Optimized gene therapy for targeting muscle in muscle diseases

Abstract

To provide an optimized gene therapy for targeting muscles of muscle diseases.SOLUTION: The present disclosure provides gene therapy vectors, such as adeno-associated virus (AAV), that are optimized to deliver transgenes to muscle. The optimized vector contains a constitutive or muscle-specific promoter that delivers systemic or skeletal / cardiac muscle-specific transgene expression, respectively, in combination with a transgene cDNA to replace a gene mutation found in a muscle disease with a normal copy of the gene, an internal ribosome entry site (IRES) to allow production of a second protein from the same transcript, and a muscle growth factor to build new muscle growth and strength.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority benefit of U.S. Provisional Patent Application No. 62 / 951,564, filed December 20, 2019, which is incorporated herein by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials This application contains a Sequence Listing in computer-readable form, which is incorporated herein by reference in its entirety as a separate part of this disclosure and identified as Filename: 54649_Sqlisting.txt, Size: 233,379 bytes, Created: December 21, 2020.

[0003] The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAVs), optimized for delivering transgenes to muscle. The optimized vectors contain a transgene cDNA to replace a gene mutation found in muscle diseases with a normal copy of the gene, an internal ribosome entry site (IRES) to enable production of a second protein from the same transcript, and a constitutive or muscle-specific promoter to deliver systemic or skeletal / cardiac muscle-specific transgene expression, respectively, in combination with a muscle growth factor gene to build new muscle growth and strength. The transgene and muscle growth factor gene are expressed from the same mRNA, expressing both proteins, due to the presence of an internal ribosome entry site (IRES) from the fibroblast growth factor 1A gene sequence, which allows the second protein to be produced from a single mRNA. [Background technology]

[0004] GNE myopathy is an adult-onset autosomal recessive disorder characterized by progressive muscle weakness that can lead to loss of ambulation and independence. As the name suggests, GNE myopathy is caused by loss-of-function pathogenic variants or mutations in the GNE gene. This disease is also known as hereditary inclusion body myopathy, quadriceps-sparing myopathy, distal myopathy with rimmed vacuoles, and Nonaka myopathy. The GNE gene encodes a bifunctional UDP-GIcNAc-epimerase / ManNAc-6 kinase, whose enzymatic activity is essential for the sialic acid biosynthesis pathway.

[0005] Sialic acid is an acidic monosaccharide that modifies the non-reducing terminal carbohydrate chains of glycoproteins and glycolipids, playing an important role in various processes, such as cell adhesion and cell-cell interactions. Sialic acid is implicated in health and disease and is found in the terminal glycans of proteins, regulating cellular functions. UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase (GNE) is a key enzyme in sialic acid biosynthesis. Furthermore, GNE expression is induced upon muscle fiber injury or regeneration, demonstrating its role in muscle regeneration. Myoblasts harboring a mutated GNE gene exhibit reduced epimerase activity, resulting in a significant reduction in overall membrane-bound sialic acid, even in cells harboring homozygous epimerase mutations (Pogoryleva et al., Orphanet J Rare Dis. 13: 70, 2018).

[0006] GNE myopathy causes muscle weakness and wasting in the legs and arms. Initial symptoms typically occur in young adults (usually in the third decade of life), although later onset has been observed in some patients. The diagnosis of GNE myopathy should be considered primarily in patients presenting with distal weakness (foot drop) in early adulthood (although other onset symptoms are possible). The disease progresses slowly, involving other lower and upper limb muscles, typically with prominent sparing of the quadriceps. Characteristic findings in biopsies of affected muscles include "rimming" (autophagic) vacuoles, aggregates of various proteins, and variations in fiber size.

[0007] Despite the fact that mutations in the GNE gene were shown to cause GNE myopathy in 2001, there is still no effective therapy for this disease. Attempts to develop sustained-release sialic acid therapy failed in phase 3 clinical trials, and ManNAc glycan therapy is currently under investigation. While developing a gene therapy approach for GNE gene replacement may seem straightforward, it is in fact complicated by several unresolved issues in GNE myopathy research, first and foremost being the lack of a robust and reproducible model of the disease. Noguchi and Nishino developed a transgenic GNED176VTg Gne gene that exhibited distinct aspects of the disease pathology. - / - Although several papers have been published on mouse models, other groups have not been able to see the same phenotype in subsequent breeding, likely a result of genetic drift in the founder transgenic lines (see Nishino et al., J. Neurol. Neurosurg. Psychiatry 86(4):385-392). M712TThe variant knock-in mouse model showed early death within a few weeks of birth due to kidney disease, a clinical phenotype not seen in GNE myopathy patients. Other strains of the same model were bred to exhibit no phenotype whatsoever, despite carrying the same genetic mutation. Second, the lack of a measurable natural history from rare and geographically diverse patient populations. Third, the slow onset of disease combined with highly variable disease progression makes it quite difficult to demonstrate clinical efficacy in GNE myopathy trials using gene replacement alone to slow or prevent disease progression.

[0008] Cells lacking GNE activity can be rescued by adding sialic acid (SA) or by adding ManNAc, which can also be converted to ManNAc-6 phosphate, the end product of GNE activity, through GlcNAc-6 kinase activity, which is not mutated in the disease. Some glycan therapies target GNE. D176V Tg Gne - / - Mouse and Gne M712T Efficacy was demonstrated in knock-in mice. This led to two sets of clinical trials: one using sustained-release SA (Phase 3 completed) (Lochmuller et al., Neurology 92(18):e2109-e17, 2019), and the other using ManNAc (Phase 1 completed) (Xu et al., Mol. Genet. Metab., 12291-2:126-34, 2017). While SA and ManNAc were shown to have significant therapeutic effects in mice, sustained-release SA therapy (ACE-ER) failed to achieve clinical milestones in a Phase 3 clinical trial in patients with GNE myopathy

[16] . There were no significant changes from placebo in any clinical measurements.

[0009] The lack of efficacy of glycan therapy in patients with GNE myopathy makes gene therapy a very attractive alternative. However, significant challenges remain: the slow and variable progression of human disease and the lack of robust short-term clinical milestones. For example, the current phase 3 clinical trial was 48 weeks in duration

[16] . At that time, there was no significant decline in any of the patient population's physical fitness measures from pretreatment baseline, although some measures tended to be lower.

[0010] The goal of the GNE treatment provided herein is to create a tandem gene therapy, utilizing a muscle-specific IRES to create a bicistronic gene therapy vector that expresses both the normal GNE gene and a known muscle growth factor. Such an AAV vector corrects the genetic defect of GNE myopathy and increases muscle strength, thus reversing rather than simply preventing the decline in clinical muscle strength. A therapy that builds new muscle and muscle strength while also preventing further disease by restoring the normal GNE gene would be of greater benefit to patients with GNE myopathy and provide a simpler means of demonstrating clinical improvement.

[0011] Given the pathophysiology of the disease, recent clinical trials have evaluated the use of sialic acid or ManNAc (a precursor of sialic acid) and early gene testing in patients with GNE myopathy. For example, AAV8 viral vectors carrying wild-type human GNE cDNA have been shown to transduce mouse muscle cells and human GNE myopathy-derived muscle cells in culture and express the transgene in these cells (Mitrani-Rosenbaum et al., Neuromuscul. Disord. 22(11):1015-24, 2012). Prior art gene therapy has focused only on delivering wild-type GNE genes and has not utilized the dual-function bicistronic technology disclosed herein. The present disclosure provides both transgenes for gene replacement that are desired to prevent further muscle damage or promote muscle growth, and genes that increase muscle strength. For example, a gene therapy vector that provides GNE gene replacement may be one of the only methods to demonstrate clinical effectiveness in GNE myopathy in less than five years, because the natural course of disease progression is slow and highly variable. It is also one of the only methods to demonstrate clinical effectiveness in all GNE myopathy patients, many of whom lose their ability to walk shortly after diagnosis but still demonstrate significant arm function, such as self-feeding, which can be preserved or improved by such therapy. Because this disease is a myopathy, not a dystrophy, the repaired muscles should remain in place permanently. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Pogoryleva et al.,Orphanet J Rare Dis.13: 70,2018 [Non-patent document 2] Nishino et al., J. Neurol. Neurosurg, Psychiatry 86(4):385-392 [Non-patent document 3] Lochmuller et al.,Neurology 92(18):e2109-e17,2019 [Non-patent document 4] Xu et al.,Mol.Genet.Metab.,12291-2:126-34,2017 [Non-Patent Document 5] Mitrani-Rosenbaum et al.,Neuromuscul.Disord.22(11):1015-24,2012 Summary of the Invention [Means for solving the problem]

[0013] The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAVs), optimized for delivering transgenes to muscle. The optimized vectors contain a transgene cDNA to replace a gene mutation found in muscle disease with a normal copy of the gene (or alternative gene replacement), an internal ribosome entry site (IRES) to enable production of a second protein from the same transcript, and a constitutive or muscle-specific promoter to deliver systemic or skeletal / cardiac muscle-specific transgene expression, respectively, in combination with a muscle growth factor gene to build new muscle growth and strength. The transgene and muscle growth factor gene are expressed from the same mRNA, expressing both proteins, due to the presence of an internal ribosome entry site (IRES) from the fibroblast growth factor 1A gene sequence, which allows the second protein to be produced from a single mRNA. For example, the present disclosure provides gene therapy vectors designed for the treatment of GNE myopathy. AAV expresses the GNE gene, which encodes the bifunctional UDP-GlcNAc-epimerase / ManNAc-6 kinase enzyme, either alone or in combination with a muscle growth factor such as follistatin (FST), a heparin-binding modified insulin-like growth factor 1 (HB-IGF), native IGF1, or SMAD7. In this scenario, the provided AAV replaces the mutated GNE gene expression in GNE myopathy patients with a normal GNE gene while simultaneously expressing a muscle growth and strength-stimulating protein that can counteract or even reverse the course of the disease. A unique aspect of tandem vectors is that they simultaneously deliver two necessary therapeutic elements: a gene replacement therapy to prevent further disease in expressing cells or tissues, and a muscle growth therapy to reverse the disease by building new muscle growth and strength. In the case of muscular dystrophies and myopathies, muscle tissue loss results from mutations in disease-causing genes. The treatment proposed here not only blocks the disease in such patients by reintroducing a non-mutated version of the disease gene, but also builds muscle and reverses ongoing muscle loss by co-expressing muscle growth factors.Such growth factors could double the amount of muscle in the tissue, potentially doubling (and thereby reversing) the weakness caused by these diseases.

[0014] The present disclosure also provides an alternative gene therapy vector for the treatment of muscular dystrophies, such as Duchenne muscular dystrophy, limb-girdle muscular dystrophy 2L (LGMD2A), and congenital muscular dystrophy 1a (MDC1A). AAV expresses the GALGT2 (B4GALNT2) gene, which encodes GalNAc transferase (beta-1,4-N-acetylgalactosamine galactosyltransferase) alone or in combination with muscle growth factors such as follistatin (FST), a heparin-binding modified insulin-like growth factor 1 (HB-IGF), natural IGF1, or SMAD7. This is an alternative gene therapy because it does not replace a mutated gene but instead provides an enzyme that transfers complex sugar molecules onto specific proteins such as dystroglycan.

[0015] Provided herein is an AAV having a genome comprising a constitutive or muscle-specific promoter that drives expression of a nucleotide sequence encoding a transgene of interest in combination with a nucleotide sequence encoding muscle growth and a muscle-specific IRES, such as the FGF IRES, or a protein that induces muscle transdifferentiation factors, such as myoD. This gene therapy approach is useful for treating any disease that requires gene replacement in combination with the need to increase muscle growth or strength, such as GNE myopathy, limb-girdle muscular dystrophy, congenital muscular dystrophy 1A, and Duchenne muscular dystrophy.

[0016] The present disclosure provides a polynucleotide comprising: 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 a muscle growth factor or muscle transdifferentiation factor (i.e., a second transgene). For example, the constitutive or muscle-specific promoter is operably linked to the transgene, and / or the IRES is operably linked to the nucleotide sequence encoding the muscle growth factor or muscle transdifferentiation factor. The fact that the elements are linked to a single mRNA allows both functions to be provided by a single AAV-mediated gene therapy product. Due to the significant cost of AAV production and safety concerns regarding AAV administration, the use of a single AAV vector with two gene therapies is far superior to achieving the same result by mixing two single-gene AAV gene therapies together, which requires producing and delivering twice (or more) the amount of AAV to the patient.

[0017] The present 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) more or more 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 operably linked to the GNE cDNA sequence, and / or the IRES is operably linked to a polynucleotide that induces muscle growth. In additional examples, the polynucleotide comprises a) more or more 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.

[0018] GNE myopathy is an adult-onset, slowly progressive muscle disease. To demonstrate therapeutic efficacy within a reasonable timeframe and maximize benefit for patients already suffering from muscle weakness at the time of diagnosis, gene therapy is needed that not only corrects the genetic defect in GNE gene function but also builds new muscle mass. Follistatin, IGF1, SMAD7, and HB-IGF are known to dramatically stimulate muscle growth in mice, macaques, and / or humans. Follistatin does this in part by inhibiting myostatin-mediated growth signaling through competitive inhibition and suppression of Smad2 / 3 signaling, whereas IGF1 does this in part by activating the muscle IGF1 receptor and activating Akt / mTOR signaling. Provided herein is a bicistronic AAV that expresses GNE using an IRES sequence from FGF1A, which is known to function most efficiently in skeletal muscle tissue. The use of a muscle-specific IRES would be ideal for follistatin, as it promotes optimal muscle growth through localized expression, whereas the use of the CMV promoter for GNE expression would be ideal, as GNE is normally expressed in all tissues.

[0019] Provided herein is an AAV having a genome comprising a promoter element, such as a constitutive promoter or a muscle-specific promoter, that drives the expression of a GNE cDNA sequence or a GALGT2 cDNA sequence. In particular, the present disclosure provides an rAAV having a genome designed to facilitate GNE gene replacement. In these AAVs, the genome comprises a) one or more muscle-specific promoter elements, and b) a GNE cDNA sequence. In another aspect, the present disclosure provides an rAAV having a genome designed to facilitate GALGT2 replacement gene therapy (expression of a replacement gene).

