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CLAIMS What is claimed: 1. A polynucleotide comprising a a) a promoter element, b) a transgene, c) internal ribosomal entry site (IRES), and d) a nucleotide sequence encoding a muscle growth factor or a muscle transdifferentiation factor. 2. A polynucleotide of clam 1 wherein the promoter element is operably linked to the transgene. 3. A polynucleotide of claim 1 or 2 wherein the IRES is operably linked to the nucleotide sequence encoding a muscle growth factor or a muscle transdifferentiation factor . 4. A polynucleotide comprising a) one or more promoter elements and b) a GNE cDNA sequence. 5. A polynucleotide comprising a) one or more promoter elements, b) a GNE cDNA sequence or a GALGT2 cDNA sequence., c) internal ribosomal entry site (IRES), and d) a nucleotide sequence that encodes a muscle growth factor or muscle transdifferentation factor . 6. A polynucleotide of clam 4 or 5 wherein the promoter element is operably linked to the GNE cDNA sequence or the GALGT2 cDNA sequence.. 7. A polynucleotide of claim 5 or 6 wherein the IRES is operably linked to the nucleotide sequence that encodes a muscle growth factor or muscle transdifferentiation factor. 8. The polynucleotide of any one claims 1-7 wherein the promoter element is a constitutive promoter or a muscle-specific promoter. 9. The polynucleotide of any one of claims 1-8 wherein the promoter element is the CMV promoter, the MCK promoter, the MHCK7 promoter, the miniCMV promoter or the GNE promoter. 10. The polynucleotide of any one of claims 4-9 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. 11. The polynucleotide sequence of any one of claims 4-10, further comprising the human GNE promoter element found between exons 1 and 2 to drive expression of the GNE cDNA. 12. The polynucleotide sequence of any one of claims 5-10 wherein the GALGT2 cDNA sequence comprises the nucleic acid sequence of SEQ ID NO: 36. 13. The polynucleotide of any one of claims 1-12 wherein the internal ribosomal entry site (IRES) is from the Fibroblast Growth Factor 1A gene. 14. The polynucleotide of claim 13 wherein the IRES comprises the nucleotide sequence of SEQ ID NO: 30 or a fragment thereof. 15. The polynucleotide of claim 13 wherein the IRES comprises the nucleotide sequence of SEQ ID NO: 8. 16. The polynucleotide of any one of claims 1-15, wherein the nucleotide sequence encodes a follistatin, SMAD7 or an Insulin Growth Factor 1 (IGF1) variant. 17. The polynucleotide of claim 16 wherein the follistatin is follistatin 344 or follistatin 314. 18. The polynucleotide of claim 16 wherein the IGF1 variant is HB-IGF1. 19. A recombinant adeno-associated virus (rAAV) having a genome comprising a polynucleotide sequence of any one of claims 1-18, wherein the polynucleotide is in a single rAAV genome. 20. The rAAV of claim 19 wherein the genome comprises a CMV promoter and a variant 2 wild type human GNE cDNA. 21. The rAAV of claim 19 wherein the genome comprises a MCK promoter and a variant 2 wild type human GNE cDNA. 22. The rAAV of claim 19 wherein the genome comprises a MHCK promoter and a variant 2 wild type human GNE cDNA. 23. The rAAV of claim 19 wherein the genome comprises the GNE promoter and a variant 2 wild type human GNE cDNA. 24. The rAAV of claim 19 wherein the genome comprises a miniCMV promoter and a variant 2 wild type human GNE cDNA. 25. The rAAV of claim 19 wherein the genome comprises the MCK7 promoter, a variant 2 wild type human cDNA, a FGF1 IRES and a nucleic acid sequence encoding follistatin 344. 26. The rAAV or claim 19 wherein the genome comprises the MHCK7 promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding HB-IGF1. 27. The rAAV of claim 19 wherein the genome comprises the comprises the CMV promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and nucleic acid sequence encoding follistatin 344. 28. The rAAV of claim 19 wherein the genome comprises the comprises the CMV promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and nucleic acid sequence encoding HB-IGF1. 29. The rAAV of claim 19 where in the genome comprises the comprises the MCK promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding follistatin 344. 30. The rAAV of claim 19 wherein the genome comprises the comprises the MCK promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and nucleic acid sequence encoding HB-IGF1. 31. The rAAV of claim 19 wherein the genome comprises the comprises the GNE promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding follistatin 344. 32. The rAAV of claim 19 wherein the genome comprises the comprises the GNE promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding HB-IGF1. 33. The rAAV of claim 19 wherein the genome comprises the comprises the miniCMV promoter, a variant 2 wild type human GNE cDNA, FGF1 IRES and a nucleic acid sequence encoding follistatin 344. 34. The rAAV of claim 19 wherein the genome comprises the comprises the miniCMV promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding HB-IGF1. 35. The rAAV of claim 19 wherein the genome comprises the comprises MHCK7 promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding SMAD7. 36. The rAAV of claim 19 wherein the genome comprises the comprises CMV promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding SMAD7. 37. The rAAV of claim 19 wherein the genome comprises the comprises MCK promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding SMAD7. 38. The rAAV of claim 19 wherein the genome comprises the comprises GNE promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding SMAD7. 39. The rAAV of claim 19 wherein the genome comprises the comprises miniCMV promoter, a variant 2 wild type human GNE cDNA, a FGF1 IRES and a nucleic acid sequence encoding SMAD7. 40. The rAAV of claim 19 wherein the genome comprises the MCK promoter, the GALGT2 cDNA, a FGFR1 IRES and a nucleic acid encoding follistatin 344. 41. The rAAV of claim 19 wherein the genome comprises the MCK promoter, the GALGT2 cDNA, a FGFR1 IRES and a nucleic acid encoding HB-IGF1. 42. The rAAV of claim 19 wherein the genome comprises the comprises MCK promoter, a the GALGT2 cDNA, a FGF1 IRES and a nucleic acid sequence encoding SMAD7. 43. The rAAV of any one of claims 19-42 wherein the rAAV is of the serotype rAAVrh.74. 44. An rAAV particle comprising the rAAV of any one of claims 19-43. 45. A method of treating GNE myopathy in a human subject in need thereof comprising the step of administering an rAAV of any one of claims 19-39 or the rAAV particle of claim 44. 46. Use of a rAAV of any one of claims 19-39 or the rAAV particle of claim 44 for the preparation of a medicament for the treatment of GNE myopathy. 47. A composition comprising the rAAV of any one of claims 19-39 or the rAAV particle of claim 44 for the treatment of GNE myopathy. 48. A method of treating muscular dystrophy in a human subject in need thereof comprising the step of administering an rAAV of any one of 40-42 or the rAAV particle of claim 44. 49. Use of a rAAV of an rAAV of any one of 40-42 or the rAAV particle of claim 44 for the preparation of a medicament for the treatment of muscular dystrophy. 50. A composition comprising the rAAV of any one of 40-42 or the rAAV particle of claim 44 for the treatment of muscular dystrophy. 51. The method, use or composition of any one of claims 48-50 wherein the muscular dystrophy is Duchene muscular dystrophy, Limb Girdle Muscular Dystrophy 2D or Congenital Muscular Dystrophy 1A. |
