitable methods for transducing and reintroducing transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells (e.g., in a suitable culture medium) and those cells with DNA of interest can be screened using conventional techniques (e.g., Southern blotting and / or PCR) or by using selection markers. The transduced cells can then be formulated into a pharmaceutical composition and the composition can be introduced into a subject using various techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth muscle and myocardium using, for example, a catheter.

[0189] Transduction of the rAAV cells disclosed herein results in sustained expression of the UDP-GlcNAc-epimerase / ManNAc-6 kinase protein. Therefore, this disclosure provides methods for administering / delivering rAAVs expressing the UDP-GlcNAc-epimerase / ManNAc-6 kinase protein to animals, preferably humans. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more of the rAAVs disclosed herein. Transduction can be performed using a gene cassette containing tissue-specific control elements. For example, one embodiment of this disclosure provides a method for transducing muscle cells and muscle tissue directed by muscle-specific promoter elements, said control elements including, but not limited to: control elements derived from actin and myosin gene families, such as those derived from the myoD gene family (see Weintraub et al., Science, 251:761-766 (1991)); muscle cell-specific enhancer-binding factor MEF-2 (Cserjesi and Olson, Molecular Cell Biology 11:4854-4862 (1991)); actin derived from human skeletal muscle genes (Muscat et al., Molecular Cell Biology 7:4089-4099 (1987)); and myocardial actin. Control elements of protein genes; muscle creatine kinase sequence elements (see Johnson et al., Molecular Cell Biology, 9:3393-3399 (1989)) and mouse creatine kinase enhancer (MCK) elements; control elements derived from the skeletal fast twitching troponin C gene, slow twitching cardiac troponin C gene, and slow twitching troponin I gene: hypoxia-induced nuclear factor (Semenza et al., Proceedings of the National Academy of Sciences, 88:5680-5684 (1991)); steroid-induced elements and promoters including glucocorticoid response elements (GRE) (see Mader and White, Proceedings of the National Academy of Sciences, 90:5603-5607 (1993)); and other control elements.

[0190] Muscle tissue is an attractive target for DNA delivery in the body because it is not a vital organ and is easily accessible. This disclosure considers the sustained expression of UDP-GlcNAc-epimerase / ManNAc-6 kinase from transduced muscle fibers.

[0191] "Muscle cells" or "muscle tissue" refers to cells or groups of cells that originate from any type of muscle (e.g., skeletal muscle and smooth muscle, such as tissue from the digestive tract, bladder, blood vessels, or heart). These muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardioblasts.

[0192] The term "transduction" is used to refer to the administration or delivery in vivo or in vitro of the coding region of GNE to recipient cells via the replication-defective rAAV of this disclosure, resulting in the expression of UDP-GlcNAc-episomerase / ManNAc-6 kinase in the recipient cells.

[0193] The examples provided are for illustrative purposes only and not for limitation. The described numerical range includes every integer value within each range, and includes the lowest and highest integers stated.

[0194] Example

[0195] Example 1

[0196] Construct or cDNA of the GalNAc transferase gene encoding GlcNAc epimerase / ManNAc kinase

[0197] The following exemplary DNA construct encoding UDP-GlcNAc-epimerase / ManNAc-6 kinase is generated as follows:

[0198] rAAVrh74.CMV.GNE (Variant 2) Figure 1A The polynucleotide listed in Figure 2 (SEQ ID NO:12) is encoded by the polynucleotide listed in Figure 2.

[0199] rAAVrh74.MCK.GNE (Variant 2) Figure 1B The polynucleotide listed in Figure 3 (SEQ ID NO:13) is encoded by the polynucleotide.

[0200] rAAVrh74.MHCK7.GNE (Variant 2) Figure 1C The polynucleotide listed in Figure 4 (SEQ ID NO:14) is encoded by the polynucleotide.

[0201] rAAVrh74.GNE promoter.GNE (variant 2) in Figure 1D The polynucleotide listed in Figure 5 is encoded by (SEQ ID NO:15).

[0202] rAAVrh74.MHCK7.GNE(variant 2).FGFIIRES.FS344 in Figure 1E The polynucleotide listed in Figure 6 (SEQ ID NO:16) is encoded by the polynucleotide.

[0203] rAAVrh74.MHCK7.GNE(variant 2).FGF1 IRES.HB-IGF1 in Figure 1F The polynucleotide listed in Figure 7 (SEQ ID NO:17) is encoded by the polynucleotide.

[0204] rAAVrh74.CVM.GNE(variant 2).FGF1IRES.FS344 in Figure 1G The polynucleotide listed in Figure 8 (SEQ ID NO:18) is encoded by the polynucleotide.

[0205] rAAVrh74.CMV.GNE(variant 2).FGF1 IRES.HB-IGF1 in Figure 1H The polynucleotide listed in Figure 9 (SEQ ID NO:19) is encoded by the polynucleotide.

[0206] rAAVrh74.MCK.GNE(variant 2).FGF1IRES.FS344 in Figure 1I The polynucleotide listed in Figure 10 is encoded by (SEQ ID NO:20).

[0207] rAAVrh74.MCK.GNE(variant 2).FGF1 IRES.HB-IGF1 in Figure 1J The polynucleotide listed in Figure 11 (SEQ ID NO:21) is encoded by the polynucleotide.

[0208] rAAVrh74.GNE promoter.GNE(variant 2).FGFIIRES.FS344 in Figure 1K The polynucleotide listed in Figure 12 is encoded by (SEQ ID NO:22).

[0209] rAAVrh74.GNE promoter.GNE (variant 2).FGF1 IRES.HB-IGFI in Figure 1L The polynucleotide listed in Figure 13 (SEQ ID NO:23) is encoded by the polynucleotide.

[0210] rAAVrh74, Mini CMV.GNE Figure 1M The polynucleotide listed in Figure 14 (SEQ ID NO:24) is encoded by the polynucleotide.

[0211] rAAVrh74.Mini CMV.GNE(Variant 2).FGF1IRES.FS344 Figure 1N The polynucleotide listed in Figure 15 is encoded by (SEQ ID NO:25).

[0212] rAAVrh74, Mini CMV.GNE (Variant 2).FGF1.IRES.HB-IGF1 in Figure 10 The polynucleotide listed in Figure 16 (SEQ ID NO:26) is encoded by the polynucleotide.

[0213] Furthermore, the following exemplary DNA construct encoding the GalNAc transferase rAAVrh74.MCK.GALGT2.FGF1IRES.FS344 was generated, which in Figure 1P The polynucleotide listed in Figure 17 (SEQ ID NO:38) is encoded by the polynucleotide.

[0214] The disclosed plasmids contain human GNE cDNA or GATGT2 expression cassettes flanked by AAV2 inverted terminal repeat (ITR) sequences. These cassettes may also contain FGFIIRES and a second transgene that induces muscle growth, such as folliculorin 344 or HB-IGF1. Expression of the GlcNAc epimerase / ManNAc kinase protein or GalNAc transferase protein is directed by CMV, MCK, MHCK7, mini CMV, or GNE promoters. CMV is a cytomegalovirus promoter (SEQ ID NO:3). MCK is a muscle creatine kinase promoter (CK7-like) (SEQ ID NO:4). MHCK7 is an MCK promoter with an additional enhancer (SEQ ID NO:5). Mini CMV is a smaller version of the CMV promoter (SEQ ID NO:7). GNE variant 2 is a cDNA variant 2 of the GlcNAc epimerase / ManNAc kinase gene that encodes a 722-amino acid protein starting from exon 3 (NM_005476; SEQ ID NO:1). GALGT2 is the cDNA of the GALGT2 (or B4GALNT2) gene (GenBank accession number #AJ517771; SEQ ID NO:36). Mini FGF1IRES represents the smallest FGFI internal ribosome entry site (SEQ ID NO:8). FS344 is the 344-amino acid form of follistatin (SEQ ID NO:10). HB-IGF1 is the signal peptide and protopeptide domain of human heparin-binding epidermal growth factor-like growth factor, which is linked to exons 1-4 of insulin-like growth factor 1 (SEQ ID NO:11). The GNE promoter (SEQ ID NO:6) represents the designated 5' sequence element immediately adjacent to exon 2, which should be used to drive the expression of variant 2GNE transcripts.

[0215] Wild-type human GNE is 2.2kB cDNA, therefore some implementations may require shortened FGF1AIRES. This shortened FGF1AIRES can be as small as 100bp to accommodate the 4.7kB encapsulation limit of AAV for the FST (1.3kB). The shortened CMV promoter (220bp instead of 800bp) is referred to as mini-CMV in this paper; if it causes problems when working, a longer IRES sequence will be required.

[0216] The GNE cDNA expression cassette or GATGT2 cDNA expression cassette contains a kanamycin resistance gene, along with an optimized Kozak sequence for more robust transcription. The rAAV vector was generated using a modified cross-packaging method, allowing the AAV type 2 vector genome to be packaged into multiple AAV capsid serotypes [Rabinowitz et al., Journal of Virology 76(2):791-801(2002)]. Production was performed using HEK293 cells via a standard three-plasmid DNA / CaPO4 precipitation method. HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin. The production plasmids were: (i) a plasmid encoding a therapeutic protein; (ii) a rep2-capX modified AAV helper plasmid encoding the cap serotype AAVrh74 isolate; and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing the adenovirus E2A, E4 ORF6, and VA I / II RNA genes. The encapsulated vector genome vg titer was determined using a qPCR-based titration method with a Prism 7500 Taqman detector system (PE Applied Biosystems). [Clark et al., Human Gene Therapy, 10(6):1031-1039(1999)]. Final titer (vg / ml) -1 Quantitative reverse transcriptase PCR was performed using specific primers and probes with a Prism 7500 real-time detector system (PE Applied Biosystems, Grand Island, NY, USA). Aliquots of virus were held at -80°C until...

[0217] All plasmids used to create the AAV genome to be encapsulated contain a kanamycin resistance gene (KanR) in addition to the ITR sequence used for encapsulation. This allows the DNA encoding the AAV genome to be converted into bacteria in the presence of kanamycin to produce a large amount of DNA, which kills all unconverted bacteria. KanR is not encapsulated into the AAV capsid in the AAV genome used to treat patients, but its presence enables the production of DNA within the bacteria.

[0218] Example 2

[0219] Expression and Testing

[0220] The vector genome of the AAV vectors was tested by transfecting the AAV vectors rAAV.CMV.GNE.mini-IRES.GFP and rAAV.mini-CMV.GNE.full-length (FL)IRES.GFP into GNE-deficient Lec3 CHO cells (Lec3) to demonstrate that the vectors described in Example 1 express GFP and a second protein. The mini-IRES is a further shortened version of the IRES, shown as SEQ ID NO:7. Figure 20 As shown, the presence of mini-IRES in the vector genome allows for the expression of a second protein (GFP) downstream of the IRES. Figure 20 In this study, GFP exhibits endogenous fluorescence, while GNE expression is revealed through immunostaining. For example... Figure 21 As shown, the full-length IRES also allows expression of a second gene (GFP). Figure 23 As shown, when GNE is introduced into Gne-deficient Lec3 cells, and IRES produces a second protein (GFP in this case), GNE can produce sialic acid.

[0221] Figure 24 This demonstrates that any transgene of appropriate size can serve as a gene substitution or replacement gene in the first position. C2C12 cells were transfected with the AAV vector rAAV.MCK.GALGT2.IRES.FS344, which expresses GALGT2, a replacement gene for the dystrophin protein in Duchenne muscular dystrophy. Expression of GALGT2 (stained green) and FST (stained red) was observed in the same cells. The inclusion of IRES allows for the production of muscle growth factors in the same cells, in this case, follicle-stimulating hormone (FS344 or FST).

[0222] For further analysis, any of the AAV vectors described in Example 1 were tested in muscle cells and GNE-deficient CHO cells (Lec3) to demonstrate their function. AAV vectors were added at varying doses, from 10 MOI (multiple of infection) to 10,000 MOI, in logarithmic increments. High MOIs are generally required for AAV to infect cultured cells because AAV functions significantly better in vivo than in vitro. C2C12 myoblasts and C2C12 myotube cultures, as well as CHO-K1 (wild-type) cells and Lec3 cells (a CHO cell variant lacking Gne activity), were infected with the provided AAV vectors.

[0223] In vivo functional tests were performed in Gne-deficient mice, where GNE gene correction was tested by demonstrating UDP-GlcNAc epimerase activity or by measuring free or membrane-bound sialic acid. These measurements were performed using known standard gas chromatography-mass spectrometry or by quantitative lectin staining with sialic acid-bound Sophora japonica or Sambuca Nigra lectin. Determination of Gne enzyme activity, such as UDP-GlcNAc epimerase activity, can also define gene substitution. The induction of muscle growth by FST and IGF1 was determined by weighing limb muscles and comparing them to the animal's total weight (e.g., see...). Figure 22 ); measuring the area and number of skeletal muscle fibers by slicing muscle and using hematoxylin and eosin staining of thin sections combined with morphometric software; and by physiological measurements of muscle strength, including, for example, in vitro measurements of grip strength, walking strength, and specific force in the tibialis anterior or extensor digitorum longus muscles.

[0224] Cells were stained with MAA or SNA (conjugated with Cy3) to assess sialylation, and GNE, FST, or IGF1 antibodies were used to assess protein co-expression. As previously described, the same constructs were infected in larger cell cultures to assess protein expression by Western blotting and ELISA. (Haidet et al., Proceedings of the National Academy of Sciences 105(11):4318-22, 2008; Hennebry et al., Journal of Molecular Endocrinology 234:187-200, 2008). As previously described, changes in signal transduction were assessed by immunostaining and Western blotting, particularly decreased phosphate-Smad 2 levels and increased phosphate-Akt (for IGF1) levels in FST (Chandraskeharen et al., Muscle Nerve 39(1):25-41, 2008; Cramer et al., Molecular Cell Biology 39(14), 2019). In all cases, gene expression was assessed by qRT-PCR, and AAV biodistribution was assessed by qPCR, as previously described by Xu et al. (Molecular Therapy, 2019). We have identified the ideal IGF1 splicing form for muscle growth (ns).

[0225] The bicistronic vector described in Example 1 allows for the expression of GNE protein as well as follicle-stimulating hormone (FSH) or IGF1 protein derived from the same mRNA. Infection with muscle cultures allowed for greater IRES-mediated bicistronic expression, as FGF1AIRES showed a greater effect in muscle than in non-muscle cell lines. GNE expression in Lec3 cells increased sialylation because these cells lack Gne enzyme activity, which is equal to or greater than SA levels in normal CHO-K1 cells.

[0226] As shown in Figure 4, both muscle- and liver-specific expression of GNE contributes to the expression of SA in muscle. In GNED176VTgGne - / - Sialic acid staining was performed on the liver and muscle of mice following intramuscular or intraperitoneal injection of rAAVrh74.MCK.GNE. Sialic acid staining in muscle and liver was shown in time-matched images 6 months after intramuscular injection of the muscle-specific GNE gene therapy vector (IM) or intraperitoneal delivery of the liver-specific GNE gene therapy vector (IP), both at a dose of 5 × 10⁻⁶. 11 vg. qRT-PCR showed that MCK expression increased 30-fold in muscle but not in the liver, while LSP expression increased 8-fold in the liver but not in muscle (ns). After 6 months, MCK increased muscle SA, but LSP increased more, possibly due to the deposition of serum glycoproteins secreted by the liver in the muscle extracellular matrix.

[0227] To demonstrate that transducing muscle cells with the rAAV vector leads to muscle growth, 1 × 10⁻⁶ rAAV vectors were injected into the anterior tibial (TA) muscle of C57Bl / 6J mice. 11 vg (vector genome), and 5 × 10⁻⁶ was injected intramuscularly into the gastrocnemius muscle. 11 vg expresses AAV in the form of insulin-like growth factor 1 (IGF1, muscle form Ea), HB-IGF1, or follicle-stimulating hormone (FST) 344. Two months post-injection, muscle dissection and weighing showed a significant increase in HB-IGF1 and FST344 in TA and in FST344 in Gastroc compared to buffer injection alone (see [link to relevant documentation]). Figure 21 ).

[0228] Example 3

[0229] Mouse model of GNE function in adult mice

[0230] A mouse model of GNE myopathy was generated by introducing the fluxed Gne allele into exon 3 of the mouse Gne gene. The introduction of this allele was sufficient to allow Cre-mediated deletion, thus producing a GNE myopathy-like phenotype. Research on GNE myopathy has long been hampered by a lack of disease models. GNED176VTgGne - / - Mice were first reported as a good late-onset model of GNE myopathy (Malicdan et al., Human Molecular Therapy 16(22):2669-82, 2007; Malicdan et al., Nature Medicine 15(6):690-5, 2009), but after further breeding, these mice lost most of the phenotype, while GNE... M712T(now GNE) M743T Knocking in the Persian Jew mutant in mice resulted in lethality, partly due to renal insufficiency, while other strains in the same lineage did not exhibit any phenotype at all (Sela et al., Neuromolecular Medicine 15(1):180-91, 2013ll). Since Gne is essential in mice, causing lethality between E8.5 and E9.5, creating a liquid oxygen allele in adult mice to delete this gene should allow for the creation of a robust systemic or muscle-specific phenotype using Cre-mediated deletion. In turn, this would allow for more reproducible evidence of therapeutic effects.

[0231] Cas9-CRISPR was used to delete exon 3 of the mouse Gne gene, the site where the UDP-GlcNAc epimerase domain begins and contains the translation initiation site of the Gne gene. Fertilized oocytes were injected with Cas9-CRISPR, associated guide RNA, and a long DNA oligonucleotide that allowed recombination to produce a new exon 3 flanked by loxP recombination sites. The resulting exon was cultured for two generations and then shipped by a supplier (the Mouse Biology Program at UC Davis) for further analysis.

[0232] In one injection session of 80 mice, two Gne-deleted exon 3 deletion founders were generated from 26 live mice (although no liquid oxygen founders were generated). Figure 19 This was followed by another round of injections into 160 mice. If successful, Cre was expressed systematically via IV tail vein injection using rAAVrh74.CMV.Cre-GFP, or rAAVrh74.MCK.Cre-GFP was used solely to delete Gne in skeletal muscle (and heart). These experiments provide a way to understand how Gne deletion in adult mice leads to a disease phenotype. Although qPCR results showed the absence of the liquid oxygen allele bordering exon 3 in these founders, they could still be used to produce Gne. - / - Mice. These mice also demonstrated that the guide RNA used could indeed achieve Cas9-CRISPR deletion of Gne exon 3.

[0233] Currently available assays for detecting disease phenotypes are available. For example, to understand the loss of sialylation, MAA and SNA lectin staining are used to visualize sialyl expression (using endogenous Cre-GFP to see which cells express Cre), which bind to α2,3- and α2,6-linked SA, respectively. qRT-PCR is used to understand the loss of Gne gene expression (and the increase of Cre-GFP gene expression). qPCR is used to understand the number of vector genomes present in each cell nucleus in each muscle tissue and the extent of gene deletion. For methods, see Kim et al., *Molecular Cell Neurosci.* 39(3):452-64, 2008) and Xu et al., *Molecular Therapy*, 2019. GC-MS / MS methods can also be used to measure total free sialyl and total glycoprotein-conjugated N- and O-linked sialyl, see Yoon et al. (*PLoS Currents*, 2013). Finally, Gne enzyme activity, whether UDP-GlcNAc epimerase activity or ManNAc6 kinase activity, can be used to measure the degree of functional gene substitution.

[0234] Analysis of muscle symptoms included staining thin sections with hematoxylin and eosin, trichrome and Congo red. Measurements included the number of inclusion bodies, myofiber size, central nucleus, changes in myofiber size, fibrosis and non-muscular areas (wasting), see Chandraskehren et al., Muscle Nerve 39(1):25-41, 2008. If inclusion bodies were found, their ultrastructure was evaluated using electron microscopy. Muscle function was assessed by measuring grip strength, walking (treadmill walking), open field test and isolated specific force and force drop during repetitive contractions (in TA and EDL), see (Chandraskehren et al., Muscle Nerve 39(1):25-41, 2008; Martin et al., American Journal of Physiology, 296:C476-88, 2009).

[0235] Liquid oxygen-treated Gne mice were injected with either a mimic (control) or 1×10 at 2 months of age. 14 Vg / kg AAV.CMV.Cre-GFP or AAV.MCK.Cre-GFP were analyzed at 1, 2, and 4 months post-injection. Six mice (3 males and 3 females) were injected in each group, with age-matched dummy mice and wild-type mice serving as controls.

[0236] If the above experiments did not produce any liquid oxygen-deficient mouse founders during these injections, then the two Gne-deficient founders were breto homozygous under the ManNAc condition at 2 g / kg / day, which is GNE M743T Model and Gne - / - Salivation and lethality were salvaged in the model. Here, mice were given 2–4 g / kg / day of ManNAc in water from conception. Once the pups were weaned, ManNAc was removed and tested via gene therapy, thus essentially creating an inducible Gne knockout model. These mice were not allowed to have muscle-specific Gne loss, but could be treated with AAV.CMV.GNE upon ManNAc withdrawal. M712T Or AAV.CMV.GNE D207V These mice can be rescued and tested for muscle-specific diseases when needed. If necessary, wild-type or GNE alleles can also be downregulated using microRNAs or siRNAs targeting mouse and / or human GNE alleles. + / - Endogenous Gne gene expression in mice. While such experiments encounter the same problems as previous transgenic and knock-in models, the ability to administer different amounts of the GNE mutant to mice allows for greater control.

[0237] Example 4

[0238] In vitro AAV.GNE potency assay

[0239] The MAA-HRP ELISA allows for comparison of sialic acid levels between Gne-expressing CHO cells and Gne-deficient Lec3 cells, and this assay should be sufficient to determine the efficacy of AAV.GNE after infecting Lec3 cells with different concentrations of AAV.GNE.

[0240] Any gene therapy clinical development program must include a potency assay that effectively describes the biological activity of the AAV vector to be used; in this case, it is a potency assay of the biological activity of the AAV.GNE gene therapy vector. This assay will be performed annually on clinical batches of AAV to demonstrate that activity has not been lost, and will also be performed to demonstrate that the AAV used in patients has the necessary biological activity at the time of administration.

[0241] Different amounts of AAV.GNE were infecting Gne-deficient Lec3 (mutant CHO) cells (Hong et al., *Journal of Biochemistry*, 278:53045-530454, 2003) to induce Lec3 sialylation to a measured level found in equivalent amounts of normal CHO cells, thereby demonstrating the bioactivity of the AAV vector. This procedure was performed using sophora japonica lectin (MAA) bound to α2,3-linked sialic acid (Song et al., 286:31610-31622, 2011). This assay can be applied to any number of GNE-containing gene therapy vectors.

[0242] In the MAA-HRP binding ELISA assay (ns), Lec3 cells fed with 10% serum-containing medium showed no difference from normal CHO cells. However, feeding Lec3 cells with Opti-MEM medium (a defined serum-free medium) for 3 days eliminated most MAA binding, while CHO cells maintained their MAA signaling. Figure 25 This is because free sialic acid (SA) in serum is absorbed by the cells and bound to lipids and glycoproteins, thus circumventing the Gne deficiency in Lec3 cells. This circumvention can only be reversed by removing serum from the culture medium used to feed the cells. For example, infecting Lec3 cells fed with Opti-MEM for two days with rAAVrh74.CMV.GNE allows for cell growth at 10... 5 Or 10 6 At MOI (multiple of infection) doses, MAA binding signals are partially restored. Figure 25 Additional optimization work may be required (i.e., variations in AAV infection time, time variations in Lec3 cells in Opti-MEM, or variations in the AAV dose used) to amplify the signal difference in this assay. In any case, the efficacy of the AAV.GNE vector can be determined by adding different amounts of AAV to Lec3 cells and defining efficacy as the dose required to restore signal in normal (or semi-normal) CHO cells. Figure 23As shown. When Lec3 cells were transfected with an AAV plasmid containing CMV.GNE and co-stained with GNE protein and MAA, we found that GNE-expressing Lec3 cells actually secreted sialylated glycoprotein cells, in which MAA could bind to non-GNE-expressing cells. Therefore, this power assay may be more sensitive than assays using GNE protein or gene levels as a standard due to the trans-effect of secreted SA-containing proteins. To test this assay, CHO cells and Lec3 cells were transferred to 96-well ELISA plates at 10,000 cells / well, with three copies of each well used for each condition. Cells were fed Opti-MEM for one day, then Opti-MEM again, and allowed to regrow for two days with or without AAV. During this period, some cells were infected with different doses of rAAV containing GNE cDNA. Note that any AAV serotype can be used for these assays. Routine measurements of MOI were used for different levels of AAV infection, including 1 × 10⁻⁶ cells / well. 4 5×10 4 1×10 5 5×10 5 1×10 6 5×10 6 and 1×10 7 It is worth noting that AAV is not very efficient at infecting cells grown in culture. This is quite different from its strong ability to infect tissue cells. Therefore, relatively high concentrations of virus are required. However, since the number of cells to be infected is small, the assay still uses only a very small amount of virus per assay.

