Compositions and methods for treating phosphomannomutase 2 (PMM2) deficiency

Abstract

Disclosed are vectors comprising a nucleic acid sequence capable of encoding Phosphomannomutase 2 (PMM2). Disclosed are vectors comprising a nucleic acid sequence of SEQ ID NO;2. Disclosed are rAAV vectors comprising at least one polynucleotide sequence encoding PMM2. Disclosed are compositions comprising a vector comprising a nucleic acid sequence capable of encoding PMM2. Disclosed are methods of increasing PMM2 expression and / or activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2. Disclosed are methods of glycosylation in cells of a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2. Disclosed are methods of treating a subject having a PMM2 deficiency comprising administering a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2 to the subject, wherein the composition increases PMM2 expression and / or activity in a cell of the subject.

Description

COMPOSITIONS AND METHODS FOR TREATING PHOSPHOMANNOMUTASE 2 (PMM2) DEFICIENCYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 586.718, filed September 29, 2023. which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Congenital disorders of glycosylation (CDG) are a group of rare genetic diseases that affect how cells add sugar building blocks, called glycans, to proteins. Glycosylation is the process of adding sugar chains, also called glycans, to the surface of proteins. More than 50% of proteins in the body are glycosylated and this process is critically important to ensure the correct structure and function of proteins. Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is the most common CDG.

[0003] PMM2-CDG, also known as congenital disorder of glycosylation type la, is an inherited condition that affects many parts of the body. The type and severity of problems associated with PMM2-CDG vary widely among affected individuals, sometimes even among members of the same family.

[0004] More than 115 mutations in the PMM2 gene have been found to cause PMM2- congenital disorder of glycosylation (PMM2-CDG, also known as congenital disorder of glycosylation type la). This is a severe condition that is characterized by developmental delay, weak muscle tone (hypotonia), abnormal distribution of fat, and various other signs and symptoms. The mutations that cause PMM2-CDG change the structure of the PMM2 enzy me in different ways; however, all of the mutations appear to result in reduced enzyme activity.Decreased activity of the PMM2 enzyme leads to a shortage of GDP-mannose within cells. As a result, there is not enough activated mannose to form oligosaccharides. Glycosylation cannot proceed normally because incorrect oligosaccharides are produced. The signs and symptoms in PMM2-CDG are likely due to the production of abnormally glycosylated proteins in many organs and tissues.

[0005] There is currently no FDA-approved treatment for this debilitating and often time lethal disease. A mannose- 1 -phosphate (M1P) replacement therapy7has been designed to deliver mannose- 1 -phosphate directly into cells and thereby bypass the PMM2 enzyme deficiency. There have also been efforts in drug repurposing that aim to rewire the metabolic pathways and there are also attempts to develop molecular chaperones to improve the efficiency of mutant PMM2 enzymes. However, none of these strategies will totally replenish the missing enzymeactivity and will require repeated dosing. PMM2-CDG comprise the largest group of patients who suffer from congenital disorders of glycosylation (CDG). Therefore, a safe and effective therapy is needed.BRIEF SUMMARY

[0006] Disclosed are vectors comprising a nucleic acid sequence capable of encoding Phosphomannomutase 2 (PMM2). Disclosed are vectors comprising a nucleic acid sequence of SEQ ID NO:2.

[0007] Disclosed are rAAV vectors comprising at least one polynucleotide sequence encoding PMM2.

[0008] Disclosed are compositions comprising a vector comprising a nucleic acid sequence capable of encoding PMM2.

[0009] Disclosed are methods of increasing PMM2 expression and / or activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2.

[0010] Disclosed are methods of glycosylation in cells of a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2.

[0011] Disclosed are methods of treating a subject having a PMM2 deficiency comprising administering a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2 to the subject, wherein the composition increases PMM2 expression and / or activity in a cell of the subject.

[0012] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary' and explanatory' only and are not restrictive of the invention as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method andcompositions.

[0014] FIGS. 1 A-1D show pharmacokinetic and pharmacodynamic studies of AAV9- PMM2 in patient-derived fibroblast cell models. (FIG. 1A) Role of phosphomannomutase 2 (PMM2) in glycan biosynthesis. (FIG. IB) Characterization of PMM2 expression in PMM2- CDG patient-derived and control fibroblasts. (1) Assessment of PMM2 protein expression through immunoblot analysis. (2) Comparative quantification of PMM2 protein levels normalized to (3 -actin protein abundance. (3) Evaluation of PMM2 enzy matic activity. Each sample represents three independent replicates of the experiment. ***, P < 0.001. ****, p < 0.0001. (FIG. 1C) Evaluation of PMM2 expression in AAV -treated and untreated PMM2- CDG patient-derived fibroblasts. (1) Assessment of PMM2 protein expression through immunoblot analysis. (2) Comparative quantification of PMM2 protein levels normalized to - actin protein abundance. (3) Evaluation of PMM2 enzymatic activity post-infection. Each sample represents pooled extracts prepared from three separate 10-cm plates of cells treated independently in the specified manners. (FIG. ID) Evaluation of glycosylation in AAV-treated and untreated PMM2-CDG patient-derived fibroblasts. (1) Assessment of IC AM-1 protein expression through immunoblot analysis. (2) Comparative quantification of IC AM-1 protein levels normalized to P-actin protein abundance. (3) Assessment of LAMP1 protein expression through immunoblot analysis. (4) Comparative quantification of LAMP1 protein levels normalized to P-actin protein abundance. Each sample represents pooled extracts prepared from three separate 10-cm plates of cells treated independently in the specified manners.

[0015] FIGS. 2A-2C shows the construction of a PMM2-deficient (PMM2 gene KO) mouse model. FIG. 2A) The application of CRISPR / Cas9 system. Two loxP sites were inserted flanking the PMM2 exon 2. FIG. 2B) Genotyping results of PMM2-floxed mice. Application of 5’ loxP site specific primers. WT allele gives a single PCR product of 206bp while the floxed allele gives a single PCR product of 246bp. FIG. 2C) Application of 3’ loxP site specific primers. WT allele gives a single PCR product of 233bp while the floxed allele gives a single PCR product of 273bp. F. forward; R, reverse.

[0016] FIGS. 3A-3E show reduced PMM2 expressio in selected tissues of PMM2-deficient (PMM2 gene KO) mouse model. FIG. 3A) brain. FIG. 3B) Cerebellum. FIG. 3C) heart. FIG. 3D) Kidney. FIG. 3E) muscle.

[0017] FIG. 4 shows phenotypic characterization of PMM2-deficient (PMM2 gene KO) mice.

[0018] FIG.5 is a western blot showing PMM2 expression in the brains of PMM2 KO mice is augmented after the tail vein injection of AAV9-PMM2 at 2.5xlOnvg / kg.

[0019] FIG. 6 shows a vector map of a pAAV-PMM2 vector.DETAILED DESCRIPTION

[0020] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0021] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0022] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a nucleic acid sequence is disclosed and discussed and a number of modifications that can be made to a number of molecules including the amino acids are discussed, each and every combination and permutation of the peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary.Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F. and C- E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to. steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.A. Definitions

[0023] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0024] It must be noted that as used herein and in the appended claims, the singular forms "a ", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" includes a plurality of such vectors, reference to "the vector” is a reference to one or more vectors and equivalents thereof known to those skilled in the art, and so forth.

