DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial Number 62/807,790, filed February 20, 2019, and U.S. Provisional Patent Application Serial Number 62/823,894, filed March 26, 2019, the contents of each of which are hereby incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED

ELECTRONICALLY

[0002] An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file is 87 kilobytes in size, and titled 19-331-WO_SequenceListing_ST25.txt.

BACKGROUND OF THE DISCLOSURE

Field

[0003] The present disclosure provides compositions comprising isolated nucleic acid molecules comprising nucleic acid sequences encoding microbial polypeptides, vectors comprising a tissue-specific promoter or an immunotolerant dual promoter system, and gene therapy methods of using the isolated nucleic acid molecules for treating glycogen storage diseases (GSDs) or other inherited diseases in a subject.

Technical Background

[0004] The present disclosure relates to gene therapy methods of treating a human genetic disease with a viral or non-viral vector expressing a therapeutic enzyme derived from bacteria or other microorganisms. Specifically, this disclosure relates to a method of treating a patient suffering from glycogen storage disease type III (GSD III) with an adeno-associated virus (AAV) vector expressing a bacterial glycogen debranching enzyme. This disclosure further relates to a method of using a tissue-specific promoter or an immunotolerant dual promoter system approach as one way to prevent bacterial enzyme induced immune responses towards gene therapy. This disclosure further relates to a method of using an immunotolerant dual promoter system to prevent a therapeutic transgene product induced immune responses towards gene therapy for a human inherited disorder.

[0005] Glycogen is a multi-branched polysaccharide consisting of a-1 , 4-linked glucose subunits with a-1 ,6-linked glucose at the branching points that serves as a form of energy storage in humans, animals, yeasts, and bacteria. Glycogen debranching enzyme (GDE) and glycogen phosphorylase are the two major enzymes responsible for glycogen breakdown. In mammals and yeast, GDE is a single polypeptide with two distinct enzyme activities: 4-a-D-glycosyltransferase (EC 2.4.1.25) and amylo-a-1 , 6-glucosidase (EC 3.2.1.33). Glycogen phosphorylase initiates the glycogen degradation process by continually cleaving a-1 , 4- glycosidic bonds to remove glucose units from the non-reducing ends of external chains until it reaches four residues from a branching point. At this stage, GDE transfers three glucose residues from the one of the four-residue branches to a nearby branch and then cleaves the a-1 , 6- glycosidic bond to release the remaining single glucose residue from the branching point, which forms a new linear chain to repeat the process. In contrast, GDE in bacteria and other microorganisms has only a single a-1 ,6-glycosidic bond hydrolyzing activity for glycogen and amylopectin, which include Pullulanase (E.C. 3.2.1.41), limit dextrin alpha-1 , 6-hydrolase (GlgX) (E.C. 3.2.1.-), and Isoamylase (E.C. 3.2.1.68).

[0006] Mutations in the human AGL gene cause a genetic deficiency of GDE in GSD III, resulting in the accumulation of abnormally structured glycogen with short outer branches (called limit dextrin) in multiple tissues. Most patients (-85%) have both muscle and liver involvement (type Ilia) while others have disease limited primarily to the liver (type 111 b) . The peripheral nervous system can also be affected by mutations in the human AGL gene. Liver symptoms including hepatomegaly, elevated aminotransferases, and hypoglycemia normally appear in infancy and childhood; progressive liver cirrhosis and hepatic failure can occur with age; hepatic adenomas and hepatocellular carcinoma have also been reported in some cases (Labrune, P., et al. (1997) J Pediatr Gastroenterol Nutr, 24(3):276-9, Demo, E. et al. (2007) J Hepatol, 46(3):492-8; Kishnani, P. et al. (2010) Genet Med, 12(7):446-63). In addition to liver disease progression, progressive myopathy and cardiomyopathy are a major cause of morbidity in adults. Muscle weakness is present during childhood and becomes more prominent in the third or fourth decade of life; some patients can become wheel chair bound due to severe impairment of skeletal muscle function. Ventricular hypertrophy is a frequent finding in GSD III. Sudden deaths caused by cardiac arrhythmias or cardiac failure have been reported (Olson, L.J., et al. (1984) Am J Cardiol, 53(7):980-81 , Moses, S.W., et al. (1986) Acta Paediatr Scand, 75(2):289-96; Labrune, P. et al. (1991) Pediatr Cardiol,

12(3): 161-3; Lee, P.J. et al. (1997) Am J Cardiol, 79(6):834-8, Mogahed, E.A. et al. (2015) Eur J Pediatr, 174(1 1): 1545-8).

[0007] To date, there is no cure for GSD III. Treatment strategies have relied on

symptomatic and dietary management to control blood glucose levels. Dietary interventions, such as controlling hypoglycemia with frequent meals high in complex carbohydrates and cornstarch supplements and a high-protein diet for patients with myopathy, do little to alter the long-term course and morbidity of the disease (Kishnani, P.S. et al. (2010) Genet Med, 12(7):446-63, Sentner, C.P et al. (2016) J Inherit Metab Dis, 39(5):697-704). There are a few case reports of improvement of cardiomyopathy on a high protein diet. The long term natural history of GSD III is evolving, and it is being recognized that long term complications are occurring, likely due to accumulation of limit dextrin in liver and muscle.

[0008] Gene therapy to replace the defective gene with a normal human gene would be an ideal treatment approach for patients with single-gene disorders like GSD III. In the past decade, AAV vectors have emerged as a promising tool for in vivo gene delivery. However, the small carrying capacity (<4.7 kb) of an AAV vector makes it impossible to deliver a gene expression cassette containing the large coding sequence for a human protein like human GDE.

[0009] Furthermore, elicitation of immune responses towards transgene products is a major concern in gene therapy. In particular, transgene-induced cytotoxic T lymphocyte (CTL) responses can result in the elimination of transgene-expressing cells after gene transfer and the loss of efficacy of gene therapy. It has been reported that tissue-restricted gene therapy using a tissue-specific promoter can prevent transgene-induced immune responses, but this gene therapy approach corrects disease only in that tissue but not in other tissues.