[0020] For example, the present disclosure provides an rAAV genome comprising a polynucleotide comprising a nucleotide sequence encoding a wild-type human GNE gene, e.g., variant 2 GNE 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 the human GNE promoter sequence (SEQ ID NO: 6). In some embodiments, the human GNE promoter element is found between exons 1 and 2 and drives expression of a variant 2 (722 amino acids) GNE cDNA comprising the nucleic acid sequence of SEQ ID NO: 1 (thereby enabling endogenous native gene expression).

[0021] The present 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) more or more 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 a cell into a muscle cell. In some embodiments, the muscle-specific regulatory element is operably linked to the GALGT2 cDNA sequence, and / or the IRES is operably linked to the polynucleotide that induces muscle growth.

[0022] For example, the present disclosure also provides an rAAV genome comprising 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).

[0023] The present disclosure also provides rAAVs with genomes designed to include a second transgene that induces muscle growth or differentiates or transforms cells into muscle. For example, the rAAVs have a genome that includes a GNE cDNA or GALGT2 cDNA sequence, an internal ribosome entry site (IRES) from the fibroblast growth factor 1A gene known to function in skeletal muscle 3' of the GNE cDNA or GALGT2 cDNA sequence, followed by a polyA sequence or a nucleotide sequence encoding a gene known to induce muscle growth, such as follistatin (e.g., follistatin 344 (FS344)) or an IGF1 variant (e.g., HB-IGF1), before SMAD7. The FGF IRES includes the nucleotide sequence of SEQ ID NO: 30 or a fragment thereof. An exemplary fragment of the FGF IRES includes the nucleotide sequence of SEQ ID NO: 8, which is also referred to herein as a "mini-IRES."

[0024] The present disclosure is directed to a gene therapy vector, such as AAV, that expresses wild-type human GNE gene in skeletal muscle to reduce or replace defective GNE gene.The gene therapy vector of the present invention can also be the AAV that expresses wild-type human GNE gene and the gene that induces muscle growth, such as follistatin, IGF1 or SMAD7, in a single rAAV genome.

[0025] The present disclosure provides a polynucleotide comprising a) one or more promoter elements, such as a constitutive or muscle-specific promoter, and b) a GNE cDNA sequence. The present disclosure also provides a polynucleotide comprising a) more or more promoter elements, such as a constitutive muscle-specific promoter, b) a GNE cDNA sequence or a GALGT2 cDNA sequence, c) an internal ribosome entry site (IRES), and d) a nucleotide sequence encoding a muscle growth factor or muscle transdifferentiation factor. The GNE cDNA is a nucleic acid sequence encoding UDP-GlcNAc-epimerase / ManNAc-6. In an exemplary embodiment, the GNE cDNA is a wild-type variant 2 GNE cDNA encoding UDP-GlcNAc-epimerase / ManNAc-6 kinase. The variant 2 wild-type GNE cDNA sequence is set forth as the nucleic acid sequence of SEQ ID NO: 1. The present disclosure also provides a polynucleotide comprising the GNE promoter element found between exons 1 and 2 to drive expression of the same variant 2 (722 amino acids) GNE cDNA. The GNE promoter sequence is set forth as SEQ ID NO: 6. GALGT2 cDNA is a nucleic acid sequence encoding GalNAc transferase. The GALGT2 cDNA sequence is shown as the nucleic acid sequence of SEQ ID NO: 36. The GalNAc transferase amino acid sequence is shown as SEQ ID NO: 37.

[0026] In some aspects, the present 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 follistatin, an insulin-like growth factor 1 (IGF1) variant, or SMAD7. For example, follistatin is follistatin 344, encoded by the nucleotide sequence of SEQ ID NO:9. Another exemplary follistatin is follistatin 317, encoded by the nucleotide sequence of SEQ ID NO:28. Additionally, an 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.

[0027] In some aspects, the present disclosure provides a polynucleotide comprising a GNE cDNA sequence or a GALGT2 cDNA sequence and a sequence encoding a protein that induces differentiation of cells into muscle (transdifferentiation factor), such as myoD (SEQ ID NO: 31).

[0028] In some embodiments, the polynucleotide comprises an internal ribosome entry site (IRES), such as the IRES from the fibroblast growth factor 1A gene (FGF IRES). The FGF IRES nucleotide sequence is set forth as SEQ ID NO: 30, or a fragment thereof. The FGF IRES can be miniaturized, such as the mini-FGR IRES set forth as SEQ ID NO: 8.

[0029] Another aspect of the present disclosure provides compositions comprising a nucleic acid molecule comprising a genome within the nucleotide sequence of any one of SEQ ID NOs: 12-26 and 36, an rAAV having a genome within the nucleic acid sequence of SEQ ID NOs: 12-12 and 36, or an rAAV particle comprising a genome within the nucleic acid sequence of any one of SEQ ID NOs: 12-26 and 36. Any of the methods disclosed herein can be performed using these compositions.

[0030] The disclosed AAV comprises a genome comprising a CMV promoter and variant 2 wild-type human GNE cDNA, for example, the genome provided in Figure 1A or the genome shown in SEQ ID NO:12.

[0031] The disclosed AAV comprises a genome comprising an MCK promoter and variant 2 wild-type human GNE cDNA, for example, the genome provided in Figure 1B or shown in SEQ ID NO:13.

[0032] The disclosed AAV comprises a genome comprising an MHCK promoter and variant 2 wild-type human GNE cDNA, for example, the genome provided in Figure 1C or the genome shown in SEQ ID NO:14.

[0033] The disclosed AAV comprises a genome comprising a GNE promoter and variant 2 wild-type human GNE cDNA, for example, the genome provided in FIG. 1D or shown in SEQ ID NO:15.

[0034] The disclosed AAV comprises a genome comprising an MHCK7 promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding a mini-FGF1 IRES, and a nucleic acid sequence encoding follistatin 344, for example, the genome provided in FIG. 1E or the genome shown in SEQ ID NO: 16.

[0035] The disclosed AAV comprises a genome comprising an MHCK7 promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding a mini-FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example, the genome provided in Figure 1F or the genome shown in SEQ ID NO: 17.

[0036] The disclosed AAVs include a genome including a CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding follistatin 344, for example, the genome provided in FIG. 1G or the genome shown in SEQ ID NO: 18.

[0037] The disclosed AAV comprises a genome comprising 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 HB-IGF1, for example, the genome provided in Figure 1H or the genome shown in SEQ ID NO: 19.

[0038] The disclosed AAV comprises a genome comprising an MCK promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding a mini-FGF1 IRES, and a nucleic acid sequence encoding follistatin 344, for example, the genome provided in FIG. 1I or the genome shown in SEQ ID NO: 20.

[0039] The disclosed AAV comprises a genome comprising an MCK promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding a mini-FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example, the genome provided in Figure 1J or the genome shown in SEQ ID NO: 21.

[0040] The disclosed AAV comprises a genome comprising a GNE promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding a mini-FGF1 IRES, and a nucleic acid sequence encoding follistatin 344, for example, the genome provided in Figure 1K or the genome shown in SEQ ID NO: 22.

[0041] The disclosed AAV comprises a genome comprising a GNE promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding a mini-FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example, the genome provided in Figure 1L or the genome shown in SEQ ID NO: 23.

[0042] The disclosed AAV comprises a mini-CMV promoter, a genome comprising variant 2 wild-type GNE cDNA, for example, the genome provided in Figure 1M or the genome shown in SEQ ID NO:24.

[0043] The disclosed AAV comprises a genome comprising a mini-CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding follistatin 344, e.g., the genome provided in FIG. 1N or the genome shown in SEQ ID NO: 25.

[0044] The disclosed AAV comprises a genome comprising a mini-CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example, the genome provided in Figure 1O or the genome shown in SEQ ID NO: 26.

[0045] The disclosed AAV comprises a genome comprising an MHCK7 promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example, the genome provided in Figure 1P.

[0046] The disclosed AAV comprises a genome comprising an MHCK7 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, the genome provided in Figure 1Q.

[0047] The disclosed AAV comprises a genome comprising a CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example, the genome provided in Figure 1R.

[0048] The disclosed AAV comprises a genome comprising 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, the genome provided in Figure 1S.

[0049] The disclosed AAV comprises a genome comprising an MCK promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example, the genome provided in Figure 1T.

[0050] The disclosed AAV comprises a genome comprising an MCK 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, the genome provided in Figure 1U.

[0051] The disclosed AAV comprises a genome comprising a GNE promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example, the genome provided in Figure 1V.

[0052] The disclosed AAV comprises a genome comprising a GNE 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, the genome provided in Figure 1W.

[0053] The disclosed AAV comprises a genome comprising a mini-CMV promoter, a variant 2 wild-type GNE cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example, the genome provided in Figure 1X.

[0054] The disclosed AAV comprises a genome comprising a mini-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, the genome provided in Figure 1Y.

[0055] The disclosed AAVs include a genome that includes an MCK promoter, a GALGT2 cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding follistatin 344, e.g., the genome provided in Figure 1Z or the genome shown in SEQ ID NO:38.

[0056] The disclosed AAV comprises a genome comprising an MCK promoter, a GALGT2 cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1, for example, the genome provided in Figure 1AA.

[0057] The disclosed AAV comprises a genome comprising an MCK promoter, a GALGT2 cDNA, a nucleic acid sequence encoding an FGF1 IRES, and a nucleic acid sequence encoding SMAD7, for example, the genome provided in Figure 1BB.

[0058] The present disclosure provides a method of treating GNE myopathy in a human subject in need thereof, comprising administering a recombinant adenovirus-associated (rAAV) or AAV as disclosed herein. Methods of treating GNE myopathy include methods of reducing, inhibiting, or slowing the muscle weakness symptoms of GNE, the progression of muscle atrophy, and / or increasing muscle strength in a subject in need thereof. The subject in need may exhibit muscle weakness symptoms of GNE myopathy. The subject in need may have a mutation in the GNE gene.

[0059] The present disclosure provides a method for treating muscular dystrophies, including Duchenne muscular dystrophy, LGMD2A, and MDC1A, in a human subject in need thereof, comprising administering a recombinant adenovirus-associated (rAAV) or AAV as disclosed herein. Methods for treating muscular dystrophy include reducing, inhibiting, or slowing the progression of muscle weakness, muscle atrophy, and / or increasing muscle strength in a subject in need thereof. The subject in need may exhibit muscle weakness symptoms of GNE myopathy. The subject in need may have a mutation in the GNE gene.

[0060] In any of the methods of the present disclosure, the dose of rAAV can be administered intramuscularly, intraperitoneally, intravenously, intraarterially, orally, orally, orally, nasally, pulmonary, intracranially, intraosseously, intraocularly, rectally, or vaginally. For example, the administration route is systemic, such as by injection, infusion, or implantation. For example, the dose of rAAV is administered by infusion over about 1 hour. In addition, the dose of rAAV is administered intravenously via a peripheral limb vein, such as a peripheral arm vein or a peripheral leg vein. Alternatively, the infusion can be administered over about 30 minutes, or about 1.5 hours, or about 2 hours, or about 2.5 hours, or about 3 hours.

[0061] In any of the methods of the present disclosure, the administered rAAV is of serotype AAVrh7.4. The rAAV vectors of the present disclosure can be of any AAV serotype, such as serotypes AAVrh.74, Anc80, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV11, AAV12, AAV13, AAVTT, AAV7m8, and derivatives thereof.

[0062] In one aspect, the present disclosure provides an rAAV comprising a muscle-specific regulatory element nucleotide sequence and a nucleotide sequence encoding UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase. For example, the nucleotide sequence encodes a functional UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase, where the nucleotide 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 typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, and wherein the encoded protein maintains kinase activity. Additionally, the nucleotide sequence comprises 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 to SEQ ID NO:2, and encodes a functional protein that retains kinase activity.

[0063] In another aspect, the disclosure provides an rAAV comprising a muscle-specific regulatory element nucleotide sequence and a nucleotide sequence encoding a GalNAc transferase, e.g., the nucleotide sequence encodes a functional GalNAc transferase, wherein the nucleotide sequence has, e.g., 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 to SEQ ID NO: 36, and wherein the encoded protein maintains transferase activity. Additionally, the nucleotide sequence comprises 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 to SEQ ID NO: 37, and encodes a functional protein that retains transferase activity.

[0064] In another aspect, the disclosure provides an rAAV comprising a muscle-specific regulatory 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, where the nucleotides have, e.g., 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 to SEQ ID NO: 9 or 28, and wherein the encoded protein maintains follistatin activity. Additionally, the nucleotide sequence comprises 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 to SEQ ID NO: 10 or 29 and encodes a functional protein that maintains follistatin activity. Follistatin activity refers to the binding of follistatin to activin and thereby antagonizing activin activity. Follistatin functions by inhibiting inhibitory growth signaling by myostatin through competitive inhibition and suppression of Smad2 / 3 signaling.

[0065] In one embodiment, the present 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 nucleotides have, 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 to SEQ ID NO: 11, and wherein the encoded protein maintains IGF activity. In addition, the nucleotide sequence comprises an amino acid sequence with sequence identity to SEQ ID NO: 27, 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%, and encodes a functional protein that maintains IGF-1 activity. IGF-1 activity refers to IGF-1 binding to and activating IGF receptor (IGFR), and / or insulin receptor IGF-1 function by activating muscle IGFR and Akt / mTOR signaling. IGF-1 activity includes stimulating cell growth and proliferation, for example, stimulating muscle cell growth, and inhibiting programmed cell death. The present disclosure also provides an rAAV comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 11, or its complement, and encodes a functional IGF variant.

[0066] The present disclosure also provides an rAAV comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 9 or 28, or its complement, and encodes a functional follistatin.