[0025] The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

[0026] As used herein, the term "therapeutically effective amount" means an amount of a therapeutic, prophylactic, and / or diagnostic agent that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition (e.g., PMM2 deficiency), to treat, alleviate, ameliorate, relieve, alleviate symptoms of, prevent, delay onset of, inhibit progression of, reduce severity of, and / or reduce incidence of the disease, disorder, and / or condition.

[0027] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. For example, "treating" PMM2 deficiency may refer to increasing the expression and / or activity of PMM2 in a subject. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0028] As used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; C, cysteine; D aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine.

[0029] As used herein, “subject” refers to the target of administration, e.g. an animal. Thusthe subject of the disclosed methods can be a vertebrate, such as a mammal. For example, the subject can be a human. The term does not denote a particular age or sex. Subject can be used interchangeably with “individual” or “patient”.

[0030] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically- contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0032] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises.” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting termsuch as “consisting of:), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.B. Vectors

[0033] Disclosed are vectors comprising a nucleic acid sequence capable of encoding Phosphomannomutase 2 (PMM2). In some aspects, the PMM2 is mammalian PMM2. In some aspects, the PMM2 is human PMM2.

[0034] In some aspects, the PMM2 is wild type PMM2. In some aspects, wild type PMM2 is any nucleic acid sequence of PMM2 that encodes a functional PMM2. In some aspects, wild type PMM2 is any nucleic acid sequence of PMM2 that does not have mutations that render the encoded protein defective.

[0035] In some aspects, the nucleic acid sequence capable of encoding PMM2 comprises the nucleic acid sequence of atggca gcgcctggcc cagcgctctg cctcttcgac gtggatggga ccctcaccgc cccgcggcag aaaattacca aagaaatgga tgacttccta caaaaattga ggcagaagat caaaatcgga gtggtaggcg gatcggactt tgagaaagtg caggagcaac tgggaaatga tgtggttgaa aaatacgatt atgtgtttcc agaaaatggc ttggtagcat acaaagatgg gaaactcttg tgtagacaga atattcaaag tcatctgggt gaggccctaa tccaagattt aatcaactac tgtctgagct acattgcgaa aattaaactc ccgaagaaga ggggtacttt cattgaattc cgaaatggga tgttaaacgt gtcccctatt ggaagaagct gcagccaaga agaacgcatt gagttctacg aactcgataa aaaagaaaat ataagacaaa agtttgtagc agatctacgg aaagagtttg ctggaaaagg cctcacgttt tccataggag gccagatcag ctttgatgtc tttcctgatg gatgggacaa gagatactgt ctgcgacatg tggaaaatga cggttataag accatttatt tctttggaga caaaactatg ccaggtggca atgaccatga gatcttcaca gaccccagaa ccatgggcta ctccgtgaca gcgcctgagg acacgcgcag gatctgtgaa ctgctgttct cctaa (SEQ ID NO: 1). SEQ ID NO: 1 is positions 45-785 of accession number NM- _000303.3. In some aspects, the PMM2 sequence can be represented as positions 1921-2661 of SEQ ID NO:2.

[0036] In some aspects, the nucleic acid sequence capable of encoding PMM2 comprises a variant of SEQ ID NO: 1, wherein the encoded PMM2 enzyme is functional. In some aspects, the variant of SEQ ID NO: 1 can be 75, 80, 85, 90. 95. or 99% identical to SEQ ID NO: 1.

[0037] Disclosed are vectors comprising a nucleic acid sequence capable of encoding the amino acid sequence of MAAPGPALCLFDVDGTLTAPRQKITKEMDDFLQKLRQKIKIGVVGGSDFEKVQEQLGN DVVEKYDYVFPENGLVAYKDGKLLCRQNIQSHLGEALIQDLINYCLSYIAKIKLPKKRG TFIEFRNGMLNVSPIGRSCSQEERIEFYELDKKENIRQKFVADLRKEFAGKGLTFSIGGQI SFDVFPDGWDKRYCLRHVENDGYKTIYFFGDKTMPGGNDHEIFTDPRTMGYSVTAPED TRRICELLFS (SEQ ID NO:3) or a functional variant thereof. In some aspects, a vectorcomprising the nucleic acid sequence of SEQ ID NO: 1 encodes the amino acid sequence of SEQ ID NO:3.

[0038] In some aspects, the vector can be an expression vector. The term "expression vector" includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). "Plasmid" and "vector" can be used interchangeably, as a plasmid is a commonly used form of vector. Moreover, the invention is intended to include other vectors which serve equivalent functions.

[0039] In some aspects, the vector can be a viral vector. In some aspects, the vector can be a non-viral vector, such as a DNA based vector. In some aspects, the viral vector is an adeno- associated viral vector, adenoviral vector, or lentiviral vector. In some aspects, the adeno- associated viral vector is AAV9.

[0040] In some aspects, the disclosed vectors comprise the nucleic acid sequence of CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT CCATCACTAGGGGTTCCTTCTAGACAACTTTGTATAGAAAAGTTGCTCGACATTGAT TATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGG GACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCT TCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAA TTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGG CGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCA ATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGG CCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCC CGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTT ACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTT GGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCG GGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCG TGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGC GGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGCAAGTTTGTACAAAAAAGCAGGCTGCCACCATGGCAGCGCCTGGCCCAGCGCTCTGCCTCTTCGACGTGGATGGGACCCTCACCGCCCCGCGGCAGAAAATTACCAAAGAAATGGATGACTTCCTACAAAAATTGAGGCAGAAGATCAAAATCGGAGTGGTAGGCGGATCGGACTTTGAGAAAGTGCAGGAGCAACTGGGAAATGATGTGGTTGAAAAATACGATTATGTGTTTCCAGAAAATGGCTTGGTAGCATACAAAGATGGGAAACTCTTGTGTAGACAGAATATTCAAAGTCATCTGGGTGAGGCCCTAATCCAAGATTTAATCAACTACTGTCTGAGCTACATTGCGAAAATTAAACTCCCGAAGAAGAGGGGTACTTTCATTGAATTCCGAAATGGGATGTTAAACGTGTCCCCTATTGGAAGAAGCTGCAGCCAAGAAGAACGCATTGAGTTCTACGAACTCGATAAAAAAGAAAATATAAGACAAAAGTTTGTAGCAGATCTACGGAAAGAGTTTGCTGGAAAAGGCCTCACGTTTTCCATAGGAGGCCAGATCAGCTTTGATGTCTTTCCTGATGGATGGGACAAGAGATACTGTCTGCGACATGTGGAAAATGACGGTTATAAGACCATTTATTTCTTTGGAGACAAAACTATGCCAGGTGGCAATGACCATGAGATCTTCACAGACCCCAGAACCATGGGCTACTCCGTGACAGCGCCTGAGGACACGCGCAGGATCTGTGAACTGCTGTTCTCCTAAACCCAGCTTTCTTGTACAAAGTGGGAATTCCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGGAATTCCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGAGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGGGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCG GAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAAT GGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCA ACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGG CCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGAAGCC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCT ACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGAT AGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACT TTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTT GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGAC CCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC TGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGA GCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATA CTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGT CGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCG GGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGA ACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTG ACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGCAGGCAG (SEQ ID N0:2).