[0243] Following infection, cells were washed in phosphate-buffered saline (PBS), fixed in PBS solution containing 4% paraformaldehyde for 20 minutes, and then washed again in PBS. Cells were then blocked in PBS containing 1% fish gelatin (without sialic acid) for 1 hour, incubated with 2 mg / mL Sophora japonica lectin conjugated with horseradish peroxidase (MAA-HRP) for 1 hour, and washed three times for 10 minutes each time in PBS. MAA-HRP binding was detected using a standard HRP activity (OPD) colorimetric assay, which involved color development for 20 minutes followed by quenching in acid for 10 minutes. Absorbance (color) was read at 450 nm using a SprectraMax plate reader.

[0244] Concentration curves (2 μg / mL) used to determine the optimal MAA-HRP concentration for this assay have been generated. This MAA-HRP concentration produced OD readings of 1.0 or higher in CHO cells, while significantly reducing OD levels in Lec3 cells (e.g., see [reference needed]). Figure 25Concentration curves were used to compare measurements in the following cell types: uninfected low-signal Lec3 cells; Lec3 cells infected with AAV.GNE, which should show a dose-responsive increase in signal; and CHO cell levels, which should have a high signal, our standard for full bioactivity. The MOI at which the signal is obtained (or halfway down the signal, depending on the ease of reproducibility) is found in CHO cells is defined as the dose required to deliver potency. These measurements were repeated at least six times, with three measurements used for each data point, and internal and inter-measurement variability was determined for each repeated measurement. AAV concentrations were adjusted as needed to more rigorously define the MOI required to deliver full potency where necessary.

[0245] When the rAAV vector contains a muscle-specific promoter, such as the MCK and GNE cDNA sequences, a GNE-deficient myoblast cell line can be used. Other “muscle-specific” promoters, such as MHCK7, will function in CHO cells, but MCK will not. Gne-deficient myoblasts can be obtained from other NDF researchers, or, if necessary, such cell lines can be generated by deleting GNE from human cells using Cas9-CRISPR. Gne-deficient myoblasts can also be generated from primary cells cultured from Gne-deficient mice using the method described by Xia et al. in Developmental Biology (Dev.Biol.) (242:58-73, 2002). Positive controls from normal wild-type mice can also be used for this assay. It is important to understand that the cells used for the potency assay do not need to be human cells; it is sufficient to define sialic acid as cells that are absent or significantly reduced due to Gne gene deficiency compared to controls.

[0246] Example 5

[0247] In vivo AAV.GNE potency assay

[0248] Wild-type mice were used to define the AAV.GNE potency in tissues by measuring UDP-GlcNAc epimerase activity. Any gene therapy clinical development program must also include a potency assay that effectively describes the bioactivity of the AAV.GNE vector to be used in tissues. Because GNE enzyme activity shows product inhibition from CMP-Neu5Ac when the enzyme is overexpressed, sialic acid measurements will saturate at normal levels and will not increase further. Therefore, the measurement of UDP-GlcNAc epimerase activity in tissue lysates, showing an increase above normal levels in tissue lysates, is one of the best methods for assessing total GNE activity. The UDP-GlcNAc epimerase assay, which can be used to measure GNE enzyme activity in mouse and human tissues, is an in vivo potency assay of the GNE gene therapy vector described herein. A dose-response study was conducted in wild-type (C57Bl / 6J) mice with the AAV.GNE vector to assess the dose and level of vector genome transduction required to double GNE enzyme activity, defined as the amount required for functional gene replacement. Even in the absence of proof-of-concept studies in GNE disease models, this information can be used to help determine the dosage.

[0249] GNE enzyme activity (UDPGlcNAc epimerase activity) was measured and compared in CHO cell lysates, Lec3 cell lysates (lacking GNE enzyme activity[2]), and Lec3 cells transfected with pAAV.CMV.GNE plasmid. GNE enzyme activity was observed in CHO cells, while almost no GNE enzyme activity was observed in Lec3 cells, and supernormal enzyme activity was observed in Lec3 cells transfected with pAAV.CMV.GNE. Figure 26 In vivo measurements of GNE enzyme activity are superior to the MAA assay for sialic acid because there is no feedback inhibition in the latter, which increases the linearity of the assay. Furthermore, performing the UDP-GlcNAc epimerase assay requires significantly more material (millions to tens of millions of CHO cells, compared to the 10,000 CHO cells used in the MAA-HRPELISA). Figure 25 Therefore, this enzyme activity assay can only be used on tissues (while MAA binding can be used for Lec3 cell ELISA). This UDP-GlcNAc epimerase assay is also applicable to mouse tissues (e.g., liver).

[0250] Since GNE genes and proteins are expressed in almost all organs, altered GNE enzyme activities (UDP-GlcNAc epimerase activities) were measured in tissues across the entire body program (liver, kidney, spleen, heart, lung, colon, and brain). However, skeletal muscle (including the diaphragm, biceps, triceps, gastrocnemius, quadriceps, and tibialis anterior) across the entire body program was a focus of this analysis because muscle pathology contributes to the disease in GNE myopathy. Tissue lysates from six mice (three males and three females) were analyzed to determine reproducibility while accounting for possible sex differences. 30–50 mg of tissue was cut and homogenized using a TissueLyser (four 30 Hz pulses, 30 seconds each) and shaken on ice for 30 minutes. Following lysis, protein levels were measured using the standard Bradford assay, and enzyme activity was normalized relative to total protein.

[0251] UDP-GlcNAc epimerase activity was determined using the Morgan-Eslon DMAB (4-dimethylaminobenzaldehyde) colorimetric method [6] with an incubation time of 30 minutes, during which ManNAc production was measured by the absorbance of the product at 578 nm using a spectrophotometer. 300 μg of total protein was used for each assay. ManNAc produced by the enzyme was determined by comparison with a ManNAc standard curve that underwent the same DMAB chemical modification protocol using concentrations of 0, 0.5, 1, 2.5, 5, 10, 25, 50, and 75 μg / mL. Next, rAAVrh74.CMV.GNE was administered intravenously to age- and sex-matched wild-type mice to determine the dose required to double endogenous GNE enzyme activity in planned tissues throughout the body. As the AAV dose increased, GNE enzyme activity was expected to increase linearly. Doses of 1×10¹¹ vg / kg, 1×10¹² vg / kg, and 1×10¹³ vg / kg were compared. Viral load in each tissue was quantified by standard qPCR, and GNE gene expression was measured by qRT-PCR, as we have done previously (Xu et al., Molecular Therapy). Protein levels could also be compared by Western blotting if reagents were available.

[0252] Most researchers define the transduction of GNE gene therapy vectors by measuring the amount of GNE cDNA introduced into tissues or the induction level of GNE mRNA expression, but these are not functional measurements of GNE bioactivity. The assay for measuring GNE enzyme activity (UDP-GlcNAc epimerase activity) described in this paper allows for a reliable functional measurement that can be normalized relative to the amount of total protein used in the assay and is reproducible between mice. It is also anticipated that by introducing different doses of GNE gene therapy, it will be demonstrated that this assay increases GNE potency and will define the minimum dose required to provide endogenous GNE enzyme activity levels (i.e., double the enzyme activity found in normal tissues). This assay provides the data needed to determine the functional GNE overexpression levels required for gene substitution in all organs and the number of vector genomes that must be transduced to accomplish such changes.

[0253] Example 6

[0254] Functional evaluation of bistronic GALGT2 and folliculorine gene therapy

[0255] The MDX muscular dystrophy model was used to evaluate the function of bistronicrAAV gene therapy expressing GALGT2 and follicle-staphylin 344 (FST). GALGT2 overexpression in the skeletal muscle of MDX mice has been reported to prevent muscle injury and inhibit muscle disease (Xu et al., Neuromusculoskeletal Disorders, 17; 209-220 (2007); Martin et al., American Journal of Physiology: Cell Physiology, 296: C476-488 (2009); Nguyen et al., Proceedings of the National Academy of Sciences, 99; 5616-5621 (2002). GALGT2 expression in MDX mice has been induced to improve even when only half of the fibers are transduced (Martin et al., (2009), ibid.).

[0256] In this experiment, 2-month-old mdx cells were compared with 1×10⁻⁶ mdx cells. 11 The vectors rAAVrh74.MCK.GALGT2.IRES.FST or single-gene vectors (rAAVrh74.MCK.GALGT2 or rAAVrh74.MCK.FST) were injected into the TA at the same dose. Phosphate-buffered saline (PBS) was injected as a negative control. Two months after injection, mice were euthanized and their muscle mass (relative to total body weight) was weighed. Figure 27As shown in Figure A, both single-gene FST and bicistronic GALGT2 / FST gene injections resulted in increased muscle size, indicating that placing the FST gene in the second position of the bicistronic vector leads to a significant FST function in inducing muscle growth.