[0010] Thus, there is a need for gene therapy treatments for inherited diseases caused by a large defective gene, such as GSD III. Described herein are compositions and methods for a novel gene therapy approach that address this need by using: (1) a smaller microbial enzyme to replace the large human GDE to overcome the limitation of the small carrying capacity of a viral vector, and optionally (2) a single or a tandem tissue-specific promoter or an immunotolerant dual promoter system as one possible way to prevent transgene-induced immune responses against the therapeutic enzyme and thereby to enhance the long-term efficacy of gene therapy. This approach can be complemented with other treatment strategies such as the use of small molecule drugs, RNA interference (RNAi)

polynucleotides and other approaches to inhibit glycogen synthase, the use of immune modulation approaches to prevent or reduce host immunity to gene therapy, or dietary approaches to address the underlying pathophysiologic issues due to GDE deficiency.

SUMMARY OF THE DISCLOSURE

[0011] The present disclosure provides compositions comprising nucleic acids encoding a microbial polypeptide for degrading glycogen and gene therapy methods for treating glycogen storage disease in a subject.

[0012] One aspect of the disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide for degrading glycogen, wherein the nucleic acid sequence is codon-optimized for expression in a mammalian cell. In some embodiments, the encoded microbial polypeptide has debranching enzyme activity that can cleave the a-1 , 6-glycosidic bonds in glycogen and/or limit dextrin or can cleave both the a-1 , 6-glycosidic bonds and a-1 , 4-glycosidic bonds in glycogen and/or limit dextrin. [0013] In some embodiments of the disclosure, the encoded microbial polypeptide is a glycogen debranching enzyme (GDE) from bacteria or another microorganism. In other embodiments of the disclosure, the encoded microbial polypeptide is a type II Pullulanase or a pullulan hydrolase type III from bacteria or other microorganism, or a type I Pullulanase from Bacillus subtilis or a limit dextrin alpha- 1 ,6-glucohydrolase (Gig X) from Escherichia coli or an isoamylase from Pseudomonas amyloderamosa.

[0014] In some embodiments of the disclosure, the encoded microbial polypeptide has an amino acid sequence set forth in SEQ ID NO:01 or SEQ ID NO:04 or SEQ ID NO:07, or has at least 50%, 90%, or 90-99% sequence identity to the amino acid sequence set forth in SEQ ID NO:01 or SEQ ID NO:04 or SEQ ID NO:07.

[0015] In some embodiments of the disclosure, the nucleic acid sequence comprises a sequence as set forth in SEQ ID NOs:03 and 08-16, or SEQ ID NOs:06 and 17-25, or has at least 50% sequence identity to the nucleic acid sequence as set forth in SEQ ID NOs:03 and 08-16, or SEQ ID NOs:06 and 17-25.

[0016] In some embodiments of the disclosure, the encoded microbial polypeptide is from bacteria, algae, protozoa, or viruses.

[0017] In some embodiments of the disclosure, the nucleic acid sequence has a coding sequence that is less than about 4.0 kilobases.

[0018] Another aspect of the disclosure provides a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide for degrading glycogen, wherein the nucleic acid sequence is codon-optimized for expression in a mammalian cell.

[0019] In some embodiments of the disclosure, the vector is a viral vector or non-viral vector. In some embodiments, the viral vector can include, but not limited to, an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease viral vector, a poxvirus vector, or a picornavirus vector. In other embodiments, the non-viral vector can include, but is not limited to, a polymer based vector, a peptide based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid based vector.

[0020] In some embodiments of the disclosure, the vector comprises a ubiquitous promoter operably linked to the nucleic acid molecule that can drive the expression of the microbial polypeptide in any tissues. In other embodiments, the ubiquitous promoter is a CMV enhancer/chicken b-actin promoter.

[0021] In some embodiments of the disclosure, the vector comprises a tissue-specific promoter operably linked to the nucleic acid molecule. In other embodiments, the tissue- specific promoter is a liver-specific promoter, a muscle-specific promoter, a neuron-specific promoter, or a combination of any of the two or more thereof. In yet other embodiments, the vector comprises a liver-specific promoter that is an a1-microglobulin/bikunin

enhancer/thyroid hormone-binding globulin promoter. In yet other embodiments, the vector comprises an immunotolerant dual promoter consisting of a liver-specific promoter and a ubiquitous promoter.

[0022] In some embodiments of the disclosure, the vector comprises a gene expression cassette comprising one or more promoters, the isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide, and a polyadenylation sequence. In other embodiments, the gene expression cassette contains a nucleotide sequence having about 4.5 kb or less.

[0023] Yet another aspect of the disclosure provides a pharmaceutical formulation comprising a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide for degrading glycogen, wherein the nucleic acid sequence is codon-optimized for expression in a mammalian cell in a pharmaceutically acceptable carrier.

[0024] Yet another aspect of the disclosure proves a method of treating a deficiency of a polypeptide for degrading glycogen in a subject, comprising administering to the subject a therapeutically effective amount of a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide for degrading glycogen, wherein the nucleic acid sequence is codon-optimized for expression in a mammalian cell.

[0025] In some embodiment of the above method, the microbial polypeptide is a type I Pullulanase, or a limit dextrin alpha-1 , 6-glucohydrolase (Gig X) or an isoamylase from bacteria or other microorganisms. In other embodiments of the above method, the microbial polypeptide is capable of cleaving a-1 , 6-glycosidic bonds in glycogen and/or limit dextrin or is capable of cleaving both a-1 , 6-glycosidic bonds and a-1 , 4- glycosidic bonds.

[0026] In some embodiments of the above method, the deficiency of a polypeptide for degrading glycogen is GSD III, GSD I, GSD II, GSD IV, GSD V, GSD VI, GSD VII, GSD IX, GSD XI, GSD XII, GSD XIII, GSD XIV, Danon disease, Lafora disease, PRKAG2 (protein kinase gamma 2 subunit) deficiency, or other condition where there is cytoplasmic accumulation of glycogen. In other embodiments, the deficiency of a polypeptide for degrading glycogen is GSD III.

[0027] In some embodiments of the above method, the microbial polypeptide is a type II Pullulanase or a pullulan hydrolase type III from bacteria or other microorganisms.

[0028] In some embodiments of the above method, the vector is administered via intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, intraventricular, or in utero administration. [0029] In some embodiments of the above method, the subject is a human subject.