[0041] In some aspects, the vector comprises a 5’ inverted terminal repeat (ITR) and 3’ ITR. In some aspects, the 5’ and / or 3’ ITR can be identical to the 5’ and / or 3’ ITR in a wild type AAV2 genome. Thus, in some aspects, the 5' and 3’ ITRs can be heterozygous to the AAV serotype of the vector. In some aspects, the 5‘ and / or 3 ’ ITR can be any functional equivalent / variant of wild type 5’ and / or 3’ ITR. In some aspects, the 5’ ITR can be represented as positions 1-130 of SEQ ID NO:2. In some aspects, the 3’ ITR can be represented complementary to positions 3535-3664 of SEQ ID NO:2.

[0042] In some aspects, the vector can further comprise conventional control elements which are operably linked with elements of the transgene in a manner that permits its transcription, translation and / or expression in a cell transfected with the vector or infected with the virus produced by the disclosure. Expression control sequences include appropriatetranscription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0043] In some aspects, the vector comprises a polyadenylation (poly A) signal. In some aspects, the polyA signal can be bovine growth hormone polyA signal. In some aspects, the polyA signal allows transcription termination and polyadenylation of mRNA transcribed by PolII RNA polymerase. In some aspects, any polyA signal can be used several of which are described herein. In some aspects, the BGH polyA can be represented as positions 3320-3527 of SEQ ID NO:2.

[0044] In some aspects, the vector comprises a promoter. In some aspects, the promoter can be a CAG promoter. In some aspects, the C AG promoter is a CMV early enhancer fused to modified chicken (3-actin promoter. In some aspects, any promoter can be used several of which are described herein. In some aspects, the CAG promoter can be represented as positions 185- 1890 of SEQ ID NO:2.

[0045] In some aspects, the vector comprises a Kozak translation initiation sequence. In some aspects, the Kozak sequence facilitates translation initiation of ATG start codon downstream of the Kozak sequence. In some aspects, the Kozak sequence can be represented as positions 1915-1920 of SEQ ID NO:2.

[0046] In some aspects, the disclosed vectors comprise a posttranscriptional response element. As used herein, the term “posttranscriptional response element” refers to a nucleic acid sequence that, when transcribed, adopts a tertiary structure that enhances expression of a gene. Examples of posttranscriptional regulator}’ elements include, but are not limited to, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), mouse RNA transport element (RTE), constitutive transport element (CTE) of the simian retrovirus type 1 (SRV-1), the CTE from the Mason-Pfizer monkey virus (MPMV), and the 5' untranslated region of the human heat shock protein 70 (Hsp70 5'UTR). In some aspects, the vector comprises a regulatory element such as woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) that enhances virus stability in packaging cells leading to higher titer of packaged virus and can enhance higher expression of transgenes. In some aspects, the WPRE can be represented as positions 2692-3289 of SEQ ID NO:2.

[0047] In some aspects, the vector comprises an antibiotic resistance gene, such as, but not limited to an ampicillin resistance gene. In some aspects, the ampicillin resistance gene can be represented as positions 4592-5452 of SEQ ID NO:2.

[0048] In some aspects, the vector comprises a pUC origin of replication that facilitates plasmid replication in E. coli and can regulate high-copy plasmid number. In some aspects, the pUC origin can be represented as positions 5623-6211 of SEQ ID NO:2.

[0049] In some aspects, the vector comprises a nucleic acid sequence that encodes a tag (purification tag or detection tag). In some aspects, the tag can be a His-tag or a fluorescent tag.

[0050] In some aspects, SEQ ID NO: 2 is the nucleic acid sequence for the vector shown in FIG. 10.Table 1: Vector Components of FIG. 10i. Viral and Non- Viral Vectors

[0051] In some aspects, the delivery vehicle or vector used to deliver PMM2 to a cell or subject can be viral or non-viral. Examples of non- viral vectors include, but are not limited to, plasmids, liposomes, microcapsules, nanoparticles, lipid nanoparticles (LNPs), highly branched poly(P-amino ester) (HPAE), single-chain cyclic polymer (SCKP), poly(amidoamine) (PAMAM) dendrimers, and polyethyleneimine (PEI).

[0052] In some cases, the vector can comprise a viral vector, a liposome, a nanoparticle, an exosome, an extracellular vesicle, or any combination thereof. In some cases, a viral vector can comprise an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a retroviral vector, a portion of any of these, or any combination thereof. In some cases, a nanoparticle vector can comprise a polymeric-based nanoparticle, an aminolipid based nanoparticle, a metallic nanoparticle (such as gold-based nanoparticle), a portion of any of these, or any combination thereof. In some cases, a vector can comprise an AAV vector. A vector can be modified to include a modified VP1 protein (such as an AAV vector modified to include a VP1 protein). An AAV can comprise a serotype — such as an AAV1 serotype, an AAV2 serotype. AAV3 serotype, an AAV4 serotype. AAV5 serotype, an AAV6 serotype, AAV7 serotype, an AAV8 serotype, an AAV9 serotype, a derivative of any of these, or any combination thereof

[0053] There are a number of compositions and methods which can be used to deliver the disclosed nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems. For example, the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection. calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes. Appropriate means for transfection, including viral vectors, chemical transfectants. or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff. J. A., et al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.

[0054] Expression vectors can be any nucleotide construction used to deliver genes or gene fragments into cells (e.g., a plasmid), or as part of a general strategy to deliver genes or gene fragments, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83- 88, (1993)). For example, disclosed herein are expression vectors comprising a nucleic acid sequence capable of encoding PMM2.

[0055] The “control elements” present in an expression vector are those non-translated regions of the vector— enhancers, promoters. 5’ and 3‘ untranslated regions-which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the pBLUESCRIPT phagemid (Stratagene, La Jolla. Calif) or pSPORTl plasmid (Gibco BRL. Gaithersburg, Md.) and the like may be used. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker.

[0056] Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3‘ (Lusky, M.L., et al. , Mol. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Baneqi, J.L. et al.. Cell 33: 729 (1983)) as wellas within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0057] The promoter or enhancer may be specifically activated either by light or specific chemical events which trigger their function. Systems can be regulated by reagents such as tetracycline and dexamethasone. There are also ways to enhance viral vector gene expression by exposure to irradiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0058] Optionally, the promoter or enhancer region can act as a constitutive promoter or enhancer to maximize expression of the polynucleotides of the invention. In certain constructs the promoter or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time.

[0059] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3’ untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contains a poly adenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs. In certain transcription units, the polyadenylation region is derived from the SV40 early poly adenylation signal and consists of about 400 bases.