[0257] After euthanasia, muscle sections were prepared from the TA, fixed in acetone, and stained with FST and WFA antibodies after injection (to identify GalNAc produced by GALGT2). Figure 27 As shown in Figure B, injection of the bicistronic vector (rAAVrh74.MCK.GALGT2.IRES.FST) resulted in the functional expression of both GALGT2 and FST. GALGT2 induced glycosylation on the muscle membrane (as shown by WFA staining), while FST was expressed in the Golgi apparatus and ultimately secreted extracellularly from the muscle cells. Note that the muscle fibers expressing GALGT2 exhibited normal muscle morphology without signs of muscle dystrophism, a known function of GALGT2 gene overexpression. Therefore, this single bicistronic AAV vector can both suppress muscle symptoms induced by GALGT2 overexpression and increase muscle size induced by FST gene expression, thus achieving a dual-function therapy. sequence list <110> National Children's Hospital Research Institute <120> Optimized gene therapy for muscle diseases <130> 28335 / 54649 <150> US 62 / 951,564 <151> 2019-12-20 <160> 40 <170> PatentIn version 3.5 <210> 1 <211> 2169 <212> DNA <213> Homo sapiens <400> 1 atggagaaga atggaaataa ccgaaagctg cgggtttgtg ttgctacttg taaccgtgca 60 gattattcta aacttgcccc gatcatgttt ggcattaaaa ccgaacctga gttctttgaa 120 cttgatgttg tggtacttgg ctctcacctg atagatgact atggaaatac atatcgaatg 180 attgaacaag atgactttga cattaacacc aggctacaca caattgtgag gggagaagat 240 gaggcagcca tggtggagtc agtaggcctg gccctagtga agctgccaga tgtccttaat 300 cgcctgaagc ctgatatcat gattgttcat ggagacaggt ttgatgccct ggctctggcc 360 acatctgctg ccttgatgaa catccgaatc cttcacattg aaggtgggga agtcagtggg 420 accattgatg actctatcag acatgccata acaaaactgg ctcattatca tgtgtgctgc 480 acccgcagtg cagagcagca cctgatatcc atgtgtgagg accatgatcg catccttttg 540 gcaggctgcc cttcctatga caaacttctc tcagccaaga acaaagacta catgagcatc 600 attcgcatgt ggctaggtga tgatgtaaaa tctaaagatt acattgttgc actacagcac 660 cctgtgacca ctgacattaa gcattccata aaaatgtttg aattaacatt ggatgcactt 720 atctcattta acaagcggac cctagtcctg tttccaaata ttgacgcagg gagcaaagag 780 atggttcgag tgatgcggaa gaagggcatt gagcatcatc ccaactttcg tgcagttaaa 840 cacgtcccat ttgaccagtt tatacagttg gttgcccatg ctggctgtat gattgggaac 900 agcagctgtg gggttcgaga agttggagct ttggacac ctgtgatcaa cctgggaaca 960 cgtcagattg gagagaac agggaat gttcttcatg tccggtgc tgacacccaa 1020 shagaaaatat tgcaagcact gcaccttcag ttgtaaac agtacccttg ttcaaagata 1080 tatgggatg gaatgctgt tccaaggatt tgaagttttc tcaatctat cgatcttcaa 1140 gagccactgc aaagaaattt ctgctttcct cctgtgaagg agatatctc tcaagatatt 1200 gaccatattc ttgaactct aagtgccttg gccgttgatc tggcggc gaacctccga 1260 gttgcaatag tcagcatgaa gggtgaaata gttaagaagt attackcagtt caatcctaaa 1320 acctatgaag agaggattaa tttaatccta cagatgtgtg tggaagctgc agcagaagct 1380 gtaaaactga actgcagaat tttggta ggcatttcca caggtggccg tgtaaatcct 1440 cgggaaggaa ttgtgctgca ttcaaccaa ctgatccaag agtggactc tgtggactt 1500 aggacccccc tttctgacac ttgcatctc cctgtgtgggg tagacaatga tggcactgt 1560 gctgccctgg cggaaaggaa atttggccaa ggaaagggac tggaaaactt tgttacactt 1620 atcacaggca caggaatcgg tggtggaatt atccatcagc atgaattgat ccacggaagc 1680 tccttctgtg ctgcagaact gggccacctt gttgtgtctc tggatgggcc tgattgttcc 1740 tgtggaagcc atgggtgcat tgaagcatac gcctctggaa tggccttgca gagggaggca 1800 aaaaagctcc atgatgagga cctgctcttg gtggaaggga tgtcagtgcc aaaagatgag 1860 gctgtgggtg cgctccatct catccaagct gcgaaacttg gcaatgcgaa ggcccagagc 1920 atcctaagaa cagctggaac agctttgggt cttggggttg tgaacatcct ccataccatg 1980 aatccctccc ttgtgatcct ctccggagtc ctggccagtc actatatcca cattgtcaaa 2040 gacgtcattc gccagcaggc cttgtcctcc gtgcaggacg tggatgtggt ggtttcggat 2100 ttggttgacc ccgccctgct gggtgctgcc agcatggttc tggactacac aacacgcagg 2160 atctactag 2169 <210> 2 <211> 753 <212> PRT <213> Homo sapiens <400> 2 Met Glu Thr Tyr Gly Tyr Leu Gln Arg Glu Ser Cys Phe Gln Gly Pro 1 5 10 15 His Glu Leu Tyr Phe Lys Asn Leu Ser Lys Arg Asn Lys Gln Ile Met 20 25 30 Glu Lys Asn Gly Asn Asn Arg Lys Leu Arg Val Cys Val Ala Thr Cys 35 40 45 Asn Arg Ala Asp Tyr Ser Lys Leu Ala Pro Ile Met Phe Gly Ile Lys 50 55 60 Thr Glu Pro Glu Phe Phe Glu Leu Asp Val Val Val Leu Gly Ser His 65 70 75 80 Leu Ile Asp Asp Tyr Gly Asn Thr Tyr Arg Met Ile Glu Gln Asp Asp 85 90 95 Phe Asp Ile Asn Thr Arg Leu His Thr Ile Val Arg Gly Glu Asp Glu 100 105 110 Ala Ala Met Val Glu Ser Val Gly Leu Ala Leu Val Lys Leu Pro Asp 115 120 125 Val Leu Asn Arg Leu Lys Pro Asp Ile Met Ile Val His Gly Asp Arg 130 135 140 Phe Asp Ala Leu Ala Leu Ala Thr Ser Ala Ala Leu Met Asn Ile Arg 145 150 155 160 Ile Leu His Ile Glu Gly Gly Glu Val Ser Gly Thr Ile Asp Asp Ser 165 170 175 Ile Arg His Ala Ile Thr Lys Leu Ala His Tyr His Val Cys Cys Thr 180 185 190 Arg Ser Ala Glu Gln His Leu Ile Ser Met Cys Glu Asp His Asp Arg 195 200 205 Ile Leu Leu Ala Gly Cys Pro Ser Tyr Asp Lys Leu Leu Ser Ala Lys 210 215 220 Asn Lys Asp Tyr Met Ser Ile Ile Arg Met Trp Leu Gly Asp Asp Val 225 230 235 240 Lys Ser Lys Asp Tyr Ile Val Ala Leu Gln His Pro Val Thr Thr Asp 245 250 255 Ile Lys His Ser Ile Lys Met Phe Glu Leu Thr Leu Asp Ala Leu Ile 260 265 270 Ser Phe Asn Lys Arg Thr Leu Val Leu Phe Pro Asn Ile Asp Ala Gly 275 280 285 Ser Lys Glu Met Val Arg Val Met Arg Lys Lys Gly Ile Glu His His 290 295 300 Pro Asn Phe Arg Ala Val Lys His Val Pro Phe Asp Gln Phe Ile Gln 305 310 315 320 Leu Val Ala His Ala Gly Cys Met Ile Gly Asn Ser Ser Cys Gly Val 325 330 335 Arg Glu Val Gly Ala Phe Gly Thr Pro Val Ile Asn Leu Gly Thr Arg 340 345 350 Gln Ile Gly Arg Glu Thr Gly Glu Asn Val Leu His Val Arg Asp Ala 355 360 365 Asp Thr Gln Asp Lys Ile Leu Gln Ala Leu His Leu Gln Phe Gly Lys 370 375 380 Gln Tyr Pro Cys Ser Lys Ile Tyr Gly Asp Gly Asn Ala Val Pro Arg 385 390 395 400 Ile Leu Lys Phe Leu Lys Ser Ile Asp Leu Gln Glu Pro Leu Gln Lys 405 410 415 Lys Phe Cys Phe Pro Pro Val Lys Glu Asn Ile Ser Gln Asp Ile Asp 420 425 430 His Ile Leu Glu Thr Leu Ser Ala Leu Ala Val Asp Leu Gly Gly Thr 435 440 445 Asn Leu Arg Val Ala Ile Val Ser Met Lys Gly Glu Ile Val Lys Lys 450 455 460 Tyr Thr Gln Phe Asn Pro Lys Thr Tyr Glu Glu Arg Ile Asn Leu Ile 465 470 475 480 Leu Gln Met Cys Val Glu Ala Ala Ala Glu Ala Val Lys Leu Asn Cys 485 490 495 Arg Ile Leu Gly Val Gly Ile Ser Thr Gly Gly Arg Val Asn Pro Arg 500 505 510 Glu Gly Ile Val Leu His Ser Thr Lys Leu Ile Gln Glu Trp Asn Ser 515 520 525 Val Asp Leu Arg Thr Pro Leu Ser Asp Thr Leu His Leu Pro Val Trp 530 535 540 Val Asp Asn Asp Gly Asn Cys Ala Ala Leu Ala Glu Arg Lys Phe Gly 545 550 555 560 Gln Gly Lys Gly Leu Glu Asn Phe Val Thr Leu Ile Thr Gly Thr Gly 565 570 575 Ile Gly Gly Gly Ile Ile His Gln His Glu Leu Ile His Gly Ser Ser 580 585 590 Phe Cys Ala Ala Glu Leu Gly His Leu Val Val Ser Leu Asp Gly Pro 595 600 605 Asp Cys Ser Cys Gly Ser His Gly Cys Ile Glu Ala Tyr Ala Ser Gly 610 615 620 Met Ala Leu Gln Arg Glu Ala Lys Lys Leu His Asp Glu Asp Leu Leu 625 630 635 640 Leu Val Glu Gly Met Ser Val Pro Lys Asp Glu Ala Val Gly Ala Leu 645 650 655 His Leu Ile Gln Ala Ala Lys Leu Gly Asn Ala Lys Ala Gln Ser Ile 660 665 670 Leu Arg Thr Ala Gly Thr Ala Leu Gly Leu Gly Val Val Asn Ile Leu 675 680 685 His Thr Met Asn Pro Ser Leu Val Ile Leu Ser Gly Val Leu Ala Ser 690 695 700 His Tyr Ile His Ile Val Lys Asp Val Ile Arg Gln Gln Ala Leu Ser 705 710 715 720 Ser Val Gln Asp Val Asp Val Val Val Ser Asp Leu Val Asp Pro Ala 725 730 735 Leu Leu Gly Ala Ala Ser Met Val Leu Asp Tyr Thr Thr Arg Arg Ile 740 745 750 Tyr <210> 3 <211> 407 <212> DNA <213> Homo sapiens <400> 3 ctcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca 60 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt 120 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 180 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag 240 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 300 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg 360 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttg 407 <210> 4 <211> 564 <212> DNA <213> Homo sapiens <400> 4 cagccactat gggtctaggc tgcccatgta aggaggcaag gcctggggac acccgagatg 60 cctggttata attaacccag acatgtggct gctccccccc cccaacacct gctgcctgag 120 cctcaccccc accccggtgc ctgggtctta ggctctgtac accatggagg agaagctcgc 180 tctaaaaata accctgtccc tggtgggctg tgggggactg agggcaggct gtaacaggct 240 tgggggccag ggcttatacg tgcctgggac tcccaaagta ttactgttcc atgttcccgg 300 cgaagggcca gctgtccccc gccagctaga ctcagcactt agtttaggaa ccagtgagca 360 agtcagccct tggggcagcc catacaaggc catggggctg ggcaagctgc acgcctgggt 420 ccggggtggg cacggtgccc gggcaacgag ctgaaagctc atctgctctc aggggcccct 480 ccctggggac agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg 540 gcacaggggc tgcccccggg tcac 564 <210> 5 <211> 810 <212> DNA <213> Homo sapiens <400> 5 gtttaaacaa gcttgcatgt ctaagctaga cccttcagat taaaaataac tgaggtaagg 60 gcctgggtag gggaggtggt gtgagacgct cctgtctctc ctctatctgc ccatcggccc 120 tttggggagg aggaatgtgc ccaaggacta aaaaaaggcc atggagccag aggggcgagg 180 gcaacagacc tttcatgggc aaaccttggg gccctgctgt ctagcatgcc ccactacggg 240 tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct ggttataatt 300 aacccagaca tgtggctgcc cccccccccc caacacctgc tgcctctaaa aataaccctg 360 tccctggtgg atcccctgca tgcgaagatc ttcgaacaag gctgtggggg actgagggca 420 ggctgtaaca ggcttggggg ccagggctta tacgtgcctg ggactcccaa agtattactg 480 ttccatgttc ccggcgaagg gccagctgtc ccccgccagc tagactcagc acttagttta 540 ggaaccagtg agcaagtcag cccttggggc agcccataca aggccatggg gctgggcaag 600 ctgcacgcct gggtccgggg tgggcacggt gcccgggcaa cgagctgaaa gctcatctgc 660 tctcaggggc ccctccctgg ggacagcccc tcctggctag tcacaccctg taggctcctc 720 tatataaccc aggggcacag gggctgccct cattctacca ccacctccac agcacagaca 780 gacactcagg agccagccag cggcgcgccc 810 <210> 6 <211> 707 <212> DNA <213> Homo sapiens <400> 6 gcttgaggcc aggagctcga gtgctgcctg gacaacatag tgagaccctt gtctcaaaaa 60 caacagcaac ccaaagtctt atttatcttc aggagctgcg caagcccttt accaccgcca 120 cgtttctccc tggatgtccc taggcctcgg gactggctgg gagttccctg tagtggaggc 180 cgccgctgaa ctgattataa agatgagaga ggctctgcca tgccttggtc atcaattcac 240 actccaccag tgtcttctag cagtcacggc aagggttaac gtcagggacc gctgtggggt 300 ggccgcgcta cacgacagta tagttgcggt cctggtttat gaaataactg agggaacaag 360 aggcgcaaga aatccctcct tgggtgcaag accaaaacaa ctacccagcg ggaagactcg 420 ggcttcagtg cgtgtgtcgc cagtggagga ggacgcttcg gggcgggagc acaagctggc 480 aggacagccc cgcagcagct ccagcgcggc agaggccatg gaagatggtc cgctggtcag 540 caccctgcct gcgcctcaaa ataccgccgg gaaggtgggg gacggggacg ggagcagcca 600 atcacgcgag ctctctcggg tggcgtcagg gcgcgatcgc ggattcggca ctccgggtgg 660 gcgtggctgg gcgagcgagg agtggggaca aggtcgagcg acgagtc 707 <210> 7 <211> 228 <212> DNA <213> Homo sapiens <400> 7 tcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccggactc 60 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa 120 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag 180 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccg 228 <210> 8 <211> 119 <212> DNA <213> Homo sapiens <400> 8 ccttctgatg gtgaatggga actcccttcc tcctgcagca gcctgcctgc agctgtcctg 60 gtagaacagt gtggacattg cagaagctgt cactgcccca gaaagaaagc accccagag 119 <210> 9 <211> 1032 <212> DNA <213> Homo sapiens <400> 9 atggtccgcg cgaggcacca gccgggtggg ctttgcctcc tgctgctgct gctctgccag 60 ttcatggagg accgcagtgc ccaggctggg aactgctggc tccgtcaagc gaagaacggc 120 cgctgccagg tcctgtacaa gaccgaactg agcaaggagg agtgctgcag caccggccgg 180 ctgagcacct cgtggaccga ggaggacgtg aatgacaaca cactcttcaa gtggatgatt 240 ttcaacgggg gcgcccccaa ctgcatcccc tgtaaagaaa cgtgtgagaa cgtggactgt 300 ggacctggga aaaaatgccg aatgaacaag aagaacaaac cccgctgcgt ctgcgccccg 360 gattgttcca acatcacctg gaagggtcca gtctgcgggc tggatgggaa aacctaccgc 420 aatgaatgtg cactcctaaa ggcaagatgt aaagagcagc cagaactgga agtccagtac 480 caaggcagat gtaaaaagac ttgtcgggat gttttctgtc caggcagctc cacatgtgtg 540 gtggaccaga ccaataatgc ctactgtgtg acctgtaatc ggatttgccc agagcctgct 600 tcctctgagc aatatctctg tgggaatgat ggagtcacct actccagtgc ctgccacctg 660 agaaaggcta cctgcctgct gggcagatct attggattag cctatgaggg aaagtgtatc 720 aaagcaaagt cctgtgaaga tatccagtgc actggtggga aaaaatgttt atgggatttc 780 aaggttggga gaggccggtg ttccctctgt gatgagctgt gccctgacag taagtcggat 840 gagcctgtct gtgccagtga caatgccact tatgccagcg agtgtgccat gaaggaagct 900 gcctgctcct caggtgtgct actggaagta aagcactccg gatcttgcaa ctccatttcg 960 gaagacaccg aggaagagga ggaagatgaa gaccaggact acagctttcc tatatcttct 1020 attctagagt gg 1032 <210> 10 <211> 344 <212> PRT <213> Homo sapiens <400> 10 Met Val Arg Ala Arg His Gln Pro Gly Gly Leu Cys Leu Leu Leu Leu 1 5 10 15 Leu Leu Cys Gln Phe Met Glu Asp Arg Ser Ala Gln Ala Gly Asn Cys 20 25 30 Trp Leu Arg Gln Ala Lys Asn Gly Arg Cys Gln Val Leu Tyr Lys Thr 35 40 45 Glu Leu Ser Lys Glu Glu Cys Cys Ser Thr Gly Arg Leu Ser Thr Ser 50 55 60 Trp Thr Glu Glu Asp Val Asn Asp Asn Thr Leu Phe Lys Trp Met Ile 65 70 75 80 Phe Asn Gly Gly Ala Pro Asn Cys Ile Pro Cys Lys Glu Thr Cys Glu 85 90 95 Asn Val Asp Cys Gly Pro Gly Lys Lys Cys Arg Met Asn Lys Lys Asn 100 105 110 Lys Pro Arg Cys Val Cys Ala Pro Asp Cys Ser Asn Ile Thr Trp Lys 115 120 125 Gly Pro Val Cys Gly Leu Asp Gly Lys Thr Tyr Arg Asn Glu Cys Ala 130 135 140 Leu Leu Lys Ala Arg Cys Lys Glu Gln Pro Glu Leu Glu Val Gln Tyr 145 150 155 160 Gln Gly Arg Cys Lys Lys Thr Cys Arg Asp Val Phe Cys Pro Gly Ser 165 170 175 Ser Thr Cys Val Val Asp Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys 180 185 190 Asn Arg Ile Cys Pro Glu Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly 195 200 205 Asn Asp Gly Val Thr Tyr Ser Ser Ala Cys His Leu Arg Lys Ala Thr 210 215 220 Cys Leu Leu Gly Arg Ser Ile Gly Leu Ala Tyr Glu Gly Lys Cys Ile 225 230 235 240 Lys Ala Lys Ser Cys Glu Asp Ile Gln Cys Thr Gly Gly Lys Lys Cys 245 250 255 Leu Trp Asp Phe Lys Val Gly Arg Gly Arg Cys Ser Leu Cys Asp Glu 260 265 270 Leu Cys Pro Asp Ser Lys Ser Asp Glu Pro Val Cys Ala Ser Asp Asn 275 280 285 Ala Thr Tyr Ala Ser Glu Cys Ala Met Lys Glu Ala Ala Cys Ser Ser 290 295 300 Gly Val Leu Leu Glu Val Lys His Ser Gly Ser Cys Asn Ser Ile Ser 305 310 315 320 Glu Asp Thr Glu Glu Glu Glu Glu Asp Glu Asp Gln Asp Tyr Ser Phe 325 330 335 Pro Ile Ser Ser Ile Leu Glu Trp 340 <210> 11 <211> 534 <212> DNA <213> Homo sapiens <400> 11 gaagctgctg ccgtcggtgg tgctgaagct ctttctggct gcagttctct cggcactggt 60 gactggcgag agcctggagc ggcttcggag agggctagct gctggaacca gcaacccgga 120 ccctcccact gtatccacgg accagctgct acccctagga ggcggccggg accggaaagt 180 ccgtgacttg caagaggcag atctggacct tttgagagtc actttatcct ccaagccaca 240 agcactggcc acaccaaaca aggaggagca cgggaaaaga aagaagaaag gcaaggggct 300 agggaagaag agggacccag gaccggagac gctctgcggg gctgagctgg tggatgctct 360 tcagttcgtg tgtggagaca ggggctttta tttcaacaag cccacagggt atggctccag 420 cagtcggagg gcgcctcaga caggcatcgt ggatgagtgc tgcttccgga gctgtgatct 480 aaggaggctg gagatgtatt gcgcacccct caagcctgcc aagtcagctt aggc 534 <210> 12 <211> 7277 <212> DNA <213> Homo sapiens <400> 12 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 60 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 120 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 180 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 240 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 300 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 360 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 420 gggcgcgtca gcgtgtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 480 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 540 ataccgcatc aggcgattcc aacatccaat aaatcataca ggcaaggcaa agaattagca 600 aaattaagca ataaagcctc agagcataa gctaaatcgg ttgtaccaaa aacattatga 660 ccctgtaata cttttgcggg agaagccttt atttcaacgc areataaaaa atttttagaa 720 ccctcatata ttttaaatgc aatgcctgag taatgtgtag gtaaagattc aaacgggtga 780 gaaaggccgg agacagtcaa atcaccatca atatgatatt caaccgttct agctgataaa 840 ttcatgccgg agagggtagc tatttttgag aggtctctac aaaggctatc aggtcattgc 900 ctgagagtct ggagcaaaca agaagaatcga tgaacggtaa tcgtaaact agcatgtcaa 960 tcatatgtac cccggttgat aatcagaaaa gccccaaaaa caggagaatt gtataagcaa 1020 atatttaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 1080 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 1140 ccgagatagg gttgagtgtt gttccagttt ggaaagag tccactatta aagaacgtgg 1200 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 1260 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 1320 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaagggga 1380 agaaaggaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 1440 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg 1500 agcacgtata acgtgctttc ctcgttagaa tcagagcggg agctaaacag gaggccgatt 1560 aaagggattt tagacaggaa cggtacgcca gaatcctgag aagtgttttt ataatcagtg 1620 aggccaccga gtaaaagagt ctgtccatca cgcaaattaa ccgttgtcgc aatacttctt 1680 tgattagtaa taacatcact tgcctgagta gaagaactca aactatcggc cttgctggta 1740 atatccagaa caatattacc gccagccatt gcaacggaat cgccattcgc cattcaggct 1800 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc 1860 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc 1920 ggcctcagtg agcgagcgag cgcgcaga gggagtggcc aactccatca ctaggggttc 1980 cttgtagtta atgattaacc cgccatgcta cttatctacg tagccatgct ctaggtcgtt 2040 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 2100 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 2160 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 2220 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 2280 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 2340 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 2400 ccaagtctcc accccattga cgtcaatggg agttgtttt ggcaccaaaa tcaacgggac 2460 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 2520 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 2580 ccacgctgtt ttgacctcca tagagacac cgggaccgat ccagcctccg gactcttagag 2640 gatccggtac tcgaggaact gaaaaaccag aaagttaact ggtaagttta gtcttttgt 2700 cttttatttc aggtcccgga tccggtggtg gtgcaaatca aagaactgct cctcagtgga 2760 tgttgccttt acttctaggc ctgtacggaa gtgttacttc tgctctaaaa gctgcggaat 2820 tgtacccgcg gccggctagc cgccaccatg gagaagaatg gaaataaccg aaagctgcgg 2880 gtttgtgttg ctacttgtaa ccgtgcagat tattctaaac ttgccccgat catgtttggc 2940 attaaaaccg aacctgagtt ctttgaactt gatgttgtgg tacttggctc tcacctgata 3000 gatgactatg gaaatacata tcgaatgatt gaacaagatg actttgacat taacaccagg 3060 ctacacacaa ttgtgagggg agaagatgag gcagccatgg tggagtcagt aggcctggcc 3120 ctagtgaagc tgccagatgt ccttaatcgc ctgaagcctg atatcatgat tgttcatgga 3180 gacaggtttg atgccctggc tctggccaca tctgctgcct tgatgaacat ccgaatcctt 3240 cacattgaag gtggggaagt cagtgggacc attgatgact ctatcagaca tgccataaca 3300 aaactggctc attatcatgt gtgctgcacc cgcagtgcag agcagcacct gatatccatg 3360 tgtgaggacc atgatcgcat ccttttggca ggctgccctt cctatgacaa acttctctca 3420 gccaagaaca aagactacat gagcatcatt cgcatgtggc taggtgatga tgtaaaatct 3480 aaagattaca ttgttgcact acagcaccct gtgaccactg acattaagca ttccataaaa 3540 atgtttgaat taacattgga tgcacttatc tcatttaaca agcggaccct agtcctgttt 3600 ccaaatattg acgcagggag caaagagatg gttcgagtga tgcggaagaa gggcattgag 3660 catcatccca actttcgtgc agttaaacac gtcccatttg accagtttat acagttggtt 3720 gcccatgctg gctgtatgat tgggaacagc agctgtgggg ttcgagaagt tggagctttt 3780 ggaacacctg tgatcaacct gggaacacgt cagattggaa gagaaacagg ggagaatgtt 3840 cttcatgtcc gggatgctga cacccaagac aaaatattgc aagcactgca ccttcagttt 3900 ggtaaacagt acccttgttc aaagatatat ggggatggaa atgctgttcc aaggattttg 3960 aagtttctca aatctatcga tcttcaagag ccactgcaaa agaaattctg ctttcctcct 4020 gtgaaggaga atatctctca agatattgac catattcttg aaactctaag tgccttggcc 4080 gttgatcttg gcgggacgaa cctccgagtt gcaatagtca gcatgaaggg tgaaatagtt 4140 aagaagtata ctcagttcaa tcctaaaacc tatgaagaga ggattaattt aatcctacag 4200 atgtgtgtgg aagctgcagc agaagctgta aaactgaact gcagaattttt gggagtaggc 4260 atttccacag gtggccgtgt aaatcctcgg gaaggaattg tgctgcattc aaccaaactg 4320 atccaagagt ggaactctgt ggaccttagg accccccttt ctgacacttt gcatctccct 4380 gtgtgggtag acaatgatgg caactgtgct gccctggcgg aaaggaaatt tggccaagga 4440 aagggactgg aaaactttgt tacacttatc acaggcacag gaatcggtgg tggaattatc 4500 catcagcatg aattgatcca cggaagctcc ttctgtgctg cagaactggg ccaccttgtt 4560 gtgtctctgg atgggcctga ttgttcctgt ggaagccatg ggtgcattga agcatacgcc 4620 tctggaatgg ccttgcagag ggaggcaaaa aagctccatg atgaggacct gctcttggtg 4680 gaagggatgt cagtgccaaa agatgaggct gtgggtgcgc tccatctcat ccaagctgcg 4740 aaacttggca atgcgaaggc ccagagcatc ctaagaacag ctggaacagc tttgggtctt 4800 ggggttgtga acatcctcca taccatgaat ccctcccttg tgatcctctc cggagtcctg 4860 gccagtcact atatccacat tgtcaaagac gtcattcgcc agcaggcctt gtcctccgtg 4920 caggacgtgg atgtggtggt ttcggatttg gttgaccccg ccctgctggg tgctgccagc 4980 atggttctgg actacacaac acgcaggatc tactagcatg cactagtgcg gccgcgggga 5040 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa 5100 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg 5160 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt 5220 gtgggaggtt ttttcggatc ctctagagtc gaaaactcta gagcatggct acgtagataa 5280 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc 5340 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 5400 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg aatcggccaa 5460 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 5520 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 5580 ttatccacag aatcaggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 5640 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 5700 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 5760 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 5820 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 5880 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 5940 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 6000 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 6060 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctcac tagaaggaca 6120 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 6180 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 6240 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 6300 cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta 6360 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag 6420 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa 6480 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt 6540 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact 6600 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga 6660 tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga 6720 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca 6780 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc 6840 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg 6900 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc 6960 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat 7020 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct 7080 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg 7140 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta 7200 agaattcgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 7260 atgtaaccca ctcgtgc 7277 <210> 13 <211> 7070 <212> DNA <213> Homo sapiens <400> 13 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 60 ccttgagagt tttcgccccg aagaacgaat tcttagaaaa actcatcgag catcaaatga 120 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt 180 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct 240 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg 300 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta 360 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc 420 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg 480 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc 540 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc 600 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg 660 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca 720 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac 780 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat 840 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata 900 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac 960 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1020 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1080 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1140 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1200 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1260 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1320 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1380 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1440 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1500 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1560 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1620 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1680 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1740 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1800 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcaagctgcgc 1860 gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc 1920 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactagggt 1980 tccttgtagt taatgattaa cccgccatgc tacttatcta cgtagccatg ctctagacag 2040 ccactatggg tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct 2100 ggttataatt aacccagaca tgtggctgct cccccccccc caacacctgc tgcctgagcc 2160 tcacccccac cccggtgcct gggtcttagg ctctgtacac catggaggag aagctcgctc 2220 taaaataac cctgtccctg gtgggctgtg ggggactgag ggcaggctgt aacaggcttg 2280 ggggccaggg cttatacgtg cctgggactc ccaaagtatt actgttccat gttcccggcg 2340 aagggccagc tgtcccccg cagctagact cagcacttag tttaggaacc agtgagcaag 2400 tcagcccttg gggcagccca tacaaggcca tggggctgggg caagctgcac gcctgggtcc 2460 ggggtgggca cggtgcccgg gcaacgagct gaaagctcat ctgctctcag gggcccctcc 2520 ctggggacag cccctcctgg ctagtcacac cctgtaggct cctctatata acccaggggc 2580 acaggggctg cccccgggtc accaccacct ccacagcaca gacagacact caggagccag 2640 ccagccaggt aagtttagtc ttttgtctt ttatttcagg tcccggatcc ggtggtggtg 2700 caaatcaaag aactgctcct cagtggatgt tgcctttact tctaggcctg tacggaagtg 2760 ttacttctgc tctaaaagct gcggaattgt acccgcggcc ggctagccgc caccatggag 2820 aagaatggaa ataaccgaaa gctgcgggtt tgtgttgcta cttgtaaccg tgcagattat 2880 tctaaacttg ccccgatcat gtttggcatt aaaaccgaac ctgagttctt tgaacttgat 2940 gttgtggtac ttggctctca cctgatagat gactatggaa atacatatcg aatgattgaa 3000 caagatgact ttgacattaa caccaggcta cacacaattg tgaggggaga agatgaggca 3060 gccatggtgg agtcagtagg cctggcccta gtgaagctgc cagatgtcct taatcgcctg 3120 aagcctgata tcatgattgt tcatggagac aggtttgatg ccctggctct ggccacatct 3180 gctgccttga tgaacatccg aatccttcac attgaaggtg gggaagtcag tgggaccatt 3240 gatgactcta tcagacatgc cataacaaaa ctggctcatt atcatgtgtg ctgcacccgc 3300 agtgcagagc agcacctgat atccatgtgt gaggaccatg atcgcatcct tttggcaggc 3360 tgcccttcct atgacaaact tctctcagcc aagaacaaag actacatgag catcattcgc 3420 atgtggctag gtgatgatgt aaaatctaaa gattacattg ttgcactaca gcaccctgtg 3480 accactgaca ttaagcattc cataaaaatg tttgaattaa cattggatgc acttatctca 3540 tttaacaagc ggaccctagt cctgtttcca aatattgacg cagggagcaa agagatggtt 3600 cgagtgatgc ggaagaaggg cattgagcat catcccaact ttcgtgcagt taaacacgtc 3660 ccatttgacc agtttataca gttggttgcc catgctggct gtatgattgg gaacagcagc 3720 tgtggggttc gagaagttgg agctttttgga acacctgtga tcaacctggg aacacgtcag 3780 attggaagag aaacagggga gaatgttctt catgtccggg atgctgacac ccaagacaaa 3840 atattgcaag cactgcacct tcagtttggt aaacagtacc cttgttcaaa gatatatggg 3900 gatggaaatg ctgttccaag gattttgaag tttctcaaat ctatcgatct tcaagagcca 3960 ctgcaaaaga aattctgctt tcctcctgtg aaggaata tctctcaaga tattgaccat 4020 attcttgaaa ctctaagtgc cttggccgtt gatcttggcg ggacgaacct ccgagttgca 4080 atagtcagca tgaagggtga aatagttaag aagtatactc agttcaatcc taaaacctat 4140 gaagagag ttaatttaat cctacagatg tgtgtggaag ctgcagcaga agctgtaaaa 4200 ctgaactgca gaattttggg agtaggcatt tccacaggtg gccgtgtaaa tcctcgggaa 4260 ggaattgtgc tgcattcaac caaactgatc caagagtgga actctgtgga ccttaggacc 4320 cccctttctg acactttgca tctccctgtg tgggtagaca atgatggcaa ctgtgctgcc 4380 ctggcggaaa ggaaatttgg ccaaggaaag ggactggaaa actttgttac acttatcaca 4440 ggcacaggaa tcggtggtgg aattatccat cagcatgaat tgatccacgg aagctccttc 4500 tgtgctgcag aactgggcca ccttgttgtg tctctggatg ggcctgattg ttcctgtgga 4560 agccatgggt gcattgaagc atacgcctct ggaatggcct tgcagaggga ggcaaaaaag 4620 ctccatgatg aggacctgct cttggtggaa gggatgtcag tgccaaaaga tgaggctgtg 4680 ggtgcgctcc atctcatcca agctgcgaaa cttggcaatg cgaaggccca gagcatccta 4740 agaacagctg gaacagcttt gggtcttggg gttgtgaaca tcctccatac catgaatccc 4800 tcccttgtga tcctctccgg agtcctggcc agtcactata tccacattgt caaagacgtc 4860 attcgccagc aggccttgtc ctccgtgcag gacgtggatg tggtggtttc ggatttggtt 4920 gaccccgccc tgctgggtgc tgccagcatg gttctggact acacaacacg caggatctac 4980 tagcatgcac tagtgcggcc gcaataaaag atctttattt tcattagatc tgtgtgttgg 5040 ttttttgtgt gtctagagca tggctacgta gataagtagc atggcgggtt aatcattaac 5100 tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact 5160 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc 5220 gagcgagcgc gcagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 5280 ttgcgcagcc tgaatggcga atggcgattc cgttgcaatg gctggcggta atattgttct 5340 ggatattacc agcaaggccg atagtttgag ttcttctact caggcaagtg atgttattac 5400 taatcaaaga agtattgcga caacggttaa tttgcgtgat ggacagactc ttttactcgg 5460 tggcctcact gattataaaa acacttctca ggattctggc gtaccgttcc tgtctaaaat 5520 ccctttaatc ggcctcctgt ttagctcccg ctctgattct aacgaggaaa gcacgttata 5580 cgtgctcgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg 5640 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 5700 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 5760 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 5820 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 5880 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 5940 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 6000 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt 6060 aaatatttgc ttatacaatc ttcctgtttt tggggctttt ctgattatca accggggtac 6120 atatgattga catgctagtt ttacgattac cgttcatcga ttctcttgtt tgctccagac 6180 tctcaggcaa tgacctgata gcctttgtag agacctctca aaaatagcta ccctctccgg 6240 catgaattta tcagctagaa cggttgaata tcatattgat ggtgatttga ctgtctccgg 6300 cctttctcac ccgtttgaat ctttacctac acattactca ggcattgcat ttaaaatata 6360 tgagggttct aaaaattttt atccttgcgt tgaaataaag gcttctcccg caaaagtatt 6420 acagggtcat