[0030] In some embodiments of the above method, the vector is delivered to areas of the body including but not limited to the liver, heart, skeletal muscle, smooth muscle, kidney, and the central and peripheral nervous systems in the subject.

[0031] In other embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immunosuppressive agent and/or a glycogen synthase inhibitor.

[0032] Yet another aspect of the present disclosure provides, a method of treating a deficiency of a polypeptide for degrading glycogen in a subject, comprising administering to the subject an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide for degrading glycogen.

[0033] In some embodiments of the above method, the nucleic acid sequence is codon- optimized for expression in human or mammalian cells.

[0034] In some embodiments of the above method, the encoded microbial polypeptide is a type I Pullulanase or a limit dextrin alpha-1 , 6-glucohydrolase (Gig X) or an isoamylase from bacteria or another microorganism, or a type II Pullulanase or a pullulan hydrolase type III from bacteria or other microorganisms.

[0035] In some embodiments of the above method, the encoded microbial polypeptide is capable of cleaving a-1 , 6-glycosidic bonds or is capable of cleaving both a-1 , 6-glycosidic bonds and a-1 , 4- glycosidic bonds.

[0036] In some embodiments of the above method, the encoded microbial polypeptide has an amino acid sequence set forth in SEQ ID NO:01 or SEQ ID NO:04 or SEQ ID NO:07, or has at least 50%, 90%, or 90-99% sequence identity to the amino acid sequence set forth in SEQ ID NO:01 or SEQ ID NO:04 or SEQ ID NO:07.

[0037] In some embodiments of the above method, the nucleic acid sequence comprises a sequence as set forth in SEQ ID NOs:03 and 08-16, or SEQ ID NOs:06 and 17-25, or has at least 50% sequence identity to the nucleic acid sequence as set forth in SEQ ID NOs:03 and 08-16, or SEQ ID NOs:6 and 17-25.

[0038] In some embodiments of the above method, the deficiency of a polypeptide for degrading glycogen is GSD III, GSD I, GSD II, GSD IV, GSD V, GSD VI, GSD VII, GSD IX, GSD XI, GSD XII, GSD XIII, GSD XIV, Danon disease, Lafora disease, PRKAG2 (protein kinase gamma 2 subunit) deficiency, or other condition where there is cytoplasmic accumulation of glycogen. In other embodiments of the above method, the deficiency of a polypeptide for degrading glycogen is GSD III.

[0039] In some embodiments of the above method, the isolated nucleic acid molecule is administered intravenously, intramuscularly, intrathecally, intraventricularly, intraarterially, intraperitoneally, or directly into utero. [0040] In some embodiments of the above method, the subject is a human subject.

[0041] In some embodiments of the above method, the isolated nucleic acid molecule is delivered to areas of the body including but not limited to the liver, heart, skeletal and smooth muscle, kidney, and/or the central and peripheral nervous systems in the subject.

[0042] In some embodiments of the above method, the isolated nucleic acid molecule is present in a vector, which can be a viral vector or non-viral vector. In some embodiments of the above method, the viral vector is selected from the group consisting of an adenovirus vector, an AAV vector, a herpes simplex virus vector, a retrovirus vector, a lentivirus vector, and alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease virus vector, a poxvirus vector, or a picornavirus vector.

[0043] In some embodiments of the above method, the vector comprises a ubiquitous promoter. In other embodiment so the above method, the ubiquitous promoter is a CMV enhancer/chicken b-actin promoter.

[0044] In some embodiments of the above-method, the vector comprises a tissue-specific promoter. In some embodiments of the above method, the tissue-specific promoter is a liver-specific promoter, a muscle-specific promoter, a neuron-specific promoter, or a combination of any of the two or more thereof. In other embodiments of the above method, the vector comprises a liver-specific promoter that is the a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter. In other embodiments of the above method, the vector comprises an immunotolerant dual promoter consisting of a liver-specific promoter and a ubiquitous promoter.

[0045] In some embodiments, the above method further comprises administering to the subject a therapeutically effective amount of an immunosuppressive agent and/or a glycogen synthase inhibitor.

[0046] In other embodiments, the above method comprises the isolated nucleic acid molecule is present in a first vector and in a second vector, wherein the first vector and second vector are administered simultaneously or wherein the second vector is administered after the first vector. In some embodiments, the second vector is administered in the minutes, hours, days, weeks, or months after the first vector is administered. In some embodiments, the first vector is selected an AAV-LSP-Pull vector or an AAV-CB-Pull vector and wherein the second vector is an AAV-LSP-Pull vector or an AAV-CB-Pull vector.

[0047] Yet another aspect of the present disclosure provides a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide having fewer than about 4.0 kilobases, wherein a counterpart human nucleic acid sequence has a coding sequence that is greater than about 4.0 kilobases, and wherein the nucleic acid sequence is codon-optimized for expression in a human cell. [0048] Yet another aspect of the present disclosure provides a vector comprising a gene expression cassette comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a therapeutic protein under the control of an immunotolerant dual promoter consisting of a liver-specific promoter and a ubiquitous promoter. In some embodiments, the immunotolerant dual promoter comprises an a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter and a CMV enhancer/beta-actin (CB) promoter. In some embodiments, the immunotolerant dual promoter has the nucleic acid sequence as set forth in SEQ ID NO:30, or has at least 50% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:30.

[0049] In some embodiments, the therapeutic protein can be a variety of therapeutic proteins/enzymes and polypeptides that are used to treat a number of diseases, including microbial polypeptides for degrading glycogen and non-microbial proteins to treat human genetic diseases that affect multiple tissues.

[0050] Yet another aspect of the present disclosure provides a method of treating a deficiency of a polypeptide for degrading glycogen in a subject, comprising administering to the subject an a vector comprising a gene expression cassette comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide for degrading glycogen under the control of an immunotolerant dual promoter consisting of a liver-specific promoter and a ubiquitous promoter

[0051] Additional features and advantages are described herein, and will be apparent from the Drawings, Detailed Description, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.