[00132] Specialized tests for LGMD are now available through a national scheme for diagnosis, the National Commissioning Group (NCG). [00133] The GALGT2 gene (otherwise known as B4GALNT2) encodes a β1-4-N- acetyl-D-galactosamine (βGalNAc) glycosyltransferase. GALGT2 overexpression 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. Path., 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)]. GALGT2 overexpression in skeletal muscles has been reported to induce the glycosylation of alpha dystroglycan with β1-4-N- acetyl-D-galactosamine (GalNAc) carbohydrate to make the CT carbohydrate antigen (Neu5Ac/Gcα2-3[GalNAcβ1-4]Galβα1-4GlcNAcβ-). The GALGT2 glycosyltransferase and the CT carbohydrate it creates are normally confined to neuromuscular and myotendinous junctions in skeletal muscles of adult humans, non- human primates, rodents and all other mammals yet studied [Martin et al., J. Neurocytol., 32: 915-929 (2003)]. Overexpression of GALGT2 in skeletal muscle has been reported to stimulate the ectopic glycosylation of the extrasynaptic membrane, stimulating the ectopic overexpression of a scaffold of normally synaptic proteins that are orthologues or homologues of proteins missing in various forms of muscular dystrophy, including dystrophin surrogates (e.g., utrophin, Plectin1) and laminin α2 surrogates (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). As a group, the induction of such surrogates by GALGT2 has been reported to strengthen sarcolemmal membrane integrity and prevent muscle injury in dystrophin-deficient muscles as well as in wild type muscles [Martin et al., supra]. GALGT2 overexpression in skeletal muscle has been reported to prevent muscle damage and inhibit muscle disease. This is true in the mdx mouse model for DMD [Xu et al., Neuromuscul. Disord.2007, supra; Martin et al.(2009), supra; Nguyen et al., supra], where improvement equal to that of micro-dystrophin gene transfer is noted even though only half the number of fibers were transduced [Martin et al.(2009), supra]. Notably, GALGT2 gene transfer has also been reported to be preventive in the dy W model for congenital muscular dystrophy 1A [Xu et al, Am. J. Path.2007, supra] and the Sgca -/- mouse model for limb girdle muscular dystrophy type 2D [Xu et al.2009, supra]. AAV Gene Therapy [00134] The present disclosure provides for gene therapy vectors, e.g. rAAV vectors, expressing the GNE gene and methods of treating GNE myopathy. [00135] As used herein, the term "AAV" is a standard 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. There are currently thirteen serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol.1, pp.169-228, and Berns, 1990, Virology, pp.1743- 1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp.165-174 of Parvoviruses and Human Disease, J. R. Pattison, 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 all bear 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 analogous self-annealing segments at the termini that correspond to "inverted terminal repeat sequences" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. [00136] An "AAV vector" as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. [00137] An "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector". Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle. AAV [00138] Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including two 145 nucleotide inverted terminal repeats (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the 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 corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos.7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); 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). Cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of Translational Medicine 5, 45 (2007). Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start 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). [00139] AAV possesses 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 of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the 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 replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56 o C to 65 o C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection. [00140] Multiple studies have demonstrated long-term (> 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). Moreover, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of transgene products in the systemic circulation following intramuscular injection as described in Herzog et al., Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94: 13921-13926 (1997). Moreover, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal myofibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. [00141] Recombinant AAV genomes of the disclosure comprise nucleic acid molecule of the disclosure and one or more AAV ITRs flanking a nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAVrh.74, AAVrh.10, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle specific expression, AAV1, AAV6, AAV8, AAV9, AAVrh10, or AAVrh.74 can be used. [00142] DNA plasmids of the disclosure comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, 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. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety. [00143] A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by 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 advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells. [00144] 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 are described in Ratschin et al., Mol. Cell. Biol.4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mo1. Cell. Biol.5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988). Samulski et al., J. Virol., 63:3822-3828 (1989); U.S. Patent No. 5,173,414; WO 95/13365 and corresponding U.S. Patent No.5,658.776 ; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. Vaccine 13:1244-1250 (1995); Paul et al. Human Gene Therapy 4:609-615 (1993); Clark et al. Gene Therapy 3:1124- 1132 (1996); U.S. Patent. No.5,786,211; U.S. Patent No.5,871,982; and U.S. Patent. No.6,258,595. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production. [00145] The disclosure thus provides packaging cells that produce infectious rAAV. In one embodiment packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), 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). [00146] The provided recombinant AAV (i.e., infectious encapsidated rAAV particles) comprise a rAAV genome. In exemplary embodiments, the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genomes. [00147] In an exemplary embodiment, the recombinant AAV is produced by the triple transfection method (Xiao et al. , J Virol 72, 2224-2232 (1998) using the AAV vector plasmid comprising the GNE gene and a muscle specific promoter element, pNLRep2-Caprh74 and pHelp, rAAV contains the GNE gene expression cassette flanked by AAV2 inverted terminal repeat sequences (ITR). It is this sequence that is encapsidated into AAVrh74 virions. The plasmid contains the GNE sequence and the muscle specific promoter element and core promoter elements of the muscle specific promoter to drive gene expression. The expression cassette may also contain an SV40 intron (SD/SA) to promote high-level gene expression and the bovine growth hormone polyadenylation signal is used for efficient transcription termination. [00148] The pNLREP2-Caprh74 is an AAV helper plasmid that encodes the 4 wild-type AAV2 rep proteins and the 3 wild-type AAV VP capsid proteins from serotype rh74. [00149] The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. The plasmid contains the regions of adenovirus genome that are important for AAV replication, namely E2A, E4ORF6, and VA RNA (the adenovirus E1 functions are provided by the 293 cells). The adenovirus sequences present in this plasmid only represents ~40% of the adenovirus genome, and does not contain the cis elements critical for replication such as the adenovirus terminal repeats. Therefore, no infectious adenovirus is expected to be generated from such a production system. [00150] The rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69427-443 (2002); U.S. Patent No.6,566,118 and WO 98/09657. [00151] In another embodiment, the disclosure contemplates compositions comprising rAAV of the present disclosure. Compositions of the disclosure comprise rAAV and a pharmaceutically acceptable carrier. The compositions may also comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed and include buffers and surfactants such as pluronics. [00152] Titers of rAAV to be administered in methods of the disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV may range from about 1x10 6 , about 1x10 7 , about 1x10 8 , about 1x10 9 , about 1x10 10 , about 1x10 11 , about 1x10 12 , about 1x10 13 to about 1x10 14 or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral vector genomes (vgs). One exemplary method of determining encapsilated vector genome titer uses quantitative PCR such as the methods described in (Pozsgai et al., Mol. Ther.25(4): 855-869, 2017). [00153] Methods of transducing a target cell with rAAV, in vivo or in vitro, are contemplated by the disclosure. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV of the disclosure to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic. In embodiments of the disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival. An example of a disease contemplated for prevention or treatment with methods of the disclosure is GNE myopathy. [00154] Combination therapies are also contemplated by the disclosure. Combination as used herein includes both simultaneous treatment and sequential treatments. Combinations of methods of the disclosure with standard medical treatments (e.g., corticosteroids) are specifically contemplated, as are combinations with novel therapies. [00155] Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravenous, intraarterial, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Route(s) of administration and serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and capsid protein) of the disclosure may be chosen and/or matched by those skilled in the art taking into account the infection and/or disease state being treated and the target cells/tissue(s) that are to express the UDP-GlcNAc-epimerase/ManNAc-6 kinase protein and either follistatin 344, follistatin 317 or insulin-like growth factor 1. [00156] The disclosure provides for local administration and systemic administration of an effective dose of rAAV and compositions of the disclosure. For example, systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parenteral administration through injection, infusion or implantation. [00157] In particular, actual administration of rAAV of the present disclosure may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal. Administration according to the disclosure includes, but is not limited to, injection into muscle and injection into the bloodstream. Simply resuspending a 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 restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV). Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as muscle. See, for example, WO 02/053703, the disclosure of which is incorporated by reference herein. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the disclosure. The rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling. [00158] The dose of rAAV to be administered in methods disclosed herein will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of each rAAV administered may range from about 1x10 6 , about 1x10 7 , about 1x10 8 , about 1x10 9 , about 1x10 10 , about 1x10 11 , about 1x10 12 , about 1x10 13 , about 1x10 14 , about 2x10 14 , or to about 1x10 15 or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) (i.e., 1x10 7 vg, 1x10 8 vg, 1x10 9 vg, 1x10 10 vg, 1x10 11 vg, 1x10 12 vg, 1x10 13 vg, 1x10 14 vg, 2x10 14 vg, 1x10 15 vg respectively). Dosages may also be expressed in units of viral genomes (vg) per kilogram (kg) of bodyweight (i.e., 1x10 10 vg/kg, 1x10 11 vg/kg, 1x10 12 vg/kg, 1x10 13 vg/kg, 1x10 14 vg/kg, 1.25x10 14 vg/kg, 1.5x10 14 vg/kg, 1.75x10 14 vg/kg, 2.0x10 14 vg/kg, 2.25x10 14 vg/kg, 2.5x10 14 vg/kg, 2.75x10 14 vg/kg, 3.0x10 14 vg/kg, 3.25x10 14 vg/kg, 3.5x10 14 vg/kg, 3.75x10 14 vg/kg, 4.0x10 14 vg/kg, 1x10 15 vg/kg respectively). Methods for titering AAV are described in Clark et al., Hum. Gene Ther., 10: 1031-1039 (1999). [00159] For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose. A dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art. [00160] The 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 must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The 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 a dispersion and by the use of surfactants. The 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 use of agents delaying absorption, for example, aluminum monostearate and gelatin. [00161] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof. [00162] Transduction with rAAV may also be carried out in vitro. In one embodiment, desired target muscle cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used where those cells will not generate an inappropriate immune response in the subject. [00163] Suitable methods for the transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle, using e.g., a catheter. [00164] Transduction of cells with rAAV of the disclosure results in sustained expression of the UDP-GIcNAc-epimerase/ManNAc-6 kinase protein. The present disclosure thus provides methods of administering/delivering rAAV which express UDP-GIcNAc-epimerase/ManNAc-6 kinase protein to an animal, preferably a human being. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more rAAV of the present disclosure. Transduction may be carried out with gene cassettes comprising tissue specific control elements. For example, one embodiment of the disclosure provides methods of transducing muscle cells and muscle tissues directed by muscle specific promoter elements, including, but not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family (See Weintraub et al., Science, 251: 761-766 (1991)), the myocyte-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11: 4854-4862 (1991)), control elements derived from the human skeletal actin gene (Muscat et al., Mol Cell Biol, 7: 4089-4099 (1987)), the cardiac actin gene, muscle creatine kinase sequence elements (See Johnson et al., Mol Cell Biol, 9:3393-3399 (1989)) and the murine creatine kinase enhancer (MCK) element, control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene: hypoxia-inducible nuclear factors (Semenza et al., Proc Natl Acad Sci USA, 88: 5680-5684 (1991)), steroid- inducible elements and promoters including the glucocorticoid response element (GRE) (See Mader and White, Proc. Natl. Acad. Sci. USA 90: 5603-5607 (1993)), and other control elements. [00165] Muscle tissue is an attractive target for in vivo DNA delivery, because it is not a vital organ and is easy to access. The disclosure contemplates sustained expression UDP-GIcNAc-epimerase/ManNAc-6 kinase of from transduced myofibers. [00166] By “muscle cell” or “muscle tissue” is meant a cell or group of cells derived from muscle of any kind (for example, skeletal muscle and smooth muscle, e.g. from the digestive tract, urinary bladder, blood vessels or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts. [00167] The term “transduction” is used to refer to the administration/delivery of the coding region of the GNE to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV of the disclosure resulting in expression of UDP-GlcNAc- epimerase/ManNAc-6 kinase by the recipient cell. [00168] The following EXAMPLES are provided by way of illustration and not limitation. Described numerical ranges are inclusive of each integer value within each range and inclusive of the lowest and highest stated integer. EXAMPLES Example 1 – Constructs Encoding GIcNAc epimerase/ManNAc kinase or GalNAc transferase Gene cDNA [00169] The following exemplary DNA constructs encoding UDP-GIcNAc- epimerase/ManNAc-6 kinase were generated as follows: rAAVrh74.CMV.GNE (variant 2) set out in Figure 1A and encoded by the polynucleotide of Figure 2 (SEQ ID NO: 12). rAAVrh74.MCK.GNE (variant 2) set out in Figure 1B and encoded by the polynucleotide of Figure 3 (SEQ ID NO: 13). rAAVrh74.MHCK7.GNE (variant 2) set out in Figure 1C and encoded by the polynucleotide of Figure 4 (SEQ ID NO: 14). rAAVrh74.GNE promoter.GNE (variant 2) set out in Figure 1D and encoded by the polynucleotide of Figure 5 (SEQ ID NO: 15). rAAVrh74.MHCK7.GNE(variant 2).FGFIIRES.FS344 set out in Figure 1E and encoded by the polynucleotide of Figure 6 (SEQ ID NO: 16). rAAVrh74.MHCK7.GNE(variant2).FGF1 IRES.HB-IGF1 set out in Figure 1F and encoded by the polynucleotide of Figure 7 (SEQ ID NO: 17). rAAVrh74.CVM.GNE(variant 2).FGF1IRES.FS344 set out in Figure 1G and encoded by the polynucleotide of Figure 8 (SEQ ID NO: 18). rAAVrh74.CMV.GNE(variant 2).FGF1 IRES.HB-IGF1 set out in Figure 1H and encoded by the polynucleotide of Figure 9 (SEQ ID NO: 19). rAAVrh74.MCK.GNE(variant 2).FGF1IRES.FS344 set out in Figure 1I and encoded by the polynucleotide of Figure 10 (SEQ ID NO: 20). rAAVrh74.MCK.GNE(variant2).FGF1 IRES.HB-IGF1 set out in Figure 1J and encoded by the polynucleotide of Figure 11 (SEQ ID NO: 21). rAAVrh74.GNE promoter.GNE(variant 2).FGFIIRES.FS344 set out 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 set out in Figure 1L and encoded by the polynucleotide of Figure 13 (SEQ ID NO: 23). rAAVrh74.mimiCMV.GNE set out in Figure 1M and encoded by the polynucleotide of Figure 14 (SEQ ID NO: 24). rAAVrh74.mimiCMV.GNE(variant 2).FGF1IRES.FS344 set out in Figure 1N and encoded by the polynucleotide of Figure 15 (SEQ ID NO: 25). rAAVrh74,miniCMV.GNE(variant 2).FGF1.IRES.HB-IGF1 set out in Figure 1O and encoded by the polynucleotide of Figure 16 (SEQ ID NO: 26). [00170] In addition, the exemplary DNA construct encoding GalNAc transferase rAAVrh74.MCK.GALGT2.FGF1IRES.FS344 set out in Figure 1P and encoded by the polynucleotide of Figure 17 (SEQ ID NO: 38) was generated as follows. [00171] The disclosed plasmid contains a human GNE cDNA or GATGT2 expression cassette flanked by AAV2 inverted terminal repeat sequences (ITR), these expression cassettes may also comprise a FGFIIRES and a second transgene that induces muscle growth such as Follistatin 344 or HB-IGF1. The expression of the GIcNAc epimerase/ManNAc kinase protein or GalNAc transferase protein is guided by either the CMV, MCK, MHCK7, miniCMV or the 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 with additional enhancer (SEQ ID NO: 5). MiniCMV 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, which encodes 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 #AJ517771; SEQ ID NO: 36). miniFGF1IRES represents a minimal FGFI internal ribosomal entry site (SEQ ID NO: 8). FS344 is follistatin 344 amino acid form (SEQ ID NO: 10). HB-IGF1 is the signal peptide and pre-pro-peptide domains 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 elements immediately 5' of exon 2, which should be used to drive expression of variant 2 GNE transcripts. [00172] Wild type human GNE is a 2.2kB cDNA, so a shortened FGF1A IRES may be required for some embodiments. This shortened FGF1A IREScan be as small as 100bp, to fit FST (1.3kB) into the 4.7kB packaging limit of AAV. A