aatgtttttg gtacaaccga tttagcttta tgctctgagg ctttattgct 6480 taattttgct aattctttgc cttgcctgta tgatttattg gatgttggaa tcgcctgatg 6540 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 6600 acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac 6660 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc 6720 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc 6780 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca 6840 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 6900 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 6960 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 7020 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 7070 <210> 14 <211> 7270 <212> DNA <213> Homo sapiens <400> 14 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 60 ttttcgcccc gaagaacgaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 120 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 180 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 240 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 300 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 360 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 420 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 480 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 540 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 600 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 660 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 720 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 780 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 840 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 900 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 960 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aggatcttc 1020 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1080 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 1140 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1200 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1260 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1320 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1380 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1440 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1500 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggttcgcc acctctgact 1560 tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1620 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 1680 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 1740 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 1800 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg 1860 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 1920 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 1980 ttaatgatta acccgccatg ctacttatct acgtagccat gctctagagt ttaaacaagc 2040 ttgcatgtct aagctagacc cttcagatta aaaataactg aggtaagggc ctgggtaggg 2100 gaggtggtgt gagacgctcc tgtctctcct ctatctgccc atcggccctt tggggaggag 2160 gaatgtgccc aaggactaaa aaaaggccat ggagccagag gggcgagggc aacagacctt 2220 tcatgggcaa accttggggc cctgctgtct agcatgcccc actacgggtc taggctgccc 2280 atgtaaggag gcaaggcctg gggacacccg agatgcctgg ttataattaa cccagacatg 2340 tggctgcccc ccccccccca acacctgctg cctctaaaaa taaccctgtc cctggtggat 2400 cccctgcatg cgaagatctt cgaacaaggc tgtgggggac tgagggcagg ctgtaacagg 2460 cttgggggcc agggcttata cgtgcctggg actcccaaag tattactgtt ccatgttccc 2520 ggcgaagggc cagctgtccc ccgccagcta gactcagcac ttagtttagg aaccagtgag 2580 caagtcagcc cttggggcag cccatacaag gccatggggc tgggcaagct gcagccctgg 2640 gtccggggtg ggcacggtgc ccgggcaacg agctgaaagc tcatctgctc tcaggggccc 2700 ctccctgggg acagcccctc ctggctagtc acaccctgta ggctcctcta tataacccag 2760 gggcacaggg gctcccctca ttctaccacc acctccacag cacacacaga cactcaggag 2820 cagccagcgg cgcgcccagg taagtttagt cttttgtct tttatttcag gtcccggatc 2880 cggtggtggt gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctaggcct 2940 gtacggaagt gttacttctg ctctaaaagc tgcggaattg tacccgcggc cggctagccg 3000 ccaccatgga gaagatgga aataccgaa agctgcgggt ttgtgttgct acttgtaacc 3060 gtgcagatta ttctaaactt gccccgatca tgtttggcat taaaaccgaa cctgagttct 3120 ttgaacttga tgttgtggta cttggctctc acctgataga tgactatgga aatacatatc 3180 gaatgattga acaagatgac tttgacatta acaccaggct acacaatt gtgaggggag 3240 aagatgaggc agccatggtg gagtcagtag gcctggccct agtgaagctg ccagatgtcc 3300 ttaatcgcct gaagcctgat atcatgattg ttcatggaga caggtttgat gccctggctc 3360 tggccacatc tgctgccttg atgaacatcc gaatccttca cattgaaggt ggggaagtca 3420 gtgggaccat tgatgactct atcagacatg ccataacaaa actggctcat tatcatgtgt 3480 gctgcacccg cagtgcagag cagcacctga tatccatgtg tgaggaccat gatcgcatcc 3540 ttttggcagg ctgcccttcc tatgacaaac ttctctcagc caagaacaaa gactacatga 3600 gcatcattcg catgtggcta ggtgatgatg taaaatctaa agattacatt gttgcactac 3660 agcaccctgt gaccactgac attaagcatt ccataaaaat gtttgaatta acattggatg 3720 cacttatctc atttaacaag cggaccctag tcctgtttcc aaatattgac gcagggagca 3780 aagagatggt tcgagtgatg cggaagaagg gcattgagca tcatcccaac tttcgtgcag 3840 ttaaacacgt cccatttgac cagtttatac agttggttgc ccatgctggc tgtatgattg 3900 ggaacagcag ctgtggggtt cgagaagttg gagcttttgg aacacctgtg atcaacctgg 3960 gaacacgtca gattggaaga gaaacagggg agaatgttct tcatgtccgg gatgctgaca 4020 cccaagacaa aatattgcaa gcactgcacc ttcagtttgg taaacagtac ccttgttcaa 4080 agatatatgg ggatggaaat gctgttccaa ggattttgaa gtttctcaaa tctatcgatc 4140 ttcaagagcc actgcaaaag aaattctgct ttcctcctgt gaaggagaat atctctcaag 4200 atattgacca tattcttgaa actctaagtg ccttggccgt tgatcttggc gggacgaacc 4260 tccgagttgc aatagtcagc atgaagggtg aatagttaa gaagtatact cagttcaatc 4320 ctaaaaccta tgaagagagg attaatttaa tcctacagat gtgtgtggaa gctgcagcag 4380 aagctgtaaa actgaactgc agaatttttgg gagtaggcat ttccacaggt ggccgtgtaa 4440 atcctcggga aggaattgtg ctgcattcaa ccaaactgat ccaagagtgg aactctgtgg 4500 accttaggac ccccctttct gacactttgc atctccctgt gtgggtagac aatgatggca 4560 actgtgctgc cctggcggaa aggaaatttg gccaaggaaa gggactggaa aactttgtta 4620 cacttatcac aggcacagga atcggtggtg gaattatcca tcagcatgaa ttgatccacg 4680 gaagctcctt ctgtgctgca gaactgggcc accttgttgt gtctctggat gggcctgatt 4740 gttcctgtgg aagccatggg tgcattgaag catacgcctc tggaatggcc ttgcagaggg 4800 aggcaaaaaa gctccatgat gaggacctgc tcttggtgga agggatgtca gtgccaaaag 4860 atgaggctgt gggtgcgctc catctcatcc aagctgcgaa acttggcaat gcgaaggccc 4920 agagcatcct aagaacagct ggaacagctt tgggtcttgg ggttgtgaac atcctccata 4980 ccatgaatcc ctcccttgtg atcctctccg gagtcctggc cagtcactat atccacattg 5040 tcaaagacgt cattcgccag caggccttgt cctccgtgca ggacgtggat gtggtggttt 5100 cggatttggt tgaccccgcc ctgctgggtg ctgccagcat ggttctggac tacacaacac 5160 gcaggatcta ctagcatgca ctagtgcggc cgcaataaaa gatctttatt ttcattagat 5220 ctgtgtgttg gttttttgtg tgtctagagc atggctacgt agataagtag catggcgggt 5280 taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 5340 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 5400 cctcagtgag cgagcgagcg cgcagctggc gtaatagcga agaggcccgc accgatcgcc 5460 cttcccaaca gttgcgcagc ctgaatggcg aatggcgatt ccgttgcaat ggctggcggt 5520 aatattgttc tggatattac cagcaaggcc gatagtttga gttcttctac tcaggcaagt 5580 gatgttatta ctaatcaaag aagtattgcg acaacggtta atttgcgtga tggacagact 5640 cttttactcg gtggcctcac tgattataaa aacacttctc aggattctgg cgtaccgttc 5700 ctgtctaaaa tccctttaat cggcctcctg tttagctccc gctctgattc taacgaggaa 5760 agcacgttat acgtgctcgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5820 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 5880 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 5940 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 6000 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 6060 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 6120 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 6180 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 6240 gcttacaatt taaatatttg cttatacaat cttcctgttt ttggggcttt tctgattatc 6300 aaccggggta catatgattg acatgctagt tttacgatta ccgttcatcg attctcttgt 6360 ttgctccaga ctctcaggca atgacctgat agcctttgta gagacctctc aaaaatagct 6420 accctctccg gcatgaattt atcagctaga acggttgaat atcatattga tggtgatttg 6480 actgtctccg gcctttctca cccgtttgaa tctttaccta cacattactc aggcattgca 6540 tttaaaatat atgagggttc taaaaatttt tatccttgcg ttgaaataaa ggcttctccc 6600 gcaaaagtat tacagggtca taatgttttt ggtacaaccg atttagcttt atgctctgag 6660 gctttattgc ttaattttgc taattctttg ccttgcctgt atgatttatt ggatgttgga 6720 atcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 6780 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 6840 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 6900 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 6960 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 7020 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 7080 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 7140 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 7200 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 7260 ctgaagatca 7270 <210> 15 <211> 7187 <212> DNA <213> Homo sapiens <400> 15 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 60 ttttcgcccc gaagaacgaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 120 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 180 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 240 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 300 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 360 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 420 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 480 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 540 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 600 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 660 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 720 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 780 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 840 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 900 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 960 ttaacgtgag tttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 1020 ttgagatcct ttttttgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1080 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 1140 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1200 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1260 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1320 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1380 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1440 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1500 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggttcgcc acctctgact 1560 tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1620 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 1680 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 1740 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 1800 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg 1860 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 1920 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 1980 ttaatgatta acccgccatg ctacttatct acgtagccat gctctagagt ttaaacaagc 2040 ttgaggccag gagctcgagt gctgcctgga caacatagtg agacccttgt ctcaaaaaca 2100 acagcaaccc aaagtcttat ttatcttcag gagctgcgca agccctttac caccgccacg 2160 tttctccctg gatgtcccta ggcctcggga ctggctggga gttccctgta gtggaggccg 2220 ccgctgaact gattataaag atgagagagg ctctgccatg ccttggtcat caattcacac 2280 tccaccagtg tcttctagca gtcacggcaa gggttaacgt cagggaccgc tgtggggtgg 2340 ccgcgctaca cgacagtata gttgcggtcc tggtttatga aataactgag ggaacaagag 2400 gcgcaagaaa tccctccttg ggtgcaagac caaaacaact acccagcggg aagactcggg 2460 cttcagtgcg tgtgtcgcca gtggaggagg acgcttcggg gcgggagcac aagctggcag 2520 gacagccccg cagcagctcc agcgcggcag aggccatgga agatggtccg ctggtcagca 2580 ccctgcctgc gcctcaaaat accgccggga aggtggggga cggggacggg agcagccaat 2640 cacgcgagct ctctcgggtg gcgtcagggc gcgatcgcgg attcggcact ccgggtgggc 2700 gtggctgggc gagcgaggag tggggacaag gtcgagcgac gagtcggcgc gcccaggtaa 2760 gtttagtctt tttgtctttt atttcaggtc ccggatccgg tggtggtgca aatcaaagaa 2820 ctgctcctca gtggatgttg cctttacttc taggcctgta cggaagtgtt acttctgctc 2880 taaaagctgc ggaattgtac ccgcggccgg ctagccgcca ccatggagaa gaatggaaat 2940 aaccgaaagc tgcgggtttg tgttgctact tgtaaccgtg cagattattc taaacttgcc 3000 ccgatcatgt ttggcattaa aaccgaacct gagttctttg aacttgatgt tgtggtactt 3060 ggctctcacc tgatagatga ctatggaaat acatatcgaa tgattgaaca agatgacttt 3120 gacattaaca ccaggctaca cacaattgtg aggggagaag atgaggcagc catggtggag 3180 tcagtaggcc tggccctagt gaagctgcca gatgtcctta atcgcctgaa gcctgatatc 3240 atgattgttc atggagacag gtttgatgcc ctggctctgg ccacatctgc tgccttgatg 3300 aacatccgaa tccttcacat tgaaggtggg gaagtcagtg ggaccattga tgactctatc 3360 agacatgcca taacaaaact ggctcattat catgtgtgct gcacccgcag tgcagagcag 3420 cacctgatat ccatgtgtga ggaccatgat cgcatccttt tggcaggctg cccttcctat 3480 gacaaacttc tctcagccaa gaacaaagac tacatgagca tcattcgcat gtggctaggt 3540 gatgatgtaa aatctaaaga ttacattgtt gcactacagc accctgtgac cactgacatt 3600 aagcattcca taaaaatgtt tgaattaaca ttggatgcac ttatctcatt taacaagcgg 3660 accctagtcc tgtttccaaa tattgacgca gggagcaaag agatggttcg agtgatgcgg 3720 aagaagggca ttgagcatca tcccaacttt cgtgcagtta aacacgtccc atttgaccag 3780 tttatacagt tggttgccca tgctggctgt atgattggga acagcagctg tggggttcga 3840 3900 acaggggaga atgttcttca tgtccgggat gctgacacc aagacaaaat attgcaagca 3960 4020 gttccaagga ttttgaagtt tctcaaatct atcgatcttc aagagccact gcaaaagaaa 4080 ttctgctttc ctcctgtgaa ggagaatatc tctcaagata ttgaccatat tcttgaaact 4140 ctaagtgcct tggccgttga tcttggcggg acgaacctcc gagttgcaat agtcagcatg 4200 aagggtgaaa tagttaagaa gtatactcag ttcaatccta aaacctatga agaggatt 4260 4320 attttgggag taggcatttc cacaggtggc cgtgtaaatc ctcgggaagg aattgtgctg 4380 cattcaacca aactgatcca agagtggaac tctgtggacc ttaggacccc cctttctgac 4440 actttgcatc tccctgtgtg ggtagacaat gatggcaact gtgctgccct ggcggaaagg 4500 aaatttggcc aaaggaaggg actggaaaac tttgttacac ttatcacagg cacaggaatc 4560 ggtggtggaa ttatccatca gcatgaattg atccacggaa gctccttctg tgctccagaa 4620 ctgggccacc ttgttgtgtc tctggatggg cctgattgtt cctgtggaag ccatgggtgc 4680 attgaagcat acgcctctgg aatggccttg cagagggagg caaaaaagct ccatgatgag 4740 gacctgctct tggtggaagg gatgtcagtg ccaaaagatg aggctgtggg tgcgctccat 4800 ctcatccaag ctgcgaaact tggcaatgcg aaggcccaga gcatcctaag aacagctgga 4860 acagctttgg gtcttggggt tgtgaacatc ctccatacca tgaatccctc ccttgtgatc 4920 ctctccggag tcctggccag tcactatatc cacattgtca aagacgtcat tcgccagcag 4980 gccttgtcct ccgtgcagga cgtggatgtg gtggtttcgg atttggttga ccccgccctg 5040 ctgggtgctg ccagcatggt tctggactac acaacacgca ggatctacta gcatgcacta 5100 gtgcggccgc aataaaagat ctttattttc attagatctg tgtgttggtt ttttgtgtgt 5160 ctagagcatg gctacgtaga tagtagcat ggcgggttaa tcattaacta caaggaaccc 5220 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 5280 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 5340 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 5400 aatggcgaat ggcgattccg ttgcaatggc tggcggtaat attgttctgg atattaccag 5460 caaggccgat agtttgagtt cttctactca ggcaagtgat gttatacta atcaaagaag 5520 tattgcgaca acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga 5580 ttataaaac acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg 5640 cctcctgttt agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa 5700 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 5760 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 5820 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcgggg ctccctttag 5880 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 5940 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 6000 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 6060 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 6120 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttaa atatttgctt 6180 atacaatctt cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca 6240 tgctagtttt acgattaccg ttcatcgatt ctcttgtttg ctccagactc tcaggcaatg 6300 acctgatagc ctttgtagag acctctcaaa aatagctacc ctctccggca tgaatttatc 6360 agctagaacg gttgaatatc atattgatgg tgatttgact gtctccggcc tttctcaccc 6420 gtttgaatct ttacctacac attactcagg cattgcattt aaaatatatg agggttctaa 6480 aaatttttat ccttgcgttg aaataaaggc ttctcccgca aaagtattac agggtcataa 6540 tgtttttggt acaaccgatt tagctttatg ctctgaggct ttattgctta attttgctaa 6600 ttctttgcct tgcctgtatg atttattgga tgttggaatc gcctgatgcg gtattttctc 6660 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 6720 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6780 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6840 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc 6900 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 6960 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 7020 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 7080 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 7140 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatca 7187 <210> 16 <211> 8423 <212> DNA <213> Homo sapiens <400> 16 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 60 ttttcgcccc gaagaacgaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 120 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 180 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 240 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 300 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 360 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 420 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 480 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 540 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 600 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 660 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 720 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 780 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 840 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 900 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 960 ttaacgtgag tttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 1020 ttgagatcct ttttttgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1080 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 1140 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1200 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1260 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1320 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1380 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1440 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1500 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggttcgcc acctctgact 1560 tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1620 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 1680 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 1740 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 1800 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg 1860 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 1920 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 1980 ttaatgatta acccgccatg ctacttatct acgtagccat gctctagagt ttaaacaagc 2040 ttgcatgtct aagctagacc cttcagatta aaaataactg aggtaagggc ctgggtaggg 2100 gaggtggtgt gagacgctcc tgtctctcct ctatctgccc atcggccctt tggggaggag 2160 gaatgtgccc aaggactaaa aaaaggccat ggagccagag gggcgagggc aacagacctt 2220 tcatgggcaa accttggggc cctgctgtct agcatgcccc actacgggtc taggctgccc 2280 atgtaaggag gcaaggcctg gggacacccg agatgcctgg ttataattaa cccagacatg 2340 tggctgcccc ccccccccca acacctgctg cctctaaaaa taaccctgtc cctggtggat 2400 cccctgcatg cgaagatctt cgaacaaggc tgtggggac tgagggcagg ctgtaacagg 2460 cttggggggcc agggcttata cgtgcctggg actcccaaag tattactgtt ccatgttccc 2520 ggcgaagggc cagctgtccc ccgccagcta gactcagcac ttagtttagg aaccagtgag 2580 caagtcagcc cttggggcag cccatacaag gccatggggc tgggcaagct gcagccctgg 2640 gtccggggtg ggcacggtgc ccgggcaacg agctgaaagc tcatctgctc tcaggggccc 2700 ctccctgggg acagcccctc ctggctagtc acaccctgta ggctcctcta tataacccag 2760 gggcacaggg gctcccctca ttctaccacc acctccacag cacacacaga cactcaggag 2820 cagccagcgg cgcgcccagg taagtttagt cttttgtct tttatttcag gtcccggatc 2880 cggtggtggt gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctaggcct 2940 gtacggaagt gttacttctg ctctaaaagc tgcggaattg tacccgcggc cggctagccg 3000 ccaccatgga gaagatgga aataccgaa agctgcgggt ttgtgttgct acttgtaacc 3060 gtgcagatta ttctaaactt gccccgatca tgtttggcat taaaaccgaa cctgagttct 3120 ttgaacttga tgttgtggta cttggctctc acctgataga tgactatgga aatacatatc 3180 gaatgattga acaagatgac tttgacatta acaccaggct acacacaatt gtgaggggag 3240 aagatgaggc agccatggtg gagtcagtag gcctggccct agtgaagctg ccagatgtcc 3300 ttaatcgcct gaagcctgat atcatgattg ttcatggaga caggtttgat gccctggctc 3360 tggccacatc tgctgccttg atgaacatcc gaatccttca cattgaaggt ggggaagtca 3420 gtgggaccat tgatgactct atcagacatg ccataacaaa actggctcat tatcatgtgt 3480 gctgcacccg cagtgcagag cagcacctga tatccatgtg tgaggaccat gatcgcatcc 3540 ttttggcagg ctgcccttcc tatgacaaac ttctctcagc caagaacaaa gactacatga 3600 gcatcattcg catgtggcta ggtgatgatg taaaatctaa agattacatt gttgcactac 3660 agcaccctgt gaccactgac attaagcatt ccataaaaat gtttgaatta acattggatg 3720 cacttatctc atttaacaag cggaccctag tcctgtttcc aaatattgac gcagggagca 3780 aagagatggt tcgagtgatg cggaagaagg gcattgagca tcatcccac ttcgtgcag 3840 ttaaacacgt cccatttgac cagttatatac agttggttgc ccatgctggc tgtatgattg 3900 ggaacagcag ctgtggggtt cgagaagttg gagctttgg aacacctgtg atcaacctgg 3960 gaacacgtca gattgaga gaacagggg agaatgttct tcatgtccgg gatgctgaca 4020 cccaagacaa atattgcaa gcactgcacc ttcagttgg taacagtac ccttgttcaa 4080 agatatgg ggatggaat gctgttccaa ggatttgaa gtttctcaa tctatcgatc 4140 ttcaagagcc actgcaaag aaattctgct ttcctcctgt gaaggagaat atctctcaag 4200 atattgacca tattcttga actctaagtg ccttggccgt tgatcttggc gggacgaacc 4260 tccgagttgc atagtcagc atgaagggtg aaatagttaa gaagtatact cagttcaatc 4320 ctaaaaccta tgaagagagg attaatttaa tcctacagat gtgtgtggaa gctgcagcag 4380 aagctgtaaa actgaactgc agaatttgg gagtaggcat ttccaggt ggccgtgtaa 4440 atcctcggga aggaattgtg ctgcattcaa ccaactgat ccaagtgg aactctgtgg 4500 accttaggac ccccctttct gacactttgc atctccctgt gtgggtagac aatgatggca 4560 actgtgctgc cctggcggaa aggaaatttg gccaaggaaa gggactggaa aactttgtta 4620 cacttatcac aggcacagga atcggtggtg gaattatcca tcagcatgaa ttgatccacg 4680 gaagctcctt ctgtgctgca gaactgggcc accttgttgt gtctctggat gggcctgatt 4740 gttcctgtgg aagccatggg tgcattgaag catacgcctc tggaatggcc ttgcagaggg 4800 aggcaaaaaa gctccatgat gaggacctgc tcttggtgga agggatgtca gtgccaaaag 4860 atgaggctgt gggtgcgctc catctcatcc aagctgcgaa acttggcaat gcgaaggccc 4920 agagcatcct aagaacagct ggaacagctt tgggtcttgg ggttgtgaac atcctccata 4980 ccatgaatcc ctcccttgtg atcctctccg gagtcctggc cagtcactat atccacattg 5040 tcaaagacgt cattcgccag caggccttgt cctccgtgca ggacgtggat gtggtggttt 5100 cggatttggt tgaccccgcc ctgctgggtg ctgccagcat ggttctggac tacacaacac 5160 gcaggatcta ctagcatgcc ttctgatggt gaatgggaac tcccttcctc ctgcagcagc 5220 ctgcctgcag ctgtcctggt agaacagtgt ggacattgca gaagctgtca ctgccccaga 5280 aagaaagcac cccagagact agtatggtcc gcgcgaggca ccagccgggt gggctttgcc 5340 tcctgctgct gctgctctgc cagttcatgg aggaccgcag tgcccaggct gggaactgct 5400 ggctccgtca agcgaagaac ggccgctgcc aggtcctgta caagaccgaa ctgagcaagg 5460 aggagtgctg cagcaccggc cggctgagca cctcgtggac cgaggaggac gtgaatgaca 5520 acacactctt caagtggatg attttcaacg ggggcgcccc caactgcatc ccctgtaaag 5580 aaacgtgtga gaacgtggac tgtggacctg ggaaaaaatg ccgaatgaac aagaagaaca 5640 aaccccgctg cgtctgcgcc ccggattgtt ccaacatcac ctggaagggt ccagtctgcg 5700 ggctggatgg gaaaacctac cgcaatgaat gtgcactcct aaaggcaaga tgtaaagagc 5760 agccagaact ggaagtccag taccaaggca gatgtaaaaa gacttgtcgg gatgttttct 5820 gtccaggcag ctccacatgt gtggtggacc agaccaataa tgcctactgt gtgacctgta 5880 atcggatttg cccagagcct gcttcctctg agcaatatct ctgtgggaat gatggagtca 5940 cctactccag tgcctgccac ctgagaaagg ctacctgcct gctgggcaga tctattggat 6000 tagcctatga gggaaagtgt atcaaagcaa agtcctgtga agatatccag tgcactggtg 6060 ggaaaaaatg tttatgggat ttcaaggttg ggagaggccg gtgttccctc tgtgatgagc 6120 tgtgccctga cagtaagtcg gatgagcctg tctgtgccag tgacaatgcc acttatgcca 6180 gcgagtgtgc catgaaggaa gctgcctgct cctcaggtgt gctactggaa gtaaagcact 6240 ccggatcttg caactccatt tcggaagaca ccgaggaaga ggaggaagat gaagaccagg 6300 actacagctt tcctatatct tctattctag agtggtaagc ggccgcaata aaagatcttt 6360 atttcatta gatctgtgtg ttggtttttt gtgtgtctag agcatggcta cgtagataag 6420 tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc 6480 tctctgcgcg ctgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgc 6540 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct ggcgtaatag cgaagaggcc 6600 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg attccgttgc 6660 aatggctggc ggtaatattg ttctggatat taccagcaag gccgatagtt tgagttcttc 6720 tactcaggca agtgatgtta tactaatca aagaagtatt gcgacaacgg ttaatttgcg 6780 tgatggacag actctttac tcggtggcct cactgattat aaaaacactt ctcaggattc 6840 tggcgtaccg ttcctgtcta aaatcccttt aatcggcctc ctgtttagct cccgctctga 6900 ttctaacgag gaaagcacgt tatacgtgct cgtcaaagca accatagtac gcgccctgta 6960 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 7020 gcgccctagc gcccgctcct ttcgctttct tccttcctt tctcgccacg ttcgccggct 7080 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 7140 acctcgacccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 7200 agacggtttt tcgccctttg acgttggagt ccacgttctt tatagtgga ctcttgttcc 7260 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 7320 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 7380 acaaaatatt aacgcttaca atttaaatat ttgcttatac aatcttcctg tttttggggc 7440 ttttctgatt atcaaccggg gtacatatga ttgacatgct agttttacga ttaccgttca 7500 tcgattctct tgtttgctcc agactctcag gcaatgacct gatagccttt gtagagacct 7560 ctcaaaaata gctaccctct ccggcatgaa tttatcagct agaacggttg aatatcatat 7620 tgatggtgat ttgactgtct ccggcctttc tcacccgttt gaatctttac ctacacatta 7680 ctcaggcatt gcatttaaaa tatatgaggg ttctaaaaat ttttatcctt gcgttgaaat 7740 aaaggcttct cccgcaaaag tattacaggg tcataatgtt tttggtacaa ccgatttagc 7800 tttatgctct gaggctttat tgcttaattt tgctaattct ttgccttgcc tgtatgattt 7860 attggatgtt ggaatcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 7920 caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 7980 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 8040 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 8100 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 8160 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 8220 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 8280 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 8340 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 8400 aaagtaaaag atgctgaaga tca 8423 <210> 17 <211> 7928 <212> DNA <213> Homo sapiens <400> 17 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 60 ttttcgcccc gaagaacgaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 120 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 180 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 240 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 300 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 360 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 420 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 480 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 540 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 600 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 660 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 720 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 780 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 840 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 900 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 960 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aggatcttc 1020 ttgagatcct ttttttgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1080 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 1140 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1200 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1260 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1320 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1380 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1440 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1500 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggttcgcc acctctgact 1560 tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1620 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 1680 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 1740 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 1800 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg 1860 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 1920 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 1980 ttaatgatta acccgccatg ctacttatct acgtagccat gctctagagt ttaaacaagc 2040 ttgcatgtct aagctagacc cttcagatta aaaataactg aggtaagggc ctgggtaggg 2100 gaggtggtgt gagacgctcc tgtctctcct ctatctgccc atcggccctt tggggaggag 2160 gaatgtgccc aaggactaaa aaaaggccat ggagccagag gggcgagggc aacagacctt 2220 tcatgggcaa accttggggc cctgctgtct agcatgcccc actacgggtc taggctgccc 2280 atgtaaggag gcaaggcctg gggacacccg agatgcctgg ttataattaa cccagacatg 2340 tggctgcccc ccccccccca acacctgctg cctctaaaaa taaccctgtc cctggtggat 2400 cccctgcatg cgaagatctt cgaacaaggc tgtgggggac tgagggcagg ctgtaacagg 2460 cttggggggcc agggcttata cgtgcctggg actcccaaag tattactgtt ccatgttccc 2520 ggcgaagggc cagctgtccc ccgccagcta gactcagcac ttagtttagg aaccagtgag 2580 caagtcagcc cttggggcag cccatacaag gccatggggc tgggcaagct gcagccctgg 2640 gtccggggtg ggcacggtgc ccgggcaacg agctgaaagc tcatctgctc tcaggggccc 2700 ctccctgggg acagcccctc ctggctagtc acaccctgta ggctcctcta tataacccag 2760 gggcacaggg gctcccctca ttctaccacc acctccacag cacacacaga cactcaggag 2820 cagccagcgg cgcgcccagg taagtttagt cttttgtct tttatttcag gtcccggatc 2880 cggtggtggt gcaaatcaaa gaactgctcc tcagtggatg ttgcctttac ttctaggcct 2940 gtacggaagt gttacttctg ctctaaaagc tgcggaattg tacccgcggc cggctagccg 3000 ccaccatgga gaagatgga aataccgaa agctgcgggt ttgtgttgct acttgtaacc 3060 gtgcagatta ttctaaactt gccccgatca tgtttggcat taaaaccgaa cctgagttct 3120 ttgaacttga tgttgtggta cttggctctc acctgataga tgactatgga aatacatatc 3180 gaatgattga acaagatgac tttgacatta acaccaggct acacacaatt gtgaggggag 3240 aagatgaggc agccatggtg gagtcagtag gcctggccct agtgaagctg ccagatgtcc 3300 ttaatcgcct gaagcctgat atcatgattg ttcatggaga caggtttgat gccctggctc 3360 tggccacatc