[0053] FIG. 1A illustrates the generation of heterozygous Agl ^+/ mice by crossing the Agl ^Tmla mice with CMV-Cre mice to delete exons 6-10 in the Agl gene. FIG. 1 B illustrates that homozygous Agl ^/_ mice were used as breeders to produce Agl knockout (Agl-KO, Ag ^/_ , or GSD Ilia) mice.

[0054] FIG. 2 illustrates Western blotting with an anti-mouse GDE antibody showing the absence of GDE protein in tissues of the homozygous Agl ^/_ mice. Data are presented as mean ± SD; n=6 for each point.

[0055] FIG. 3 illustrates patterns of glycogen accumulation in tissues of GSD Ilia mice with age. Wild-type (Wt) values were indicated. Diaph, diaphragm; Gast, gastrocnemius; Quad, quadriceps. Data are presented as mean ± SD; n=6 for each point. [0056] FIG. 4 illustrates impaired muscle function of GSD Ilia mice compared with wild-type (WT) mice assessed by Treadmill, Wire-hang, and Rota-rod tests at 6 months of age. Data shown as Mean ± SD and analyzed with equal variance, unpaired, two-tailed Student's t test. n=5 each group. * indicates p<0.05; *** indicates p<0.001.

[0057] FIG. 5A - FIG. 5B show biochemical correction of glycogen storage in liver of GSD Ilia mice two or seven weeks following intravenous administration of the indicated AAV vectors. FIG.5A shows pullulanase activity. FIG. 5B shows glycogen content. UT, untreated GSD Ilia mice; AAV-CB-Pull or AAV-LSP-Pull, AAV-treated GSD Ilia mice; WT, age- matched wild-type control mice. Data shown as mean ± SD and analyzed with one-way ANOVA. n=5 for each GSD III group; n=4 for WT. **** indicates p<0.0001 vs UT.

[0058] FIG. 6 shows representative PAS-stained liver sections showing clearance of glycogen storage in livers of GSD Ilia mice two weeks following AAV treatment. UT, untreated GSD Ilia mice; AAV-CB-Pull or AAV-LSP-Pull, AAV-treated GSD Ilia mice; WT, wild-type mice.

[0059] FIG. 7A - FIG. 7B illustrate correction of liver symptoms in GSD Ilia mice two weeks after AAV treatment. FIG. 7A shows the ratios of liver to body weight. FIG. 7B shows liver enzyme ALT activities in plasma. UT, untreated GSD Ilia mice; AAV-CB-Pull or AAV-LSP- Pull, AAV-treated GSD Ilia mice; WT, wild-type mice. Data shown as Mean ± SD. n=5 for each GSD III group; n=4 for WT. *, p<0.05; ****, p<0.0001 vs UT.

[0060] FIG. 8 shows immunohistochemical detection of CD8 ⁺ and CD4 ⁺ lymphocytes in the liver of GSD Ilia mice two weeks after AAV administration. Sections of the paraffin- embedded livers from untreated (UT), AA9-CB-Pull treated, and AAV9-LSP-Pull treated GSD Ilia mice were stained with an anti-CD4 or anti-CD8a monoclonal antibody (Abeam). Foci of lymphocytes are indicated (arrows).

[0061] FIG. 9 illustrates AAV vector genome copy numbers in liver of GSD Ilia mice two or seven weeks after AAV treatment. UT, untreated GSD Ilia mice; AAV-CB-Pull or AAV-LSP- Pull, AAV-treated GSD Ilia mice. Data represent mean ± SD. n=5 for each condition. ***, pO.001 ; ****, pO.0001 vs UT.

[0062] FIG. 10A - FIG. 10B illustrate the correction of liver symptoms in GSD Ilia mice seven weeks after AAV treatment. FIG. 10A shows the ratios of liver to body weight. FIG. 10B shows plasma alanine aminotransferase (ALT) activities. UT, untreated GSD Ilia mice; AAV-CB-Pull or AAV-LSP-Pull, AAV-treated GSD Ilia mice; WT, wild-type mice. Data shown as Mean ± SD. n=5 for each GSD III group; n=4 for WT. *, p<0.05; ****, p<0.0001 vs UT.

[0063] FIG. 11 shows histological correction of liver disease in GSD Ilia mice seven weeks after AAV treatment. Upper panel: PAS stained liver sections show clearance of glycogen storage by the AAV-LSP-Pull treatment. Lower panel: Trichrome stained liver sections show the absence of blue-stained fibrotic tissues (arrows) in the AAV-LSP-Pull treated liver. [0064] FIG. 12A - FIG. 12B show AAV9-LSP-Pull reduced glycogen contents in liver of GSD Ilia dogs two weeks after vector injection. FIG. 12A shows detection of Pullulanase expression in liver by Western blot with an anti-HA antibody. FIG. 12B is a graph showing glycogen content in the liver biopsies was measured. Data shown are mean ± SD. Student's t-test. ***p<0.001.

[0065] FIG. 13A - FIG. 13B show AAV9-LSP-Pull significantly reduced liver size in GSD Ilia dogs two weeks after AAV treatment. FIG. 13A is radiographic images of a GSD Ilia dog showing reduced liver size after two weeks of treatment. The shape of the abdomen (dot lines) showed unusual curvatures before treatment due to hepatomegaly. However, the shape had less curvature after two weeks of treatment. FIG. 13B is a graph of the liver size during the pre-treatment and post-treatment periods. The length of the right liver lobe in dorsoventral radiographs was normalized with the length of the T11 bone. The normalized liver size was significantly reduced after two weeks of treatment. The graph represents the mean ± SD of n=2 GSD Ilia dogs. Student's t-test. **p<0.01.

[0066] FIG. 14A - FIG. 14B show biochemical correction of glycogen storage in tissues of GSD Ilia mice ten weeks after intravenous administration of AAV vectors at two weeks of age. FIG. 14A shows pullulanase activity. FIG. 14B shows glycogen content. UT, untreated GSD III mice (n=7); AAV-CB-Pull, AAV-treated GSD Ilia mice (n=8). Data shown as mean ± SD and analyzed with one-way ANOVA. n.s., no significant difference; ****, p<0.0001 vs UT.