shortened CMV promoters (220bp instead of 800bp) is denoted herein as the miniCMV, that works very well if this is an issue, which would allow for a longer IRES sequence to be used. [00173] The GNE cDNA expression cassette or the GATGT2 cDNA expression cassette had a Kanamycin resistance gene, and an optimized Kozak sequence an optimized Kozak sequence, which allows for more robust transcription. rAAV vectors were produced by a modified cross-packaging approach whereby the AAV type 2 vector genome can be packaged into multiple AAV capsid serotypes [Rabinowitz et al., J Virol.76 (2):791-801 (2002)]. Production was accomplished using a standard three plasmid DNA/CaPO4 precipitation method using HEK293 cells. HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and streptomycin. The production plasmids were: (i) plasmids encoding the therapeutic proteins, (ii) rep2-capX modified AAV helper plasmids encoding cap serotype AAVrh74 isolate, and (iii) an adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6, and VA I/II RNA genes. A quantitative PCR-based titration method was used to determine an encapsidated vector genome (vg) titer utilizing a Prism 7500 Taqman detector system (PE Applied Biosystems). [Clark et al., Hum Gene Ther.10 (6): 1031-1039 (1999)]. A final titer (vg ml −1 ) was determined by quantitative reverse transcriptase PCR using the specific primers and probes utilizing a Prism 7500 Real-time detector system (PE Applied Biosystems, Grand Island, NY, USA). Aliquoted viruses were kept at −80 °C until [00174] All plasmids used to make AAV genomes to be packaged also contain a Kanamycin resistance gene (KanR) outside of the ITR sequences used for packaging of the genome. This allows for the DNA encoding the AAV genome to be transformed into bacteria to produce large amounts of DNA in the presence of Kanamycin, which will kill all non-transformed bacteria. KanR is not packaged into the AAV capsid in the AAV genome used to treat patients, but its presence allows for DNA production in bacteria. Example 2 Expression and Testing [00175] The vector genomes for 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) cells to demonstrate that the vectors described in Example 1 express both GFP and a second protein. The miniIRES is a further shortened version of the IRES and it is set out as SEQ ID NO: 7. As shown in Figures 20, the presence of the mini-IRES in the vector genome allowed for expression of the second protein downstream of the IRES (GFP). In Figure 20, the GFP shows endogenous fluorescence, while GNE expression is demonstrated by immunostaining. As shown in Figure 21, the full-length IRES also allowed for expression of the second gene (GFP). Figure 23 shows that GNE can allow for sialic acid production when introduced into Gne-deficient Lec3 cells at the same time that the IRES produces a second protein, in this case GFP. [00176] Figure 24 shows that any transgene of an appropriate size can be in the first position as a gene replacement or surrogate gene replacement. C2C12 cells were transfected with the AAV vector rAAV.MCK.GALGT2.IRES.FS344, which expresses GALGT2, a surrogate gene replacement for dystrophin in Duchenne Muscular Dystrophy. Expression of both GALGT2 (stained green) and FST (stained in red) was observed in the same cell. Inclusion of the IRES allows for production of a muscle growth factor in the same cells, in this case follistatin (FS344 or FST). [00177] For additional analysis, any of the AAV vectors described in Example 1 are tested in muscle cells and in GNE-deficient CHO cells (Lec3) cells to demonstrate their function. AAV vectors are added at different doses, from 10 MOI (multiplicity of infection) to 10,000 MOI in log increments. High MOI are typically needed for AAVs to infect cells in culture, as AAV works far better in vivo than in vitro. C2C12 myoblast and C2C12 myotube cultures as well as CHO-K1 (wild type) cells and Lec3 cells, a CHO cell variant that lacks Gne activity are infected with the provided AAV vectors. [00178] In vivo tests of function are carried out in Gne deficient mice, where GNE gene correction is tested either by demonstration of UDP-GlcNAc epimerase enzyme activity or by measurement of free or membrane bound sialic acid. These measurements are carried out either by gas chromatorgraphy-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, for example UDP-GlcNAc epimerase activity, may also define gene replacement. FST and IGF1 induction of muscle growth is assayed by weighing limb muscles and comparing them to total animal weight (e.g., see Figure 22), by sectioning muscles and measuring the area and number of skeletal myofibers present using hematoxylin and eosin staining of thin sections, coupled with morphometric software, or by physiological measures of muscle strength, including grip strength, ambulation, and ex vivo measures of specific force, for example in the tibialis anterior or extensor digitorum longus muscle. [00179] Cells are stained for MAA or SNA (conjugated to Cy3) to assess sialylation, and with antibodies to GNE, FST, or IGF1 to assess protein co-expression. The same constructs are infected in larger cell cultures to assess protein expression by Western blotting and ELISA, as previously described (Haidet et al., Proc. Natl. Acad. Sci.105(11): 4318-22, 2008; Hennebry et al., J. Endocrinolgy 234: 187-200, 2008). Changes in signaling, in particular reduced phosphor-Smad 2 levels for FST and increased phosphor-Akt (for IGF1) is assessed with immunostaining and Western blotting, as done previously (Chandraskeharen et al. Muscle Nerve 39(1):25-41, 2008; Cramer et al., Mol. Cell. Biol.39(14), 2019). In all cases, for gene expression is assessed by qRT-PCR and for AAV biodistribution by qPCR, as previously described in Xu et al. (Mol. Ther.2019). We have already identified the ideal IGF1 splice form for muscle growth (ns). [00180] The bicistronic vectors described in Example 1 allow for GNE protein expression and either follistatin or IGF1 protein expression from the same mRNA. Infection of muscle cultures allows for greater IRES-mediated bicistronic expression, as the FGF1A IRES shows much greater effects in muscle than in non-muscle cell lines. GNE expression in Lec3 cells increase sialylation, as these cells are deficient in Gne enzyme activity, and this is equal to or exceeds SA levels in normal CHO-K1 cells. [00181] As shown in Figure 4, both muscle and liver specific expression of GNE contributed to muscle SA expression. 