tgctgccttg atgaacatcc gaatccttca cattgaaggt ggggaagtca 3420 gtgggaccat tgatgactct atcagacatg ccataacaaa actggctcat tatcatgtgt 3480 gctgcacccg cagtgcagag cagcacctga tatccatgtg tgaggaccat gatcgcatcc 3540 ttttggcagg ctgcccttcc tatgacaaac ttctctcagc caagaacaaa gactacatga 3600 gcatcattcg catgtggcta ggtgatgatg taaaatctaa agattacatt gttgcactac 3660 agcaccctgt gaccactgac attaagcatt ccataaaaat gtttgaatta acattggatg 3720 cacttatctc atttaacaag cggaccctag tcctgtttcc aaatattgac gcagggagca 3780 aagagatggt tcgagtgatg cggaagaagg gcattgagca tcatcccaac tttcgtgcag 3840 ttaaacacgt cccatttgac cagtttatac agttggttgc ccatgctggc tgtatgattg 3900 ggaacagcag ctgtggggtt cgagaagttg gagctttttgg aacacctgtg atcaacctgg 3960 gaacacgtca gattggaaga gaaacagggg agaatgttct tcatgtccgg gatgctgaca 4020 cccaagacaa aatattgcaa gcactgcacc ttcagtttgg taaacagtac ccttgttcaa 4080 agatatatgg ggatggaaat gctgttccaa ggattttgaa gtttctcaaa tctatcgatc 4140 ttcaagagcc actgcaaaag aaattctgct ttcctcctgt gaaggagaat atctctcaag 4200 atattgacca tattcttgaa actctaagtg ccttggccgt tgatcttggc gggacgaacc 4260 tccgagttgc aatagtcagc atgaagggtg aatagttaa gaagtatact cagttcaatc 4320 ctaaaaccta tgaagagagg attaatttaa tcctacagat gtgtgtggaa gctgcagcag 4380 aagctgtaaa actgaactgc agaatttttgg gagtaggcat ttccacaggt ggccgtgtaa 4440 atcctcggga aggaattgtg ctgcattcaa ccaaactgat ccaagagtgg aactctgtgg 4500 accttaggac ccccctttct gacactttgc atctccctgt gtgggtagac aatgatggca 4560 actgtgctgc cctggcggaa aggaaatttg gccaaggaaa gggactggaa aactttgtta 4620 cacttatcac aggcacagga atcggtggtg gaattatcca tcagcatgaa ttgatccacg 4680 gaagctcctt ctgtgctgca gaactgggcc accttgttgt gtctctggat gggcctgatt 4740 gttcctgtgg aagccatggg tgcattgaag catacgcctc tggaatggcc ttgcagaggg 4800 aggcaaaaaa gctccatgat gaggacctgc tcttggtgga agggatgtca gtgccaaaag 4860 atgaggctgt gggtgcgctc catctcatcc aagctgcgaa acttggcaat gcgaaggccc 4920 agagcatcct aagaacagct ggaacagctt tgggtcttgg ggttgtgaac atcctccata 4980 ccatgaatcc ctcccttgtg atcctctccg gagtcctggc cagtcactat atccacattg 5040 tcaaagacgt cattcgccag caggccttgt cctccgtgca ggacgtggat gtggtggttt 5100 cggatttggt tgaccccgcc ctgctgggtg ctgccagcat ggttctggac tacacaacac 5160 gcaggatcta ctagcatgcc ttctgatggt gaatgggaac tcccttcctc ctgcagcagc 5220 ctgcctgcag ctgtcctggt agaacagtgt ggacattgca gaagctgtca ctgccccaga 5280 aagaaagcac cccagagact ggccgcacca tgaagctgct gccgtcggtg gtgctgaagc 5340 tctttctggc tgcagttctc tcggcactgg tgactggcga gagcctggag cggcttcgga 5400 gagggctagc tgctggaacc agcaacccgg accctcccac tgtatccacg gaccagctgc 5460 tacccctagg aggcggccgg gaccgggaaag tccgtgactt gcaagaggca gatctggacc 5520 ttttgagagt cactttatcc tccaagccac aagcactggc cacaccaaac aaggaggac 5580 acgggaaaag aaagagaaa ggcaaggggc tagggagaa gagggaccca ggaccggaga 5640 cgctctgcgg ggctgagctg gtggatgctc ttcagttcgt gtgtggagac aggggctttt 5700 atttcaacaa gcccacaggg tatggctcca gcagtcggag ggcgcctcag acaggcatcg 5760 tggatgagtg ctgcttccgg agctgtgatc taaggaggct ggagatgtat tgcgcacccc 5820 tcaagcctgc caagtcagct taggcggccg aataaaga tctttattt cattagatct 5880 gtgtgttggt tttttgtgtg tctagagcat ggctacgtag ataagtagca tggcgggtta 5940 atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 6000 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 6060 tcagtgagcg agcgagcgcg cagctggcgt aatagcgaag aggcccgcac cgatcgccct 6120 tcccaacagt tgcgcagcct gaatggcgaa tggcgattcc gttgcaatgg ctggcggtaa 6180 tattgttctg gatattacca gcaaggccga tagtttgagt tcttctactc aggcaagtga 6240 tgttattact aatcaaagaa gtattgcgac aacggttaat ttgcgtgatg gacagactct 6300 tttactcggt ggcctcactg attataaaaa cacttctcag gattctggcg taccgttcct 6360 gtctaaaatc cctttaatcg gcctcctgtt tagctcccgc tctgattcta acgaggaaag 6420 cacgttatac gtgctcgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 6480 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 6540 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 6600 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 6660 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 6720 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 6780 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt 6840 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 6900 ttacaattta aatatttgct tatacaatct tcctgttttt ggggcttttc tgattatcaa 6960 ccggggtaca tatgattgac atgctagttt tacgattacc gttcatcgat tctcttgttt 7020 gctccagact ctcaggcaat gacctgatag cctttgtaga gacctctcaa aaatagctac 7080 cctctccggc atgaatttat cagctagaac ggttgaatat catattgatg gtgatttgac 7140 tgtctccggc ctttctcacc cgtttgaatc tttacctaca cattactcag gcattgcatt 7200 taaaatatat gagggttcta aaaattttta tccttgcgtt gaaataaagg cttctcccgc 7260 aaaagtatta cagggtcata atgtttttgg tacaaccgat ttagctttat gctctgaggc 7320 tttattgctt aattttgcta attctttgcc ttgcctgtat gatttattgg atgttggaat 7380 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc 7440 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 7500 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 7560 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 7620 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 7680 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 7740 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 7800 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 7860 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 7920 gaagatca 7928 <210> 18 <211> 8430 <212> DNA <213> Homo sapiens <400> 18 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 60 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 120 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 180 atttgaatgt atttagaaa aaaacaat aggggttccg cgcacattc cccgaaagt 240 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 300 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gandacatgca 360 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 420 gggcgcgtca gcgtgtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 480 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taggagaaa 540 ataccgcatc aggcgattcc aacatccaat aaatcataca ggcaaggcaa agattagca 600 aaattaagca aaagccctc agagcataa gctaaatcgg ttgtaccaa aacattatga 660 ccctgtataa cttttgcgggg agaagcctttt atttcaacgc aaggataaaa atttttagaa 720 ccctcatata ttttaaatgc atgcctgag taatgtgtag gtaagattc aaacggggtga 780 gaaaggccgg agacagtca atcaccatca atatgatatt caacgttct agctgataaa 840 ttcatgccgg agagggtagc tattttgag aggtctctac aaggctatc aggtcattgc 900 ctgagagtct ggagcaaaca agaagaatcga tgaacggtaa tcgtaaact agcatgtcaa 960 tcatatgtac cccggttgat aatcagaaaa gccccaaaaa caggagaatt gtataagcaa 1020 atatttaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 1080 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 1140 ccgagatagg gttgagtgtt gttccagttt ggaaagag tccactatta aagaacgtgg 1200 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 1260 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 1320 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaagggga 1380 agaaaggaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 1440 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg 1500 agcacgtata acgtgctttc ctcgttagaa tcagagcggg agctaaacag gaggccgatt 1560 aaagggattt tagacaggaa cggtacgcca gaatcctgag aagtgttttt ataatcagtg 1620 aggccaccga gtaaaagagt ctgtccatca cgcaaattaa ccgttgtcgc aatacttctt 1680 tgattagtaa taacatcact tgcctgagta gaagaactca aactatcggc cttgctggta 1740 atatccagaa caatattacc gccagccatt gcaacggaat cgccattcgc cattcaggct 1800 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc 1860 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc 1920 ggcctcagtg agcgagcgag cgcgcaga gggagtggcc aactccatca ctaggggttc 1980 cttgtagtta atgattaacc cgccatgcta cttatctacg tagccatgct ctaggtcgtt 2040 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 2100 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 2160 gtggagtatt tacggtaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 2220 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 2280 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 2340 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 2400 ccaagtctcc accccattga cgtcaatggg agttgtttt ggcaccaaaa tcaacgggac 2460 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 2520 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 2580 ccacgctgtt ttgacctcca tagagacac cgggaccgat ccagcctccg gactcttagag 2640 gatccggtac tcgaggaact gaaaaaccag aaagttaact ggtaagttta gtcttttgt 2700 cttttatttc aggtcccgga tccggtggtg gtgcaaatca aagaactgct cctcagtgga 2760 tgttgccttt acttttaggc ctgtacggaa gtgttacttc tgctctaaaa gctcggaat 2820 tgtacccgcg gccggctagc cgccaccatg gagaagaatg gaaataaccg aaagctgcgg 2880 gtttgtgttg ctacttgtaa ccgtgcagat tattctaaac ttgccccgat catgtttggc 2940 attaaaaccg aacctgagtt ctttgaactt gatgttgtgg tacttggctc tcacctgata 3000 gatgactatg gaaatacata tcgaatgatt gaacaagatg actttgacat taacaccagg 3060 ctacacacaa ttgtgagggg agaagatgag gcagccatgg tggagtcagt aggcctggcc 3120 ctagtgaagc tgccagatgt ccttaatcgc ctgaagcctg atatcatgat tgttcatgga 3180 gacaggtttg atgccctggc tctggccaca tctgctgcct tgatgaacat ccgaatcctt 3240 cacattgaag gtggggaagt cagtgggacc attgatgact ctatcagaca tgccataaca 3300 aaactggctc attatcatgt gtgctgcacc cgcagtgcag agcagcacct gatatccatg 3360 tgtgaggacc atgatcgcat ccttttggca ggctgccctt cctatgacaa acttctctca 3420 gccaagaaca aagactacat gagcatcatt cgcatgtggc taggtgatga tgtaaaatct 3480 aaagattaca ttgttgcact acagcaccct gtgaccactg acattaagca ttccataaaa 3540 atgtttgaat taacattgga tgcacttatc tcatttaaca agcggaccct agtcctgttt 3600 ccaaatattg acgcagggag caaagagatg gttcgagtga tgcggaagaa gggcattgag 3660 catcatccca actttcgtgc agttaaacac gtcccatttg accagtttat acagttggtt 3720 gcccatgctg gctgtatgat tgggaacagc agctgtgggg ttcgagaagt tggagctttt 3780 ggaacacctg tgatcaacct gggaacacgt cagattggaa gagaaacagg ggagaatgtt 3840 cttcatgtcc gggatgctga cacccaagac aaaatattgc aagcactgca ccttcagttt 3900 ggtaaacagt acccttgttc aaagatatat ggggatggaa atgctgttcc aaggattttg 3960 aagttctca aatctatcga tcttcaagag ccactgcaaa agaaattctg ctttcct 4020 gtgaaggaga atactctca agatattgac catattcttg aaactctaag tgccttggcc 4080 gttgatcttg gcgggacgaa cctccgagtt gcaatagtca gcatgaaggg tgaaatagtt 4140 aagaagtata ctcagttcaa tcctaaaacc tatgaagaga ggattaattt aatcctacag 4200 atgtgtgtgg aagctgcagc agaagctgta aaactgaact gcagaattttt gggagtaggc 4260 atttccacag gtggccgtgt aaatcctcgg gaaggaattg tgctgcattc aaccaaactg 4320 atccaagagt ggaactctgt ggaccttagg accccccttt ctgacacttt gcatctccct 4380 gtgtgggtag acaatgatgg caactgtgct gccctggcgg aaaggaaatt tggccaagga 4440 aagggactgg aaaactttgt tacacttatc acaggcacag gaatcggtgg tggaattatc 4500 catcagcatg aattgatcca cggaagctcc ttctgtgctg cagaactggg ccaccttgtt 4560 gtgtctctgg atgggcctga ttgttcctgt ggaagccatg ggtgcattga agcatacgcc 4620 tctggaatgg ccttgcagag ggaggcaaaa aagctccatg atgaggacct gctcttggtg 4680 gaagggatgt cagtgccaaa agatgaggct gtgggtgcgc tccatctcat ccaagctgcg 4740 aaacttggca atgcgaaggc ccagagcatc ctaagaacag ctggaacagc tttgggtctt 4800 ggggttgtga acatcctcca taccatgaat ccctcccttg tgatcctctc cggagtcctg 4860 gccagtcact atatccacat tgtcaaagac gtcattcgcc agcaggcctt gtcctccgtg 4920 caggacgtgg atgtggtggt ttcggatttg gttgaccccg ccctgctggg tgctgccagc 4980 atggttctgg actacacaac acgcaggatc tactagcatg ccttctgatg gtgaatggga 5040 actcccttcc tcctgcagca gcctgcctgc agctgtcctg gtagaacagt gtggacattg 5100 cagaagctgt cactgcccca gaaagaaagc accccagaga ctagtatggt ccgcgcgagg 5160 caccagccgg gtgggctttg cctcctgctg ctgctgctct gccagttcat ggaggaccgc 5220 agtgcccagg ctgggaactg ctggctccgt caagcgaaga acggccgctg ccaggtcctg 5280 tacaagaccg aactgagcaa ggaggagtgc tgcagcaccg gccggctgag cacctcgtgg 5340 accgaggagg acgtgaatga caacacactc ttcaagtgga tgattttcaa cgggggcgcc 5400 cccaactgca tcccctgtaa agaaacgtgt gagaacgtgg actgtggacc tgggaaaaaa 5460 tgccgaatga acaagaagaa caaaccccgc tgcgtctgcg ccccggattg ttccaacatc 5520 acctggaagg gtccagtctg cgggctggat gggaaaacct accgcaatga atgtgcactc 5580 ctaaaggcaa gatgtaaaga gcagccagaa ctggaagtcc agtaccaagg cagatgtaaa 5640 aagacttgtc gggatgtttt ctgtccaggc agctccacat gtgtggtgga ccagaccaat 5700 aatgcctact gtgtgacctg taatcggatt tgcccagagc ctgcttcctc tgagcaatat 5760 ctctgtggga atgatggagt cacctactcc agtgcctgcc acctgagaaa ggctacctgc 5820 ctgctgggca gatctattgg attagcctat gagggaaagt gtatcaaagc aaagtcctgt 5880 gaagatatcc agtgcactgg tgggaaaaaa tgtttatggg atttcaaggt tgggagaggc 5940 cggtgttccc tctgtgatga gctgtgccct gacagtaagt cggatgagcc tgtctgtgcc 6000 agtgacaatg ccacttatgc cagcgagtgt gccatgaagg aagctgcctg ctcctcaggt 6060 gtgctactgg aagtaaagca ctccggatct tgcaactcca tttcggaaga caccgaggaa 6120 gaggaggaag atgaagacca ggactacagc tttcctatat cttctattct agagtggtaa 6180 gcggccgcgg ggatccagac atgataagat acattgatga gtttggacaa accacaacta 6240 gaatgcagtg aaaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttattgttaa 6300 ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg 6360 ttcaggggga ggtgtgggag gttttttcgg atcccttaga gtcgaaaact ctagagcatg 6420 gctacgtaga taagtagcat ggcggggttaa tcattaacta caaggaaccc ctagtgatgg 6480 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg 6540 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcatta 6600 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttctct 6660 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 6720 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 6780 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 6840 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 6900 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 6960 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 7020 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 7080 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 7140 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 7200 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 7260 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 7320 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 7380 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 7440 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa 7500 aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa 7560 cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat 7620 gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg 7680 atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg 7740 agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta 7800 tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc 7860 aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc 7920 tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc 7980 gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg 8040 acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat 8100 tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg 8160 aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg 8220 atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt 8280 ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg 8340 atgagttttt ctaagaattc gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 8400 gagatccagt tcgatgtaac ccactcgtgc 8430 <210> 19 <211> 7937 <212> DNA <213> Homo sapiens <400> 19 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 60 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 120 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 180 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 240 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 300 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 360 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 420 gggcgcgtca gcgtgtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 480 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg tagggaagaaa 540 ataccgcatc aggcgattcc aacatccaat aaatcataca ggcaaggcaa agaattagca 600 aaattaagca ataaagcctc agagcataa gctaaatcgg ttgtaccaaa aacattatga 660 720 ccctcatata ttttaaatgc aatgcctgag taatgtgtag gtaaagattc aaacgggtga 780 gaaaggccgg agacagtcaa atcaccatca atatgatatt caaccgttct agctgataaa 840 ttcatgccgg agagggtagc tatttttgag aggtctctac aaaggctatc aggtcattgc 900 ctgagagtct ggagcaaaca agaagaatcga tgaacggtaa tcgtaaact agcatgtcaa 960 tcatatgtac cccggttgat aatcagaaaa gccccaaaaa caggagaatt gtataagcaa 1020 atatttaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 1080 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 1140 ccgagatagg gttgagtgtt gttccagttt ggaaagag tccactatta aagaacgtgg 1200 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 1260 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 1320 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaagggga 1380 agaaaggaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 1440 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg 1500 agcacgtata acgtgctttc ctcgttagaa tcagagcggg agctaaacag gaggccgatt 1560 aaagggattt tagacaggaa cggtacgcca gaatcctgag aagtgttttt ataatcagtg 1620 aggccaccga gtaaaagagt ctgtccatca cgcaaattaa ccgttgtcgc aatacttctt 1680 tgattagtaa taacatcact tgcctgagta gaagaactca aactatcggc cttgctggta 1740 atatccagaa caatattacc gccagccatt gcaacggaat cgccattcgc cattcaggct 1800 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc 1860 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc 1920 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc 1980 cttgtagtta atgattaacc cgccatgcta cttatctacg tagccatgct ctaggtcgtt 2040 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 2100 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 2160 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 2220 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 2280 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 2340 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 2400 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 2460 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 2520 tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 2580 ccacgctgtt ttgacctcca tagagacac cgggaccgat ccagcctccg gactcttagag 2640 gatccggtac tcgaggaact gaaaaaccag aaagttaact ggtaagttta gtcttttgt 2700 cttttatttc aggtcccgga tccggtggtg gtgcaaatca aagaactgct cctcagtgga 2760 tgttgccttt acttttaggc ctgtacggaa gtgttacttc tgctctaaaa gctcggaat 2820 tgtacccgcg gccggctagc cgccaccatg gagaagaatg gaaataaccg aaagctgcgg 2880 gtttgtgttg ctacttgtaa ccgtgcagat tattctaaac ttgccccgat catgtttggc 2940 attaaaaccg aacctgagtt ctttgaactt gatgttgtgg tacttggctc tcacctgata 3000 gatgactatg gaaatacata tcgaatgatt gaacaagatg actttgacat taacaccagg 3060 ctacacacaa ttgtgagggg agaagatgag gcagccatgg tggagtcagt aggcctggcc 3120 ctagtgaagc tgccagatgt ccttaatcgc ctgaagcctg atatcatgat tgttcatgga 3180 gacaggtttg atgccctggc tctggccaca tctgctgcct tgatgaacat ccgaatcctt 3240 cacattgaag gtggggaagt cagtgggacc attgatgact ctatcagaca tgccataaca 3300 aaactggctc attatcatgt gtgctgcacc cgcagtgcag agcagcacct gatatccatg 3360 tgtgaggacc atgatcgcat ccttttggca ggctgccctt cctatgacaa acttctctca 3420 gccaagaaca aagactacat gagcatcatt cgcatgtggc taggtgatga tgtaaaatct 3480 aaagattaca ttgttgcact acagcaccct gtgaccactg acattaagca ttccataaaa 3540 atgtttgaat taacattgga tgcacttatc tcatttaaca agcggaccct agtcctgttt 3600 ccaaatattg acgcagggag caaagagatg gttcgagtga tgcggaagaa gggcattgag 3660 catcatccca actttcgtgc agttaaacac gtcccatttg accagtttat acagttggtt 3720 gcccatgctg gctgtatgat tgggaacagc agctgtgggg ttcgagaagt tggagctttt 3780 ggaacacctg tgatcaacct gggaacacgt cagattggaa gagaaacagg ggagaatgtt 3840 cttcatgtcc gggatgctga cacccaagac aaaatattgc aagcactgca ccttcagttt 3900 ggtaaacagt acccttgttc aaagatatat ggggatggaa atgctgttcc aaggattttg 3960 aagttctca aatctatcga tcttcaagag ccactgcaaa agaaattctg ctttcct 4020 gtgaaggaga atactctca agatattgac catattcttg aaactctaag tgccttggcc 4080 gttgatcttg gcgggacgaa cctccgagtt gcaatagtca gcatgaaggg tgaaatagtt 4140 aagaagtata ctcagttcaa tcctaaaacc tatgaagaga ggattaattt aatcctacag 4200 atgtgtgtgg aagctgcagc agaagctgta aaactgaact gcagaattttt gggagtaggc 4260 atttccacag gtggccgtgt aaatcctcgg gaaggaattg tgctgcattc aaccaaactg 4320 atccaagagt ggaactctgt ggaccttagg accccccttt ctgacacttt gcatctccct 4380 gtgtgggtag acaatgatgg caactgtgct gccctggcgg aaaggaaatt tggccaagga 4440 aagggactgg aaaactttgt tacacttatc acaggcacag gaatcggtgg tggaattatc 4500 catcagcatg aattgatcca cggaagctcc ttctgtgctg cagaactggg ccaccttgtt 4560 gtgtctctgg atgggcctga ttgttcctgt ggaagccatg ggtgcattga agcatacgcc 4620 tctggaatgg ccttgcagag ggaggcaaaa aagctccatg atgaggacct gctcttggtg 4680 gaagggatgt cagtgccaaa agatgaggct gtgggtgcgc tccatctcat ccaagctgcg 4740 aaacttggca atgcgaaggc ccagagcatc ctaagaacag ctggaacagc tttgggtctt 4800 ggggttgtga acatcctcca taccatgaat ccctcccttg tgatcctctc cggagtcctg 4860 gccagtcact atatccacat tgtcaaagac gtcattcgcc agcaggcctt gtcctccgtg 4920 caggacgtgg atgtggtggt ttcggatttg gttgaccccg ccctgctggg tgctgccagc 4980 atggttctgg actacacaac acgcaggatc tactagcatg ccttctgatg gtgaatggga 5040 actcccttcc tcctgcagca gcctgcctgc agctgtcctg gtagaacagt gtggacattg 5100 cagaagctgt cactgcccca gaaagaaagc accccagaga ctagggccgc accatgaagc 5160 tgctgccgtc ggtggtgctg aagctctttc tggctccagt tctctcggca ctggtgactg 5220 gcgagagcct ggagcggctt cggagagggc tagctgctgg aaccagcaac ccggaccctc 5280 ccactgtatc cacggaccag ctgctacccc taggaggcgg ccgggaccgg aaagtccgtg 5340 acttgcaaga ggcagatctg gaccttttga gagtcacttt atcctccaag ccacaagcac 5400 tggccacacc aaacaaggag gagcacggga aaagaaagaa gaaaggcaag gggctaggga 5460 agaagaggga cccaggaccg gagacgctct gcggggctga gctggtggat gctcttcagt 5520 tcgtgtgtgg agacaggggc ttttatttca acaagcccac agggtatggc tccagcagtc 5580 ggagggcgcc tcagacaggc atcgtggatg agtgctgctt ccggagctgt gatctaagga 5640 ggctggagat gtattgcgca cccctcaagc ctgccaagtc agcttaggcg gccgcgggga 5700 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa 5760 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg 5820 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt 5880 gtgggaggtt ttttcggatc ctctagagtc gaaaactcta gagcatggct acgtagataa 5940 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc 6000 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 6060 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg aatcggccaa 6120 cgcgcggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg 6180 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 6240 ttatccacag aatcaggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 6300 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 6360 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 6420 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 6480 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 6540 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 6600 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 6660 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 6720 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctcac tagaaggaca 6780 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 6840 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 6900 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 6960 cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta 7020 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag 7080 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa 7140 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt 7200 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact 7260 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga 7320 tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga 7380 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca 7440 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc 7500 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg 7560 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc 7620 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat 7680 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct 7740 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg 7800 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta 7860 agaattcgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 7920 atgtaaccca ctcgtgc 7937 <210> 20 <211> 8223 <212> DNA <213> Homo sapiens <400> 20 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 60 ccttgagagt tttcgccccg aagaacgaat tcttagaaaa actcatcgag catcaaatga 120 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt 180 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct 240 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg 300 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta 360 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc 420 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg 480 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc 540 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc 600 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg 660 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca 720 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac 780 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat 840 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata 900 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac 960 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1020 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1080 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1140 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1200 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1260 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1320 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1380 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1440 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1500 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1560 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1620 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1680 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1740 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1800 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcaagctgcgc 1860 gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc 1920 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactagggt 1980 tccttgtagt taatgattaa cccgccatgc tacttatcta cgtagccatg ctctagacag 2040 ccactatggg tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct 2100 ggttataatt aacccagaca tgtggctgct cccccccccc caacacctgc tgcctgagcc 2160 tcacccccac cccggtgcct gggtcttagg ctctgtacac catggaggag aagctcgctc 2220 taaaataac cctgtccctg gtgggctgtg ggggactgag ggcaggctgt aacaggcttg 2280 ggggccaggg cttatacgtg cctgggactc ccaaagtatt actgttccat gttcccggcg 2340 aagggccagc tgtccccgc cagctagact cagcacttag tttaggaacc agtgagcaag 2400 tcagcccttg gggcagccca tacaaggcca tggggctgggg caagctgcac gcctgggtcc 2460 ggggtgggca cggtgcccgg gcaacgagct gaaagctcat ctgctctcag gggcccctcc 2520 ctggggacag cccctcctgg ctagtcacac cctgtaggct cctctatata acccaggggc 2580 acaggggctg cccccgggtc accaccacct ccacagcaca gacagacact caggagccag 2640 ccagccaggt aagtttagtc ttttgtctt ttatttcagg tcccggatcc ggtggtggtg 2700 caaatcaaag aactgctcct cagtggatgt tgcctttact tctaggcctg tacggaagtg 2760 ttacttctgc tctaaaagct gcggaattgt acccgcggcc ggctagccgc caccatggag 2820 aagaatggaa ataaccgaaa gctgcgggtt tgtgttgcta cttgtaaccg tgcagattat 2880 tctaaacttg ccccgatcat gtttggcatt aaaaccgaac ctgagttctt tgaacttgat 2940 gttgtggtac ttggctctca cctgatagat gactatggaa atacatatcg aatgattgaa 3000 caagatgact ttgacattaa caccaggcta cacacaattg tgaggggaga agatgaggca 3060 gccatggtgg agtcagtagg cctggcccta gtgaagctgc cagatgtcct taatcgcctg 3120 aagcctgata tcatgattgt tcatggagac aggtttgatg ccctggctct ggccacatct 3180 gctgccttga tgaacatccg aatccttcac attgaaggtg gggaagtcag tgggaccatt 3240 gatgactcta tcagacatgc cataacaaaa ctggctcatt atcatgtgtg ctgcacccgc 3300 agtgcagagc agcacctgat atccatgtgt gaggaccatg atcgcatcct tttggcaggc 3360 tgcccttcct atgacaaact tctctcagcc aagaacaaag actacatgag catcattcgc 3420 atgtggctag gtgatgatgt aaaatctaaa gattacattg ttgcactaca gcaccctgtg 3480 accactgaca ttaagcattc cataaaaatg tttgaattaa cattggatgc acttatctca 3540 tttaacaagc ggaccctagt cctgtttcca aatattgacg cagggagcaa agagatggtt 3600 cgagtgatgc ggaagaaggg cattgagcat catcccaact ttcgtgcagt taaacacgtc 3660 ccatttgacc agtttataca gttggttgcc catgctggct gtatgattgg gaacagcagc 3720 tgtggggttc gagaagttgg agcttttgga acacctgtga tcaacctggg aacacgtcag 3780 attggagag aacagggga gatgttctt catgtccgggg atgctgacac ccaagacaaa 3840 atattgcaag cactgcacct tcagttggt aaacagtacc cttgttcaaa gatatatggg 3900 gatggaaatg ctgttccaag gatttgaag tttctcaat cttcgatct tcaagagcca 3960 ctgcaaaga aattctgctt tcctcctgtg aaggagata tctccaaga tattgaccat 4020 attcttgaaa ctctaagtgc cttggccgtt gatcttggcg ggacgaacct ccgagttgca 4080 atagtcagca tgaagggtga atagttaag aagtatactc agttcaatcc taaaaacctat 4140 gagagagga taatttaat cctacagatg tgtgtggaag ctgcagcaga agctgtaaaa 4200 ctgaactgca gatttttgggg agtaggcatt tccaggtg gccgtgtaaa tcctcgggaa 4260 ggaattgtgc tgcattcac caactgatc cagagtgga actctgtgga ccttaggacc 4320 cccctttctg acactttgca tctcctgtg tgggtagaca atgatggcaa ctgtgctgcc 4380 ctggcggaaa ggaatttgg ccaggaaag ggactggaaa acttgttac acttatcaca 4440 ggcacaggaa tcggtggtgg aattatchat cagcatgaat tgatccacgg aagctccttc 4500 tgtgctgcag aactgggcca ccttgttgtg tctctggatg ggcctgattg ttcctgtgga 4560 agccatgggt gcattgaagc atacgcctct ggaatggcct tgcagaggga ggcaaaaaag 4620 ctccatgatg aggacctgct cttggtggaa gggatgtcag tgccaaaaga tgaggctgtg 4680 ggtgcgctcc atctcatcca agctgcgaaa cttggcaatg cgaaggccca gagcatccta 4740 agaacagctg gaacagcttt gggtcttggg gttgtgaaca tcctccatac catgaatccc 4800 tcccttgtga tcctctccgg agtcctggcc agtcactata tccacattgt caaagacgtc 4860 attcgccagc aggccttgtc ctccgtgcag gacgtggatg tggtggtttc ggatttggtt 4920 gaccccgccc tgctgggtgc tgccagcatg gttctggact acacaacacg caggatctac 4980 tagcatgcct tctgatggtg aatgggaact cccttcctcc tgcagcagcc tgcctgcagc 5040 tgtcctggta gaacagtgtg gacattgcag aagctgtcac tgccccagaa agaaagcacc 5100 ccagagacta gtatggtccg cgcgaggcac cagccgggtg ggctttgcct cctgctgctg 5160 ctgctctgcc agttcatgga ggaccgcagt gcccaggctg ggaactgctg gctccgtcaa 5220 gcgaagaacg gccgctgcca ggtcctgtac aagaccgaac tgagcaagga ggagtgctgc 5280 agcaccggcc ggctgagcac ctcgtggacc gaggaggacg tgaatgacaa cacactcttc 5340 aagtggatga ttttcaacgg gggcgccccc aactgcatcc cctgtaaaga aacgtgtgag 5400 aacgtggact gtggacctgg gaaaaaatgc cgaatgaaca agaagaacaa accccgctgc 5460 gtctgcgccc cggattgttc caacatcacc tggaagggtc cagtctgcgg gctggatggg 5520 aaaacctacc gcaatgaatg tgcactccta aaggcaagat gtaaagagca gccagaactg 5580 gaagtccagt accaaggcag atgtaaaaag acttgtcggg atgttttctg tccaggcagc 5640 tccacatgtg tggtggacca gaccaataat gcctactgtg tgacctgtaa tcggatttgc 