[0060] The expression vectors can include a nucleic acid sequence encoding a marker product. This marker product can be used to determine if the gene has been delivered to the cell and once delivered is being expressed. Marker genes can include, but are not limited to the E. coli lacZ gene, which encodes B-galactosidase, and the gene encoding the green fluorescent protein.

[0061] In some aspects, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category' is based on a cell’s metabolism and the use of a mutant cell line which lacks the ability7to grow independent of a supplemented media. Two examples are CHO DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

[0062] Another type of selection that can be used with the composition and methods disclosed herein is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0063] As used herein, plasmid (e.g., non-viral vectors) or viral vectors are agents that transport the disclosed nucleic acids, such as a nucleic acid sequence capable of encoding SMYD1 into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. In some aspects, the nucleic acid sequences disclosed herein are derived from either a virus or a retrovirus. Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Lentivirus, Vaccinia virus, Polio virus. AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemiavirus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect nondividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.

[0064] Viral vectors can have higher transaction abilities (i.e., ability to introduce genes) than chemical or physical methods of introducing genes into cells. Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation. and promoters to control the transcription and replication of the viral genome. When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.

[0065] Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer. In Microbiology, Amer. Soc. for Microbiology, pp. 229-232. Washington. (1985). w hich is hereby incorporated by reference in its entirety. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO 90 / 02806 and WO 89 / 07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference in their entirety for their teaching of methods for using retroviral vectors for gene therapy.

[0066] A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell.Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serves as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

[0067] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

[0068] The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology' 61 : 1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj- Ahmad et al., J. Virology' 57:267-274 (1986); Davidson et al., J. Virology' 61: 1226-1239 (1987): Zhang "‘Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis” BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell ty pes, since they can replicate within an initial infected cell but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest.92: 1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92: 1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988- 990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73: 1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5: 1287-1291 (1993); and Ragot, J. Gen. Virology74:501-507 (1993)) the teachings of which are incorporated herein by reference in their entirety for their teaching of methods for using retroviral vectors for gene therapy. Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales. Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, etal., J. Virol. 51:650-655 (1984); Seth, e / al.. Mol. Cell. Biol., 4:1528-1533 (1984); Varga et al. . Virology 65:6061-6070 (1991); Wickham et al.. Cell 73:309-319 (1993)).

[0069] A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. Optionally, both the El and E3 genes are removed from the adenovirus genome.

[0070] Another type of viral vector that can be used to introduce the polynucleotides of the invention into a cell is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4. 1 C vector produced by Avigen. San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0071] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus. Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United States Patent No. 6,261,834 is herein incorporated by reference in its entirety for material related to the AAV vector. In some aspects, the AAV vector can be any one of AAV1-9.

[0072] The inserted genes in viral and retroviral vectors usually contain promoters, or enhancers to help control the expression of the desired gene product. A promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and responseelements.

[0073] Other useful systems include, for example, replicating and host-restricted nonreplicating vaccinia virus vectors. In addition, the disclosed nucleic acid sequences can be delivered to a target cell in a non-nucleic acid based system. For example, the disclosed polynucleotides can be delivered through electroporation, or through lipofection. or through calcium phosphate precipitation. The deliver}' mechanism chosen will depend in part on the type of cell targeted and whether the deliver}' is occurring for example in vivo or in vitro.

[0074] Thus, the compositions can comprise, in addition to the disclosed expression vectors, lipids such as liposomes, such as cationic liposomes (e.g.. DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a nucleic acid sequences, nucleic acid constructs, or vectors and a cationic liposome can be administered to the blood, to a target organ, or inhaled into the respiratory tract to target cells of the respirator}’ tract. For example, a composition comprising a nucleic acid sequences, nucleic acid constructs, or vectors described herein and a cationic liposome can be administered to a subjects lung cells. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Patent No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage. ii. Adeno-associated virus vector (AAV vector)

[0075] As disclosed herein, the vector can be an AAV vector, In some aspects, any serotype of AAV can be used. In some aspects, the vector is an AAV9. In some aspects, AAV vector and recombinant AAV (rAAV) can be used interchangeably.

[0076] Disclosed are rAAV vectors comprising at least one polynucleotide sequence encoding PMM2.

[0077] In some aspects, the polynucleotide sequence encoding PMM2 comprises the nucleic acid sequence of SEQ ID NO: 1 or a variant thereof.

[0078] Disclosed are rAAV vectors comprising, in the 5' to 3' direction: a first ITR sequence; a promoter sequence;, a polynucleotide sequence encoding PMM2 (e.g., polynucleotide sequence encoding PMM2 comprising the nucleic acid sequence of SEQ ID NO: 1); a termination sequence; a polyA sequence; and a second ITR sequence. In some aspects, the first and second ITR sequences are AAV2 ITR sequences.

[0079] In some aspects, the rAAV vector comprises the sequence set forth in SEQ ID NO:2.

[0080] Disclosed are rAAV viral vectors comprising an AAV capsid protein; and any one of rAAV vector sequences described herein.

[0081] In some aspects, the AAV capsid protein is an AAV9 capsid protein.

[0082] In some aspects, the promoter is CMV early enhancer fused to modified chicken [3- actin promoter. In some aspects, the promoter can be any promoter disclosed herein.

[0083] In some aspect, the promoter can be a tissue-specific promoter such as, but not limited to, the liver-specific human thyroxine-binding globulin (TBG) promoter or CNS-specific promoter. In some aspects, PMM2-CDG is a multi-organ disorder and therefore, it can be best to have a promoter that acts on every’ cell typesC. Nucleic Acid Sequences

[0084] Disclosed are nucleic acid sequences capable of encoding PMM2. In some aspects, the nucleic acid sequences can comprise SEQ ID NO:1 or a functional variant thereof. In some aspects, the nucleic acid sequences can encode the amino acid sequence of SEQ ID NO:3 or a functional vanant thereof.

[0085] In some aspects, the nucleic acid sequences can be part of a construct, such as a vector.

[0086] In some aspects, the nucleic acid sequence further comprises a promoter sequence, kozak sequence, inverted terminal repeats, and / or a polyA tail. In some aspects, the nucleic acid sequences can further comprise conventional control elements which are operably linked with elements of the transgene (e.g., PMM2) in a manner that permits its transcription, translation and / or expression in a cell. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0087] In some aspects, all of the elements present in the disclosed vectors can be present in a nucleic acid sequence prior to being inserted into a vector. In some aspects, the vector carrying the nucleic acid sequence capable of encoding PMM2 can be a viral (e.g., adeno- associated viral vector) or non-viral vector (e.g., lipid nanoparticle, nucleic acid based vector).D. Compositions

[0088] Disclosed are compositions comprising any of the disclosed vectors or nucleic acid sequences. In some instances, disclosed are compositions comprising a vector comprising anucleic acid sequence capable of encoding PMM2. In some aspects, the disclosed compositions can comprise two or more different vectors, each comprising a nucleic acid sequence capable of encoding PMM2. In some aspects, at least two different AAV seroty pes, each comprising a nucleic acid sequence capable of encoding PMM2, can be present in the composition.