[0067] The present disclosure also provides an rAAV comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 11, or its complement, and encodes a functional IGF.

[0068] The term "stringent" refers to conditions generally understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°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, NY 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agents) can also be used, but the hybridization rate will be affected. Where deoxyoligonucleotide hybridization is involved, examples of additional stringent hybridization conditions include washing in 6×SSC, 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).

[0069] Other agents can be included in the hybridization and wash buffers to reduce nonspecific and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4 (SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, although other suitable agents may also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8-7.4, although the rate of hybridization is largely independent of pH under typical ionic strength conditions. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one skilled in the art to take these variables into account and allow DNAs of different sequence similarities to form hybrids.

[0070] The term "muscle-specific promoter element" refers to a nucleotide sequence that regulates the expression of a coding sequence specific for expression in muscle tissue. These control elements include enhancers and promoters. The present disclosure provides a polynucleotide or AAV having a genome containing one or more of the muscle-specific control elements MCKH7 promoter, MCK promoter, or MCK enhancer. The GNE promoter can be the promoter of the human wild-type GNE gene. Other promoter elements, such as CMV, mini-CMV, and GNE promoters, enable expression in almost all tissues and are referred to as "constitutive promoters."

[0071] The term "constitutive promoter element" refers to an unregulated promoter that allows for continuous transcription of its associated gene. Examples of constitutive promoter elements include the hACTB, hEF-1α, CAG, CMV, herpes simplex virus thymidine kinase (HSV-TK), SP1, c-FOS, or c-MYC promoters.

[0072] The term "operably linked" refers to an arrangement of regulatory element nucleotide sequences, such as a promoter nucleotide sequence, that confers expression of the nucleotide sequence by the regulatory element.

[0073] For example, the muscle-specific promoter element is the MHCK7 promoter nucleotide sequence of SEQ ID NO: 5, or the muscle-specific promoter element is the CMV promoter nucleic acid sequence of SEQ ID NO: 3, or the muscle-specific promoter element is the MCK nucleotide sequence of SEQ ID NO: 4, or the muscle-specific promoter element is the GNE promoter nucleotide sequence of SEQ ID NO: 6, or the muscle-specific promoter element is the mini-CMV nucleotide sequence of SEQ ID NO: 7. Additionally, in any of the rAAV vectors of the present disclosure, the muscle-specific promoter element nucleotide sequence is operably linked to the GNE cDNA sequence. (SEQ ID NO: 1)

[0074] In a further aspect, the present disclosure provides an rAAV construct contained in a plasmid comprising the nucleotide sequence of any one of SEQ ID NOs: 12-26 and 38, or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleotide sequences of SEQ ID NOs: 12-26.

[0075] The present disclosure also provides pharmaceutical compositions (or sometimes simply referred to herein as "compositions") comprising any of the rAAV vectors or rAAV particles of the present disclosure.

[0076] In another embodiment, the present disclosure provides a method for producing rAAV particles, comprising culturing cells transfected with any of the rAAV vectors disclosed herein and recovering the rAAV particles from the supernatant of the transfected cells. The present disclosure also provides viral particles comprising any of the disclosed recombinant AAV vectors.

[0077] In any of the methods for treating GNE myopathy, the level of GNE gene expression in the cells of a subject is increased after administration of rAAV. The expression of the GNE gene in cells is detected by measuring the level of UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase by Western blot, immunohistochemistry, or enzymes in various tissues (e.g., muscle, heart, liver, kidney, brain, colon assay) before and after administration of rAAV. [Brief explanation of the drawings]

[0078] [Figure 1A] 1 provides a schematic diagram of the AAV genome provided herein. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 1D] Same as above. [Figure 1E] Same as above. [Figure 1F] Same as above. [Figure 1G] Same as above. [Figure 1H] Same as above. [Figure 1I] Same as above. [Figure 1J] Same as above. [Figure 1K] Same as above. [Figure 1L] Same as above. [Figure 1M] Same as above. [Figure 1N] Same as above. [Figure 1O] Same as above. [Figure 1P] Same as above. [Figure 1Q] Same as above. [Figure 1R] Same as above. [Figure 1S] Same as above. [Figure 1T] Same as above. [Figure 1U] Same as above. [Figure 1V] Same as above. [Figure 1W] Same as above. [Figure 1X] Same as above. [Figure 1Y] Same as above. [Figure 1Z] Same as above. [Figure 1AA] Same as above. [Figure 1BB] Same as above. [Figure 2A] The plasmid sequence containing the genome of rAAVrh74.CMV.GNE (variant 2) (SEQ ID NO: 12) shown in Figure 1A is provided. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E] Same as above. [Figure 2F] Same as above. [Figure 2G] Same as above. [Figure 2H] Same as above. [Figure 2I] Same as above. [Figure 2J] Same as above. [Figure 2K] Same as above. [Figure 2L] Same as above. [Figure 3A] The plasmid sequence containing the genome of rAAVrh74.MCK.GNE (variant 2) (SEQ ID NO: 13) shown in Figure 1B is provided. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above. [Figure 3J] Same as above. [Figure 3K] Same as above. [Figure 3L] Same as above. [Figure 4A] The plasmid sequence containing the genome of rAAVrh74.MHCK7.GNE (variant 2) (SEQ ID NO: 14) shown in Figure 1C is provided. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 4H] Same as above. [Figure 4I] Same as above. [Figure 4J] Same as above. [Figure 4K] Same as above. [Figure 4L] Same as above. [Figure 5A] The plasmid sequence containing the genome of rAAVrh74.GNEpromoter.GNE(variant 2) (SEQ ID NO: 15) shown in Figure ID is provided. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 5H] Same as above. [Figure 5I] Same as above. [Figure 5J] Same as above. [Figure 5K] Same as above. [Figure 5L] Same as above. [Figure 6A]The plasmid sequence containing the genome of rAAVrh74.MHCK7.GNE(variant 2).FGF1IRES.FS344 (SEQ ID NO: 16) shown in Figure IE is provided. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 6G] Same as above. [Figure 6H] Same as above. [Figure 6I] Same as above. [Figure 6J] Same as above. [Figure 6K] Same as above. [Figure 6L] Same as above. [Figure 6M] Same as above. [Figure 6N] Same as above. [Figure 7A] The plasmid sequence containing the genome of rAAVrh74.MHCK7.GNE(variant 2).FGF1IRES.HB-IGF1 (SEQ ID NO: 17) shown in Figure IF is provided. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above. [Figure 7G] Same as above. [Figure 7H] Same as above. [Figure 7I] Same as above. [Figure 7J] Same as above. [Figure 7K] Same as above. [Figure 7L] Same as above. [Figure 7M] Same as above. [Figure 7N] Same as above. [Figure 8A]The plasmid sequence containing the genome of rAAVrh74.CVM.GNE(variant 2).FGF1IRES.FS344 (SEQ ID NO: 18) shown in Figure 1G is provided. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F] Same as above. [Figure 8G] Same as above. [Figure 8H] Same as above. [Figure 8I] Same as above. [Figure 8J] Same as above. [Figure 8K] Same as above. [Figure 8L] Same as above. [Figure 8M] Same as above. [Figure 8N] Same as above. [Figure 9A] The plasmid sequence containing the genome of rAAVrh74.CMV.GNE(variant 2).FGF1IRES.HB-IGF1 (SEQ ID NO: 19) shown in Figure 1H is provided. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above. [Figure 9G] Same as above. [Figure 9H] Same as above. [Figure 9I] Same as above. [Figure 9J] Same as above. [Figure 9K] Same as above. [Figure 9L] Same as above. [Figure 9M] Same as above. [Figure 9N] Same as above. [Figure 10A]The plasmid sequence containing the genome of rAAVrh74.MCK.GNE(variant 2).FGF1IRES.FS344 (SEQ ID NO: 20) shown in Figure 1I is provided. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Figure 10F] Same as above. [Figure 10G] Same as above. [Figure 10H] Same as above. [Figure 10I] Same as above. [Figure 10J] Same as above. [Figure 10K] Same as above. [Figure 10L] Same as above. [Figure 10M] Same as above. [Figure 11A] The plasmid sequence containing the genome of rAAVrh74.MCK.GNE(variant 2).FGF1IRES.HB-IGF1 (SEQ ID NO: 21) shown in Figure 1J is provided. [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 11G] Same as above. [Figure 11H] Same as above. [Figure 11I] Same as above. [Figure 11J] Same as above. [Figure 11K] Same as above. [Figure 11L] Same as above. [Figure 11M] Same as above. [Figure 12A] The plasmid sequence containing the genome of rAAVrh74.GNEpromoter.GNE(variant 2).FGF1IRES.FS344 (SEQ ID NO: 22) shown in Figure 1K is provided. [Figure 12B]Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above. [Figure 12F] Same as above. [Figure 12G] Same as above. [Figure 12H] Same as above. [Figure 12I] Same as above. [Figure 12J] Same as above. [Figure 12K] Same as above. [Figure 12L] Same as above. [Figure 12M] Same as above. [Figure 12N] Same as above. [Figure 13A] The plasmid sequence containing the genome of rAAVrh74.GNE promoter.GNE(variant 2).FGF1IRES.HB-IGFI (SEQ ID NO: 23) shown in Figure 1L is provided. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 13F] Same as above. [Figure 13G] Same as above. [Figure 13H] Same as above. [Figure 13I] Same as above. [Figure 13J] Same as above. [Figure 13K] Same as above. [Figure 13L] Same as above. [Figure 13M] Same as above. [Figure 13N] Same as above. [Figure 14A] The plasmid sequence containing the genome of rAAVrh74.miniCMV.GNE (SEQ ID NO: 24) shown in Figure 1M is provided. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 14E] Same as above. [Figure 14F] Same as above. [Figure 14G] Same as above. [Figure 14H] Same as above. [Figure 14I] Same as above. [Figure 14J] Same as above. [Figure 14K] Same as above. [Figure 14L] Same as above. [Figure 14M] Same as above. [Figure 15A] The plasmid sequence containing the genome of rAAVrh74.miniCMV.GNE(variant 2).FGF1IRES.FS344 (SEQ ID NO: 25) shown in Figure 1N is provided. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above. [Figure 15G] Same as above. [Figure 15H] Same as above. [Figure 15I] Same as above. [Figure 15J] Same as above. [Figure 15K] Same as above. [Figure 15L] Same as above. [Figure 15M] Same as above. [Figure 16A] The plasmid sequence containing the genome of rAAVrh74.miniCMV.GNE(variant 2).FGF1IRES.HB-IGF1 (SEQ ID NO: 26) shown in Figure 1O is provided. [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 16D] Same as above. [Figure 16E] Same as above. [Figure 16F] Same as above. [Figure 16G] Same as above. [Figure 16H] Same as above. [Figure 16I] Same as above. [Figure 16J] Same as above. [Figure 16K] Same as above. [Figure 17A] The plasmid sequence containing the genome of rAAVrh74.MCK.GALGT2.FGF1IRES.FS344 (SEQ ID NO: 38) shown in Figure 1Z is provided. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 17E] Same as above. [Figure 17F] Same as above. [Figure 17G] Same as above. [Figure 17H] Same as above. [Figure 17I] Same as above. [Figure 17J] Same as above. [Figure 17K] Same as above. [Figure 17L] Same as above. [Figure 17M] Same as above. [Figure 18] Sialic acid staining of liver and muscle after intramuscular injection of rAAVrh74.MCK.GNE or IP injection of rAAVrh74.LSP.GNE in GNED176V TgGne mice. Bars are 100 μm. [Figure 19] Genotyping data for founder mice in a Cas9-CRISPR Gne exon 3 deletion / loxP recombination experiment are provided. Founders CR10646-8 and -9 contain a genomic deletion in Gne exon 3. [Figure 20] Figure 1 shows staining of Gne-deficient Lec3 CHO cells with rAAV.CMV.GNE.mini-IRES.GFP, demonstrating expression of a second protein using a mini-IRES sequence. GFP indicates endogenous fluorescence, Gne indicates immunostaining, and DAPI is used as a nuclear stain in a triple exposure. [Figure 21]Shown is a triple exposure of Gne-deficient Lec3 CHO cells after transfection with rAAV.miniCMV.GNE. Full-length (FL)-IRES.GFP, demonstrating expression of a second protein using the full-length IRES sequence. GFP indicates endogenous fluorescence, Gne indicates immunostaining, and DAPI indicates nuclear staining. [Figure 22] Muscle growth after intramuscular injection of IGF1, HB-IGF1, or FST344 using rAAVrh74 is shown. 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 follistatin (FST) form 344. Muscles were dissected and weighed 2 months after injection, demonstrating a significant increase in HB-IGF1 and FST344 in the TA and FST344 in the gastrocnemius compared to injection with buffer alone. Error bars are SEM for n = 12 muscles per group. *p < 0.05, ***p < 0.001. [Figure 23] This figure shows that CMV.GNE.IRES.GFP allows for the induction of sialic acid expression on the membrane of Lec3 Gne-deficient CHO cells, and the IRES allows for the expression of a second protein (in this case, GFP). Endogenous GFP expression is shown in the green channel, and MAA staining for sialic acid is shown in red. Normal CHO cells have normal Gne function and therefore MAA staining, but Lec3 cells lack functional Gne and therefore do not normally express MAA. Introduction of CMV.GNE.IRES.GFP allows for functional Gne expression in Lec3 cells due to the presence of the IRES, as well as the expression of a second protein, GFP. DAPI is shown in a triple exposure to show the nuclei stained blue. [Figure 24]Muscle cells (C2C12 cells) transfected with MCK.GALGT2.IRES.FS344 (or FST) demonstrate the ability to express GALGT2 (stained green) and FST (stained red) in the same cells due to the presence of the IRES sequence in the bicistronic vector. C2 cells mock-transfected without the bicistronic DNA demonstrate low or no expression of either protein using time-matched images. [Figure 25] Figure 1 shows the change in MAA signal in Lec3 cells after infection with rAAVrh74.CMV.GNE. Maackia amurensis agglutinin (MAA) conjugated to horseradish peroxidase (HRP) was used to assay sialic acid expression in CHO or Lec3 cells in a 96-well ELISA plate assay using colorimetric analysis of HRP activity as the output. Lec3 cells grown in Opti-MEM for 3 days showed reduced MAA binding relative to CHO cells, and this binding could be partially rescued by adding rAAVrh74.CMV.GNE for 2 days. Errors represent SD, n=2 per group. MOI, multiplicity of infection; OD, optical density; **p<0.01. [Figure 26] Figure 1 shows GNE enzyme activity in CHO cells, Lec3 cells, and CLec3 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 exhibit significantly more UDP-GlcNAc epimerase activity than Lec3 cells, which lack functional Gne enzyme. Lec3 cells transfected with pAAV.CMV.GNE exhibit GNE enzyme activity above the levels seen in CHO cells. Errors are SD, n=2 per group. **p<0.01, ***p<0.001 [Figure 27]Figure 27A shows the function of bistronic GALGT2 and follistatin 344 (FST) gene therapy in mdx mice. Figure 27A shows that injection of 1 x 10 vg of rAAVrh74.MCK.GALGT2.IRES.FST or the single gene vector rAAVrh74.MCK.FST at the same dose into the TA muscle increased muscle size, measured as muscle weight relative to total body weight (mg / g). Error bars are SD, n = 4 / group. *p<0.05, **p<0.01. Figure 27B provides images of TA muscles stained with antibodies against FST and WFA (to recognize GalNAc generated by GALGT2) after injection. DETAILED DESCRIPTION OF THE INVENTION