[0067] FIG. 15 shows representative PAS-stained tissue sections showing clearance of glycogen storage in the heart and skeletal muscle of GSD Ilia mice ten weeks following AAV administration at two weeks of age. UT, untreated GSD Ilia mice; AAV-CB-Pull, AAV- treated GSD Ilia mice; WT, wild-type mice; Quad, quadriceps.

[0068] FIG. 16A - FIG. 16C show the effect of AAV-CB-Pull treatment on plasma enzyme activities and urinary Glc4 levels in the GSD Ilia mice ten weeks after intravenous administration of AAV vectors at two weeks of age. FIG. 16A shows plasma alanine aminotransferase (ALT) activity. FIG. 16B shows plasma creatine kinase (CK) activity. FIG. 16C shows urinary Glc4 concentration. UT, untreated GSD Ilia mice (n=7); AAV-CB-Pull, AAV-treated GSD Ilia mice (n=8). Data shown as mean ± SD. *, p<0.05 vs UT.

[0069] FIG. 17A - FIG. 17B show biochemical correction of muscle defects in GSD Ilia mice by AAV9-MHCK7-Pull treatment. FIG. 17A is a graph showing pullulanase activities in the liver, heart and quadriceps two weeks following AAV injection. FIG. 17B is a graph showing glycogen content in the liver, heart, at quadriceps two weeks following AAV injection. UT, untreated GSD Ilia mice (n=9); AAV, AAV9-MHCK7-Pull treated GSD Ilia mice (n=5). Data shown as mean ± SD. Student's test. **p<0.01 , ****p<0.0001.

[0070] FIG. 18A - FIG. 18B show improvement of muscle function by the AAV-MHCK7-Pull treatment. FIG. 18A is a graph showing the maximum running of mice treated with AAV- MHCK7-Pull (AAV) and untreated (UT) mice as evaluated by a treadmill test to assess exercise intolerance. FIG. 18B is a graph showing the time to latency of mice treated with AAV-MHCK7-Pull (AAV) and untreated (UT) mice as evaluated by a wire-hand test to assess limb muscle strength.

[0071] FIG. 19A - FIG. 19E show AAV-mediated gene delivery of Pullulanase in the liver and muscles of GSD Ilia mice. FIG. 19A is a schematic that shows the constructs of AAV vector containing the 2.2 kb codon-optimized Pullulanase cDNA under the control of the ubiquitous CMV enhancer/chicken b-actin promoter (CB) and the a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin liver specific promoter (LSP). ITR: inverted terminal repeats; polyA: human growth hormone polyadenylation signal sequence. FIG. 19B is a schematic that shows an experimental flow chart of AAV treatments in GSD Ilia mice. Fifteen mice were injected with AAV9-CB-Pull at two weeks of age at a dose of 1x10 ¹³ vg/kg. Eight mice were sacrificed after 10 weeks of treatment (3-month-old, gray arrow) for sample collection; the remaining seven mice received a second AAV injection with AAV8-LSP-Pull at the same dose. Tissues and plasma were analyzed after another 10 weeks (5-month-old, black arrow). Behavioral tests were performed at ages 2, 2.5, 3, 4, and 5 months old (open arrows). FIG. 19C is a graph showing the AAV genome copy numbers of AAV9-CB-Pull (CB) treated mice at 3 months of age and after subsequent treatment with AAV8-LSP-Pull 10 weeks later (CB+LSP). AAV genome copy numbers were evaluated by real-time-PCR using primers for Pullulanase. The graph represents the mean ± SD. n=5 for each group vg/genome: vector genome copies per nucleus (genome). FIG. 19D is a Western blot analysis using an anti-HA antibody confirmed the expression of Pullulanase in tissues of the AAV treated GSD Ilia mice. Pullulanase was undetectable in any tissues of the untreated (UT) mice. Pullulanase was not detectable in the liver of the CB treated mice but was highly expressed in the liver of CB+LSP treated mice. The heart showed a high level and skeletal muscle showed a low level (but detectable) of Pullulanase in both CB and CB+LSP treated mice. b-Actin was used as a loading control. FIG. 19E is a graph showing pullulanase activity in the liver, heart, and skeletal muscle in mice treated with AAV9-CB-Pull alone (CB) and with an additional treatment with AAV8-LSP-Pull 10 weeks later (CB+LSP) as compared to untreated (UT) mice using the Pullulanase activity kit (Megazyme). Consistent with the western blot results, the enzyme activity was not detectable in the CB treated liver but drastically increased in the CB+LSP treated liver. The enzyme activity was significantly elevated in the heart and skeletal muscle of both CB and CB+LSP treated mice compared to the UT mice. The graph represents the mean ± SD. n=5 for UT, n=8 for CB, and n=7 for CB+LSP; Student's test. *p<0.05, ***p<0.001 , and ****p<0.0001.

[0072] FIG. 20A - FIG. 20D show AAV-mediated pullulanase expression reduced glycogen accumulation in the liver and muscles and reversed liver fibrosis in GSD Ilia mice. FIG. 20A is a graph showing glycogen contents in the liver, heart, and skeletal muscle of mice treated with AAV9-CB-Pull alone (CB) or with an additional treatment with AAV8-LSP-Pull

(CB+LSP). Consistent with the enzyme activity results, there is no difference in liver glycogen content between the UT and CB treated GSD Ilia mice at 3 months of age, but glycogen level was reduced significantly in the CB+LSP treated liver at 5 months of age. Glycogen content was profoundly decreased in the heart and skeletal muscle of both CB and CB+LSP treated mice. The graph represents the mean ± SD. n=5 for UT, n=8 for CB, and n=7 for CB+LSP; Student's test. ***p<0.001 and ****p<0.0001. FIG. 20B is histology images of wild type, untreated (UT), and mice treated with AAV9-CB-Pull alone (CB) or with a subsequent treatment with AAV8-LSP-Pull (CB+LSP). PAS staining was used for histological detection of glycogen accumulation in tissues. Untreated GSD Ilia mice showed intense PAS-positive glycogen accumulation (dark staining) in the liver, heart, and skeletal muscle. CB treatment had no effect on liver glycogen accumulation; in contrast, CB+LSP treatment markedly reduced glycogen accumulation in the liver. Glycogen buildup was profoundly cleared in the heart and skeletal muscle by both CB and CB+LSP treatments. At least 3 mice were examined in each group, and representative images are shown. Scale bar=50 pm. FIG. 20C is histology images of wild type (WT), untreated (UT), and mice treated with AAV9-CB-Pull (CB). Glycogen level was assessed by PAS staining in tissues of WT mice and UT and CB treated GSD Ilia mice at 3 months of age. Untreated GSD Ilia mice showed massive glycogen staining in the skeletal muscles (gastrocnemius, soleus, diaphragm, and tongue) and smooth muscle (bladder) and a mild but significant amount of glycogen accumulation in some regions of the brain (cerebellum) and spinal cord. CB treatment markedly reduced glycogen accumulation in all the skeletal muscles but had no effect on the smooth muscle, brain, and spinal cord. At least 3 mice were examined in each group, and representative images are shown. Scale bar=50 pm. FIG. 20D is histology images showing trichrome staining, which is used for detection of liver fibrosis. The blue staining (arrows) indicates the presence of fibrotic tissues in the liver of UT and CB treated GSD Ilia mice. CB+LSP treatment successfully reversed liver fibrosis. At least 3 mice were examined in each group, and representative images are shown. Scale bar=50 pm.