[0089] In some instances, the compositions can further comprise a pharmaceutically acceptable carrier. By ‘'pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subj ect, as would be well know n to one of skill in the art. Examples of carriers include dimyristoylphosphatidyl choline (DMPC), phosphate buffered saline or a multivesicular liposome. For example, PG:PC:Cholesterol:peptide or PC:peptide can be used as carriers in this invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0090] Pharmaceutical compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the vectors of the invention is not compromised. Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

[0091] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyloleate. Aqueous earners include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0092] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0093] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0094] The disclosed vectors can be formulated and / or administered in or with a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the inj ectable pharmaceutical form can be brought about by theinclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug (e.g. vector) in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0095] Thus, the compositions disclosed herein can comprise lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising a vector and a cationic liposome can be administered to the blood, to a target organ, or inhaled into the respiratory' tract to target cells of the respiratory tract. For example, a composition comprising a vector described herein and a cationic liposome can be administered to a subject's lung cells. Regarding liposomes, see, e.g., Brigham et al. Am. J.Resp. Cell. Mol. Biol. 1:95 100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413 7417 (1987); U.S. Patent No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0096] In some instances, disclosed are pharmaceutical compositions comprising any of the disclosed vectors described herein, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, buffer, or diluent. In various aspects, the vector of the pharmaceutical composition is encapsulated in a delivery vehicle. In a further aspect, the delivery' vehicle is a liposome, a microcapsule, or a nanoparticle. In a still further aspect, the delivery' vehicle is PEG-ylated.

[0097] In the methods described herein, delivery of the compositions to cells can be via a variety of mechanisms. As defined above, disclosed herein are compositions comprising any one or more of the vectors described herein and can also include a carrier such as a pharmaceutically acceptable carrier. For example, disclosed are pharmaceutical compositions, comprising thevectors disclosed herein, and a pharmaceutically acceptable carrier. In one aspect, disclosed are pharmaceutical compositions comprising the disclosed vectors. That is, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed vectors or at least one product of a disclosed method and a pharmaceutically acceptable carrier.

[0098] In certain aspects, the disclosed pharmaceutical compositions comprise the disclosed vectors as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for nasal, oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

[0099] In practice, the vectors described herein, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a pow der, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, and / or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and / or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

[0100] By "‘pharmaceutically acceptable” is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. The vectors described herein can also be included in pharmaceutical compositions in combination with one or moreother therapeutically active compounds.

[0101] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil. olive oil. and water. Examples of gaseous carriers include carbon dioxide and nitrogen. Other examples of carriers include dimyristoylphosphatidyl choline (DMPC), phosphate buffered saline or a multivesicular liposome. For example, PG:PC:Cholesterol: peptide or PC:peptide can be used as carriers in this invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer’s solution and dextrose solution. The pH of the solution can be from about 5 to about 8, or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.

[0102] In order to enhance the solubility and / or the stability of the disclosed vectors in pharmaceutical compositions, it can be advantageous to employ a-, (3- or y-cyclodextrins or their derivatives, in particular hydroxyalkyl substituted cyclodextrins, e.g. 2-hydroxypropyl-P- cyclodextrin or sulfobutyl-P-cyclodextrin. Also, co-solvents such as alcohols may improve the solubility and / or the stability of the vectors according to the invention in pharmaceutical compositions.

[0103] Pharmaceutical compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the vector of the invention is not compromised. Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.Tl

[0104] Because of the ease in administration, oral administration can be used, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.

[0105] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and aryl amines and substituted ethanolamines.

[0106] A tablet containing the compositions of the present invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

[0107] The pharmaceutical compositions of the present invention comprise a disclosed vector as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art of pharmacy.

[0108] Pharmaceutical compositions of the present invention suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.

[0109] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. Typically, the final injectable form should be sterile and should be effectively fluid for easy syringability. The pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should 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 and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0110] Injectable solutions, for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations.

[0111] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0112] Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As anexample, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.

[0113] In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and / or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and / or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot on. as an ointment.

[0114] Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.

[0115] Formulations for optical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be desirable.

[0116] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient.Compositions containing a disclosed vector can also be prepared in powder or liquid concentrate form.

[0117] The exact dosage and frequency of administration depends on the particular disclosed vector, a product of a disclosed method of making, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the medical history' of the subject to whom the dosage is administered such as the age; weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medication the individual may be taking; as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and / or depending on the evaluation of the physician prescribing the compositions.

[0118] Depending on the mode of administration, the pharmaceutical composition will comprise from 0.05 to 99 % by weight, preferably from 0.1 to 70 % by weight, more preferably from 0.1 to 50 % by weight of the active ingredient, and, from 1 to 99.95 % by weight, preferably from 30 to 99.9 % by weight, more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.E. Cells

[0119] Disclosed are cells comprising one or more of the vectors disclosed herein. In some aspects, the cells are prokaryotic or eukaryotic. In some aspects, the cells are mammalian cells.

[0120] In some aspects, the cells can be a cell line.F. Methods

[0121] Disclosed are methods of using any of the disclosed compositions or vectors.

[0122] In some aspects, the composition used in any of the disclosed methods is a vector comprising a nucleic acid sequence encoding a PMM2. In some aspects, the PMM2 is wild type PMM2 that is functional. In some aspects, the nucleic acid sequence capable of encoding PMM2 comprises the nucleic acid sequence of SEQ ID NO: 1 or a functional variant thereof. In some aspects, the composition comprises a vector comprising a nucleic acid sequence capable of encoding the amino acid sequence of SEQ ID NO: 3 or a functional variant thereof.

[0123] In some aspects, the vector is a viral vector. In some aspects, the viral vector is an adeno-associated viral vector, for example AAV9. In some aspects, the vector comprises the sequence of SEQ ID NO:2.

[0124] In some aspects, the disclosed methods comprise administering a therapeutically effective amount of a vector, for example a rAAV, to a subject in need thereof. In some aspects, a therapeutically effective amount of an rAAV is an amount sufficient to infect an animal and / or target a desired tissue. The therapeutically effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, a therapeutically effective amount of the rAAV can be in the range from about 1 ml to about 100 ml of solution containing from about 106to 1016genome copies (e.g., from I xlO6to I xlO16, inclusive). In methods disclosed herein, the therapeutically effective dose is between 6xl013gc / kg to 6x 1014gc / kg, including 7xl013gc / kg, 8x l013gc / kg, 9xl013gc / kg, I x lO14gc / kg. 2x l014gc / kg, 3 xl014gc / kg, 4x l014gc / kg. or 5x l014gc / kg (or alternatively, genome copies per heart volume or other measurement appropriate for intracardiac delivery). In some aspects, a dosage between about 1011to 1012per kg or appropriate measurement rAAV genome copies can be appropriate. In some aspects, a dosage ofbetween about 1011to 1013per kg or appropriate measurement rAAV genome copies can be appropriate. In some aspects, a dosage of between about 1011to 1014per kg or appropriate measurement rAAV genome copies can be appropriate. In some aspects, a dosage of between about 1011to 1015per kg or appropriate measurement rAAV genome copies can be appropriate. In some aspects, a dosage of about IxlO14vector genome (vg) copies per kg or appropriate measurement can be appropriate. In some aspects, the dosage can vary or be reduced when specifically targeting one or more organs (e.g., liver, brain, skeletal muscle, or heart). In some aspects, a dosage between about 107to 108rAAV genome copies per kg or appropriate measurement can be appropriate. In some aspects, a dosage of between about 108to 109rAAV genome copies per kg or appropriate measurement can be appropriate. In some aspects, a dosage of between about 109to 1010rAAV genome copies per kg or appropriate measurement can be appropriate. In some aspects, a dosage of between about IO10to 1011rAAV genome copies per kg or other appropriate measurement can be appropriate.