[0079] The present disclosure provides gene therapy vectors, such as adeno-associated viruses (AAVs), optimized for delivering transgenes to muscle. The optimized vectors contain a transgene cDNA to replace gene mutations found in muscle diseases with a normal copy of the gene or to provide alternative gene therapy, an internal ribosome entry site (IRES) to enable the production of a second protein from the same transcript, and a constitutive or muscle-specific promoter to deliver the expression of a systemic or skeletal / cardiac muscle-specific transgene, respectively, in combination with a muscle growth factor gene to build new muscle growth and strength. The transgene and muscle growth factor gene are expressed from the same mRNA, expressing both proteins, due to the presence of an internal ribosome entry site (IRES) from the fibroblast growth factor 1A gene sequence, which allows the second protein to be produced from a single mRNA.

[0080] This disclosure provides a gene therapy vector, such as an adeno-associated virus (AAV), designed for the treatment of GNE myopathy. The AAV expresses UDP-GlcNAc-epimerase / ManNAc-6 alone or in combination with follistatin or IGF1. The provided AAV expresses a protein that stimulates muscle growth while replacing the mutated GNE gene expression. A strategy that combines gene replacement (either direct gene replacement or replacement with an alternative gene function) to prevent further disease with muscle growth or muscle transdifferentiation therapy to build new muscle mass and strength may not only halt the disease process, but also reverse it by stimulating new muscle growth and strength while simultaneously halting disease pathogenesis.

[0081] The present invention provides a gene therapy vector that 1) provides a transgene for gene replacement or as replacement gene therapy, 2) provides a gene encoding growth factor that induces muscle growth or increases muscle strength.This gene therapy is encoded by a single gene therapy genome, for example, a single AAV genome.This combined therapy can not only prevent the progression of disease, but also reverse it by stimulating new muscle growth and muscle strength, and at the same time stopping the pathogenesis of disease.

[0082] The gene therapies offered are for GNAE myopathy, Duchenne and Becker muscular dystrophy (DMD and BMD), and LGMD2A (CAPN3), LGMD2C (SGCG), LGMD2D (SGCA), LGMD2E (SGCB), LGMD2F (SGCD), LGMD2G (TCAP), LGMD2H (TRIM32), LGMD2I (FKRP), LGMD2K (POMT1), LGMD2L (ANO5), LGMD2M (F KTN), LGMD2O(POMT2), LGMD2P(DAG1), LGMD2R(DES), LGMD2T(GMPPB), LGMD2U(ISPD), LGMD2X(BVES), LGMD2Y(TOR1AIP1) , LGMD2Z(POGLUT1), LGMD1A(TTID, MYOT), LGMD1B(LMNA), LGMD1C(Cav3), LGMD1D(DES), LGMD1F(TNPO3), LGMD1G(HNRPDL) These transgenes are useful for treating limb-girdle muscular dystrophies (LGMDs), such as BMJ, BMDC1A, and MDC1A. In each case, the first transgene can be used for gene replacement of a gene missing in the disease, or for a replacement gene replacement such as GALGT2 or B4GALNT2, while the second transgene is a muscle growth factor, such as FS344, HB-IGF1, IGF1, or SMAD7, which reverses the symptoms of the disease by building new muscle growth and strength. The gene therapy methods of the present disclosure can also be used to treat diseases in which a replacement gene is used to prevent the disease instead of a gene replacement as the first transgene, and in which muscle growth from the second transgene replacement is not driven by muscle growth factors but by muscle transdifferentiation factors (e.g., MyoD), and apply to therapies in which muscle is built by the conversion of fat or fibroblasts into muscle, rather than by the support of muscle growth factors.

[0083] In some embodiments where the construct has available space, the AAV genome also contains a second IRES and a third transgene, providing three gene therapies simultaneously.

[0084] AAVs have a genome containing a muscle-specific promoter driving expression of a nucleotide sequence encoding a transgene of interest in combination with a nucleotide sequence encoding a muscle growth factor, such as a protein that induces muscle growth, and a muscle-specific IRES, such as the FGF IRES. This gene therapy approach is useful for treating any disease requiring gene replacement in combination with the need to increase muscle growth or strength, such as GNAE myopathy, limb-girdle muscular dystrophy, and Duchenne muscular dystrophy.

[0085] Growth Factors and Transdifferentiation Factors 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 (mother for decapentadecalytic homolog 7 (MADH7)).

[0086] Growth factors that induce muscle growth or increase muscle strength also include follistatin. Follistatin is a secreted protein that inhibits the activity of TGF-β family members, such as GDF-11 / BMP-11. Follistatin-344 is a follistatin precursor that undergoes peptide cleavage to form the circulating follistatin-315 isoform, which contains a C-terminal acidic region. It circulates with the myostatin propeptide in a complex containing two other proteins, follistatin-related gene (FLRG) and GDF-associated serum protein-1 (GASP-1). Follistatin-317 is another follistatin precursor that undergoes peptide cleavage to form the membrane-bound follistatin-288 isoform.

[0087] The DNA and amino acid sequences of the follistatin-344 precursor are set forth in SEQ ID NOS: 9 and 10, respectively. The follistatin-288 isoform, which lacks the C-terminal acidic region, exhibits strong affinity for heparin sulfate proteoglycans, is a potent inhibitor of pituitary follicle-stimulating hormone, is found in ovarian follicular fluid, and exhibits high affinity for ovarian granulosa cells. The testes also produce follistatin-288. The DNA and amino acid sequences of the follistatin-317 precursor are set forth in SEQ ID NOS: 28 and 29, respectively. Lack of follistatin results in reduced muscle mass at birth.

[0088] Examples of follistatin are provided in Shimasaki et al., U.S. Pat. No. 5,041,538; other follistatin-like proteins are provided in U.S. Pat. Nos. 5,942,420, 6,410,232, 6,537,966, and 6,953,662); FLRG (SEQ ID NO: 33, the corresponding nucleotide sequence is SEQ ID NO: 32) is provided in Hill et al., J. Biol. Chem., 277(43):40735-40741 (2002); and GASP-1 (SEQ ID NO: 35, the corresponding nucleotide sequence is SEQ ID NO: 34) is provided in Hill et al. al., Mol Endocrinol, 17:1144-1154 (2003).

[0089] SMAD7 is known to inhibit TGF-β-activated signaling responses by associating with the active TGF-β complex, thereby reducing TGF-β signaling. Myostatin and TGF-β signaling induce SMAD7 expression, establishing a negative feedback loop that inhibits TGF-β signaling. In particular, SMAD7 is known to regulate myogenesis using this negative feedback loop (Kollias et al. Mol. Cell Biol. 26(16):6248-6260, 2006). The nucleotide sequence encoding the SMAD7 protein is shown as SEQ ID NO: 39 (GenBank Accession No. NM_005904.4), and the amino acid sequence is shown as SEQ ID NO: 40 (GenBank Accession No. NP_005895).

[0090] Transdifferentiation factors are factors that convert non-muscle cells into muscle cells or induce their differentiation. For example, MyoD is known to convert several cell types, including skin fibroblasts, chondrocytes, smooth muscle cells, retinal pigment epithelial cells, adipocytes, and cells of melanoma, neuroblastoma, osteosarcoma, and hepatoma, into muscle cells (Abraham & Tapscott, Curr. Opin. Genet. Dev. 23(5):568-573, 2013). Other examples of transdifferentiation factors include Myocd (myocardin), Mef2C (myocyte enhancer factor 2C), Mef2B (myocyte enhancer factor 2B), Mkl1 (MKL [megakaryoblastic leukemia] / Myocd-like 1), Gata4 (GATA-binding protein 4), Gata5 (GATA-binding protein 5), Gata6 (GATA-binding protein 6), and Ets1 (E26 avian leukemia oncogene 1, 5' domain).

[0091] GNE myopathy GNE myopathy is characterized by progressive muscle atrophy and weakness. Age at onset typically occurs in the third decade of life, beginning with weakness of the tibialis anterior (TA) and femoral flexor muscles. By 20 years after diagnosis, patients are often wheelchair-bound. Patients may eventually require assistance with daily living functions, such as eating. Muscle biopsies typically demonstrate rimmed vacuoles and inclusions. 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 acids (SAs). The SA biosynthetic pathway culminates in the production of CMP-SA, which is utilized by sialyltransferases to transfer SA onto glycoproteins and glycolipids in all mammalian cells.

[0092] The incidence of GNE myopathy has recently been estimated at 1 to 6 per million, making it a rare disease. However, in certain human populations, such as Japanese (D176V, D207V in the new nomenclature) and Middle Eastern (M712T, M743T in the new nomenclature) patients, founder effect mutations exist that cause GNE myopathy at a very high incidence. One study of 1,000 Iranian Jews found the mutation carrier frequency to be 1 in 11. Partial reduction in GNE activity in patients leads to reduced, but not absent, SA expression.

[0093] Decreased IGF1R signaling has been shown to underlie muscle stem cell death in models of GNE myopathy, making IGF1 an 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 replacement) but also induce new muscle growth (thereby increasing muscle strength) and potentially prevent stem cell death. Because patients with GNE myopathy lose muscle and strength over decades, these vectors are highly unique, and the provided AAVs are expected to not only slow this progression but actually reverse it. The provided dual-function AAVs can demonstrate clinical efficacy because this disease exhibits high clinical variability (between patient disease mutations and even between patients with the same disease mutation) and slow progression (significant clinical changes occur over decades).

[0094] GNE myopathy mutations In any of the provided methods, the subject suffers from GNE myopathy. For example, the subject has a mutation in the GNE gene that results in reduced expression of UDP-N-acetylglucosamine 2-epimerase / N-acetylmannosamine kinase. The diagnosis of GNE myopathy in a subject is confirmed by the presence of pathogenic (mainly missense) mutations in both alleles of the GNE gene. Table 1 below provides known mutations in the GNE gene associated with GNE myopathy. The subject of the claimed method may have the mutations shown in this table.

[0095] Bold text in Table 1 indicates cDNA or protein-truncating variants. Italics and dark gray highlighting indicate "mild" variants. A question mark (?) indicates that the exact nomenclature could not be derived from the reference. The DNA numbering system is based on the cDNA sequence. Nucleotide numbering uses +1 as the A of the ATG translation start codon of the reference sequence, and the start codon as codon 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0096] GNE mouse model Gne is an essential gene in mice. Its deletion causes embryonic lethality between embryonic days (E) 8.5 and 9.5. The most prominent model of Gne myopathy was created by Malicdan et al. (Hum. Mol. Genet. 16(22):2669-82, 2007). This model uses mouse Gne - / - Mutant human GNE background D207V The transgene (Tg) was constitutively expressed. By 30 weeks, GNE D207V Tg Gne - / -Mice were reported to exhibit a significant shortening of lifespan, reduced scores on rod climbing and constant-speed treadmill walking, and modest increases in serum CK activity and muscle production of Aβ1-42 peptide. By 42 weeks, muscles displayed rimmed vacuoles with congophilic inclusions, as well as respiratory and cardiac pathology not seen in human GNE myopathy patients. Unfortunately, as these mice have been bred, most of these phenotypes have been lost from the line, and therefore, evidence of muscle pathology or muscle defects cannot be found at 64 weeks.

[0097] A second model, a knock-in of the Persian founder GNE mutation M712T (now called M743T), showed perinatal lethality (by P3) due to kidney disease (Galeno et al., Clin. Invest. 117(6):1585-94, 2007). It was subsequently discovered that this homozygous knock-in line could be bred to generate a subpopulation of phenotype-free animals (Sela et al., Neuromuscular Med. 15(1):180-91, 2013). Therefore, the robustness of all preclinical data regarding this disease has been called into question due to the high phenotypic variability of the models used.

[0098] All preclinical data are highly convoluted due to the fact that all current mouse models of GNE myopathy display complex and overly variable phenotypes. M743T The knockin model exhibits early death due to renal complications, which can be counteracted by ManNAc. Other strains with the same knockin do not exhibit this phenotype. D207V Tg Gne - / - In early studies, mouse models showed clear disease phenotypes at 1 year of age, but none of these phenotypes can be replicated in currently living mice. Because Gne deficiency causes embryonic death at E8.5–E9.5 in mice, pure gene-deficient mice are not useful. However, foxed mice, which allow for more precise gene deletion, have been generated by several groups, including ours.