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 was carried out. Sialic acid staining in muscle and liver was shown for time-matched images 6 months after IM injection of a muscle-specific GNE gene therapy vector in muscle or IP delivery of a liver-specific GNE gene therapy vector in liver, both at a dose of 5x10 11 vg. qRT-PCR showed a 30-fold increase in muscle expression for MCK, with no expression in liver, while LSP showed an 8-fold increase in liver expression, with no increase in muscle (ns). After 6 months, MCK increased muscle SA, but LSP increased it even more so, likely the result deposition of serum glycoprotein secreted by the liver in the muscle extracellular matrix. [00182] To demonstrate that transduction of muscles cells using a rAAV vector results in muscle growth, the tibialis anterior (TA) muscle of C57Bl/6J mice as injected with 1x10 11 vg (vector genomes) and the gastrocnemius (Gastroc) muscle was injected with 5x10 11 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 at 2 months post-injection, showing significant increases for HB-IGF1 and FST344 in the TA and for FST344 in the Gastroc compared to injection of buffer alone (see Figure 21). Example 3 Mouse Model for GNE Function in Adult Mice [00183] A mouse model of GNE myopathy is generated by introducing a floxed Gne allele into exon 3 of the mouse Gne gene, and introduction of this allelle is sufficient to allow for Cre-mediated deletion, yielding a GNE myopathy-like phenotype. The field of GNE myopathy research has been plagued by the inadequacies of the diseases models that have been made. GNED176VTgGne -/- mice were first reported to be a good late onset model for 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 have lost much of their phenotype, while a mouse knock-in of the GNEM712T (now GNE M743T) Persian Jewish mutation led to lethality[10], in part due to kidney dysfunction, while other strains of the same line show no phenotype at all (Sela et al., Neuromolecular medicine 15(1): 180-91, 2013ll. As Gne is essential in mice, leading to lethality between E8.5 and E9.5, creation of a floxed allele to delete the gene in the adult mouse should allow for creation of a robust body-wide or muscle-specific phenotypes using Cre-mediated deletion. This, in turn, allows for more reproducible demonstrations of therapeutic efficacy. [00184] Cas9-CRISPR is used to make a deletion in exon 3 on the mouse Gne gene, the exon where the functional domain for UDP-GlcNAc epimerase begins and which contains the translation start site for the Gne gene. Fertilized oocytes are injected with Cas9-CRISPR, relevant guide RNAs, and a long DNA oligonucleotide that allows for recombination to create a new exon 3 flanked by loxP recombination sites. Founders are bred out over two generations and then shipped from vendor (Mouse Biology Program at UC Davis) for subsequent analysis. [00185] An 80-mouse injection session has yielded two Gne deletion exon 3 deletion founders (though no floxed founders) from 26 live mice (Figure 19). This is followed by another injection round of 160 mice. If successful, rAAVrh74.CMV.Cre-GFP is used to express Cre systemically via IV tail vein injection, or use rAAVrh74.MCK.Cre-GFP is used to delete Gne only in skeletal muscle (and heart). These experiments provide a means of understanding how deletion of Gne in the adult mouse cause disease phenotypes. While qPCR results showed no floxed allele was present bordering exon 3 in these founders, they can nevertheless be used to make Gne -/- mice. These mice also demonstrate that the guide RNAs used do allow for Cas9-CRISPR deletion of Gne exon 3. [00186] Assays for detecting disease phenotypes are currently available. For example, to understand loss of sialylation MAA and SNA lectin staining is used to visualize sialic acid expression (with endogenous Cre-GFP used to see which cells Cre is expressed in), which bind α2,3- and α2,6-linked SAs respectively. qRT-PCR is used to understand loss of Gne gene expression (and 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). The GC-MS/MS method is 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). Last, Gne enzyme activity, either UDP-GlcNAc epimerase activity or ManNAc 6 kinase activity, may be used to measure the degree of functional gene replacement. [00187] Muscle pathology analysis includes staining of thin sections with hematoxylin and eosin, trichrome, and Congo Red. Measures include numbers of inclusion bodies, myofiber size, central nuclei, variance in myofiber size, fibrosis, and non-muscle area (wasting), see Chandraskeharen et al. (Muscle Nerve 39(1):25-41, 2008). If inclusion bodies are found, their ultrastructure using electron microscopy is assessed. Muscle function is determined by measuring grip strength, ambulation (treadmill walking), open field tests, and ex vivo specific force and force drop during repeated contractions (in TA and EDL), see (Chandraskeharen et al. (Muscle Nerve 39(1):25-41, 2008; Martin et al., Am. J. Physiol. Cell Physiol., 296:C476-88, 2009). [00188] Floxed Gne mice are mock-injected (control) or 1x10 14 vg/kg AAV.CMV.Cre-GFP or AAV.MCK.Cre-GFP at 2 months of age, with analysis at 1, 2 and 4 months post-injection. Six mice (3 males and 3 females) per group are injected, and age-matched mock-injected mice and wild type mice are used as controls. [00189] If the above experiments do not generate any floxed mouse founders from these injection sessions, two Gne deletion founders are breto homozygosity in the presence of 2g/kg/day ManNAc, which rescues sialylation and lethality in the GNE M743T model and in Gne -/- model. Here, mice are given ManNAc at 2-4g/kg/day in water from conception onward. Once the pups are weaned, ManNAc can be removed and gene therapies tested, essentially creating an inducible Gne knock-out model. These mice do not allow for a muscle-specific Gne deletion, one could rescue such mice at the time of ManNAc withdrawal with AAV.CMV.GNEM712T or AAV.CMV.GNED207V and test for a muscle-specific disease if needed. If needed, one could also down-regulate endogenous Gne gene expression in wild type or in Gne +/- mice using a micro-RNA or siRNA targeted to the mouse and/or human GNE allele. While such experiments would be subject to the same issues as the previous transgenic and knock-in models, the ability to dose the mouse with different amounts of the GNE mutant allows for more control. Example 4 In Vitro AAV.GNE Potency Assay [00190] An MAA-HRP ELISA allows for a comparison of sialic acid levels between Gne-expressing CHO cells and Gne-deficient Lec3 cells, and this assay should be sufficient to define AAV.GNE potency after infecting Lec3 cells with different concentrations of AAV.GNE. [00191] Any gene therapy clinical development plan must contain a potency assay that effectively describes the biological activity of the AAV vector to be used, in this case a AAV.GNE gene therapy vector. This assay will be