[0125] In some aspects, a potential side-effect for administering an AAV (or any other viral vector) to a subject can be an immune response in the subject to the AAV, including inflammation, and, and may depend on the route of administration, and in particularly, when the administration of an AAV is systemic. Thus, in some aspects of any of the disclosed methods, a subject can be immunosuppressed prior to administration of one or more rAAVs as described herein.

[0126] As used herein, “immunosuppressed” or “immunosuppression” refers to a decrease in the activation or efficacy of an immune response in a subject. Immunosuppression can be induced in a subject using one or more (e.g., multiple, such as 2, 3, 4. 5, or more) agents, including, but not limited to, rituximab, methylprednisolone, prednisolone, sirolimus, immunoglobulin injection, prednisone, methotrexate, and any combination thereof.

[0127] In some aspects, methods disclosed herein can further comprise the step of inducing immunosuppression (e.g., administering one or more immunosuppressive agents) in a subject prior to the subject being administered an rAAV (e.g.. an rAAV or pharmaceutical composition as disclosed herein). In some aspects, a subject can be immunosuppressed (e.g., immunosuppression is induced in the subject) between about 30 days and about 0 days (e.g., any time between 30 days until administration of the rAAV. inclusive) prior to administration of the rAAV to the subject. In some aspects, the subject can be pretreated with immune suppression agent (e g., rituximab, sirolimus, and / or prednisone) for at least 7 days. In some aspects, immunosuppression of a subject maintained during and / or after administration of a rAAV or pharmaceutical composition. In some aspects, a subject can be immunosuppressed (e.g.,administered one or more immunosuppressants) for between 1 day and 1 year after administration of the rAAV or pharmaceutical composition.

[0128] 1. Methods of increasing PMM2 expression and / or activity

[0129] Disclosed are methods of increasing PMM2 expression and / or activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more of the compositions disclosed herein. For example, disclosed are methods of increasing PMM2 expression and / or activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2. In some aspects, the composition further comprises a pharmaceutically acceptable carrier.

[0130] In some aspects, wherein the subject in need thereof has a mutant (or defective) PMM2 gene. In some aspects, the subject in need thereof has a PMM2 deficiency. In some aspects, the PMM2 deficiency is a PMM2-Congenital Disorder of Glycosylation (PMM2-CDG).

[0131] In some aspects, the subject in need thereof has a condition where the enzymes downstream of PMM2 are absent.

[0132] In some aspects, the PMM2 expression and / or activity is increased in fibroblasts of the subject. In some aspects, the PMM2 expression and / or activity is restored to levels of a control. In some aspects, a control is a standard amount of PMM2 previously established. In some aspects, a control is a known amount of PMM2 found in subjects with no PMM2-CDG. In some aspects, a control is a known amount of PMM2 found in subjects with no known health conditions. In some aspects, a control is a known amount of PMM2 found in subjects that are heterozygous carriers. In some aspects, the heterozygous carrier has half the amount of a healthy subject but still has half the amount of a subject with PMM2-CDG.

[0133] In some aspects, a cell receptor for the vector is not altered by the mutant (or defective) PMM2 gene. In some aspects, this allows the vector to bind to the cell receptor which results in expression of PMM2 in the cell.

[0134] In some aspects of the disclosed methods, protein and lipid glycosylation is improved in the subject. In some aspects, an improvement of protein and lipid glycosylation increases the abundance of the glycoproteins that were otherwise mis-glycosylated and decreased in abundance.

[0135] In some aspects, the methods can be performed in vitro. Thus, disclosed are methods of increasing PMM2 expression and / or activity in a cell comprising contacting to the cell a therapeutically effective amount of a composition comprising a vector comprising a nucleic acidsequence capable of encoding PMM2. In some aspects, the cells are in culture. In some aspects, the cells are derived from a subject having a PMM2 deficiency. In some aspects, the cells are in a subject.2. Methods of increasing glycosylation in cells

[0136] Disclosed are methods of increasing glycosylation in cells of a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more of the compositions disclosed herein. For example, disclosed are methods of glycosylation in cells of a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2. In some aspects, the composition further comprises a pharmaceutically acceptable carrier.

[0137] In some aspects, the subject in need thereof has a Congenital Disorder of Glycosylation (CDG). In some aspects, the CDG is inherited Phosphomannomutase 2 (PMM2) deficiency -CDG (PMM2-CDG).

[0138] In some aspects, the subject in need thereof has a condition where the enzymes downstream of PMM2 are absent.

[0139] In some aspects, the cells are fibroblasts. In some aspects, the cells can be any cell type. In some aspects, the cells can be any cell deficient in PMM2.

[0140] In some aspects, the methods can be performed in vitro. Thus, disclosed are methods of increasing glycosylation in a cell comprising contacting the cell with a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2. In some aspects, the cells are in culture. In some aspects, the cells are derived from a subject having a PMM2 deficiency. In some aspects, the cells are in a subject.3. Methods of treating a subject having a phosphomannomutase 2 (PMM2) deficiency

[0141] Disclosed are methods of treating a subject having a PMM2 deficiency comprising administering a therapeutically effective amount of one or more compositions disclosed herein. For example, disclosed are methods of treating a subject having a PMM2 deficiency comprising administering a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2 to the subject, wherein the composition increases PMM2 expression and / or activity in a cell of the subject.

[0142] In some aspects, the PMM2 deficiency is phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG). In some aspects, the PMM2 deficiency is a loss-of- function mutation in PMM2.

[0143] In some aspects, the PMM2 expression and / or activity levels are restored to levels found in healthy subjects. In some aspects, the PMM2 expression and / or activity levels are restored to levels found in a control. In some aspects, a control is a standard amount of PMM2 previously established. In some aspects, a control is a known amount of PMM2 found in subjects with no PMM2-CDG. In some aspects, a control is a known amount of PMM2 found in subjects with no known health conditions. In some aspects, a control is a known amount of PMM2 found in subjects that are heterozy gous carriers. In some aspects, the heterozygous carrier has half the amount of a healthy subject but still has half the amount of a subject with PMM2-CDG.G. Combination Therapy

[0144] In one aspect of the disclosed methods, the compositions can be administered alone or in combination with one or more additional therapeutic agents. The additional therapeutic agents are selected based on the disease or symptom to be treated. A description of the various classes of suitable pharmacological agents and drugs may be found in Goodman and Gilman, The Pharmacological Basis of Therapeutics, (11th Ed., McGraw-Hill Publishing Co.) (2005).