[0099] The mouse model is described in Example 3 herein. This mouse model was generated using Cas9-CRISPR, ultimately enabling the creation of a floxed allele in exon 3 of the mouse Gne gene. This allele is sufficient to allow Cre-mediated deletion, resulting in a Gne myopathy-like phenotype. Because Gne is essential in mice and causes lethality between E8.5 and E9, the creation of a floxed allele to delete the gene in adult mice allows for the generation of robust systemic or muscle-specific phenotypes using Cre-mediated deletion. This allows for reproducible demonstration of therapeutic efficacy.

[0100] muscular dystrophy Muscular dystrophies (MD) are a group of genetic disorders characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD begin in infancy or childhood, while others may not appear until middle age or later. The disorders differ in the distribution and degree of muscle weakness (some forms of MD also affect the heart muscle), age of onset, rate of progression, and mode of inheritance.

[0101] One type of MD is Duchenne muscular dystrophy (DMD). It is the most common severe childhood form of muscular dystrophy, affecting 1 in 5,000 newborn boys. Inheritance follows an X-linked recessive pattern. DMD is caused by mutations in the DMD gene, resulting in the absence of the dystrophin protein (427 kDa) in skeletal and cardiac muscles, as well as in the gastrointestinal tract and retina. Dystrophin not only protects the sarcolemma from eccentric contractions but also anchors numerous signaling proteins in the immediate vicinity of the sarcolemma. Clinical symptoms of DMD are usually first observed between the ages of 3 and 5 years, with changes in gait and decreased motor skills typically leading to diagnostic evaluation. DMD progresses relentlessly, resulting in loss of walking ability by age 12. Historically, patients died from respiratory complications in the late 20s, but improvements in supportive care, particularly the judicious use of nocturnal ventilatory support, have extended life expectancy by nearly 10 years. The increased life expectancy reveals nearly universal cardiac decline, accompanied by complications of dilated cardiomyopathy. This raises additional clinical challenges and a need for previously unmet medical needs. Non-progressive cognitive impairment may also be present in DMD. Despite virtually hundreds of clinical trials in DMD, corticosteroid treatment remains the only consistently effective treatment. Current standard treatments for DMD involve the use of prednisone or deflazacort, which may extend walking ability by several years at the expense of significant side effects and have limited evidence of an impact on survival.

[0102] Another type of MD is congenital muscular dystrophy type 1A (MCD1A). MCD1A belongs to a group of neuromuscular disorders that begin at birth or in infancy and are characterized by hypotonia, muscle weakness, and muscle wasting. MCD1A accounts for 30-40% of congenital muscular dystrophies, with some regional variation. The prevalence is estimated at 1 / 30,000. This disorder manifests at birth or within the first few months of life as hypotonia and limb and trunk weakness. Respiratory and feeding disorders may also occur. Motor development is delayed and limited (sitting or standing is impossible without assistance). Infants exhibit early spinal rigidity, scoliosis, and respiratory insufficiency. Facial defects are present with a typical elongated, myopathic facies, and ophthalmoplegic disorders may develop later. Epileptic seizures may occur, but occur in less than one-third of cases. Intellectual development is normal. MCD1A is caused by mutations in the LAMA2 gene, which encodes the alpha-2 laminin chain. Transmission is autosomal recessive. Current treatment is symptomatic. It consists of a multidisciplinary approach involving physical, occupational, and speech therapists, with the goal of optimizing each patient's abilities. Seizures or other neurological complications require specific treatment. The prognosis for MDC1A is extremely severe, as the majority of affected children do not reach adolescence. Currently, the prognosis can only be improved with careful multidisciplinary (especially orthopedic and respiratory) management.

[0103] Yet another type of MD is limb-girdle muscular dystrophy (LGMD). LGMD is a rare condition, and symptoms vary from person to person in terms of age of onset, areas of muscle weakness, cardiac and respiratory involvement, rate of progression, and severity. LGMD can begin in childhood, adolescence, young adulthood, or later. Both genders are affected equally. LGMD causes weakness in the shoulders and pelvic girdle, and nearby muscles in the upper limbs and arms may also weaken over time. Leg weakness often precedes arm weakness. Facial muscles are usually unaffected. As the condition progresses, people may have trouble walking and may need to use a wheelchair over time. Involvement of shoulder and arm muscles can make it difficult to lift the arms overhead or lift objects. Depending on the type of LGMD, cardiac and respiratory muscles may also be involved.

[0104] There are at least 19 forms of LGMD, which are classified according to the genetic defect involved. [Table 2]

[0105] Specialist tests for LGMD are now available through the National Commissioning Group (NCG), the national scheme for diagnosis.

[0106] The GALGT2 gene (also known as B4GALNT2) encodes β1-4-N-acetyl-D-galactosamine (βGalNAc) glycosyltransferase. Overexpression of GALGT2 has been studied in three different models of muscular dystrophy: DMD, LGMD2D, and MDC1A [Xu et al., Am. J. Pathol, 175:235-247 (2009), Xu et al., Am. J. Pathol, 171:181-199 (2007), Xu et al., Neuromuscul. Disord., 17:209-220 (2007), Martin et al., Am. J. Physiol. Cell. Physiol., 296:C476-488 (2009), and Nguyen et al., Proc. Natl. Acad. Sci. USA, 99:5616-5621 (2002)]. Overexpression of GALGT2 in skeletal muscle induces glycosylation of alpha-dystroglycan with β1-4-N-acetyl-D-galactosamine (GalNAc) carbohydrates to produce the CT carbohydrate antigen (Neu5Ac / Gcα2-3[GalNAcβ1-4]Galβα1-4GlcNAcβ-). The GALGT2 glycosyltransferase and the CT carbohydrates it produces are normally restricted to the neuromuscular and myotendinous junctions of skeletal muscle in adults, nonhuman primates, rodents, and all other mammals yet to be studied [Martin et al., J. Neurocytol., 32:915-929 (2003)]. Overexpression of GALGT2 in skeletal muscle has been reported to stimulate ectopic glycosylation of synaptic outer membranes, as well as ectopic overexpression of normal synaptic protein scaffolds that are orthologs or homologs of proteins missing in various forms of muscular dystrophies, including dystrophin substitutes (e.g., utrophin, plectin 1) and laminin α2 substitutes (laminin α5 and agrin) [Xu et al. 2009, supra; Xu et al., Am. J. Path. 2007, supra; Xu et al., Neuromuscul. Disord. 2007, supra; Nguyen et al., supra; Chicoine et al., Mol. Ther. 22:713-724. (2014)].Collectively, induction of such substitutes by GALGT2 has been reported to strengthen sarcolemmal integrity and prevent muscle damage in dystrophin-deficient and wild-type muscles [Martin. et al., supra]. Overexpression of GALGT2 in skeletal muscle has been reported to prevent muscle damage and inhibit muscle disease. This is also true in the mdx mouse model of DMD [Xu et al., Neuromuscul. Disord. 2007, supra; Martin et al. (2009), supra; Nguyen et al., supra], where improvements comparable to those observed with microdystrophin gene transfer were observed, despite the number of transduced fibers being half [Martin et al. (2009), supra]. In particular, GALGT2 gene transfer significantly improved the dystrophin function of congenital muscular dystrophy 1A. W model [Xu et al, Am. J. Path. 2007, supra] and Sgca of limb-girdle muscular dystrophy type 2D. - / - It has also been reported to be preventative in a mouse model [Xu et al. 2009, supra].

[0107] AAV gene therapy The present disclosure provides gene therapy vectors, e.g., rAAV vectors, that express the GNE gene and methods for treating GNE myopathy.

[0108] As used herein, the term "AAV" is a general abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. Currently, there are 13 characterized serotypes of AAV. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes, and they all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0109] As used herein, "AAV vector" refers to one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0110] "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and a polynucleotide AAV vector enclosed in the capsid.When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to mammalian cells), it is typically referred to as an "AAV vector particle" or simply an "AAV vector".Therefore, since such a vector is contained within the AAV vector particle, the production of AAV vector particles necessarily includes the production of AAV vectors.

[0111] AAV Adeno-associated virus (AAV) is a replication-deficient parvovirus, whose single-stranded DNA genome is approximately 4.7 kb long and contains two 145-nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983), as revised by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077, the complete genome of AAV-3 is provided under GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829, the AAV-5 genome is provided under GenBank Accession No. AF085716, the complete genome of AAV-6 is provided under GenBank Accession No. NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are provided under GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the AAV-9 genome is disclosed in Gao et al. The AAV-10 genome is provided in Mol. Ther., 13(1):67-76(2006), and the AAV-11 genome is provided in Virology, 330(2):375-383(2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 5, 45(2007). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs.Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Coupled with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 in AAV2), the two rep promoters (p5 and p19) result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins possess multiple enzymatic properties ultimately involved in viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0112] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, allowing the potential for targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially throughout the lifespan of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV can be lyophilized. Finally, AAV-infected cells do not tolerate superinfection.

[0113] Several studies have demonstrated long-term (more than 1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996), and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000), and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscle is highly vascularized, recombinant AAV transduction resulted in the appearance of the transgene product in the systemic circulation following intramuscular injection, as described by 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). Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the necessary cellular factors for proper antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics.

[0114] The recombinant AAV genome of the present disclosure comprises the nucleic acid molecule of the present disclosure and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA of the rAAV genome can be derived from any AAV serotype capable of inducing recombinant virus, 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 production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. 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. AAV1, AAV6, AAV8, AAV9, AAVrhlO, or AAVrhl.74 can be used to promote skeletal muscle-specific expression.

[0115] The DNA plasmid of the present disclosure contains the rAAV genome of the present disclosure. The DNA plasmid is transferred to a cell permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided in the cell, are standard in the art. rAAV production requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and helper virus functions. The AAV rep and cap genes can be derived from any AAV serotype capable of deriving recombinant virus, 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, and can be derived from an AAV serotype different from the rAAV genome ITRs. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0116] The method for generating packaging cells is to create a cell line that stably expresses all the components necessary for the production of AAV particles. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the genome of the cell. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus instead of a plasmid to introduce the rAAV genome and / or rep and cap genes into the packaging cell.

[0117] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches include Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984), Tratschin et al., Mo 1. Cell. Biol. al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al. al., J. Virol., 63:3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365, and corresponding U.S. Pat. Nos. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO 97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. Vaccine 13:1244-1250 (1995), Paul et al. al. Human Gene Therapy 4:609-615 (1993), Clark et al. Gene Therapy 3:1124-1132 (1996), U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The foregoing documents are incorporated herein by reference in their entireties, with particular emphasis placed on the portions of the documents relating to rAAV production.

[0118] Thus, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 cells). In another embodiment, the packaging cells are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells).

[0119] The provided recombinant AAV (i.e., infectious, encapsidated rAAV particles) comprise a rAAV genome. In exemplary embodiments, the rAAV genome lacks both AAV rep and cap DNA, i.e., no AAV rep or cap DNA is present between the ITRs of the rAAV genome.

[0120] In one exemplary embodiment, recombinant AAV is produced by the triple transfection method_ENREF_1 (Xiao et al., J Virol 72, 2224-2232 (1998)) using an AAV vector plasmid containing the GNE gene and muscle-specific promoter elements pNLRep2-Caprh74 and pHelp; the rAAV contains the GNE gene expression cassette flanked by AAV2 inverted terminal repeats (ITRs). It is this sequence that is encapsidated into AAVrh74 virions. The plasmid contains the GNE sequence, as well as muscle-specific and muscle-specific promoter elements that drive gene expression. The expression cassette also contains an SV40 intron (SD / SA) that promotes high-level gene expression, and the bovine growth hormone polyadenylation signal is used for efficient transcription termination.

[0121] pNLREP2-Caprh74 is an AAV helper plasmid encoding four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins from serotype rh74.

[0122] The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. This plasmid contains regions of the adenovirus genome important for AAV replication, namely E2A, E4 ORF6, and VA RNA (the adenovirus E1 function is provided by 293 cells). The adenovirus sequences present in this plasmid represent only approximately 40% of the adenovirus genome and do not contain cis-elements important for replication, such as the adenovirus terminal repeats. Therefore, infectious adenovirus is not expected to be generated from such a production system.

[0123] rAAV can be purified by standard methods in the art, such as by column chromatography or cesium chloride gradient. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0124] In another embodiment, the present disclosure contemplates a composition comprising the rAAV of the present disclosure. The composition of the present disclosure comprises rAAV and a pharmaceutically acceptable carrier. The composition may also contain other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients, preferably inert at the dosages and concentrations employed, and include buffers and surfactants such as Pluronic®.

[0125] The titer of the rAAV administered in the methods of the present disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the therapeutic goal, the targeted individual, and the cell type, and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per ml. 6 , about 1×107 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 The dosage may range from 0.01 to 0.1 mg / mL of DNase-resistant particles (DRP) or more. Dosages may also be expressed in units of viral vector genomes (vg). One exemplary method for determining the encapsulated vector genome titer uses quantitative PCR, such as the method described in (Pozsgai et al., Mol. Ther. 25(4):855-869, 2017).

[0126] Methods for transducing target cells with rAAV in vivo or in vitro are contemplated by the present disclosure. In vivo methods include administering an effective dose or effective multiple doses of a composition comprising an rAAV of the present disclosure to an animal (including a human) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present disclosure, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the extent of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival. An example of a disease contemplated for prevention or treatment by the methods of the present disclosure is GNE myopathy.

[0127] Combination therapy is also contemplated by the present disclosure. As used herein, combination includes both simultaneous treatment and sequential treatment. The combination of the method of the present disclosure with standard medical treatment (e.g., corticosteroids), including combination with new therapies, is particularly contemplated.

[0128] Administration of an effective dose of the composition can be by any route standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, intraarterial, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or intravaginal. The route of administration and serotype of the AAV components (particularly the AAV ITRs and capsid proteins) of the rAAV of the present disclosure can be selected and / or adapted by one skilled in the art taking into consideration the infection and / or disease state to be treated and the target cells / tissues expressing UDP-GlcNAc-epimerase / ManNAc-6 kinase protein and either follistatin 344, follistatin 317, or insulin-like growth factor 1.