carried out annually on clinical lots of AAV to demonstrate that activity has not been lost, and it will be carried out to demonstrate that the AAV to be used in patients has the necessary biological activity when it is administered. [00192] Infection of different amounts of AAV.GNE into Gne-deficient Lec3 (mutant CHO) cells (Hong et al. J. Biol. Chem.278:53045-530454, 2003) is carried out to bring Lec3 sialylation up to a defined amount found in equivalent numbers of normal CHO cells, thus demonstrating the potency of the AAV vector’s biological activity. This is carried out using Maackia amurensis agglutinin (MAA), which binds a2,3-linked sialic acids (Song et al.286: 31610-31622, 2011). Such an assay could be applied to any number of GNE-containing gene therapy vectors. [00193] Lec3 cells fed 10% serum-containing media did not show a difference from normal CHO cells in MAA-HRP-binding ELISA assays (ns), but feeding of Lec3 cells for 3 days in Opti-MEM media, a defined serum-free media, eliminated most MAA binding, while CHO cells maintain their MAA signal (Figure 25). This is because free sialic acid (SA) from serum was taken up into cells and incorporated into lipids and glycoproteins, bypassing the Gne deficiency in Lec3 cells. This bypass can only be removed by eliminating serum from the media used to feed the cells. For example, infection of rAAVrh74.CMV.GNE into Lec3 cells fed in Opti-MEM for two days allowed for partial recovery of a MAA-binding signal at 10 5 or 10 6 MOI (multiplicity of infection) doses (Figure 25). Some additional optimization work, (i.e., varying time of AAV infection, varying time of Lec3 cells in Opti-MEM, or varying AAV dose used) may need to be carried out to expand signal differences in this assay. Regardless, this assay is able to determine the potency of AAV.GNE vectors by adding different amounts of AAV to Lec3 cells and defining potency as the dose required to recover a normal (or half-normal) CHO cell signal. As shown in Figure 23. when an AAV plasmid containing CMV.GNE was transfected into Lec3 cells and co-stain for GNE protein and MAA, we find that GNE-expressing Lec3 cells actually secrete sialylated glycoproteins that MAA can bind on non-GNE expressing cells . Thus, this potency assay may be more sensitive than assays where GNE protein or gene levels are used as the standard due to trans effects from secreted SA- containing proteins. To test this assay, CHO cells and Lec3 cells are transferred at 10,000 cells/well into 96-well ELISA plates, with triplicate wells being used for each condition. Cells are fed Opti-MEM for one day, after which cells will be re-fed Opti- MEM and allowed to grow for two more days with or without AAV. During that period, some cells are infected with different doses of an rAAV comprising the GNE cDNA . Note that any serotype of AAV could be used in these assays. The conventional measure of MOI is used to carry out different levels of AAV infectivity, including 1x10 4 , 5x10 4 , 1x10 5 , 5 x10 5 , 1x10 6 , 5x10 6 , and 1x10 7 . It is important to note that AAV is not very efficient at infecting cells grown in culture. This differs very significantly from its robust ability to infect cells in tissues. As such, a relatively large concentration of virus needs to be used. Because so few cells need to be infected, however, this assay still utilizes only a very small amount of virus per assay. [00194] After infection, cells are washed in phosphor-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 20 minutes, and washed again in PBS. Cells are then blocked in PBS containing 1% fish gelatin (which contains no sialic acid) for 1 hour, incubated with 2mg/mL Maackia ameurensus agglutinin linked to horseradish peroxidase (MAA-HRP) for one hour, and washed 3 times for 10 minutes each in PBS. Bound MAA-HRP is detected using standard HRP activity (OPD) colorimetric assay, which are developed for 20 minutes followed by quenching in acid for 10 minutes. Absorbance (color) is read at 450nm on a SprectraMax plate reader. [00195] A concentration curve to determine the optimal MAA-HRP concentration to use in this assay (2µg/mL) has been generated. This MAA-HRP concentration yields OD readings at or above 1.0 for CHO cells and significantly reduced OD levels for Lec3 cells (e.g., see Figure 25). The concentration curve is used to compare measures for uninfected Lec3 cells, which will have a low signal, AAV.GNE-infected Lec3 cells, which should show a dose-responsive increase in signal, and CHO cell levels, which should have a high signal that is our standard for full biological activity. The MOI that achieves a signal at the signal found in CHO cells (or half that signal, depending on ease of reproducibility) is the dose defined as giving potency. These measurements are repeated at least 6 times, using triplicate measures per data point, and determine intra- and inter-assay variability of repeated measures. AAV concentrations are adjusted as needed to more narrowly define the MOI required to give full potency if necessary. When a rAAV vector comprises a muscle specific promoter. e.g. MCK, and the GNE cDNA sequence, a myoblast cell line where GNE has been deleted may be used. . Other “muscle-specific” promoters, e.g. MHCK7, will work in CHO cells, but MCK does not . Gne-deficient myoblasts could be obtained from other NDF investigators, or if necessary, such a cell line is generated by deleting GNE in human cells using Cas9-CRISPR. Gne-deficient myoblasts may also be generated from primary cells cultured from Gne-deficient mice using methods described in Xia et al., Dev. Biol. 242: 58-73, 2002. Positive controls from normal wild type mice may also be used in this assay. It is important to understand that the cells used for the potency assay need not be human cells, just cells where sialic acid is defined as being absent or very reduced compared to a control as the result of Gne gene deficiency. Example 5 In Vivo AAV.GNE Potency Assay [00196] Wild type mice are used to define AAV.GNE potency in tissues using a measure of UDP-GlcNAc epimerase activity. Any gene therapy clinical development plan must also contain a potency assay that effectively describes the biological activity of the AAV.GNE vector to be used in tissues. Because GNE enzyme activity displays product inhibition from CMP-Neu5Ac when the enzyme is overexpressed, measures of sialic acid will saturate at normal levels and not increase further. As such, measures of UDP-GlcNAc epimerase activity in tissue lysates, which show increases beyond normal levels in tissue lysates, is one of the best means of assessing total GNE activity. An UDP-GlcNAc epimerase assay that can be used to measure GNE enzyme activity in mouse