[0073] FIG. 21 A - FIG. 21 D show that pullulanase improved liver and muscle function. FIG. 21 A is a graph showing liver to body weight (%). The ratio of liver to body weight was measured to determine hepatomegaly. The ratio did not change by the CB alone treatment. However, the liver size was significantly reduced by the combination CB+LSP treatment.

The dotted line represents the level of liver to body weight ratio in WT mice (4.5±0.2%, n=6). The graph represents the mean ± SD. n=7 for UT (3mo) and CB+LSP, n=8 for CB, and n=5 for UT (5mo). Each dot/triangle represents an individual mouse. Student's test. *p<0.05. FIG. 21 B is a graph showing activity of alanine aminotransferase (ALT). The activity of alanine aminotransferase (ALT) was measured in the plasma from UT and AAV treated GSD Ilia mice to evaluate liver function. The ALT level was slightly reduced (p=0.09) in the CB treated mice compared to UT. The ALT level was remarkably increased in UT mice from age 3 to 5 months. CB+LSP treatment significantly reduced plasma ALT activity compared with UT mice. The graph represents the mean ± SD. n=7 for UT (3mo) and CB+LSP, n=8 for CB, and n=5 for UT (5mo). Each dot/triangle represents an individual mouse. The dotted line represents the ALT level in WT mice (98.1 ±33.24 U/L, n=4). Student's test. *p<0.05 and ****p<0.0001. FIG. 21 C is a graph showing the Glc4 urinary concentrations. The

concentrations of urinary Glc4 were assessed in the UT and AAV-CB-Pull (CB) treated GSD Ilia mice. The CB treated mice showed a significant decrease in the level of urinary Glc4 compared to the UT mice. The urinary creatinine level was used for normalization. The graph represents the mean ± SD. n=6 for UT and n=8 for CB. Each dot represents an individual mouse. The dotted line represents the WT level (4.4±1.7 mmol/mol of creatinine, n=7). Student's test. *p<0.05. FIG. 21 D is graphs showing the results of the behavioral tests in untreated (UT) and AAV treated mice. Treadmill test was used for evaluating exercise intolerance in UT and AAV treated GSD Ilia mice. The graph represents the maximum running distance. The running distance gradually declined in the UT mice with time (UT, dots). However, the running distance was steady in the AAV treated mice (AAV, squares). Wire hang test was used to assess limb muscle strength. The time of latency to fall was measured and the maximum time from three independent trials was used for comparison. The hanging time was also progressively decreased in UT mice with age (UT, dots), but AAV treatments significantly improved the hanging time (AAV, squares). Rota-rod test was used to measure muscle strength, motor coordination, and balance. The mice were tested during three sessions using the accelerating Rota-rod protocol (4.0-40 rpm), and the latency to fall was recorded. AAV treated mice also showed significant improvement in Rota-rod performance compared to the UT mice. The data represents the mean ± SD. n=7 for both groups. Student's test. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001.

[0074] FIG. 22 shows the protein sequence of pullulanase derived from Bacillus subtilis strain 168 (718 amino acids). Gene: amyX. UniProtKB # C0SPA0; NCBI Database # NP_390871.2 (SEQ ID NO:01).

[0075] FIG. 23 shows the cDNA sequence of pullulanase derived from Bacillus subtilis strain 168 (2157 bp). Gene: amyX. NCBI Reference # NC_000964.3 (SEQ ID NO:02).

[0076] FIG. 24 shows the codon optimized coding sequence for expressing pullulanase derived from Bacillus subtilis strain 168 in human cells (SEQ ID NO:03).

[0077] FIG. 25 shows the protein sequence of the bacterial limit dextrin alpha-1 , 6- glucohydrolase (GlgX) derived from E. coli strain K-12 (657 amino acids). Gene: glgX. UniProt # P15067. NCBI Database # NP_417889.1 (SEQ ID NQ:04). [0078] FIG. 26 shows the cDNA sequence of the limit dextrin alpha-1 , 6-glucohydrolase (GlgX) derived from E. coli strain K-12 (1974 bp). NCBI Reference # NP_417889.1 (SEQ ID NO:05).

[0079] FIG. 27 shows the codon optimized coding sequence for expressing the bacterial limit dextrin alpha-1 , 6-glucohydrolase (GlgX) derived from Escherichia coli strain K-12 in human cells (SEQ ID NO:06).

[0080] FIG. 28 shows the protein sequence of isoamylase derived from Pseudomonas amyloderamosa SB-15 (776 amino acids). Uniprot # P10342.3 (SEQ ID NO:07).

[0081] FIG. 29A - FIG. 29B show AAV treatment groups and AAV vector bio-distribution. FIG. 29A is a schematic that shows the constructs of indicated AAV vectors and the treatment groups with these AAV vectors. FIG. 29B is graphs showing the AAV copy numbers in the liver, heart, and quadriceps of GSD Ilia mice ten weeks after AAV treatment. CB, AAV9-CB-Pull injection; LSP, AAV9-LSP-Pull injection; Co, AAV9-CB-Pull + AAV9-LSP- Pull co-injection; Dual, AAV9-LSP-CB-Pull injection. Data shown as mean ± SD. n=5 for each group.