5700 ccagagcctg cttcctctga gcaatatctc tgtgggaatg atggagtcac ctactccagt 5760 gcctgccacc tgagaaaggc tacctgcctg ctgggcagat ctattggatt agcctatgag 5820 ggaaagtgta tcaaagcaaa gtcctgtgaa gatatccagt gcactggtgg gaaaaaatgt 5880 ttatgggatt tcaaggttgg gagaggccgg tgttccctct gtgatgagct gtgccctgac 5940 agtaagtcgg atgagcctgt ctgtgccagt gacaatgcca cttatgccag cgagtgtgcc 6000 atgaaggaag ctgcctgctc ctcaggtgtg ctactgggaag taagcactc cggatcttgc 6060 6120 cctatatctt ctattctaga gtggtaagcg gccgcaataa aagatcttta ttttcattag 6180 atctgtgtgt tggttttttg tgtgtctaga gcatggctac gtagataagt agcatggcgg 6240 gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc 6300 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 6360 ggcctcagtg agcgagcgag cgcgcagctg gcgtaatagc gaagaggccc gcaccgatcg 6420 cccttcccaa cagttgcgca gcctgaatgg cgaatggcga ttccgttgca atggctggcg 6480 gtaatattgt tctggatatt accagcaagg ccgatagttt gagttcttct actcaggcaa 6540 gtgatgttat tactaatcaa agaagtattg cgacaacggt taatttgcgt gatggagaga 6600 ctcttttact cggtggcctc actgattata aaaacacttc tcaggattct ggcgtaccgt 6660 tcctgtctaa aatcccttta atcggcctcc tgtttagctc ccgctctgat tctaacgagg 6720 aaagcacgtt atacgtgctc gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 6840 cccgctcctt tcgctttctt cccttcctttt ctcgccacgt tcgccggctt tccccgtcaa 6900 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gattcggcc tattggttaa aaaatgagct gattaca aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaaatatt tgcttataca atcttcctgt ttttggggct tttctgatta tcaaccgggg tcatatgat tgacatgcta gttttacgat taccgttcat cgattctctt gtttgctcca gactctcagg caatgacctg atagccttttg tagagacctc tcaaaaatag ctaccctctc cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt 7440 tgactgtctc cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg 7500 catttaaaat atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc 7560 ccgcaaaagt attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg 7620 aggctttatt gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg 7680 gaatcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 7740 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc 7800 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 7860 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 7920 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 7980 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 8040 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 8100 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 8160 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 8220 tgc 8223 <210> 21 <211> 7726 <212> DNA <213> Homo sapiens <400> 21 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 60 ccttgagagt tttcgccccg aagaacgaat tcttagaaaa actcatcgag catcaaatga 120 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt 180 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct 240 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg 300 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta 360 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc 420 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg 480 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc 540 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc 600 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg 660 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca 720 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac 780 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat 840 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata 900 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac 960 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1020 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1080 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1140 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1200 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1260 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1320 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1380 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 1440 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 1500 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1560 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 1620 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 1680 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 1740 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 1800 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctgcgc 1860 gctcgctcgc tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc 1920 ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactagggt 1980 tccttgtagt taatgattaa cccgccatgc tacttatcta cgtagccatg ctctagacag 2040 ccactatggg tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct 2100 ggttataatt aacccagaca tgtggctgct cccccccccc caacacctgc tgcctgagcc 2160 tcacccccac cccggtgcct gggtcttagg ctctgtacac catggaggag aagctcgctc 2220 taaaataac cctgtccctg gtgggctgtg ggggactgag ggcaggctgt aacaggcttg 2280 ggggccaggg cttatacgtg cctgggactc ccaaagtatt actgttccat gttcccggcg 2340 aagggccagc tgtcccccg cagctagact cagcacttag tttaggaacc agtgagcaag 2400 tcagcccttg gggcagccca tacaaggcca tggggctgggg caagctgcac gcctgggtcc 2460 ggggtgggca cggtgcccgg gcaacgagct gaaagctcat ctgctctcag gggcccctcc 2520 ctggggacag cccctcctgg ctagtcacac cctgtaggct cctctatata acccaggggc 2580 acaggggctg cccccgggtc accaccacct ccacagcaca gacagacact caggagccag 2640 ccagccaggt aagtttagtc ttttgtctt ttatttcagg tcccggatcc ggtggtggtg 2700 caaatcaaag aactgctcct cagtggatgt tgcctttact tctaggcctg tacggaagtg 2760 ttacttctgc tctaaaagct gcggaattgt acccgcggcc ggctagccgc caccatggag 2820 aagaatggaa ataaccgaaa gctgcgggtt tgtgttgcta cttgtaaccg tgcagattat 2880 tctaaacttg ccccgatcat gtttggcatt aaaaccgaac ctgagttctt tgaacttgat 2940 gttgtggtac ttggctctca cctgatagat gactatggaa atacatatcg aatgattgaa 3000 caagatgact ttgacattaa caccaggcta cacacaattg tgaggggaga agatgaggca 3060 gccatggtgg agtcagtagg cctggcccta gtgaagctgc cagatgtcct taatcgcctg 3120 aagcctgata tcatgattgt tcatggagac aggtttgatg ccctggctct ggccacatct 3180 gctgccttga tgaacatccg aatccttcac attgaaggtg gggaagtcag tgggaccatt 3240 gatgactcta tcagacatgc cataacaaaa ctggctcatt atcatgtgtg ctgcacccgc 3300 agtgcagagc agcacctgat atccatgtgt gaggaccatg atcgcatcct tttggcaggc 3360 tgcccttcct atgacaaact tctctcagcc aagaacaaag actacatgag catcattcgc 3420 atgtggctag gtgatgatgt aaaatctaaa gattacattg ttgcactaca gcaccctgtg 3480 accactgaca ttaagcattc cataaaaatg tttgaattaa cattggatgc acttatctca 3540 tttaacaagc ggaccctagt cctgtttcca aatattgacg cagggagcaa agagatggtt 3600 cgagtgatgc ggaagaaggg cattgagcat catcccaact ttcgtgcagt taaacacgtc 3660 ccatttgacc agtttataca gttggttgcc catgctggct gtatgattgg gaacagcagc 3720 tgtggggttc gagaagttgg agctttttgga acacctgtga tcaacctggg aacacgtcag 3780 attggaagag aaacagggga gaatgttctt catgtccggg atgctgacac ccaagacaaa 3840 atattgcaag cactgcacct tcagtttggt aaacagtacc cttgttcaaa gatatatggg 3900 gatggaaatg ctgttccaag gattttgaag tttctcaaat ctatcgatct tcaagagcca 3960 ctgcaaaaga aattctgctt tcctcctgtg aaggagaata tctctcaaga tattgaccat 4020 attcttgaaa ctctaagtgc cttggccgtt gatcttggcg ggacgaacct ccgagttgca 4080 atagtcagca tgaagggtga aatagttaag aagtatactc agttcaatcc taaaacctat 4140 gaagagag ttaatttaat cctacagatg tgtgtggaag ctgcagcaga agctgtaaaa 4200 ctgaactgca gaattttggg agtaggcatt tccacaggtg gccgtgtaaa tcctcgggaa 4260 ggaattgtgc tgcattcaac caaactgatc caagagtgga actctgtgga ccttaggacc 4320 cccctttctg acactttgca tctccctgtg tgggtagaca atgatggcaa ctgtgctgcc 4380 ctggcggaaa ggaaatttgg ccaaggaaag ggactggaaa actttgttac acttatcaca 4440 ggcacaggaa tcggtggtgg aattatccat cagcatgaat tgatccacgg aagctccttc 4500 tgtgctgcag aactgggcca ccttgttgtg tctctggatg ggcctgattg ttcctgtgga 4560 agccatgggt gcattgaagc atacgcctct ggaatggcct tgcagaggga ggcaaaaaag 4620 ctccatgatg aggacctgct cttggtggaa gggatgtcag tgccaaaaga tgaggctgtg 4680 ggtgcgctcc atctcatcca agctgcgaaa cttggcaatg cgaaggccca gagcatccta 4740 agaacagctg gaacagcttt gggtcttggg gttgtgaaca tcctccatac catgaatccc 4800 tcccttgtga tcctctccgg agtcctggcc agtcactata tccacattgt caaagacgtc 4860 attcgccagc aggccttgtc ctccgtgcag gacgtggatg tggtggtttc ggatttggtt 4920 gaccccgccc tgctgggtgc tgccagcatg gttctggact acaacacg caggatctac 4980 tagcatgcct tctgatggtg aatgggaact cccttctcc tgcagcagcc tgcctgcagc 5040 tgtcctggta gaacagtgtg gacattgcag aagctgtcac tgccccagaa agaaagcacc 5100 ccagagaggc cgcaccatga agctgctgcc gtcggtggtg ctgaagctct ttctggctgc 5160 agttctctcg gcactggtga ctggcgagag cctggagcgg cttcggagag ggctagctgc 5220 tggaaccagc aacccggacc ctcccactgt atccacggac cagctgctac ccctaggagg 5280 cggccgggac cggaaagtcc gtgacttgca agaggcagat ctggaccttt tgagagtcac 5340 tttatcctcc aagccacaag cactggccac accaaacaag gaggagcacg ggaaaagaaa 5400 gaaaaggc aaggggctag ggaaagag ggacccagga ccggagacgc tctgcggggc 5460 tgagctggtg gatgctcttc agttcgtgtg tggagacagg ggctttttt tcaacaagcc 5520 cacagggtat ggctccagca gtcggagggc gcctcagaca ggcatcgtgg atgagtgctg 5580 cttccggagc tgtgatctaa ggaggctgga gatgtattgc gcacccctca agcctgccaa 5640 gtcagcttag gcggccgcaa taaaagatct ttattttcat tagatctgtg tgttggtttt 5700 ttgtgtgtct agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca 5760 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 5820 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 5880 gagcgcgcag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 5940 gcagcctgaa tggcgaatgg cgattccgtt gcaatggctg gcggtaatat tgttctggat 6000 attaccagca aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat 6060 caaagaagta ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc 6120 ctcactgatt ataaaaacac ttctcaggat tctggcgtac cgttcctgtc taaaatccct 6180 ttaatcggcc tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg 6240 ctcgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 6300 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 6360 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 6420 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 6480 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 6540 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 6600 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 6660 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaaat 6720 atttgcttat acaatcttcc tgtttttggg gcttttctga ttatcaaccg gggtacatat 6780 gattgacatg ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc 6840 aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg 6900 aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt 6960 tctcacccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag 7020 ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 7080 ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 7140 tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaatcgc ctgatgcggt 7200 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 7260 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 7320 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 7380 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 7440 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 7500 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 7560 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 7620 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 7680 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgc 7726 <210> 22 <211> 8340 <212> DNA <213> Homo sapiens <400> 22 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 60 ttttcgcccc gaagaacgaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 120 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 180 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 240 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 300 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 360 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 420 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 480 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 540 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 600 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 660 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 720 ctacctttgc catgtttcag aaaaactct ggcgcatcgg gcttcccata caatcgatag 780 attgcgcac ctgattgccc gacattatcg cgagcccatt tataccccata taaatcagca 840 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 900 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 960 ttaacgtgag tttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 1020 ttgagatcct ttttttgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1080 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 1140 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1200 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1260 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1320 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1380 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1440 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1500 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 1560 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1620 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 1680 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 1740 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 1800 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg 1860 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 1920 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 1980 ttaatgatta acccgccatg ctacttatct acgtagccat gctctagagt ttaaacaagc 2040 ttgaggccag gagctcgagt gctgcctgga caacatagtg agacccttgt ctcaaaaaca 2100 acagcaaccc aaagtcttat ttatcttcag gagctgcgca agccctttac caccgccacg 2160 tttctccctg gatgtcccta ggcctcggga ctggctggga gttccctgta gtggaggccg 2220 ccgctgaact gattataaag atgagagagg ctctgccatg ccttggtcat caattcacac 2280 tccaccagtg tcttctagca gtcacggcaa gggttaacgt cagggaccgc tgtggggtgg 2340 ccgcgctaca cgacagtata gttgcggtcc tggtttatga aataactgag ggaacaagag 2400 gcgcaagaaa tccctccttg ggtgcaagac caaaacaact acccagcggg aagactcggg 2460 cttcagtgcg tgtgtcgcca gtggaggagg acgcttcggg gcgggagcac aagctggcag 2520 gacagccccg cagcagctcc agcgcggcag aggccatgga agatggtccg ctggtcagca 2580 ccctgcctgc gcctcaaaat accgccggga aggtggggga cggggacggg agcagccaat 2640 cacgcgagct ctctcgggtg gcgtcagggc gcgatcgcgg attcggcact ccgggtgggc 2700 gtggctgggc gagcgaggag tggggacaag gtcgagcgac gagtcggcgc gcccaggtaa 2760 gtttagtctt tttgtctttt atttcaggtc ccggatccgg tggtggtgca aatcaaagaa 2820 ctgctcctca gtggatgttg cctttacttc taggcctgta cggaagtgtt acttctgctc 2880 taaaagctgc ggaattgtac ccgcggccgg ctagccgcca ccatggagaa gaatggaaat 2940 aaccgaaagc tgcgggtttg tgttgctact tgtaaccgtg cagattattc taaacttgcc 3000 ccgatcatgt ttggcattaa aaccgaacct gagttctttg aacttgatgt tgtggtactt 3060 ggctctcacc tgatagatga ctatggaaat acatatcgaa tgattgaaca agatgacttt 3120 gacattaaca ccaggctaca cacaattgtg aggggagaag atgaggcagc catggtggag 3180 tcagtaggcc tggccctagt gaagctgcca gatgtcctta atcgcctgaa gcctgatatc 3240 atgattgttc atggagacag gtttgatgcc ctggctctgg ccacatctgc tgccttgatg 3300 aacatccgaa tccttcacat tgaaggtggg gaagtcagtg ggaccattga tgactctatc 3360 agacatgcca taacaaaact ggctcattat catgtgtgct gcacccgcag tgcagagcag 3420 cacctgatat ccatgtgtga ggaccatgat cgcatccttt tggcaggctg cccttcctat 3480 vakaaacttc tctcagccaa gaacaagac tacatgagca tcattcgcat gtggctaggt 3540 gatgatgtaa atctaaga ttacattgtt gcactacagc accctgtgac cactgacatt 3600 aagcattcca taaaaatgtt tgaatcaca ttggatgcac ttatctcatt taacaagcgg 3660 accctagtcc tgttttccaaa tattgacgca gggagcaaag agatggttcg agtgatgcgg 3720 aagaagggca ttgagcatca tcccactt cgtgcagtta aacacgtccc atttgaccag 3780 tttatacagt tggttgccca tgctggctgt atgattggt acagcagctg tggggttcga 3840 gaagttggag cttttggaac acctgtgatc aacctgggaa cacgtcagat tggagagaa 3900 acaggggaga atgttcttca tgtccgggat gctgacaccc agacaaat attgcaagca 3960 ctgcaccttc agtttggtaa acagccct tgttcaaaga tatatgggga tggatgct 4020 gttccaagga tttgaagtt tctcaatct atcgatctc agagccact gcaaagaaa 4080 ttctgctttc ctcctgtgaa ggagaatatc tccaagata tgaccatat tcttgaact 4140 ctaagtgcct tggccgttga tcttggcggg acgaacctcc gagttgcaat attcagcatg 4200 aagggtgaaa tagttaagaa gtatactcag ttcaatccta aaacctatga agaggatt 4260 4320 attttgggag taggcatttc cacaggtggc cgtgtaaatc ctcgggaagg aattgtgctg 4380 cattcaacca aactgatcca agagtggaac tctgtggacc ttaggacccc cctttctgac 4440 actttgcatc tccctgtgtg ggtagacaat gatggcaact gtgctgccct ggcggaaagg 4500 aaatttggcc aaaggaaggg actggaaaac tttgttacac ttatcacagg cacaggaatc 4560 ggtggtggaa ttatccatca gcatgaattg atccacggaa gctccttctg tgctccagaa 4620 ctgggccacc ttgttgtgtc tctggatggg cctgattgtt cctgtggaag ccatgggtgc 4680 attgaagcat acgcctctgg aatggccttg cagagggagg caaaaaagct ccatgatgag 4740 gacctgctct tggtggaagg gatgtcagtg ccaaaagatg aggctgtggg tgcgctccat 4800 ctcatccaag ctgcgaaact tggcaatgcg aaggcccaga gcatcctaag aacagctgga 4860 acagctttgg gtcttggggt tgtgaacatc ctccatacca tgaatccctc ccttgtgatc 4920 ctctccggag tcctggccag tcactatatc cacattgtca aagacgtcat tcgccagcag 4980 gccttgtcct ccgtgcagga cgtggatgtg gtggtttcgg atttggttga ccccgccctg 5040 ctgggtgctg ccagcatggt tctggactac acaacacgca ggatctacta gcatgccttc 5100 tgatggtgaa tgggaactcc cttctctcctg cagcagcctg cctgcagctg tcctggtaga 5160 5220 atggtccgcg cgaggcacca gccgggtggg ctttgcctcc tgctgctgct gctctgccag 5280 ttcatggagg accgcagtgc ccaggctggg aactgctggc tccgtcaagc gaaacggc 5340 cgctgccagg tcctgtacaa gaccgaactg agcaaggagg agtgctgcag caccggccgg 5400 ctgagcacct cgtggaccga ggaggacgtg aatgacaaca cactcttcaa gtggatgatt 5460 ttcaacgggg gcgccccccaa ctgcatcccc tgtaaagaaa cgtgtgagaa cgtggactgt 5520 ggacctggga aaaaatgccg aatgaaaag aagaaaac cccgctgcgt ctgcgccccg 5580 gattgttcca acatcacctg gaagggtcca gtctgcgggc tggatgggaa aacctaccgc 5640 aatgaatgtg cactcctaaa ggcaagatgt aaagagcagc cagaactgga agtccagtac 5700 caaggcagat gtaaaaagac ttgtcgggat gttttctgtc caggcagctc cacatgtgtg 5760 gtggaccaga ccaataatgc ctactgtgtg acctgtaatc ggatttgccc agagcctgct 5820 tcctctgagc aatatctctg tgggaatgat ggagtcacct actccagtgc ctgccacctg 5880 agaaaggcta cctgcctgct gggcagatct attggattag cctatgaggg aaagtgtatc 5940 aaagcaaagt cctgtgaaga tatccagtgc actggtggga aaaaatgttt atgggatttc 6000 aaggttggga gaggccggtg ttccctctgt gatgagctgt gccctgacag taagtcggat 6060 gagcctgtct gtgccagtga caatgccact tatgccagcg agtgtgccat gaaggaagct 6120 gcctgctcct caggtgtgct actggaagta aagcactccg gatcttgcaa ctccatttcg 6180 gaagacaccg aggaagagga ggaagatgaa gaccaggact acagctttcc tatatcttct 6240 attctagagt ggtaagcggc cgcaataaaa gatctttatt ttcattagat ctgtgtgttg 6300 gttttttgtg tgtctagagc atggctacgt agataagtag catggcgggt taatcattaa 6360 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 6420 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag 6480 cgagcgagcg cgcagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 6540 gttgcgcagc ctgaatggcg aatggcgatt ccgttgcaat ggctggcggt aatattgttc 6600 tggatattac cagcaaggcc gatagtttga gttcttctac tcaggcaagt gatgttatta 6660 ctaatcaaag aagtattgcg acaacggtta atttgcgtga tggacagact cttttactcg 6720 gtggcctcac tgattataaa aacacttctc aggattctgg cgtaccgttc ctgtctaaaa 6780 tccctttaat cggcctcctg tttagctccc gctctgattc taacgaggaa agcacgttat 6840 acgtgctcgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt 6900 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 6960 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 7020 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 7080 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 7140 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 7200 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 7260 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 7320 taaatatttg cttatacaat cttcctgttt ttggggcttt tctgattatc aaccggggta 7380 catatgattg acatgctagt tttacgatta ccgttcatcg attctcttgt ttgctccaga 7440 ctctcaggca atgacctgat agcctttgta gagacctctc aaaaatagct accctctccg 7500 gcatgaattt atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg 7560 gcctttctca cccgtttgaa tctttaccta cacattactc aggcattgca tttaaaatat 7620 atgagggttc taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat 7680 tacagggtca taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc 7740 ttaattttgc taattctttg ccttgcctgt atgatttatt ggatgttgga atcgcctgat 7800 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag 7860 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 7920 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 7980 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 8040 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 8100 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 8160 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 8220 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 8280 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 8340 <210> 23 <211> 7843 <212> DNA <213> Homo sapiens <400> 23 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 60 ttttcgcccc gaaacgaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 120 ttatcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 180 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 240 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 300 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 360 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 420 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 480 ggacaattac aaacaggaat cgaatgcaac cggcgcag acactgccag cgcatcaaca 540 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 600 660 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 720 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttccata caatcgatag 780 attgcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 840 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 900 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 960 ttaacgtgag tttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 1020 ttgagatcct ttttttgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1080 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 1140 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1200 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1260 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1320 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1380 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1440 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1500 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 1560 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1620 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 1680 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 1740 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 1800 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg 1860 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 1920 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 1980 ttaatgatta acccgccatg ctacttatct acgtagccat gctctagagt ttaaacaagc 2040 ttgaggccag gagctcgagt gctgcctgga caacatagtg agacccttgt ctcaaaaaca 2100 acagcaaccc aaagtcttat ttatcttcag gagctgcgca agccctttac caccgccacg 2160 tttctccctg gatgtcccta ggcctcggga ctggctggga gttccctgta gtggaggccg 2220 ccgctgaact gattataaag atgagagagg ctctgccatg ccttggtcat caattcacac 2280 tccaccagtg tcttctagca gtcacggcaa gggttaacgt cagggaccgc tgtggggtgg 2340 ccgcgctaca cgacagtata gttgcggtcc tggtttatga aataactgag ggaacaagag 2400 gcgcaagaaa tccctccttg ggtgcaagac caaaacaact acccagcggg aagactcggg 2460 cttcagtgcg tgtgtcgcca gtggaggagg acgcttcggg gcgggagcac aagctggcag 2520 gacagccccg cagcagctcc agcgcggcag aggccatgga agatggtccg ctggtcagca 2580 ccctgcctgc gcctcaaaat accgccggga aggtggggga cggggacggg agcagccaat 2640 cacgcgagct ctctcgggtg gcgtcagggc gcgatcgcgg attcggcact ccgggtgggc 2700 gtggctgggc gagcgaggag tggggacaag gtcgagcgac gagtcggcgc gcccaggtaa 2760 gtttagtctt tttgtctttt atttcaggtc ccggatccgg tggtggtgca aatcaaagaa 2820 ctgctcctca gtggatgttg cctttacttc taggcctgta cggaagtgtt acttctgctc 2880 taaaagctgc ggaattgtac ccgcggccgg ctagccgcca ccatggagaa gaatggaaat 2940 aaccgaaagc tgcgggtttg tgttgctact tgtaaccgtg cagattattc taaacttgcc 3000 ccgatcatgt ttggcattaa aaccgaacct gagttctttg aacttgatgt tgtggtactt 3060 ggctctcacc tgatagatga ctatggaaat acatatcgaa tgattgaaca agatgacttt 3120 gacattaaca ccaggctaca cacaattgtg aggggagaag atgaggcagc catggtggag 3180 tcagtaggcc tggccctagt gaagctgcca gatgtcctta atcgcctgaa gcctgatatc 3240 atgattgttc atggagacag gtttgatgcc ctggctctgg ccacatctgc tgccttgatg 3300 aacatccgaa tccttcacat tgaaggtggg gaagtcagtg ggaccattga tgactctatc 3360 agacatgcca taacaaaact ggctcattat catgtgtgct gcacccgcag tgcagagcag 3420 cacctgatat ccatgtgtga ggaccatgat cgcatccttt tggcaggctg cccttcctat 3480 gacaaacttc tctcagccaa gaacaaagac tacatgagca tcattcgcat gtggctaggt 3540 gatgatgtaa aatctaaaga ttacattgtt gcactacagc accctgtgac cactgacatt 3600 aagcattcca taaaaatgtt tgaattaaca ttggatgcac ttatctcatt taacaagcgg 3660 accctagtcc tgtttccaaa tattgacgca gggagcaaag agatggttcg agtgatgcgg 3720 aagaagggca ttgagcatca tcccaacttt cgtgcagtta aacacgtccc atttgaccag 3780 tttatacagt tggttgccca tgctggctgt atgattggga acagcagctg tggggttcga 3840 gaagttggag cttttggaac acctgtgatc aacctgggaa cacgtcagat tggaagagaa 3900 acaggggaga atgttcttca tgtccgggat gctgacaccc aagacaaaat attgcaagca 3960 ctgcaccttc agtttggtaa acagtaccct tgttcaaaga tatatgggga tggaaatgct 4020 gttccaagga ttttgaagtt tctcaaatct atcgatcttc aagagccact gcaaaagaaa 4080 ttctgctttc ctcctgtgaa ggagaatatc tctcaagata ttgaccatat tcttgaaact 4140 ctaagtgcct tggccgttga tcttggcggg acgaacctcc gagttgcaat agtcagcatg 4200 aagggtgaaa tagttaagaa gtatactcag ttcaatccta aaacctatga agagaggatt 4260 aatttaatcc tacagatgtg tgtggaagct gcagcagaag ctgtaaaact gaactgcaga 4320 attttgggag taggcatttc cacaggtggc cgtgtaaatc ctcgggaagg aattgtgctg 4380 cattcaacca aactgatcca agagtggaac tctgtggacc ttaggacccc cctttctgac 4440 actttgcatc tccctgtgtg ggtagacaat gatggcaact gtgctgccct ggcggaaagg 4500 aaatttggcc aaaggaaggg actggaaaac tttgttacac ttatcacagg cacaggaatc 4560 ggtggtggaa ttatccatca gcatgaattg atccacggaa gctccttctg tgctccagaa 4620 ctgggccacc ttgttgtgtc tctggatggg cctgattgtt cctgtggaag ccatgggtgc 4680 attgaagcat acgcctctgg aatggccttg cagagggagg caaaaaagct ccatgatgag 4740 gacctgctct tggtggaagg gatgtcagtg ccaaaagatg aggctgtggg tgcgctccat 4800 ctcatccaag ctgcgaaact tggcaatgcg aaggcccaga gcatcctaag aacagctgga 4860 acagctttgg gtcttggggt tgtgaacatc ctccatacca tgaatccctc ccttgtgatc 4920 ctctccggag tcctggccag tcactatatc cacattgtca aagacgtcat tcgccagcag 4980 gccttgtcct ccgtgcagga cgtggatgtg gtggtttcgg atttggttga ccccgccctg 5040 ctgggtgctg ccagcatggt tctggactac acaacacgca ggatctacta gcatgccttc 5100 tgatggtgaa tgggaactcc cttcctcctg cagcagcctg cctgcagctg tcctggtaga 5160 acagtgtgga cattgcagaa gctgtcactg ccccagaaag aaagcacccc agagaggccg 5220 caccatgaag ctgctgccgt cggtggtgct gaagctcttt ctggctgcag ttctctcggc 5280 actggtgact ggcgagagcc tggagcggct tcggagaggg ctagctgctg gaaccagcaa 5340 cccggaccct cccactgtat ccacggacca gctgctaccc ctaggaggcg gccgggaccg 5400 gaaagtccgt gacttgcaag aggcagatct ggaccttttg agagtcactt tatcctccaa 5460 gccacaagca ctggccacac caaacaagga ggagcacggg aaaagaaaga agaaaggcaa 5520 ggggctaggg aagaagaggg acccaggacc ggagacgctc tgcggggctg agctggtgga 5580 tgctcttcag ttcgtgtgtg gagacagggg cttttatttc aacaagccca cagggtatgg 5640 ctccagcagt cggagggcgc ctcagacagg catcgtggat gagtgctgct tccggagctg 5700 tgatctaagg aggctggaga tgtattgcgc acccctcaag cctgccaagt cagcttaggc 5760 ggccgcaata aaagatcttt attttcatta gatctgtgtg ttggtttttt gtgtgtctag 5820 agcatggcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 5880 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 5940 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct 6000 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 6060 gcgaatggcg attccgttgc aatggctggc ggtaatattg ttctggatat taccagcaag 6120 gccgatagtt tgagttcttc tactcaggca agtgatgtta tactaatca aagaagtatt 6180 gcgacaacgg ttaatttgcg tgatggacag actctttac tcggtggcct cactgattat 6240 aaaaacactt ctcaggattc tggcgtaccg ttcctgtcta aaatcccttt aatcggcctc 6300 ctgtttagct cccgctctga ttctaacgag gaaagcacgt tatacgtgct cgtcaaagca 6360 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 6420 cgtgaccgct acacttgcca gcgccctagc gccgctcct ttcgctttct tcccttcctt 6480 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 6540 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 6600 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 6660 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 6720 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 6780 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaaatat ttgcttatac 6840 aatcttcctg tttttggggc ttttctgatt atcaaccggg gtacatatga ttgacatgct 6900 agttttacga ttaccgttca tcgattctct tgtttgctcc agactctcag gcaatgacct 6960 gatagccttt gtagagacct ctcaaaaata gctaccctct ccggcatgaa tttatcagct 7020 agaacggttg aatatcatat tgatggtgat ttgactgtct ccggcctttc tcacccgttt 7080 gaatctttac ctacacatta ctcaggcatt gcatttaaaa tatatgaggg ttctaaaaat 7140 ttttatcctt gcgttgaaat aaaggcttct cccgcaaaag tattacaggg tcataatgtt 7200 tttggtacaa ccgatttagc tttatgctct gaggctttat tgcttaattt tgctaattct 7260 ttgccttgcc tgtatgattt attggatgtt ggaatcgcct gatgcggtat tttctcctta 7320 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 7380 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 7440 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 7500 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 7560 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 7620 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 7680 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 7740 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 7800 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tca 7843 <210> 24 <211> 6594 <212> DNA <213> Homo sapiens <400> 24 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 60 aaggcaaat gccgcaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 120 cttccttttt caatattatt gagcatttta tgtctcatga gcggatacat 180 atttgaatgt atttagaaa aaaacaat aggggttccg cgcacattc cccgaaagt 240 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 300 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gandacatgca 360 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 420 gggcgcgtca gcgtgtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 480 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taggagaaa 540 ataccgcatc aggcgattcc aacatccaat aaatcataca ggcaaggcaa agattagca 600 