and human tissues is an in vivo potency assay for the GNE gene therapy vectors described herein. A dose-response study in wild type (C57Bl/6J) mice with AAV.GNE vector is carried out to assess the dose and level of vector genome transduction needed to provide a one-fold elevation in GNE enzyme activity, which is defined as the amount required for functional gene replacement. This information may be used to help define dosage even in the absence of proof of concept studies in a GNE disease model. [00197] GNE enzyme activity (UDP-GlcNAc epimerase activity) was measured and compared in CHO cell lysates, Lec3 cell lysates (which are deficient in Gne enzyme activity[2]), and Lec3 cells transfected with pAAV.CMV.GNE plasmid. GNE enzyme activity was demonstrated in CHO cells, while almost no GNE enzyme activity was observed in Lec3 cells, and supernormal enzyme activity was observed in Lec3 cells transfected with pAAV.CMV.GNE (Figure 26). In vivo measures of GNE enzyme activity are superior to MAA assay of sialic acid because of the absence of feedback inhibition in this assay, which will increase the assay’s linear read-out. In addition, significantly more material is required to carry out the UDP-GlcNAc epimerase enzyme assay (millions to tens of millions of CHO cells instead of 10,000 CHO cells used for the MAA-HRP ELISA (Fig.25)). As such, this enzyme activity assay should only be used with tissues (while MAA binding can be used for Lec3 cell ELISAs). This UDP-GlcNAc epimerase assay also works in mouse tissues (e.g., in liver ). [00198] As the GNE gene and protein are expressed in almost all organs, the changed GNE enzyme activity (UDP-GlcNAc epimerase activity) is measured in tissues throughout the body plan (liver, kidney, spleen, heart, lung, colon, brain). However, skeletal muscles throughout the body plan (including diaphragm, biceps brachii, triceps brahii, gastrocnemius, quadriceps and tibialis anterior) are a focus for this analysis, as muscle pathology causes disease in GNE myopathy. Tissue lysates from 6 mice (3 male and 3 female) are analyzed, allowing for determinations of reproducibility while accounting for possible gender differences.30-50mg of tissue will be cut and homogenized using a TissueLyser (430Hz pulses of 30 seconds each) and allowed to shake on ice for 30 minutes. Once lysed, protein levels are measured by standard Bradford assay and to allow enzyme activity to be normalized to total protein. [00199] UDP-GlcNAc epimerase activity is assayed using the Morgan-Eslon DMAB (4-di-methylamino benzaldeyde) colorimetric method[6] with a 30-minute incubation time, where ManNAc production will be measured by product absorbance on a spectrophotometer at 578 nm.300µg of total protein will be used per assay. ManNAc produced by the enzyme is determined by comparison with a ManNAc standard curve undergoing the same DMAB chemical modification protocol, using concentrations of 0, 0.5, 1, 2.5, 5, 10, 25 ,50 and 75 µg/mL. Next, IV injection of rAAVrh74.CMV.GNE in age and gender-matched wild type mice is carried out to determine the dose required to double endogenous GNE enzyme activity in tissues throughout the body plan. A linear increase in GNE enzyme activity is expected as the dose of AAV increases. Dose of 1x1011vg/kg, 1x1012vg/kg, and 1x1013vg/kg doses are compared. The amount of virus in each tissue is quantified by standard qPCR measures and the amount of GNE gene expression will be measured by qRT- PCR, as we have done previously (Xu et al., Mol. Ther.) . Protein levels are also be compared by Western blot should reagents become available. [00200] Most investigators have defined transduction of GNE gene therapy vectors by measuring the amount of GNE cDNA introduced into a tissue or the level of induction of GNE mRNA expression, but neither of these are functional measures of GNE biological activity. The assay described herein which measures GNE enzyme activity (UDP-GlcNAc epimerase activity) allows for a robust functional measure that can be normalized to the amount of total protein used in the assay, and that this assay will be reproducible between mice. It is also expected that by introducing GNE gene therapy at different doses, will demonstrate increases in GNE potency using this assay, and the minimal dose needed to provide an endogenous level of GNE enzyme activity (i.e., a doubling of enzyme activity found in normal tissue) will be defined. This assay provides data needed to determine levels of functional GNE overexpression required for gene replacement in all organs and the number of vector genomes that must be transduced to accomplish such changes. Example 6 Functional Assessment of Bistronic GALGT2 and Follistatin Gene Therapy [00201] The mdx model of muscular dystrophy was used to assess the function of the bistronic rAAV gene therapy expressing GALGT2 and follistatin 344 (FST). It is known that GALGT2 overexpression in 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 has induced improvement equal to that of micro-dystrophin gene transfer even though only half the number of fibers were transduced (Martin et al.(2009), supra). [00202] In the present experiment, 2-month-old mdx were injected in the TA with 1x10 11 vg of rAAVrh74.MCK.GALGT2.IRES.FST or with single gene vectors (rAAVrh74.MCK.GALGT2 or rAAVrh74.MCK.FST) at the same dose. Phosphobuffered saline (PBS) was injected as a negative control. Two months after injection, the mice were euthanized and muscles weighed, relative to total body weight. As shown in Figure 27A, both single gene FST and bicistronic GALGT2/FST gene injection led to an increase in muscle size, showing that the placement of the FST gene in the second position of bicistronic vectors leads allows for significant FST function in inducing muscle growth. [00203] After euthanization, the TA muscles were sectioned, fixed in acetone, and stained with antibodies to FST and WFA (to recognize GalNAc made by GALGT2) after injection. As shown in Figure 27B, injection with the bicistronic vector (rAAVrh74.MCK.GALGT2.IRES.FST) led to functional expression of both GALGT2, which induces glycosylation on the muscle membrane (shown by WFA staining), and FST, which is expressed in the Golgi apparatus, from where it is ultimately secreted outside the muscle cell. Note that the myofibers expressing GALGT2 show normal muscle morphology, showing no signs of muscular dystrophy, a function known for GALGT2 gene overexpression. Thus, this single bicistronic AAV vector can both inhibit muscle pathology, which results from GALGT2 overexpression, and increase muscle size, which results from FST gene expression, allowing a dual function therapy.
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