[0082] FIG. 30A - FIG. 30B show long-term Pullulanase expression and glycogen reduction by the LSP-CB dual promoter in the major affected tissues of GSD Ilia mice. FIG. 30A is a graph of pullulanase activities in liver, heart, and skeletal of GSD Ilia mice ten weeks after AAV treatment. FIG. 30B is a graph of glycogen contents in tissues in FIG. 30A. UT, untreated GSD Ilia mice; CB, AAV9-CB-Pull injection; LSP, AAV9-LSP-Pull injection; Co, AAV9-CB-Pull and AAV9-LSP-Pull co-injection; Dual, AAV9-LSP-CB-Pull injection. Data shown as mean ± SD. n=5 for each group. Student's test. *p<0.05,**p<0.01 , ***p<0.001 , and ****p<0.0001 vs UT.

[0083] FIG. 31 shows PAS-stained tissue sections, which confirmed the clearance of glycogen accumulation in tissues of GSD Ilia mice ten weeks after AAV-LSP-CB-Pull treatment. UT, untreated GSD Ilia mice; CB, AAV9-CB-Pull injection; LSP, AAV9-LSP-Pull injection; Co, AAV9-CB-Pull + AAV9-LSP-Pull co-injection; Dual, AAV9-LSP-CB-Pull injection. Scale bar = 50 pm. The images represent at least three mice in each group

[0084] FIG. 32A - FIG. 32D show the LSP-CB dual promoter is as effective as the LSP promoter in correcting liver abnormalities in GSD Ilia mice. FIG. 32A is a graph of the ratio of liver to body weight, which was measured to determine liver size. FIG. 32B is a graph showing the activity of alanine aminotransferase (ALT) in plasma, which were measured ten weeks after treatment to evaluate liver damage. FIG. 32C is a graph showing the activity of aspartate aminotransferase (AST) in plasma, which were measured ten weeks after treatment to evaluate liver damage. Data shown as mean ± SD. n=5 for each group.

Student's test. *p<0.05, **p<0.01 , and ***p<0.001 vs UT. FIG. 32D is trichrome staining of liver sections for the detection of liver fibrosis. Stained fibrotic tissues is observed in livers of UT, CB-treated, and Co-treated mice but invisible in livers of LSP-treated and LSP-CB (Dual)-treated mice. Scale bar = 50 pm. The images represent at least three mice in each group.

[0085] FIG. 33 is a graph showing that AAV-LSP-CB-Pull treatment significantly improved muscle function in GSD Ilia mice as evidenced by treadmill test. The graph represents the maximum running distance. Data shown as mean ± SD. n=5 for each group at each time point. Student's test. ***p<0.001 , and ****p<0.0001 vs UT.

[0086] FIG. 34 shows immunohistochemical detection of cytotoxic T cell immune responses in liver of GSD Ilia mice. Paraffin-embedded liver sections two weeks following AAV treatment were stained with anti-CD4 or CD8a antibodies. Infiltration of CD4+ or CD8+ lymphocytes (black arrows) increased in the CB-treated and Co-treated livers but barely visible in LSP- and LSP-CB- treated livers (arrowheads). The images represent at least three mice in each group. Scale bar = 50 pm.

[0087] FIG. 35 is a schematic depicting the AAV vectors used in Example 9. The AAV-CB- hGBE vector contains a ubiquitous CMV-enhanced chicken beta-actin hybrid promoter (CB) and the AAV-LSPCB-hGBE vector contains a dual LSP and CB fusion promoter. ITR, inverted repeats; polyA, human growth hormone polyA signal sequence.

[0088] FIG. 36A - FIG. 36B show AAV-CB-hGBE and AAV-LSPCB-hGBE plasmids are over-expressed hGBE in HEK293 cells. Cells in 10-cm plates were transfected with 10 pg plasmid per plate. Cells were harvested 48h after transfection. FIG. 36A is a Western blot showing expression of hGBE from the two plasmids. Twenty pg total protein was loaded per lane. FIG. 36B is a graph of GBE activity in cell lysates. UT, untreated HEK293 cells.

[0089] FIG. 37 is graphs that show expression of hGBE in the liver, heart, and muscle of adult Gbe1 ^ys/ys mice. Liver, heart and muscle (quadriceps) tissues were collected 2 weeks after AAV injection and GBE activities were measured in tissue lysates. N=3 mice for each group; *p<0.01.

[0090] FIG. 38 shows immunohistochemical detection of cellular immune responses in liver of Gbe1 ^ys/ys mice two weeks post AAV administration. AAV-CB-hGBE treatment apparently induced significant cellular immune response as shown by heavily stained CD4 ⁺ and CD8 ⁺ foci of lymphocytes (arrows), while AAV-LSPCB-hGBE showed apparently fewer CD4 ⁺ and CD8 ⁺ cells. The images represent at least three mice in each group.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0091] Before the disclosed processes and materials are described, it is to be understood that the aspects described herein are not limited to specific embodiments, apparati, or configurations, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0092] Throughout this specification, unless the context requires otherwise, the word “comprise” and“include” and variations (e.g.,“comprises,”“comprising,”“includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.

[0093] As used in the specification and the appended claims, the singular forms“a,”“an” and“the” include plural referents unless the context clearly dictates otherwise.

[0094] Ranges can be expressed herein as from“about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another aspect. 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.

[0095] The term“about” in association with a numerical value means that the numerical value can vary plus or minus by 5% or less of the numerical value.

[0096] The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and“consisting of” can be replaced with either of the other two terms, while retaining their ordinary meanings.

[0097] As used herein, the term“contacting” includes the physical contact of at least one substance to another substance.

[0098] As used herein,“treatment,”“therapy” and/or“therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.