aaattaagca aaagccctc agagcataa gctaaatcgg ttgtaccaa aacattatga 660 ccctgtataa cttttgcgggg agaagcctttt atttcaacgc aaggataaaa atttttagaa 720 ccctcatata ttttaaatgc atgcctgag taatgtgtag gtaagattc aaacggggtga 780 gaaaggccgg agacagtcaa atcaccatca atatgatatt caaccgttct agctgataaa 840 ttcatgccgg agagggtagc tatttttgag aggtctctac aaaggctatc aggtcattgc 900 ctgagagtct ggagcaaaca agaagaatcga tgaacggtaa tcgtaaact agcatgtcaa 960 tcatatgtac cccggttgat aatcagaaaa gccccaaaaa caggagaatt gtataagcaa 1020 atatttaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 1080 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 1140 ccgagatagg gttgagtgtt gttccagttt ggaaagag tccactatta aagaacgtgg 1200 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 1260 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 1320 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaagggga 1380 agaaaggaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 1440 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg 1500 agcacgtata acgtgctttc ctcgttagaa tcagagcggg agctaaacag gaggccgatt 1560 aaagggattt tagacaggaa cggtacgcca gaatcctgag aagtgttttt ataatcagtg 1620 aggccaccga gtaaaagagt ctgtccatca cgcaaattaa ccgttgtcgc aatacttctt 1680 tgattagtaa taacatcact tgcctgagta gaagaactca aactatcggc cttgctggta 1740 atatccagaa caatattacc gccagccatt gcaacggaat cgccattcgc cattcaggct 1800 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc 1860 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc 1920 ggcctcagtg agcgagcgag cgcgcaga gggagtggcc aactccatca ctaggggttc 1980 ctaggaagct ttcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 2040 cccccggact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 2100 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 2160 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgc 2220 tcgaccgggg tacccggccg gctagccgcc accatggaga agaatggaa taaccgaaag ctgcgggttt gtgttgctac ttgtaaccgt gcagattatt ctaaacttgc cccgatcatg 2340. tttggcatta aaaccgaacc tgagttcttt gaacttgatg ttgtggtact tggctctcac ctgatagatg actatggaaa tacatatcga atgattgaac aagatgactt tgacattaac accaggctac acacaattgt gaggggaga gatgaggcag ccatggtgga gtcagtaggc ctggccctag tgaagctgcc agatgtcctt aatcgcctga agcctgatat catgattgtt 2580. catggagaca ggtttgatgc cctggctctg gccacatctg ctgccttgat gaacatccga atccttcaca ttgaaggtgg ggaagtcagt gggaccattg atgactctat cagacatgcc ataacaaaac tggctcatta tcatgtgtgc tgcacccgca gtgcagagca gcacctgata 2760 tccatgtgtg aggaccatga tcgcatcctt ttggcaggct gcccttccta tgacaaactt 2820 ctctcagcca agaacaaaga ctacatgagc atcattcgca tgtggctagg tgatgatgta aaatctaaag attacattgt tgcactacag caccctgtga ccactgacat tagcattcc aaaaatgt ttgaattaac attggatgca cttatctcat ttaacaagcg gacctagtc 3000 ctgtttccaa atttgacgc agggagcaaa gagatggttc gagtgatgcg gagaagggc 3060 attgagcatc atcccaactt tcgtgcagtt aacacgtcc catttgacca gtttatacag 3120 ttggttgccc atgctggctg tatgattggg aacagcagct gtggggttcg agaagttgga 3180 gcttttggaa cacctgtgat siacctggga acacgtcaga ttggagaga aacaggggag 3240 aatgttcttc atgtccggga tgctgacacc caacaaaa tattgcaagc actgcacctt 3300 cagttttggta aacagtaccc ttgttcaag atatatgggg atggaatgc tgttccaagg 3360 attttgaagt ttctcaatc tatcgattt CAagagccac tgcaaagaa attctgcttt 3420 cctcctgtga aggaatat ctctcagat attgaccata ttctgaaac tctaagtgcc 3480 ttggccgttg atctttggcgg gacgaacctc cgagttgcaa tagtcagcat gaagggtgaa 3540 aatagttaga agtatactca gttcaatcct aaaacctag aagaggaggat taatttaatc 3600 ctacagatgt gtgtggaagc tgcagcagaa gctgtaaaac tgaacgcag aattttggga 3660 gtaggcattt ccacaggtgg ccgtgtaaat cctcgggaag gaattgtgct gcattcaacc 3720 aaactgatcc aagagtggaa ctctgtggac cttaggaccc ccctttctga cactttgcat 3780 3840 caaggaaagg gactggaaaa ctttgttaca cttatcacag gcacaggaat cggtggtgga 3900 attatccatc agcatgaatt gatccacgga agctccttct gtgctccaga actgggccac 3960 cttgttgtgt ctctggatgg gcctgattgt tcctgtggaa gccatgggtg cattgaagca 4020 tacgcctctg gaatggcctt gcagagggag gcaaaaaagc tccatgatga ggacctgctc 4080 ttggtggaag ggatgtcagt gccaaaagat gaggctgtgg gtgcgctcca tctcatccaa 4140 gctgcgaaac ttggcaatgc gaaggcccag agcatcctaa gaacagctgg aacagctttg 4200 ggtcttgggg ttgtgaacat cctccatacc atgaatccct cccttgtgat cctctccgga 4260 gtcctggcca gtcactatat ccacattgtc aaagacgtca ttcgccagca ggccttgtcc 4320 tccgtgcagg acgtggatgt ggtggtttcg gatttggttg accccgccct gctgggtgct 4380 gccagcatgg ttctggacta cacaacacgc aggatctact agcatgcact agtgcggccg 4440 cggatctcag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag 4500 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 4560 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 4620 gaggtgtggg aggtttttta ggtttaaaacc ccactccctc tctgcgcgct cgctcgctca 4680 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 4740 gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 4800 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 4860 gcgagcggta tcagctcact caaaaggcggt aatacggtta tccacagaat caggggataa 4920 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 4980 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 5040 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 5100 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 5160 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 5220 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 5280 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 5340 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 5400 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 5460 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 5520 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 5580 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 5640 agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg 5700 ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg 5760 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa 5820 tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta 5880 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc 5940 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg 6000 cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata 6060 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc 6120 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt 6180 tggttgatgc gagtgattttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga 6240 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct 6300 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag 6360 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt 6420 ctccttcatt acagaaacgg cttttcaaa aatatggtat tgataatcct gatatgaata 6480 aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attcgttctt cggggcgaaa 6540 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgc 6594 <210> 25 <211> 8340 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (6)..(6) <223> n is a, c, g or t <220> <221> misc_feature <222> (15)..(15) <223> n is a, c, g or t <220> <221> misc_feature <222> (20)..(20) <223> n is a, c, g or t <400> 25 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 60 ttttcgcccc gaagaacgaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 120 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 180 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 240 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 300 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 360 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 420 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 480 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 540 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 600 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 660 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 720 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 780 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 840 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 900 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 960 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aggatcttc 1020 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1080 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 1140 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1200 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1260 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1320 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1380 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1440 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1500 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggttcgcc acctctgact 1560 tgagcgtcga ttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1620 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 1680 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 1740 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 1800 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg 1860 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 1920 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 1980 ttaatgatta acccgccatg ctacttatct acgtagccat gctctagagt ttaaacaagc 2040 ttgaggccag gagctcgagt gctgcctgga caacatagtg agacccttgt ctcaaaaaca 2100 acagcaaccc aaagtcttat ttatcttcag gagctgcgca agccctttac caccgccacg 2160 tttctccctg gatgtcccta ggcctcggga ctggctggga gttccctgta gtggaggccg 2220 ccgctgaact gattataaag atgagagagg ctctgccatg ccttggtcat caattcacac 2280 tccaccagtg tcttctagca gtcacggcaa gggttaacgt cagggaccgc tgtggggtgg 2340 ccgcgctaca cgacagtata gttgcggtcc tggtttatga aataactgag ggaacaagag 2400 gcgcaagaaa tccctccttg ggtgcaagac caaaacaact acccagcggg aagactcggg 2460 cttcagtgcg tgtgtcgcca gtggaggagg acgcttcggg gcgggagcac aagctggcag 2520 gacagccccg cagcagctcc agcgcggcag aggccatgga agatggtccg ctggtcagca 2580 ccctgcctgc gcctcaaaat accgccggga aggtggggga cggggacggg agcagccaat 2640 cacgcgagct ctctcgggtg gcgtcagggc gcgatcgcgg attcggcact ccgggtgggc 2700 gtggctgggc gagcgaggag tggggacaag gtcgagcgac gagtcggcgc gcccaggtaa 2760 gtttagtctt tttgtctttt atttcaggtc ccggatccgg tggtggtgca aatcaaagaa 2820 ctgctcctca gtggatgttg cctttacttc taggcctgta cggaagtgtt acttctgctc 2880 taaaagctgc ggaattgtac ccgcggccgg ctagccgcca ccatggagaa gaatggaaat 2940 aaccgaaagc tgcgggtttg tgttgctact tgtaaccgtg cagattattc taaacttgcc 3000 ccgatcatgt ttggcattaa aaccgaacct gagttctttg aacttgatgt tgtggtactt 3060 ggctctcacc tgatagatga ctatggaaat acatatcgaa tgattgaaca agatgacttt 3120 gacattaaca ccaggctaca cacaattgtg aggggagaag atgaggcagc catggtggag 3180 tcagtaggcc tggccctagt gaagctgcca gatgtcctta atcgcctgaa gcctgatatc 3240 atgattgttc atggagacag gtttgatgcc ctggctctgg ccacatctgc tgccttgatg 3300 aacatccgaa tccttcacat tgaaggtggg gaagtcagtg ggaccattga tgactctatc 3360 agacatgcca taacaaaact ggctcattat catgtgtgct gcacccgcag tgcagagcag 3420 cacctgatat ccatgtgtga ggaccatgat cgcatccttt tggcaggctg cccttcctat 3480 gacaaacttc tctcagccaa gaacaaagac tacatgagca tcattcgcat gtggctaggt 3540 gatgatgtaa aatctaaaga ttacattgtt gcactacagc accctgtgac cactgacatt 3600 aagcattcca taaaaatgtt tgaattaaca ttggatgcac ttatctcatt taacaagcgg 3660 accctagtcc tgtttccaaa tattgacgca gggagcaaag agatggttcg agtgatgcgg 3720 aagaagggca ttgagcatca tcccaacttt cgtgcagtta aacacgtccc atttgaccag 3780 tttatacagt tggttgccca tgctggctgt atgattggga acagcagctg tggggttcga 3840 gaagttggag cttttggaac acctgtgatc aacctgggaa cacgtcagat tggaagagaa 3900 acaggggaga atgttcttca tgtccgggat gctgacaccc aagacaaaat attgcaagca 3960 4020 gttccaagga ttttgaagtt tctcaaatct atcgatcttc aagagccact gcaaaagaaa 4080 ttctgctttc ctcctgtgaa ggagaatatc tctcaagata ttgaccatat tcttgaaact 4140 ctaagtgcct tggccgttga tcttggcggg acgaacctcc gagttgcaat agtcagcatg 4200 aagggtgaaa tagttaagaa gtatactcag ttcaatccta aaacctatga agaggatt 4260 4320 attttgggag taggcatttc cacaggtggc cgtgtaaatc ctcgggaagg aattgtgctg 4380 cattcaacca aactgatcca agagtggaac tctgtggacc ttaggacccc cctttctgac 4440 actttgcatc tccctgtgtg ggtagacaat gatggcaact gtgctgccct ggcggaaagg 4500 aaatttggcc aaaggaaggg actggaaaac tttgttacac ttatcacagg cacaggaatc 4560 ggtggtggaa ttatccatca gcatgaattg atccacggaa gctccttctg tgctccagaa 4620 ctgggccacc ttgttgtgtc tctggatggg cctgattgtt cctgtggaag ccatgggtgc 4680 attgaagcat acgcctctgg aatggccttg cagagggagg caaaaaagct ccatgatgag 4740 gacctgctct tggtggaagg gatgtcagtg ccaaaagatg aggctgtggg tgcgctccat 4800 ctcatccaag ctgcgaaact tggcaatgcg aaggcccaga gcatcctaag aacagctgga 4860 acagctttgg gtcttggggt tgtgaacatc ctccatacca tgaatccctc ccttgtgatc 4920 ctctccggag tcctggccag tcactatatc cacattgtca aagacgtcat tcgccagcag 4980 gccttgtcct ccgtgcagga cgtggatgtg gtggtttcgg atttggttga ccccgccctg 5040 ctgggtgctg ccagcatggt tctggactac acaacacgca ggatctacta gcatgccttc 5100 tgatggtgaa tgggaactcc cttctctcctg cagcagcctg cctgcagctg tcctggtaga 5160 5220 atggtccgcg cgaggcacca gccgggtggg ctttgcctcc tgctgctgct gctctgccag 5280 ttcatggagg accgcagtgc ccaggctggg aactgctggc tccgtcaagc gaaacggc 5340 cgctgccagg tcctgtacaa gaccgaactg agcaaggagg agtgctgcag caccggccgg 5400 ctgagcacct cgtggaccga ggaggacgtg aatgacaaca cactcttcaa gtggatgatt 5460 ttcaacgggg gcgcccccaa ctgcatcccc tgtaaagaaa cgtgtgagaa cgtggactgt 5520 ggacctggga aaaaatgccg aatgaacaag aagaacaaac cccgctgcgt ctgcgccccg 5580 gattgttcca acatcacctg gaagggtcca gtctgcgggc tggatgggaa aacctaccgc 5640 aatgaatgtg cactcctaaa ggcaagatgt aaagagcagc cagaactgga agtccagtac 5700 caaggcagat gtaaaaagac ttgtcgggat gttttctgtc caggcagctc cacatgtgtg 5760 gtggaccaga ccaataatgc ctactgtgtg acctgtaatc ggatttgccc agagcctgct 5820 tcctctgagc aatatctctg tgggaatgat ggagtcacct actccagtgc ctgccacctg 5880 agaaaggcta cctgcctgct gggcagatct attggattag cctatgaggg aaagtgtatc 5940 aaagcaaagt cctgtgaaga tatccagtgc actggtggga aaaaatgttt atgggatttc 6000 aaggttggga gaggccggtg ttccctctgt gatgagctgt gccctgacag taagtcggat 6060 gagcctgtct gtgccagtga caatgccact tatgccagcg agtgtgccat gaaggaagct 6120 gcctgctcct caggtgtgct actggaagta aagcactccg gatcttgcaa ctccatttcg 6180 gaagacaccg aggaagagga ggaagatgaa gaccaggact acagctttcc tatatcttct 6240 attctagagt ggtaagcggc cgcaataaaa gatctttatt ttcattagat ctgtgtgttg 6300 gttttttgtg tgtctagagc atggctacgt agataagtag catggcgggt taatcattaa 6360 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 6420 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag 6480 cgagcgagcg cgcagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 6540 gttgcgcagc ctgaatggcg aatggcgatt ccgttgcaat ggctggcggt aatattgttc 6600 tggatattac cagcaaggcc gatagtttga gttcttctac tcaggcaagt gatgttatta 6660 ctaatcaaag aagtattgcg acaacggtta atttgcgtga tggacagact cttttactcg 6720 gtggcctcac tgattataaa aacacttctc aggattctgg cgtaccgttc ctgtctaaaa 6780 tccctttaat cggcctcctg tttagctccc gctctgattc taacgaggaa agcacgttat 6840 acgtgctcgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt 6900 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 6960 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 7020 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 7080 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 7140 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 7200 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 7260 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 7320 taaatatttg cttatacaat cttcctgttt ttggggcttt tctgattatc aaccggggta 7380 catatgattg acatgctagt tttacgatta ccgttcatcg attctcttgt ttgctccaga 7440 ctctcaggca atgacctgat agcctttgta gagacctctc aaaaatagct accctctccg 7500 gcatgaattt atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg 7560 gcctttctca cccgtttgaa tctttaccta cacattactc aggcattgca tttaaaatat 7620 atgagggttc taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat 7680 tacagggtca taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc 7740 ttaattttgc taattctttg ccttgcctgt atgatttatt ggatgttgga atcgcctgat 7800 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag 7860 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 7920 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 7980 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 8040 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 8100 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 8160 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 8220 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 8280 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 8340 <210> 26 <211> 7539 <212> DNA <213> Homo sapiens <400> 26 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 60 ttttcgcccc gaagaacgaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 120 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 180 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 240 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 300 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 360 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 420 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 480 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 540 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 600 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 660 ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 720 ctacctttgc catgtttcag aaaaactct ggcgcatcgg gcttcccata caatcgatag 780 attgcgcac ctgattgccc gacattatcg cgagcccatt tataccccata taaatcagca 840 tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 900 acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 960 ttaacgtgag tttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 1020 ttgagatcct ttttttgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 1080 agcggtggtt tgtttgccgg atcaagagct accaactctt ttccgaagg taactggctt 1140 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 1200 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 1260 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 1320 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 1380 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 1440 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 1500 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 1560 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 1620 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 1680 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 1740 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 1800 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg 1860 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 1920 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 1980 ttaatgatta acccgccatg ctacttatct acgtagccat gctctagagt ttaaacaagc 2040 tttcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg accccccggac 2100 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa 2160 aatcaacggg actttccaaa atgtcgtaac aactccgcccc cattgacgca aatgggcggt 2220 aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg ctcgaccggg 2280 gtaccggcc ggctagccgc caccatggag aagaatggaa ataaccgaaa gctgcgggtt 2340 tgtgttgcta cttgtaaccg tgcagattat tctaaacttg ccccgatcat gtttggcatt 2400 aaaaccgaac ctgagttctt tgaacttgat gttgtggtac ttggctctca cctgatagat 2460 gactatggaa atacatatcg aatgattgaa caagatgact ttgacattaa caccaggcta 2520 cacacaattg tgaggggaga agatgaggca gccatggtgg agtcagtagg cctggcccta 2580 gtgaagctgc cagatgtcct taatcgcctg aagcctgata tcatgattgt tcatggagac 2640 aggtttgatg ccctggctct ggccacatct gctgccttga tgaacatccg aatccttcac 2700 attgaaggtg gggaagtcag tgggaccatt gatgactcta tcagacatgc cataacaaaa 2760 ctggctcatt atcatgtgtg ctgcacccgc agtgcagagc agcacctgat atccatgtgt 2820 gaggaccatg atcgcatcct tttggcaggc tgcccttcct atgacaaact tctctcagcc 2880 aagaacaaag actacatgag catcattcgc atgtggctag gtgatgatgt aaaatctaaa 2940 gattacattg ttgcactaca gcaccctgtg accactgaca ttaagcattc cataaaaatg 3000 tttgaattaa cattggatgc acttatctca tttaacaagc ggaccctagt cctgtttcca 3060 aatattgacg cagggagcaa agagatggtt cgagtgatgc ggaagaaggg cattgagcat 3120 catcccaact ttcgtgcagt taaacacgtc ccatttgacc agtttataca gttggttgcc 3180 catgctggct gtatgattgg gaacagcagc tgtggggttc gagaagttgg agctttttgga 3240 acacctgtga tcaacctggg aacacgtcag attggaagag aaacagggga gaatgttctt 3300 catgtccggg atgctgacac ccaagacaaa atattgcaag cactgcacct tcagtttggt 3360 aaacagtacc cttgttcaaa gatatatggg gatggaaatg ctgttccaag gattttgaag 3420 tttctcaaat ctatcgatct tcaagagcca ctgcaaaaga aattctgctt tcctcctgtg 3480 your area tctctcaaa tattgaccat attcttgaaa ctctaagtgc cttggccgtt 3540 gatcttggcg ggacgaacct ccgagttgca atagtcagca tgaagggtga aatagttaag 3600 aagtatctc agttcaatcc taaaacctat gaagagag ttaatttaat cctacagatg 3660 tgtgtggaag ctgcagcaga agctgtaaaa ctgaactgca gaattttgg agtaggcatt 3720 tccacaggtg gccgtgtaaa tcctcgggaa ggaattgtgc tgcattcaac caaactgatc 3780 caagagtgga actctgtgga ccttaggacc cccctttctg acactttgca tctccctgtg 3840 tgggtagaca atgatggcaa ctgtgctgcc ctggcggaaa ggaaatttgg ccaaggaaag 3900 ggactggaaa actttgttac acttatcaca ggcacaggaa tcggtggtgg aattatccat 3960 cagcatgaat tgatccacgg aagctccttc tgtgctgcag aactgggcca ccttgttgtg 4020 tctctggatg ggcctgattg ttcctgtgga agccatgggt gcattgaagc atacgctctct 4080 ggaatggcct tgcagaggga ggcaaaaaag ctccatgatg aggacctgct cttggtggaa 4140 gggatgtcag tgccaaaaga tgaggctgtg ggtgcgctcc atctcatcca agctctcgaaa 4200 cttggcaatg cgaaggccca gagcatccta agaacagctg gaacagcttt gggtcttggg 4260 gttgtgaaca tcctccatac catgaatccc tcccttgtga tcctctccgg agtcctggcc 4320 agtcactata tccacattgt caaagacgtc attcgccagc aggccttgtc ctccgtgcag 4380 gacgtggatg tggtggtttc ggatttggtt gaccccgccc tgctgggtgc tgccagcatg 4440 gttctggact acacaacacg caggatctac tagcatgcgg gcataccagt gtcagctgca 4500 cttgtagggg cccaagtgcc tcatgaccca ctcggcagcc ttcctctcca ggatccccaa 4560 ggctaggagg ccaacctact aacaggtggg tgggtatggt gtgtggtttc actcagttct 4620 tctcatgggg tttctctgag ctccattcat accagaaagg gagcaggaga gagaggacaa 4680 gtggatccaa cagccttcgc tccaggggaa tcagggcatc gcctcctttt ctgggaggac 4740 actcccttct gatggtgaat gggaactccc ttcctcctgc agcagcctgc ctgcagctgt 4800 cctggtagaa cagtgtggac attgcagaag ctgtcactgc cccagaaaga aagcacccca 4860 gagccaaggc aaagagtctt gaaagcgcca caagcagcag ctgctgagac tggccgcacc 4920 atgaagctgc tgccgtcggt ggtgctgaag ctctttctgg ctgcagttct ctcggcactg 4980 gtgactggcg agagcctgga gcggcttcgg agagggctag ctgctggaac cagcaacccg 5040 gaccctccca ctgtatccac ggaccagctg ctacccctag gaggcggccg ggaccggaaa 5100 gtccgtgact tgcaagaggc agatctggac cttttgagag tcactttatc ctccaagcca 5160 caagcactgg ccacaccaaa caaggaggag cacgggaaaa gaaagaagaa aggcaagggg 5220 ctagggaaga agagggaccc aggaccggag acgctctgcg gggctgagct ggtggatgct 5280 cttcagttcg tgtgtggaga caggggcttt tatttcaaca agcccacagg gtatggctcc 5340 agcagtcgga gggcgcctca gacaggcatc gtggatgagt gctgcttccg gagctgtgat 5400 ctaaggaggc tggagatgta ttgcgcaccc ctcaagcctg ccaagtcagc ttaggcggcc 5460 gcaataaaag atctttattt tcattagatc tgtgtgttgg ttttttgtgt gtctagagca 5520 tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat 5580 ggagtggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt 5640 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctggcg 5700 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 5760 atggcgattc cgttgcaatg gctggcggta atattgttct ggatattacc agcaaggccg 5820 atagtttgag ttcttctact caggcaagtg atgttattac taatcaaaga agtattgcga 5880 caacggttaa tttgcgtgat ggacagactc ttttactcgg tggcctcact gattataaaa 5940 acacttctca ggattctggc gtaccgttcc tgtctaaaat ccctttaatc ggcctcctgt 6000 ttagctcccg ctctgattct aacgaggaaa gcacgttata cgtgctcgtc aaagcaacca 6060 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 6120 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 6180 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 6240 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 6300 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 6360 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 6420 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 6480 tttaacgcga attttaacaa aatattaacg cttacaattt aaatatttgc ttatacaatc 6540 ttcctgtttt tggggctttt ctgattatca accggggtac atatgattga catgctagtt 6600 ttacgattac cgttcatcga ttctcttgtt tgctccagac tctcaggcaa tgacctgata 6660 gcctttgtag agacctctca aaaatagcta ccctctccgg catgaattta tcagctagaa 6720 cggttgaata tcatattgat ggtgatttga ctgtctccgg cctttctcac ccgtttgaat 6780 ctttacctac acattactca ggcattgcat ttaaaatata tgagggttct aaaaattttt 6840 atccttgcgt tgaaataaag gcttctcccg caaaagtatt acagggtcat aatgtttttg 6900 gtacaaccga tttagcttta tgctctgagg ctttattgct taattttgct aattctttgc 6960 cttgcctgta tgatttattg gatgttggaa tcgcctgatg cggtattttc tccttacgca 7020 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc 7080 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 7140 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 7200 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 7260 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 7320 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 7380 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 7440 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 7500 cccagaaacg ctggtgaaag taaaagatgc tgaagatca 7539 <210> 27 <211> 178 <212> PRT <213> Homo sapiens <400> 27 Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Phe Leu Ala Ala Val 1 5 10 15 Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly 20 25 30 Leu Ala Ala Gly Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr Asp 35 40 45 Gln Leu Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu 50 55 60 Gln Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro 65 70 75 80 Gln Ala Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg Lys Lys 85 90 95 Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Gly Pro Glu Thr Leu 100 105 110 Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe Val Cys Gly Asp Arg 115 120 125 Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser Arg Arg 130 135 140 Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg Ser Cys Asp 145 150 155 160 Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser 165 170 175 Ala Val <210> 28 <211> 2549 <212> DNA <213> Homo sapiens <400> 28 gtccattgaa tcgcgggggc ggccggcggc gagcgcgggg ctgcgccggg atcgctgcgc 60 cctccgccgc tggcctgc gacgcgcgcc gctcgcccga gccaccgcc gccgcgccgg 120 ctcccgcgc cgctgcgctc ctcgccccgc gcctgccccc aggatgtcc gcgcgaggca 180 ccagccgggt gggctttgcc tcctgctgct gctgctgc cagttcatgg aggaccgcag 240 tgcccaggct gggaacctgct ggctccgtca agcgagaac ggccgctgcc aggtcctgta 300 caaccgaa ctgagcagg aggagtgctg cagcaccggc cggctgagca cctcgtggac 360 cgaggaggac gtgaatgaca acacactt caagtggatg atttcaacg ggggcgcccc 420 caactgcatc ccctgtaaag aaacgtgtga gaacgtggac tgtggacctg ggaaaaaatg 480 ccgaatgaac agagaaca aaccccgctg cgtctgcgcc ccggattgtt ccacacac 540 ctggaagggt ccagtctgcg ggctggatgg gaaaacctac cgcaatgaat gtgcactcct 600 aaaggcaga tgtaagagc agccagaact ggaagtccag taccaggca gatgtaaaaa 660 gacttgtcgg gatgttttct gtccaggcag ctccacatgt gtggtggacc agaccataa 720 tgcctactgt gtgacctgta atcggatttg cccagagcct gcttcctctg agcaatatct 780 ctgtgggaat gatggagtca cctactccag tgcctgccac ctgagaaagg ctacctgcct 840 gctgggcaga tctattggat tagcctatga gggaaagtgt atcaaagcaa agtcctgtga 900 agatatccag tgcactggtg ggaaaaaatg tttatgggat ttcaaggttg ggagaggccg 960 gtgttccctc tgtgatgagc tgtgccctga cagtaagtcg gatgagcctg tctgtgccag 1020 tgacaatgcc acttatgcca gcgagtgtgc catgaaggaa gctgcctgct cctcaggtgt 1080 gctactggaa gtaaagcact ccggatcttg caactgaatc tgcccgtaaa acctgagcca 1140 ttgattcttc agaactttct gcagtttttg acttcataga tttgcttta aaaaaatttt 1200 tttaacttat tgcataacag cagatgccaa aaacaaaaaa agcatctcac tgcaagtcac 1260 ataaaaatgc aacgctgtaa tatggctgta tcagagggct ttgaaaacat acactgagct 1320 gcttctgcgc tgttgttgtc cgtatttaaa caacagctcc cctgtattc cccatctagc 1380 cattcggaa gacaccgagg aagaggagga agatgaagac caggactaca gctttcctat 1440 atcttctatt ctagagtggt aaactctcta taagtgttca gtgttgacat agcctttgtg 1500 1560 aataagtgta tgcttattta tttgggggga aaactataca ttaaaggacc tttgtcctaa 1620 agctctctcc caggccacct tgttactcat tggacacgga gaggcattca ttgtgaggtc 1680 tactggatga ggcccatagt tgagacttgt agacatttat ttatactgtg tcatgtttta 1740 taatttatac ataaaatgtc tggttgactg tataccttgt ttttgaagaa atttattcgt 1800 gaaaggaaga gcagttgtta tttattgtga ggtctcttgc ttgtaaagta aaagcttttt 1860 ttccttgtaa accatttaag tccattcctt actattcact cactcatctg tctcccttca 1920 tttcactgtt agactctttt ccactttcaa caaacttgca tgtcagtttc tgtcatgttt 1980 atttattgga ttctctgctg cctgatctgt acatacatga tccctcgggt tttgtttaca 2040 aggaaccttg actgaccaaa aggcattata actctgactc aaatacaagg tacagaagat 2100 aagcatcttt gaggaaactc ctacttcagt tctttttgtta tgatgaagac atttgtgaga 2160 gaggagatga ttagaattct agtaatgtac ttttaagatg ttacagatac aaagaaatga 2220 tgtgggtgtc aggagactaa aggatgttga aggctacaca ttcaaccttt tgttaggtgt 2280 ttcctttaag ctactcagct gtacctttta aattagttct ttttcaacca gtatatcact 2340 aaaagttata tcaaagcttt atcagttcaa gtttcttgct tttcataata cttttttctg 2400 atgcaatttt atattttcaa acatggcaag ttaaaatata aattcattta aatatatagt 2460 tttgtacttt tctaccatgt aaatgtgcaa tgtatataaa agttataatg tgtatttgta 2520 aataaatgat gagtgaaaaa ataaaaaaa 2549 <210> 29 <211> 317 <212> PRT <213> Homo sapiens <400> 29 Met Val Arg Ala Arg His Gln Pro Gly Gly Leu Cys Leu Leu Leu Leu 1 5 10 15 Leu Leu Cys Gln Phe Met Glu Asp Arg Ser Ala Gln Ala Gly Asn Cys 20 25 30 Trp Leu Arg Gln Ala Lys Asn Gly Arg Cys Gln Val Leu Tyr Lys Thr 35 40 45 Glu Leu Ser Lys Glu Glu Cys Cys Ser Thr Gly Arg Leu Ser Thr Ser 50 55 60 Trp Thr Glu Glu Asp Val Asn Asp Asn Thr Leu Phe Lys Trp Met Ile 65 70 75 80 Phe Asn Gly Gly Ala Pro Asn Cys Ile Pro Cys Lys Glu Thr Cys Glu 85 90 95 Asn Val Asp Cys Gly Pro Gly Lys Lys Cys Arg Met Asn Lys Lys Asn 100 105 110 Lys Pro Arg Cys Val Cys Ala Pro Asp Cys Ser Asn Ile Thr Trp Lys 115 120 125 Gly Pro Val Cys Gly Leu Asp Gly Lys Thr Tyr Arg Asn Glu Cys Ala 130 135 140 Leu Leu Lys Ala Arg Cys Lys Glu Gln Pro Glu Leu Glu Val Gln Tyr 145 150 155 160 Gln Gly Arg Cys Lys Lys Thr Cys Arg Asp Val Phe Cys Pro Gly Ser 165 170 175 Ser Thr Cys Val Val Asp Gln Thr Asn Asn Ala Tyr Cys Val Thr Cys 180 185 190 Asn Arg Ile Cys Pro Glu Pro Ala Ser Ser Glu Gln Tyr Leu Cys Gly 195 200 205 Asn Asp Gly Val Thr Tyr Ser Ser Ala Cys His Leu Arg Lys Ala Thr 210 215 220 Cys Leu Leu Gly Arg Ser Ile Gly Leu Ala Tyr Glu Gly Lys Cys Ile 225 230 235 240 Lys Ala Lys Ser Cys Glu Asp Ile Gln Cys Thr Gly Gly Lys Lys Cys 245 250 255 Leu Trp Asp Phe Lys Val Gly Arg Gly Arg Cys Ser Leu Cys Asp Glu 260 265 270 Leu Cys Pro Asp Ser Lys Ser Asp Glu Pro Val Cys Ala Ser Asp Asn 275 280 285 Ala Thr Tyr Ala Ser Glu Cys Ala Met Lys Glu Ala Ala Cys Ser Ser 290 295 300 Gly Val Leu Leu Glu Val Lys His Ser Gly Ser Cys Asn 305 310 315 <210> 30 <211> 427 &...