[0129] The present disclosure provides for local administration and systemic administration of effective doses of the rAAV and compositions of the present disclosure.For example, systemic administration refers to administration into the circulatory system so that the whole body is affected.Systemic administration includes enteral administration, such as absorption through the digestive tract, and parenteral administration through injection, infusion or implantation.

[0130] In particular, the actual administration of the rAAV of the present disclosure can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present disclosure includes, but is not limited to, injection into the muscle and the bloodstream. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, 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 be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as local formulations delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in practicing the present disclosure. The rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0131] The dose of rAAV administered in the methods disclosed herein will vary depending, for example, on the particular rAAV, the method of administration, the therapeutic goal, the individual, and the targeted cell type, and can be determined by standard methods in the art. The titer of each rAAV administered is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 , about 1×10 14 , 2 × 10 14 to, or about 1 × 10 15 Dosages may also range from 1 x 10 to 1 x 10 or 1 x 10 DNase-resistant particles (DRP). 7 vg, 1×10 8 vg, 1×10 9vg, 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 Dosages may also be expressed in units of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1 x 10 10 vg / kg, 1 × 10 11 vg / 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 AAV titration may be expressed as a percentage of the total antibody titer (vg / kg). Methods for titrating AAV are described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0132] For intramuscular injection, solutions in adjuvants such as sesame or peanut oil, or aqueous propylene glycol solutions, and sterile aqueous solutions can be used. Such aqueous solutions can be buffered, if necessary, and the liquid diluent is first rendered isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary storage and use conditions, these formulations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed are readily available by standard techniques well known to those skilled in the art.

[0133] Pharmaceutical carriers, diluents, or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. The form must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0134] Sterile injectable solution is prepared by incorporating the required amount of rAAV into a suitable solvent, and then sterilizing by filtration, as necessary, with various other components listed above.Generally, dispersion is prepared by mixing sterilized active ingredient into a sterile vehicle that contains a basic dispersion medium and other required components listed above.For the preparation of sterile powder for sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces powder of active ingredient plus any additional desired components from their solution that has been previously sterilized and filtered.

[0135] Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV, and then reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used if they do not generate an inappropriate immune response in the subject.

[0136] Suitable methods for transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in an appropriate medium and screening for cells carrying the DNA of interest using conventional techniques such as Southern blot and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, and the composition can be introduced into a subject by a variety of techniques, including intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth muscle and cardiac muscle, for example, using a catheter.

[0137] Transduction of cells with the rAAV of the present disclosure results in sustained expression of the UDP-GIcNAc-epimerase / ManNAc-6 kinase protein. The present disclosure therefore provides methods for administering / delivering rAAVs expressing the UDP-GIcNAc-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 rAAVs of the present disclosure. Transduction can be performed with a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present disclosure includes, but is not limited to, genes encoding actin and myosin gene families, such as those 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, Mol Cell Biol 11:4854-4862 (1991)), the human skeletal actin gene (Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)), the cardiac actin gene, muscle creatine kinase sequence elements (Johnson et al., Mol Cell Biol, 7:4099-4099 (1987)), and the myoD gene family. Biol, 9:3393-3399 (1989)), and regulatory elements derived from the mouse creatine kinase enhancer (mCK) element, regulatory elements derived from the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow troponin I gene; hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)), promoters containing steroid-inducible elements, and glucocorticoid response elements (GREs) (Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), as well as other regulatory elements.

[0138] Because muscle tissue is not a vital organ and is easily accessible, it is an attractive target for in vivo DNA delivery. The present disclosure contemplates persistent expression of UDP-GIcNAc-epimerase / ManNAc-6 kinase from transduced muscle fibers.

[0139] "Muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle (e.g., skeletal and smooth muscle derived from the digestive tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0140] The term "transduction" is used to refer to the administration / delivery of the coding region of GNE to recipient cells either in vivo or in vitro via the replication-deficient rAAV of the present disclosure, resulting in expression of UDP-GlcNAc-epimerase / ManNAc-6 kinase by the recipient cells.

[0141] The following examples are offered by way of illustration and not by way of limitation: The numerical ranges listed include each integer value within each range, including the stated integer minimum and maximum. [Example]

[0142] Example 1 Constructs encoding GIcNAc epimerase / ManNAc kinase or GalNAc transferase gene cDNA The following exemplary DNA construct encoding UDP-GIcNAc-epimerase / ManNAc-6 kinase was generated as follows: rAAVrh74.CMV.GNE (Variant 2), shown in Figure 1A and encoded by the polynucleotide of Figure 2 (SEQ ID NO: 12). rAAVrh74.MCK.GNE (Variant 2), shown in Figure 1B and encoded by the polynucleotide of Figure 3 (SEQ ID NO: 13). rAAVrh74.MHCK7.GNE (Variant 2), shown in Figure 1C and encoded by the polynucleotide of Figure 4 (SEQ ID NO: 14). rAAVrh74.GNEpromoter.GNE(variant 2), shown in Figure 1D and encoded by the polynucleotide of Figure 5 (SEQ ID NO: 15). rAAVrh74.MHCK7.GNE(variant 2).FGFIIRES.FS344, shown in Figure 1E and encoded by the polynucleotide of Figure 6 (SEQ ID NO: 16). rAAVrh74.MHCK7.GNE(variant 2).FGF1 IRES.HB-IGF1, shown in Figure IF and encoded by the polynucleotide of Figure 7 (SEQ ID NO: 17). rAAVrh74.CVM.GNE(variant 2).FGF1IRES.FS344, shown in Figure 1G and encoded by the polynucleotide of Figure 8 (SEQ ID NO: 18). rAAVrh74.CMV.GNE(Variant 2).FGF1 IRES.HB-IGF1, shown in Figure 1H and encoded by the polynucleotide of Figure 9 (SEQ ID NO: 19). rAAVrh74.MCK.GNE(variant 2).FGF1IRES.FS344, shown in Figure 1I and encoded by the polynucleotide of Figure 10 (SEQ ID NO: 20). rAAVrh74.MCK.GNE(variant 2).FGF1 IRES.HB-IGF1, shown in Figure 1J and encoded by the polynucleotide of Figure 11 (SEQ ID NO:21). rAAVrh74.GNEpromoter.GNE(variant 2).FGFIIRES.FS344, shown in Figure 1K and encoded by the polynucleotide of Figure 12 (SEQ ID NO:22). rAAVrh74.GNE promoter.GNE(variant 2).FGF1 IRES.HB-IGFI, as shown in Figure 1L and encoded by the polynucleotide of Figure 13 (SEQ ID NO:23). rAAVrh74.miniCMV.GNE, shown in Figure 1M and encoded by the polynucleotide of Figure 14 (SEQ ID NO:24). rAAVrh74.miniCMV.GNE(variant 2).FGF1IRES.FS344, shown in Figure 1N and encoded by the polynucleotide of Figure 15 (SEQ ID NO:25). rAAVrh74, miniCMV.GNE(variant 2).FGF1.IRES.HB-IGF1, shown in Figure 1O and encoded by the polynucleotide of Figure 16 (SEQ ID NO:26).

[0143] Additionally, an exemplary DNA construct encoding the GalNAc transferase rAAVrh74.MCK.GALGT2.FGF1IRES.FS344, shown in Figure 1P and encoded by the polynucleotide of Figure 17 (SEQ ID NO:38), was generated as follows.

[0144] The disclosed plasmids contain a human GNE cDNA or GATGT2 expression cassette flanked by AAV2 inverted terminal repeats (ITRs); these expression cassettes may also contain a second transgene that induces muscle growth, such as FGFIIRES and follistatin 344 or HB-IGF1. Expression of the GIcNAc epimerase / ManNAc kinase or GalNAc transferase protein is driven by either the CMV, MCK, MHCK7, mini-CMV, or GNE promoter. CMV is the cytomegalovirus promoter (SEQ ID NO: 3). MCK is the muscle creatine kinase promoter (CK7-like) (SEQ ID NO: 4). MHCK7 is the MCK promoter (SEQ ID NO: 5) with an additional enhancer. Mini-CMV is a smaller version of the CMV promoter (SEQ ID NO: 7). GNE variant 2 is the GIcNAc epimerase / ManNAc kinase gene cDNA variant 2, encoding a 722-amino acid protein beginning within exon 3 (NM_005476; SEQ ID NO: 1). GALGT2 is the GALGT2 (or B4GALNT2) gene cDNA (Genbank accession number AJ517771; SEQ ID NO: 36). miniFGF1IRES represents a minimal FGF1 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 pre-pro-peptide domain of human heparin-binding epidermal growth factor-like growth factor linked to exons 1-4 of insulin-like growth factor 1 (SEQ ID NO: 11). GNE promoter (SEQ ID NO: 6) represents the indicated sequence element immediately 5' of exon 2, which should be used to drive expression of the variant 2 GNE transcript.

[0145] Because wild-type human GNE is a 2.2 kB cDNA, a truncated FGF1A IRES may be required in some embodiments. This truncated FGF1A IRES may be as small as 100 bp to fit the FST (1.3 kB) into the 4.7 kB packaging limit of AAV. A truncated CMV promoter (220 bp instead of 800 bp), referred to herein as mini-CMV, works very well if this is an issue, allowing for the use of a longer IRES sequence.

[0146] The GNE or GATGT2 cDNA expression cassette contained a kanamycin resistance gene and an optimized Kozak sequence, which allows for stronger transcription. rAAV vectors were produced using a modified cross-packaging approach, which allows packaging of AAV type 2 vector genomes into multiple AAV capsid serotypes [Rabinowitz et al., J Virol. 76(2):791-801(2002)]. Production was achieved using the standard three-plasmid DNA / CaPO4 precipitation method in HEK293 cells. 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 the 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. A quantitative PCR-based titration method was used to determine encapsidated vector genome (vg) titers utilizing a Prism 7500 Taqman detector system (PE Applied Biosystems) [Clark et al., Hum Gene Ther. 10 (6):1031-1039 (1999)]. The final titer (vg ml -1) was determined by quantitative reverse transcriptase PCR using specific primers and probes utilizing a Prism 7500 real-time detector system (PE Applied Biosystems, Grand Island, NY, USA). The split viruses were kept at -80°C until 2020.

[0147] All plasmids used to generate packaged AAV genomes also contain a kanamycin resistance gene (KanR) outside of the ITR sequences used for genome packaging. This allows DNA encoding the AAV genome to be transformed into bacteria and, in the presence of kanamycin, produce large amounts of DNA, which kills all untransformed bacteria. KanR is not packaged into the AAV capsid of the AAV genome used to treat patients, but its presence enables DNA production in bacteria.

[0148] Example 2 Expression and Testing The vector genomes of the AAV vectors rAAV.CMV.GNE.mini-IRES.GFP and rAAV.miniCMV.GNE.full-length(FL)-IRES.GFP were tested by transfecting them into GNE-deficient Lec3 CHO cells (Lec3) to demonstrate that the vectors described in Example 1 express both GFP and a second protein. The mini-IRES is a further shortened version of the IRES and is shown as SEQ ID NO: 7. As shown in Figure 20, the presence of the mini-IRES in the vector genome allows for the expression of a second protein downstream of the IRES (GFP). In Figure 20, GFP exhibits intrinsic fluorescence, and the expression of GNE is shown by immunostaining. As shown in Figure 21, the full-length IRES also allowed for the expression of a second gene (GFP). Figure 23 shows that the IRES produces a second protein (in this case, GFP) while simultaneously allowing the production of sialic acid when GNE is introduced into Gne-deficient Lec3 cells.

[0149] Figure 24 shows that any transgene of appropriate size can be put into the initial position as a gene replacement or alternative gene replacement. C2C12 cells were transfected with the AAV vector rAAV.MCK.GALGT2.IRES.FS344, which expresses GALGT2, an alternative gene replacement for dystrophin in Duchenne muscular dystrophy. Expression of both GALGT2 (stained green) and FST (stained red) was observed in the same cells. Inclusion of an IRES allows the same cells to produce muscle growth factor, in this case follistatin (FS344 or FST).

[0150] For further analysis, some of the AAV vectors described in Example 1 were tested in muscle cells and Gne-deficient CHO cells (Lec3) to demonstrate their functionality. AAV vectors were added at different doses, ranging from 10 MOI (multiplicity of infection) to 10,000 MOI, in logarithmic increments. Because AAV functions much better in vivo than in vitro, a high MOI is typically required for AAV to infect cells in culture. C2C12 myoblast 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.

[0151] In vivo tests of function are performed in Gne-deficient mice, and Gne gene correction is tested by either demonstrating UDP-GlcNAc epimerase enzyme activity or measuring free or membrane-bound sialic acid. These measurements are performed either by gas chromatography-mass spectrometry using known standards or by quantitative lectin staining using Maackia amurensis agglutinin or Sambuca nigra agglutinin, which bind sialic acid. Assays of Gne enzyme activity, such as UDP-GlcNAc epimerase activity, can also define gene replacement. FST and IGF1 induction of muscle growth are assessed by weighing limb muscles and comparing them to the animal's total weight (see, e.g., Figure 22), by sectioning the muscles and measuring the area and number of skeletal muscle fibers present using hematoxylin and eosin staining of thin sections in combination with morphometry software, or by physiological measurements of grip strength, walking ability, and muscle strength, including ex vivo measurements of specific force, e.g., in the tibialis anterior or extensor digitorum longus muscles.

[0152] Cells are stained with MAA or SNA (conjugated to Cy3) to assess sialylation, and with antibodies against GNE, FST, or IGF1 to assess protein co-expression. As previously described (Haidet et al., Proc. Natl. Acad. Sci. 105(11):4318-22, 2008; Hennebry et al., J. Endocrinolgy 234:187-200, 2008), the same constructs are transfected into larger cell cultures, and protein expression is assessed by Western blotting and ELISA. Signaling changes, particularly a decrease in phosphor-Smad2 levels in FST and an increase in phosphor-Akt (in the case of IGF1), will be assessed by immunostaining and Western blotting, as previously described (Chandraskeharen et al. Muscle Nerve 39(1):25-41, 2008; Cramer et al., Mol. Cell. Biol. 39(14), 2019). In all cases, gene expression will be assessed by qRT-PCR and AAV biodistribution will be assessed by qPCR, as previously described by Xu et al. (Mol. Ther. 2019). The ideal IGF1 splice form for muscle growth has already been identified (ns).