[0145] Disclosed are methods of treating a subject having a PMM2 deficiency comprising administering a therapeutically effective amount of a composition comprising a vector comprising a nucleic acid sequence capable of encoding PMM2 and a therapeutic agent to the subject, wherein the composition increases PMM2 expression and / or activity in a cell of the subject. In some aspects, the therapeutic agent is an agent, other than the composition, that can be used to treat PMM2 deficiency. In some aspects, a therapeutic agent can be any agent used to manage symptoms and prevent complications such as, but not limited to, hormones or anticonvulsants.H. Kits

[0146] The compositions and materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. Disclosed are kits comprising one or more of the vectors described herein. For example disclosed are kits comprising vectors comprising a nucleic acid sequence capable of encoding PMM2.

[0147] Also disclosed herein are kits for producing a rAAV.ExamplesA. AAV9-based PMM2 gene replacement augments PMM2 expression and improves glycosylation in primary fibroblasts of patients with phosphomannomutase 2 deficiency (PMM2-CDG)1. Introduction

[0148] Congenital disorders of glycosylation (CDG) are a group of inherited metabolic disorders characterized by glycoprotein and glycolipid hypoglycosylation .Phosphomannomutase 2 (PMM2) deficiency (aka PMM2-CDG, MIM# 212065), the most common CDG. is caused by recessive pathogenic mutations in the PMM2 gene, which effectively and significantly diminished cellular PMM2 activity, thereby reducing the conversion rate of mannose 6-phosphate into mannose 1 -phosphate. The latter is used to synthesize GDP-mannose, a crucial mannose donor for gly can synthesis (Fig. 1A). Patients with PMM2-CDG present with variable features ranging from isolated neurologic involvement to severe multi-organ dysfunction and early death.

[0149] In the absence of curative treatments and a high mortality before two years of age, not to mention the severe morbidity throughout life, it is imperative to explore safe and effective therapeutic strategies. We hypothesized that AAV9-based PMM2 gene replacement could restore significant PMM2 expression in patient cells and improve the long-term outcome of this disease.2. Materials and methods i. Cell culture

[0150] PMM2-CDG patient-derived fibroblasts (GM27226, GM27386) were obtained from the Coriell Institute for Medical Research. De-identified control fibroblasts were derived from normal healthy individuals. Cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution.

[0151] ii. AAV9-PMM2 infection

[0152] AAV9-PMM2 expression vector was designed by the Lai Lab and synthesized by VectorBuilder Inc. (IL, U.S.A.). Infection of fibroblasts was performed using VectorBuilder's recommended protocol. iii. PMM2 activity assay

[0153] Fibroblasts were assayed for PMM2 activity as previously described iv. Immunoblot analysis

[0154] PMM2 protein abundance in fibroblasts was evaluated with primary antibody againstPMM2 (Proteintech, #10666-l-AP) using our laboratory's published protocol. Assessment of the abundances of glycoproteins ICAM-1 and LAMP1 was performed using primary antibodies against ICAM-l(Santa Cruz Biotechnology7, #sc-8439) and LAMPl(Cell Signaling Technology, #3243), respectively. P-actin detected by anti- -actin antibody (Cell Signaling Technology, #CST-3700) was used as loading control.3. Results

[0155] We conducted in vitro studies by infecting patient fibroblasts with AAV9-based vector expressing the human PMM2 gene. The patient 1 fibroblast strain was compound heterozygous for R141H and N216I mutations, while patient 2 fibroblast strain was compound heterozygous for R141H and E139K mutations. Patient 1 presented with a range of symptoms, including global developmental delay, oculomotor apraxia, cerebellar hypoplasia, feeding difficulties, abnormal coagulation, muscular w eakness, ataxia, strabismus, growth hormone deficiency, eczema, hypothyroidism, iron deficiency, transaminitis. weekly episodes of epistaxis and mild myopia. Patient 2 had developmental delay, muscle weakness, noticeable mild plagiocephaly, dysarthria, strabismus, difficulty chewing and swallowing, severe central sleep apnea with mild hypoxemia. Both patient fibroblast strains expressed residual PMM2 activity7(9.22% and 6.85%, respectively of Control) (Fig. 1 B3) and residual immunoreactive PMM2 protein (37.43% and 11.37%, respectively of control) (Fig. 1B1, 2). Infection of the AAV9- PMM2 at multiplicity of infection (MOI) of 10,000 augmented PMM2 protein levels by 2.87 and 2.60-fold, respectively (Fig. 1C1, 2). This w7as accompanied by an increase in activity72.50 and 1.67-fold, respectively (Fig. 1C3). Additionally, augmentation of PMM2 activity in the patient fibroblast strain increased ICAM-1 and LAMP1 expression (Fig. ID). Interestingly, supplementation of the patient cells with 1 mM D (+) mannose did not appear to improve the infection efficiency.4. Discussion

[0156] With an estimated prevalence of 1:20,000 in some studies, PMM2-CDG is the most prevalent CDG. Therefore, it is not surprising that different therapeutic strategies have been tested to address the great unmet needs of patients. These approaches include, but are not limited to, the use of phosphomannose isomerase inhibitors, aldose reductase inhibitors, pharmacological chaperones, and acetazolamide supplementation. None of these approaches restore near-normal PMM2 activity in patient cells. The recent Food and Drug Administration (FDA)-approved gene-based therapies like Zolgensma®14and Hemgenix® indicated that AAV- mediated gene replacement therapy could be a promising modality7for PMM2-CDG. Although gene-based therapy like Zolgenma® is an intuitively sound approach for many recessive geneticdisorders, we were originally concerned if it would work for CDG because cell receptors for AAV vectors are glycoproteins that could be so severely altered due to hypoglycosylation in PMM2-CDG that AAV infection is no longer feasible. Consequently, it is crucial to test the viability of such approach in pilot Proof-of-Concepts (POC) studies in patient cells early in the therapeutic development programs. Our results shown here are not only reassuring, but they also demonstrate that AAV9-mediated PMM2 replacement augmented PMM2 expression and improved glycosylation in the patient cells (Fig. 1), thus paving the way for in vivo preclinical studies.