Claims

1. A polynucleotide comprising: a) One or more promoter elements, b) GNE cDNA sequence, c) Internal ribosome entry site (IRES), and d) Nucleotide sequences encoding muscle growth factor or muscle transdifferentiation factor.

2. The polynucleotide of claim 1, wherein the promoter element is operatively linked to the GNE cDNA sequence.

3. The polynucleotide of claim 1 or 2, wherein the IRES is operatively linked to the nucleotide sequence encoding muscle growth factor or muscle transdifferentiation factor.

4. The polynucleotide according to any one of claims 1 to 3, wherein the promoter element is a constitutive promoter or a muscle-specific promoter.

5. The polynucleotide according to any one of claims 1 to 4, wherein the promoter element is a CMV promoter, an MCK promoter, an MHCK7 promoter, a mini CMV promoter, or a GNE promoter.

6. The polynucleotide according to any one of claims 1 to 5, wherein the GNE cDNA sequence is a variant 2 GNE wild-type human GNE gene comprising the nucleic acid sequence of SEQ ID NO:

1.

7. The polynucleotide sequence according to any one of claims 1 to 6, further comprising the human GNE promoter element found between exons 1 and 2 to drive the expression of the GNE cDNA.

8. The polynucleotide according to any one of claims 1 to 7, wherein the internal ribosome entry site (IRES) is derived from the fibroblast growth factor 1A gene.

9. The polynucleotide of claim 8, wherein the IRES comprises the nucleotide sequence of SEQ ID NO:30 or a fragment thereof.

10. The polynucleotide of claim 8, wherein the IRES comprises the nucleotide sequence of SEQ ID NO:

8.

11. The polynucleotide according to any one of claims 1 to 10, wherein the nucleotide sequence encodes a variant of follicle-staphylin, SMAD7, or insulin-like growth factor 1 (IGF1).

12. The polynucleotide of claim 11, wherein the follicle-stabilizing hormone is follicle-stabilizing hormone 344 or follicle-stabilizing hormone 314.

13. The polynucleotide of claim 11, wherein the IGF1 variant is HB-IGF1.

14. A recombinant adeno-associated virus (rAAV) whose genome comprises the polynucleotide sequence of any one of claims 1 to 13, wherein said polynucleotide is located in a single rAAV genome.

15. The rAAV of claim 14, wherein the genome comprises a CMV promoter and variant 2 wild-type human GNEcDNA.

16. The rAAV of claim 14, wherein the genome comprises the MCK promoter and variant 2 wild-type human GNEcDNA.

17. The rAAV of claim 14, wherein the genome comprises the MHCK promoter and variant 2 wild-type human GNE cDNA.

18. The rAAV of claim 14, wherein the genome comprises a GNE promoter and variant 2 wild-type human GNE cDNA.

19. The rAAV of claim 14, wherein the genome comprises a mini CMV promoter and variant 2 wild-type human GNE cDNA.

20. The rAAV of claim 14, wherein the genome comprises the MCK7 promoter, variant 2 wild-type human cDNA, FGF1 IRES, and a nucleic acid sequence encoding follistatin 344.

21. The rAAV of claim 14, wherein the genome comprises the MHCK7 promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1.

22. The rAAV of claim 14, wherein the genome comprises the CMV promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344.

23. The rAAV of claim 14, wherein the genome comprises the CMV promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1.

24. The rAAV of claim 14, wherein the genome comprises the MCK promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344.

25. The rAAV of claim 14, wherein the genome comprises the MCK promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1.

26. The rAAV of claim 14, wherein the genome comprises the GNE promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344.

27. The rAAV of claim 14, wherein the genome comprises the GNE promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1.

28. The rAAV of claim 14, wherein the genome comprises the mini CMV promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding follicle-stimulating hormone 344.

29. The rAAV of claim 14, wherein the genome comprises the mini CMV promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1.

30. The rAAV of claim 14, wherein the genome comprises the MHCK7 promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES and a nucleic acid sequence encoding SMAD7.

31. The rAAV of claim 14, wherein the genome comprises a CMV promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding SMAD7.

32. The rAAV of claim 14, wherein the genome comprises the MCK promoter, variant 2 wild-type human GNEcDNA, FGF1 IRES and a nucleic acid sequence encoding SMAD7.

33. The rAAV of claim 14, wherein the genome comprises a GNE promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES and a nucleic acid sequence encoding SMAD7.

34. The rAAV of claim 14, wherein the genome comprises a mini CMV promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding SMAD7.

35. The rAAV according to any one of claims 14 to 34, wherein the rAAV is serotype AAVrh.

74.

36. An rAAV particle comprising rAAV according to any one of claims 14 to 35.

37. Use of the rAAV particles according to any one of claims 14 to 34 or according to claim 36 in the preparation of a medicament for treating GNE myopathy.

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