[0153] The bicistronic vector described in Example 1 allows expression of GNE protein and either follistatin or IGF1 protein from the same mRNA. Because the FGF1A IRES has a much greater effect in muscle than in non-muscle cell lines, infection of muscle cultures results in greater IRES-mediated bicistronic expression. GNE expression in Lec3 cells increases sialylation, which equals or exceeds SA levels in normal CHO-K1 cells, because these cells lack Gne enzyme activity.

[0154] As shown in Figure 4, both muscle- and liver-specific expression of GNE contributed to the expression of muscle SA. - / -Sialic acid staining of the liver and muscle was performed after intramuscular injection of rAAVrh74.MCK.GNE or IP injection of rAAVrh74.LSP.GNE in mice. Sialic acid staining in the muscle and liver was compared with IM injection of the muscle-specific GNE gene therapy vector in the muscle or IP delivery of the liver-specific GNE gene therapy vector in the liver (both 5 x 10 11 Time-matched images were shown after 6 months of treatment with 100mg of MCK (at a dose of 100mg / kg). qRT-PCR showed a 30-fold increase in muscle expression of MCK but not in liver, whereas LSP showed an 8-fold increase in liver expression but not in muscle (ns). After 6 months, MCK increased muscle SA, but LSP further increased it. This may be the result of serum glycoproteins secreted by the liver being deposited in the muscle extracellular matrix.

[0155] To demonstrate that transduction of muscle cells with rAAV vectors results in muscle growth, 1 × 10 cells were transfected into the tibialis anterior (TA) muscle of C57Bl / 6J mice. 11 vg (vector genome) into the gastrocnemius muscle, 5 × 10 11 vg of AAV expressing insulin-like growth factor 1 (IGF1, muscle form Ea), HB-IGF1, or follistatin (FST) form 344. Two months after injection, muscles were dissected and weighed, showing a significant increase in HB-IGF1 and FST344 in the TA and FST344 in the gastrocnemius compared to injection of buffer alone (see Figure 21).

[0156] Example 3 Mouse model of GNE function in adult mice A mouse model of GNE myopathy was generated by introducing a loxP-transfected Gne allele into exon 3 of the mouse Gne gene. The introduction of this allele is sufficient to allow Cre-mediated deletion, resulting in a GNE myopathy-like phenotype. The field of GNE myopathy research has been plagued by the inadequacy of disease models. GNED176VTgGne - / -Mice were first reported to be an excellent late-onset model of GNE myopathy (Malicdan et al., Hum. Mol. Ther. 16(22):2669-82, 2007; Malicdan et al. Nat. Med. 15(6):690-5, 2009), but upon further breeding, these mice lose much of their phenotype, but retain the GNE myopathy. M712T (Currently GNE M743T ) Mouse knock-in of a Persian-Jewish mutation results in lethality, in part due to kidney dysfunction

[10] , while other strains of the same lineage show no phenotype at all (Sela et al., Neuromolecular medicine 15(1):180-91, 2013). Because Gne is essential in mice and causes lethality between E8.5 and E9.5, creating loxP-transfected alleles to delete the gene in adult mice allows for the generation of robust systemic or muscle-specific phenotypes using Cre-mediated deletion, allowing for reproducible demonstration of therapeutic efficacy.

[0157] Cas9-CRISPR was used to delete exon 3 of the mouse Gne gene, the exon where the functional domain of UDP-GlcNAc epimerase begins and which contains the translation start site of the Gne gene. Fertilized oocytes were injected with Cas9-CRISPR, associated guide RNAs, and long DNA oligonucleotides that enable recombination to create a new exon 3 flanked by loxP recombination sites. Founders were bred for two generations and shipped by a supplier (Mouse Biology Program at UC Davis) for subsequent analysis.

[0158] An injection session of 80 mice yielded two Gne-deleted exon 3-deleted founders (but no loxP-transfected founders) from 26 live mice (Figure 19). This was followed by another round of injections of 160 mice. If successful, we will use rAAVrh74.CMV.Cre-GFP to express Cre systemically via IV tail vein injection, or rAAVrh74.MCK.Cre-GFP to delete only Gne in skeletal muscle (and heart). These experiments provide a means of understanding how Gne deletion in adult mice causes disease phenotypes. qPCR results showed that these founders lacked the loxP-transfected allele in the adjacent exon 3, yet still retained Gne. - / - These mice can be used to generate mice that also demonstrate that the guide RNAs used enable Cas9-CRISPR deletion of Gne exon 3.

[0159] Assays for detecting disease phenotypes are now available. For example, to understand the loss of sialylation, MAA and SNA lectin staining are used to visualize sialic acid expression bound to α2,3- and α2,6-linked SA, respectively (endogenous Cre-GFP is used to confirm Cre-expressing cells). qRT-PCR is used to understand the loss of Gne gene expression (and the increase in Cre-GFP gene expression). qPCR is used to understand the number of vector genomes present per nucleus in each muscle tissue and the extent of gene deletion. For methods, see Kim et al. (Mol. Cell Neurosci. 39(3):452-64, 2008) and Xu et al. (Mol. Ther. 2019). GC-MS / MS methods are also used to measure total free sialic acid and total glycoprotein-conjugated N- and O-linked sialic acid. See Yoon et al. (PLoS Currents 2013). Finally, Gne enzymatic activity, either UDP-GlcNAc epimerase activity or ManNAc 6-kinase activity, can be used to measure the degree of functional gene replacement.

[0160] Muscle pathology analysis includes staining of thin sections with hematoxylin and eosin, trichrome, and Congo red. Measurements include the number of inclusions, myofiber size, central nuclei, myofiber size variation, fibrosis, and non-muscle area (atrophy). See Chandraskeharen et al. (Muscle Nerve 39(1):25-41, 2008). If inclusions are found, their ultrastructure is evaluated using electron microscopy. Muscle function is determined by measuring grip strength, locomotor activity (treadmill walking), open field testing, and ex vivo specific force and force decline (in the TA and EDL) during repeated contractions (Chandraskeharen et al. (Muscle Nerve 39(1):25-41, 2008; Martin et al., Am. J. Physiol. Cell Physiol., 296:C476-88, 2009).

[0161] LoxP-transfected Gne mice were mock-injected (control) or injected with 1 × 10 14 Mice were injected with 100 mg / kg of AAV.CMV.Cre-GFP or AAV.MCK.Cre-GFP and analyzed at 1, 2, and 4 months post-injection. Six mice (3 males and 3 females) were injected per group, and age-matched mock-injected and wild-type mice served as controls.

[0162] If no loxP-transfected founders were generated from these injection sessions in the above experiment, the two Gne-deficient founders were M743T Models and Gne - / - These mice are bred to homozygosity in the presence of 2 g / kg / day ManNAc, which rescues sialylation and lethality in the model. Here, mice are fed 2-4 g / kg / day ManNAc in water from conception onward. Once the pups are weaned, ManNAc is withdrawn and gene therapy is tested, essentially creating an inducible Gne knockout model. These mice do not allow for muscle-specific Gne deletion and, upon ManNAc withdrawal, AAV.CMV.GNE M712Tor AAV.CMV.GNE D207V Such mice can be rescued using microRNA or siRNA targeting mouse and / or human GNE alleles and tested for muscle-specific disease if desired. + / - It is also possible to downregulate endogenous Gne gene expression in mice. Such experiments are subject to the same issues as previous transgenic and knock-in models, but the ability to administer different amounts of Gne mutants to mice allows for more control.

[0163] Example 4 In vitro AAV.GNE potency assay The MAA-HRP ELISA allows for the comparison of sialic acid levels between Gne-expressing CHO cells and Gne-deficient Lec3 cells, and this assay should be sufficient to define the potency of AAV.GNE after infecting Lec3 cells with different concentrations of AAV.GNE.

[0164] Any gene therapy clinical development plan must include a potency assay that effectively describes the biological activity of the AAV vector used (in this case, the AAV.GNE gene therapy vector). This assay is performed annually on clinical lots of AAV to demonstrate that activity has not been lost, and to demonstrate that the AAV used in patients has the required biological activity upon administration.

[0165] Infection of Gne-deficient Lec3 (mutant CHO) cells with different amounts of AAV.GNE (Hong et al. J. Biol. Chem. 278:53045-530454, 2003) was performed to achieve a defined level of Lec3 sialylation found in the same number of normal CHO cells, thus demonstrating the efficacy of the AAV vector's biological activity. This was performed using Maackia amurensis agglutinin (MAA), which binds α2,3-linked sialic acid (Song et al. 286:31610-31622, 2011). Such assays can be applied to gene therapy vectors containing any number of GNEs.

[0166] Lec3 cells fed with 10% serum-containing medium showed no difference from normal CHO cells in the MAA-HRP binding ELISA assay (ns), whereas feeding Le3 cells for 3 days in Opti-MEM medium, a defined serum-free medium, eliminated most MAA binding, although CHO cells maintained MAA signal (Figure 25). This is because free sialic acid (SA) from serum is taken up by the cells and incorporated into lipids and glycoproteins, circumventing the Gne deficiency in Le3 cells. This circumvention can only be removed by excluding serum from the medium used to feed the cells. For example, infection of Le3 cells fed with Opti-MEM for 2 days with rAAVrh74.CMV.GNE resulted in 10% MAA binding. 5 or 10 6A high MOI (multiplicity of infection) dose allowed partial recovery of the MAA binding signal (Figure 25). To expand the signal differences in this assay, some additional optimization work (i.e., varying the time of AAV infection, varying the time of Lec3 cells in Opti-MEM, or varying the AAV dose used) may be necessary. Regardless, this assay can determine the potency of AAV.GNE vectors by adding different amounts of AAV to Lec3 cells and defining potency as the dose required to restore normal (or semi-normal) CHO cell signal. As shown in Figure 23, transfecting Lec3 cells with an AAV plasmid containing CMV.GNE and co-staining for GNE protein and MAA reveals that GNE-expressing Lec3 cells indeed secrete a sialylated glycoprotein that allows MAA to bind to non-GNE-expressing cells. Therefore, this potency assay may be more sensitive than assays in which GNE protein or gene levels are used as a standard, due to transactivation effects from secreted SA-containing proteins. To test this assay, CHO and Lec3 cells were transferred to 96-well ELISA plates at 10,000 cells / well, with triplicate wells for each condition. After feeding the cells with Opti-MEM for 1 day, they were re-fed with Opti-MEM and allowed to grow for an additional 2 days with or without AAV. During that time, some cells were infected with different doses of rAAV containing GNE cDNA. Note that any serotype of AAV can be used in these assays. The conventional measurement of MOI is 1 x 10. 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , and 1 × 10 7It is used to perform different levels of AAV infection, including in vitro. It is important to note that AAV is not very efficient at infecting cells grown in culture. This is in stark contrast to its robust ability to infect cells in tissues. Therefore, relatively high concentrations of virus must be used. However, because so few cells need to be infected, this assay utilizes only a very small amount of virus per assay.

[0167] After infection, cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 20 minutes, and washed again with PBS. Cells were then blocked for 1 hour with 1% fish gelatin (sialic acid-free) in PBS, incubated with 2 mg / mL Maackia ameurensus agglutinin linked to horseradish peroxidase (MAA-HRP) for 1 hour, and washed three times for 10 minutes each with PBS. Bound MAA-HRP was detected using a standard HRP activity (OPD) colorimetric assay, which was developed for 20 minutes and then quenched with acid for 10 minutes. Absorbance (color) was read at 450 nm on a SprectraMax plate reader.

[0168] A concentration curve was generated to determine the optimal MAA-HRP concentration (2 μg / mL) to use in this assay. This MAA-HRP concentration yields an OD reading of 1.0 or greater in CHO cells and significantly reduced OD levels in Lec3 cells (see, for example, Figure 25). The concentration curve was used to compare measurements from uninfected Le3 cells, which have a low signal, AAV.GNE-infected Le3 cells, which should show a dose-responsive signal increase, and CHO cells, which should have a high signal—our criterion for full biological activity. The MOI (or half of that signal, depending on ease of reproducibility) that achieves the signal seen in CHO cells is the dose defined as conferring potency. These measurements were repeated at least six times, using triplicate measurements per data point, to determine intra- and interassay variability of repeated measurements. The AAV concentration was adjusted as needed to more narrowly define the MOI required to confer full potency, if necessary. If the rAAV vector contains a muscle-specific promoter, such as MCK and GNE cDNA sequences, a myoblast cell line lacking GNE can be used. Other "muscle-specific" promoters, such as MHCK7, function in CHO cells, but MCK does not. Gne-deficient myoblasts can be obtained from other NDF researchers, or, if necessary, such cell lines can be generated by deleting GNE in human cells using Cas9-CRISPR. Gne-deficient myoblasts can also be generated from primary cells cultured from Gne-deficient mice using the method described in Xia et al., Dev. Biol. 242:58-73, 2002. A positive control of normal wild-type mice can also be used in this assay. It is important to understand that the cells used in the efficacy assay do not have to be human cells; they can simply be cells defined as having no or greatly reduced sialic acid content compared to controls as a result of a Gne gene deficiency.