[0157] Yet, we are aware that patients with PMM2-CDG suffer liver disease and neurological deficits, which could not be fully modeled in patient fibroblasts. However, we believe that construction of immortalized patient cell lines of liver and central nervous system (CNS) origins is outside the scope of this study, not to mention how achievability it will be. Moreover, we would like to emphasize that the focus this Short Communication is to test whether any residual PMM2 activity in patient cells will be sufficient for AAV9 infection. As we now demonstrate that it is feasible, we will proceed to in vivo testing in animal models of the disorder in the future, so that we can address the tissue-specific phenoty pes more effectively. Furthermore, we have chosen in this study the AAV9 serotype because of its documented ability to cross the blood-brain barrier, which has led to successful, non-invasive intravascular delivery in animal models of several diseases. In fact, the Zolgensma® gene therapy approved by FDA to treat Spinal Muscular Atropy (SMA), a neurological disease, also employed AAV9 as the delivery agent. Therefore, we are confident that the AAV9-PMM2 vector we designed and tested in this pilot study will allow us to further evaluate the efficacy of this modality’ in alleviating the neurological disease of PMM2-CDG in our ongoing studies.5. Conclusion

[0158] AAV9-based PMM2 gene replacement is a promising therapeutic strategy to tackle the unmet medical needs for the patients with PMM2-CDG.B. In vivo augmentation of brain PMM2 expression by an AAV-based vector in a Pmm2- deficient mouse model1. Introduction

[0159] A novel PMM2-CDG mouse model was constructed and characterization assays were performed.2. Methods and Results

[0160] Cre-Lox approach was adopted to construct the conditional Pmm2 gene knockout (KO) mice (FIG. 2A). Two loxP sites were inserted flanking exon 2 of the mouse Pmm2 geneusing CRISPR / Cas9 technique. Next, mice were genotyped using primers specific to either the 5’ loxP site or the 3’ loxP site (FIGs. 2B-C).

[0161] A tamoxifen-inducible transgene was introduced in mice that are homozy gous for the Pmm2-floxed allele through a meticulous breeding scheme. When this is accomplished, the mice were treated orally with a selected, daily dose of tamoxifen for five consecutive days to induce the excision of exon 2 of the Pmm2 genes in widespread tissues.

[0162] To assess the degree of Pmm2 knockout / knockdown. the mice were euthanized one week after the end of the tamoxifen treatment and selected tissues were harvested for immunoblot analysis using anti-Pmm2 antibody. Significant reduced Pmm2 protein abundance was found in brain, heart, kidney, and muscle (FIGs. 3A-E).

[0163] Since debilitating neurological deficits are highly prevalent in human patients with PMM2-CDG, the mutant KO mice were tested with a battery of tests that collectively comprise the Composite Scoring Test. Out of the four component tests, the Pmm2-K0 animals yield scores that were significantly higher than the control animals (FIG. 4) (the higher the score yielded for each test, the poorer the performance), indicating that the animals do have a relevant CNS phenotype.

[0164] Lastly, a few KO mice were injected with 2.5 x 1013vg / kg body weight of AAV9- PMM2 vector expressing the HUMAN PMM2 gene through tail vein (i.e., systemic dosing). Immunoblot in FIG. 5 showed that even at this relatively moderate dosage, PMM2 protein expression was almost restored to control levels. This indicated that the vector can penetrate brain tissues and express high levels of PMM2 protein in vivo.

[0165] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWe claim:

1. A vector comprising a nucleic acid sequence capable of encoding Phosphomannomutase 2 (PMM2).

2. The vector of claim 1, wherein the PMM2 is human PMM2.

3. The vector of any one of claims 1-2, wherein the PMM2 is wild ty pe.

4. The vector of any one of claims 1-3, wherein the nucleic acid sequence capable of encoding PMM2 comprises the nucleic acid sequence of SEQ ID NO:1.

5. The vector of any one of claims 1-4, wherein the vector is an expression vector.

6. The vector of claim 5, wherein the expression vector is a viral vector.

7. The vector of claim 6, wherein the viral vector is an adeno-associated viral vector, adenoviral vector, or lentiviral vector.

8. The vector of claim 7, wherein the adeno-associated viral vector is AAV9.

9. The vector of claim 8, wherein the vector consists of the nucleic acid sequence of SEQ ID NO:2.

10. The vector of any one of claims 1-9, wherein the vector further comprises a promoter, poly A signal, and / or posttranscri phonal regulatory element.

11. A recombinant adeno-associated virus (rAAV) vector comprising at least one polynucleotide sequence encoding Phosphomannomutase 2 (PMM2).

12. The rAAV vector of claims 1, wherein the polynucleotide sequence encoding Phosphomannomutase 2 (PMM2) comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: XXX13. An rAAV vector comprising, in the 5' to 3' direction: a. a first AAV2 ITR sequence; b. a promoter sequence; c. a polynucleotide sequence encoding Phosphomannomutase 2 (PMM2), polynucleotide sequence encoding Phosphomannomutase 2 (PMM2) comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: XXX; d. atermination sequence; e. a synthetic poly A sequence; and f. a second AAV2 ITR sequence.

14. The rAAV vector of claim 13, wherein the rAAV vector comprises the sequence set forth in SEQ ID NO: XXX.

15. An rAAV viral vector comprising: (a) an AAV capsid protein; and (b) an rAAV vector of any one of claims 11-12.

16. The rAAV viral vector of claim 15, wherein the AAV capsid protein is an AAV9 capsid protein.

17. The rAAV viral vector of any one of claims 13-14, wherein the promoter is CMV early enhancer fused to modified chicken 0-actin promoter.

18. A composition comprising the vector of any one of claims 1-10 or the rAAV vector of any one of claims 11-17.

19. The composition of claim 18 further comprising a pharmaceutically acceptable carrier.

20. A method of increasing PMM2 expression and / or activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of claim 19.

21. The method of claim 20, wherein the subject in need thereof has a mutant (or defective) PMM2 gene.

22. The method of any one of claims 20-21, wherein the subject in need thereof has a Phosphomannomutase 2 (PMM2) deficiency.

23. The method of claim 22, wherein the PMM2 deficiency is PMM2-Congenital Disorder of Glycosylation (PMM2-CDG)24. The method of any one of claims 20-23, wherein the PMM2 expression and / or activity is increased in fibroblasts of the subject.

25. The method of any one of claims 20-24, wherein the PMM2 expression and / or activity is restored to levels of a control.

26. The method of any one of claims 20-25, wherein a cell receptor for the vector is not altered by the mutant (or defective) PMM2 gene.

27. The method of any one of claims 20-26, wherein protein and lipid glycosylation is improved in the subject.

28. A method of increasing glycosylation in cells of a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of claim 19.

29. The method of claim 28, wherein the subject in need thereof has a Congenital Disorder of Gly cosylation (CDG).

30. The method of claim 29, wherein the CDG is inherited Phosphomannomutase 2 (PMM2) deficiency-CDG (PMM2-CDG).

31. The method of any one of claims 28-30, wherein the cells are fibroblasts.

32. A method of treating a subject having a phosphomannomutase 2 (PMM2) deficiency comprising administering a therapeutically effective amount of a composition to the subject, wherein the composition increases PMM2 expression and / or activity in a cell of the subject.

33. The method of claim 32, wherein the composition is a vector comprising a nucleic acid sequence encoding a wild type PMM2.

34. The method of claim 33, wherein the vector is a viral vector.

35. The method of claim 34, wherein the viral vector is an adeno-associated viral vector.

36. The method of any one of claims 32-35, wherein the composition is the composition of claim 19.

37. The method of any one of claims 32-36, wherein the PMM2 deficiency is phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG).

38. The method of any one of claims 32-37, wherein the PMM2 deficiency is a loss-of- function mutation in PMM2.

39. The method of any one of claims 32-38, wherein PMM2 expression and / or activity levels are restored to levels found in healthy subjects.

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