[0169] Example 5 In vivo AAV.GNE potency assay Wild-type mice are used to define AAV.GNE potency in tissues using measurements of UDP-GlcNAc epimerase activity. Any gene therapy clinical development plan must include a potency assay that effectively describes the biological activity of the AAV.GNE vector used in tissues. Because GNE enzyme activity exhibits product inhibition from CMP-Neu5Ac when the enzyme is overexpressed, measurements of sialic acid saturate at normal levels and do not increase further. Therefore, measurements of UDP-GlcNAc epimerase activity in tissue lysates, which show increases above normal levels in tissue lysates, are one of the best means to assess 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 for the GNE gene therapy vector described herein. Dose-response studies in wild-type (C57Bl / 6J) mice using the AAV.GNE vector are performed to assess the dose and level of vector genome transduction required to produce a one-fold increase in GNE enzyme activity, defined as the amount required for functional gene replacement. This information can be used to help define dosage even in the absence of proof of concept studies in GNE disease models.

[0170] GNE enzyme activity (UDP-GlcNAc epimerase activity) was measured and compared in CHO cell lysates, Lec3 cell lysates (which lack GNE enzyme activity [2]), and Lec3 cells transfected with the pAAV.CMV.GNE plasmid. GNE enzyme activity was demonstrated in CHO cells, but little GNE enzyme activity was observed in Lec3 cells, and ultranormal enzyme activity was observed in Lec3 cells transfected with pAAV.CMV.GNE (Figure 26). In vivo measurement of GNE enzyme activity is superior to the MAA assay for sialic acid because this assay lacks feedback inhibition, resulting in an increased linear readout of the assay. Additionally, performing a UDP-GlcNAc epimerase enzyme assay requires significantly more material (millions to tens of millions of CHO cells, rather than the 10,000 CHO cells used for the MAA-HRP ELISA (Figure 25)). Therefore, this enzyme activity assay should only be used in tissues (MAA conjugation can be used for Lec3 cell ELISA). This UDP-GlcNAc epimerase assay also works in mouse tissues (e.g., liver).

[0171] Because the GNE gene and protein are expressed in almost all organs, altered GNE enzyme activity (UDP-GlcNAc epimerase activity) is measured in tissues from the entire body (liver, kidney, spleen, heart, lung, colon, and brain). However, because muscle pathology drives GNE myopathy, skeletal muscle from the entire body (including the diaphragm, biceps, triceps, gastrocnemius, quadriceps, and tibialis anterior) is the focus of this analysis. Tissue lysates from six mice (three males and three females) are analyzed to allow for reproducible determinations while accounting for potential sex differences. Using a TissueLyser (four 30 Hz pulses of 30 s each), 30–50 mg of tissue is cut, homogenized, and shaken on ice for 30 min. Once lysed, protein levels are measured by a standard Bradford assay, and enzyme activity is normalized to total protein.

[0172] UDP-GlcNAc epimerase activity is assayed using a Morgan-Eslon DMAB (4-dimethylaminobenzaldehyde) colorimetric method [6] with a 30-minute incubation time. ManNAc production is measured by product absorbance at 578 nm in a spectrophotometer. 300 μg of total protein is used per assay. ManNAc produced by the enzyme is 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. Age- and sex-matched wild-type mice are then intravenously injected with rAAVrh74.CMV.GNE to determine the dose required to double the tissue's endogenous GNE enzyme activity across regimes. A linear increase in GNE enzyme activity is expected as the AAV dose increases. Doses of 1 x 10 vg / kg, 1 x 10 vg / kg, and 1 x 10 vg / kg will be compared. The amount of virus in each tissue will be quantified by standard qPCR assays, and the amount of GNE gene expression will be measured by qRT-PCR, as previously performed (Xu et al., Mol. Ther.). Protein levels will also be compared by Western blot, when reagents become available.

[0173] Most researchers define transduction of GNE gene therapy vectors by measuring the amount of GNE cDNA introduced into tissues or the level of induction of GNE mRNA expression, but neither of these measures is a functional measure of GNE biological activity. The assay described herein, which measures GNE enzyme activity (UDP-GlcNAc epimerase activity), can be normalized to the amount of total protein used in the assay, allowing for a robust functional measurement that is reproducible across mice. Furthermore, by introducing GNE gene therapy at different doses, this assay is expected to demonstrate increasing GNE efficacy and define the minimum dose required to produce endogenous levels of GNE enzyme activity (i.e., a doubling of the enzyme activity seen in normal tissues). This assay provides the data necessary to determine the level of functional GNE overexpression required for gene replacement in all organs and the number of vector genomes that must be transduced to achieve such a change.

[0174] Example 6 Functional evaluation of bistronic GALGT2 and follistatin gene therapy We evaluated the efficacy of bistronic rAAV gene therapy expressing GALGT2 and follistatin 344 (FST) using the mdx model of muscular dystrophy. Overexpression of GALGT2 in the skeletal muscle of mdx mice has been reported to prevent muscle damage and inhibit muscle disease (Xu et al., Neuromuscul. Disord. 17: 209-220 (2007); Martin et al., Am. J. Physiol. Cell. Physiol., 296: C476-488 (2009); Nguyen et al., Proc. Natl. Acad. Sci. USA, 99: 5616-5621 (2002)). GALGT2 expression in mdx mice induced improvements comparable to those achieved by microdystrophin gene transfer, even though only half the number of fibers were transduced (Martin et al. (2009) supra).

[0175] In the current experiment, 2-month-old mdx mice were treated with 1 × 10 11 The same doses of rAAVrh74.MCK.GALGT2.IRES.FST or single-gene vectors (rAAVrh74.MCK.GALGT2 or rAAVrh74.MCK.FST) were injected into the TA. Phosphate-buffered saline (PBS) was injected as a negative control. Two months after injection, mice were euthanized, and muscle was weighed relative to total body weight. As shown in Figure 27A, both single-gene FST and bicistronic GALGT2 / FST gene injections resulted in increased muscle size, indicating that placing the FST gene at the second position of the bicistronic vector lead to significant FST function in inducing muscle growth.

[0176] After euthanasia, TA muscles were sectioned, fixed in acetone, and stained with antibodies against FST and WFA (to recognize GalNAc produced by GALGT2) after injection. As shown in Figure 27B, injection of the bicistronic vector (rAAVrh74.MCK.GALGT2.IRES.FST) resulted in functional expression of both GALGT2, which induces glycosylation in the muscle membrane (as indicated by WFA staining), and FST, which is expressed in the Golgi apparatus and ultimately secreted outside the muscle cell. Note that GALGT2-expressing muscle fibers exhibited normal muscle morphology and no signs of muscular dystrophy, a known feature of GALGT2 gene overexpression. Thus, this single bicistronic AAV vector can both inhibit muscle pathology caused by GALGT2 overexpression and increase muscle size caused by expression of the FST gene, enabling a dual-function therapy. In certain embodiments, for example, the following items are provided: (Item 1) A polynucleotide comprising: a) a promoter element; b) a transgene; c) internal ribosome entry site (IRES), and d) A polynucleotide comprising a nucleotide sequence encoding a muscle growth factor or a muscle transdifferentiation factor. (Item 2) 2. The polynucleotide of claim 1, wherein the promoter element is operably linked to the transgene. (Item 3) 3. The polynucleotide of item 1 or 2, wherein the IRES is operably linked to a nucleotide sequence encoding a muscle growth factor or a muscle transdifferentiation factor. (Item 4) A polynucleotide comprising: a) one or more promoter elements, and b) A polynucleotide comprising a GNE cDNA sequence. (Item 5) A polynucleotide comprising: a) one or more promoter elements; b) the GNE cDNA sequence or the GALGT2 cDNA sequence; c) internal ribosome entry site (IRES), and d) A polynucleotide comprising a nucleotide sequence encoding a muscle growth factor or a muscle transdifferentiation factor. (Item 6) 6. The polynucleotide of item 4 or 5, wherein the promoter element is operably linked to the GNE cDNA sequence or the GALGT2 cDNA sequence. (Item 7) 7. The polynucleotide of item 5 or 6, wherein the IRES is operably linked to the nucleotide sequence encoding a muscle growth factor or a muscle transdifferentiation factor. (Item 8) 8. The polynucleotide of any one of items 1 to 7, wherein the promoter element is a constitutive promoter or a muscle-specific promoter. (Item 9) 9. The polynucleotide of any one of items 1 to 8, wherein the promoter element is a CMV promoter, an MCK promoter, an MHCK7 promoter, a mini-CMV promoter, or a GNE promoter. (Item 10) 10. The polynucleotide according to any one of items 4 to 9, 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. (Item 11) 11. The polynucleotide sequence of any one of items 4 to 10, further comprising a human GNE promoter element found between exons 1 and 2 to drive expression of the GNE cDNA. (Item 12) 11. The polynucleotide sequence of any one of items 5 to 10, wherein the GALGT2 cDNA sequence comprises the nucleic acid sequence of SEQ ID NO: 36. (Item 13) 13. The polynucleotide of any one of items 1 to 12, wherein the internal ribosome entry site (IRES) is derived from the fibroblast growth factor 1A gene. (Item 14) 14. The polynucleotide of item 13, wherein the IRES comprises the nucleotide sequence of SEQ ID NO: 30 or a fragment thereof. (Item 15) 14. The polynucleotide of item 13, wherein the IRES comprises the nucleotide sequence of SEQ ID NO:8. (Item 16) 16. The polynucleotide of any one of items 1 to 15, wherein the nucleotide sequence encodes follistatin, SMAD7, or an insulin growth factor 1 (IGF1) variant. (Item 17) 17. The polynucleotide of item 16, wherein the follistatin is follistatin 344 or follistatin 314. (Item 18) 17. The polynucleotide of item 16, wherein the IGF1 variant is HB-IGF1. (Item 19) 19. A recombinant adeno-associated virus (rAAV) having a genome comprising the polynucleotide sequence of any one of items 1 to 18, wherein the polynucleotide is present within a single rAAV genome. (Item 20) 20. The rAAV of item 19, wherein the genome comprises a CMV promoter and a variant 2 wild-type human GNE cDNA. (Item 21) 20. The rAAV of item 19, wherein the genome comprises an MCK promoter and a variant 2 wild-type human GNE cDNA. (Item 22) 20. The rAAV of item 19, wherein the genome comprises an MHCK promoter and a variant 2 wild-type human GNE cDNA. (Item 23) 20. The rAAV of item 19, wherein the genome comprises the GNE promoter and variant 2 wild-type human GNE cDNA. (Item 24) 20. The rAAV of item 19, wherein the genome comprises a mini-CMV promoter and a variant 2 wild-type human GNE cDNA. (Item 25) 20. The rAAV of item 19, wherein the genome comprises an MCK7 promoter, a variant 2 wild-type human cDNA, an FGF1 IRES, and a nucleic acid sequence encoding follistatin 344. (Item 26) 19. The rAAV or item 19, wherein the genome comprises the MHCK7 promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1. (Item 27) 20. The rAAV of item 19, wherein the genome comprises the CMV promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding follistatin 344. (Item 28) 20. The rAAV of item 19, wherein the genome comprises the CMV promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1. (Item 29) 20. The rAAV of item 19, wherein the genome comprises the MCK promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding follistatin 344. (Item 30) 20. The rAAV of item 19, wherein the genome comprises the MCK promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1. (Item 31) 20. The rAAV of item 19, wherein the genome comprises the GNE promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding follistatin 344. (Item 32) 20. The rAAV of item 19, wherein the genome comprises the GNE promoter, variant 2 wild-type human GNE cDNA, FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1. (Item 33) 20. The rAAV of item 19, wherein the genome comprises the mini-CMV promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding follistatin 344. (Item 34) 20. The rAAV of item 19, wherein the genome comprises the mini-CMV promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding HB-IGF1. (Item 35) 20. The rAAV of item 19, wherein the genome comprises an MHCK7 promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding SMAD7. (Item 36) 20. The rAAV of item 19, wherein the genome comprises a CMV promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding SMAD7. (Item 37) 20. The rAAV of item 19, wherein the genome comprises an MCK promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding SMAD7. (Item 38) 20. The rAAV of item 19, wherein the genome comprises a GNE promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding SMAD7. (Item 39) 20. The rAAV of item 19, wherein the genome comprises a mini-CMV promoter, a variant 2 wild-type human GNE cDNA, an FGF1 IRES, and a nucleic acid sequence encoding SMAD7. (Item 40) 20. The rAAV of item 19, wherein the genome comprises the MCK promoter, the GALGT2 cDNA, an FGFR1 IRES, and a nucleic acid encoding follistatin 344. (Item 41) 20. The rAAV of item 19, wherein the genome comprises the MCK promoter, the GALGT2 cDNA, an FGFR1 IRES, and a nucleic acid encoding HB-IGF1. (Item 42) 20. The rAAV of item 19, wherein the genome comprises an MCK promoter, the GALGT2 cDNA, an FGF1 IRES, and a nucleic acid sequence encoding SMAD7. (Item 43) 43. The rAAV of any one of items 19 to 42, wherein the rAAV is of the serotype rAAVrh.74. (Item 44) An rAAV particle comprising the rAAV described in any one of items 19 to 43. (Item 45) A method for treating GNE myopathy in a human subject in need thereof, comprising administering an rAAV described in any one of items 19 to 39 or an rAAV particle described in item 44. (Item 46) Use of the rAAV described in any one of items 19 to 39 or the rAAV particle described in item 44 for the preparation of a medicament for the treatment of GNE myopathy. (Item 47) A composition comprising the rAAV of any one of items 19 to 39 or the rAAV particle of item 44 for the treatment of GNE myopathy. (Item 48) A method for treating muscular dystrophy in a human subject in need thereof, comprising administering an rAAV described in any one of items 40 to 42 or an rAAV particle described in item 44. (Item 49) Use of the rAAV described in any one of items 40 to 42 or the rAAV particle described in item 44 for the preparation of a medicament for the treatment of muscular dystrophy. (Item 50) A composition comprising the rAAV described in any one of items 40 to 42 or the rAAV particle described in item 44 for the treatment of muscular dystrophy. (Item 51) 51. The method, use, or composition of any one of items 48 to 50, wherein the muscular dystrophy is Duchenne muscular dystrophy, limb-girdle muscular dystrophy 2D, or congenital muscular dystrophy 1A.

Claims

[Claim 1] The invention described in the specification.

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