[0099] The term“effective amount” or“therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results. Alternatively stated, a“therapeutically effective” amount is an amount that will provide some alleviation, mitigation or decrease in at least one clinical symptom in the subject. For example, in the case of a deficiency of a polypeptide for degrading glycogen, an amount that provides some alleviation, mitigation or decrease in at least one clinical symptom of a deficiency of a polypeptide for degrading glycogen (e.g., reduced glycogen stores in liver, skeletal, cardiac muscles, nervous system, prevented hepatic fibrosis and cirrhosis, improved muscle strength and function, improved motor development or attainment of motor developmental milestones, prevention of cardiac arrhythmias and cardiac failure, prevention of neuropathy and the like). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0100] A“reduction in glycogen stores” in a tissue is intended to indicate about a 2%, 5%, 10%, 15%, 20%, 25%, 35%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, or more reduction in total glycogen in a particular tissue, unless otherwise indicated.

[0101] As used herein, the terms“express” or“expression” of a nucleic acid coding sequence, in particular a coding sequence for a microbial polypeptide for degrading glycogen, it is meant that the sequence is transcribed, and optionally, translated. Generally, however, according to the present disclosure, the term“express” or“expression” is intended to refer to transcription and translation of the coding sequence resulting in production of the encoded polypeptide.

[0102] By“enhanced” or“enhancement” with respect to nucleic acid expression or polypeptide production, it is meant an increase and/or prolongation of steady-state levels of the indicated nucleic acid or polypeptide, e.g., by at least about 2%, 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 2-fold, 2.5-fold, 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 100-fold or more.

[0103] As used herein, the term“subject” and“patient” are used interchangeably herein and refer to both human and nonhuman animals. The term“nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The subject can be a human patient that is at risk for, or suffering from, a GSD, in particular GSD III or any other conditions where there is an abnormal accumulation of glycogen (both in terms of quality and quantity). The subject can also be a human patient that is at risk for, or suffering from, a disease caused by a deficiency of a protein or enzyme in the body caused by a large defective gene (e.g., a gene having greater than about 5 kilobases). The human patient can be of any age (e.g., an infant, child, or adult). The human patient can also be an infant that has an underdeveloped immune system. The human patient can also be a fetus in utero.

[0104]“Recombinant” is used herein to refer to new combinations of genetic material as a result of genetic engineering. For instance, a recombinant organism (e.g., bacteria) can be an organism that contains different genetic material from either of its parents as a result of genetic modification, recombinant DNA can be a form of artificial DNA, a recombinant protein or enzyme can be an artificially produced and purified form of the protein or enzyme, and a recombinant virus can be a virus formed by recombining genetic material. [0105] As used herein, the term“disease” refers to any condition that is abnormal, such as a disorder or a structure or function that affects part or all of a subject.

[0106] Gene Therapy Using Microbial Glycogen Debranchinq Enzyme (GDE)

[0107] The inventors have discovered a gene therapy method of treating a glycogen storage disease by administering a vector containing a coding sequence codon optimized for expressing a therapeutic microbial GDE in human cells to patients suffering from the disease. These vectors can further comprise a tissue-specific promoter (e.g., a liver-specific promoter, a muscle-specific promoter, a neuron-specific promoter, or a combination of any of the two or more thereof) or an immunotolerant dual promoter consisting of a liver-specific promoter and a ubiquitous promoter to reduce the risk of an immune response to the microbial protein.

[0108] As described in more detail below, the present disclosure provides improved polypeptides having glycogen degrading enzymatic activity to treat GSDs, which are autosomal recessive disorders or X-linked disorders. The problem underlying GSDs is that the subject has an absence or deficiency in an enzyme that is responsible for making or breaking down glycogen in the body. The enzyme deficiency in GSDs causes either abnormal concentrations of glycogen or abnormally formed glycogen or both in the affected tissues. Depending on the type of GSD, the subject can have an enzyme deficiency in all parts of the body, or only in some parts of the body (e.g., liver, muscle, heart tissues, or nervous system).

[0109] Notably, the data described herein indicate that gene therapy with vectors expressing a microbial GDE can be successfully used to treat GSDs and the cellular immune responses induced by the microbial polypeptides can be overcome by using a tissue-specific promoter or an immunotolerant dual promoter.

[0110] Furthermore, the present disclosure provides for successful transduction of vectors carrying a therapeutic bacterial GDE or other therapeutic proteins that are capable of being packaged in a vector, without eliciting any immune responses against the therapeutic protein.

[0111] GSDs include, but are not limited to, GSD III, GSD II or Pompe Disease, GSD I, GSD IV, GSD V, GSD VI, GSD VII, GSD IX, GSD XI, GSD XII, GSD XIII, GSD XIV, Danon disease, Lafora disease, or PRKAG2 (protein kinase gamma 2 subunit) deficiency. Glycogen storage diseases can also include any other condition where there is cytoplasmic

accumulation of glycogen.

[0112] The gene therapy methods described herein can offer several advantages over enzyme replacement therapy (ERT). ERT involves treating a patient with an intravenous infusion of a solution containing the enzyme that is deficient in the patient, whereas gene therapy involves delivering a gene (e.g., cDNA) encoding the deficient enzyme into the affected cells of a patient via a delivery vector where the gene can then express a functioning enzyme in the patient. Unlike ERT or small molecule therapy, gene therapy can provide equal or better outcomes while requiring typically only a single administration, which further reduces the risk of an immune response. Furthermore, ERT/small molecule therapy could be used in combination with gene therapy at a lower dose. Finally, gene therapy provides a cost savings to the patient and the convenience of fewer administrations.

[0113] Unlike its human counterpart, bacterial GDE does not have glycosyltransferase activity, but has only amylo-a-1 , 6-glucosidase activity, and can directly hydrolyze a-1 , 6- glycosidic bonds at the branching points in limit dextrin to release maltotetraose (4-glucose) molecules, thus performing a similar function as human GDE.

[0114] GDEs can be found in many species and strains of bacteria. GDEs can differ in sequence, but they have the same amylo-a-1 , 6-glucosidase hydrolyzing activity.

[0115] A GDE can be derived from any microorganism. As used herein, the term

“microorganism” refers to an organism that can only be seen through a microscope. A microorganism can include bacteria, protozoa, algae, fungi (e.g., yeast), and viruses.

[0116] Examples of GDE, can include, but are not limited to, pullulanase (EC 3.2.1.41) (e.g., type I pullulanase, type II pullulanase (amylopullalanase), type III pullulan hydrolase), limit dextrin alpha-1 , 6-hydrolase (GlgX) (E.C.3.2.1.-) encoded by the gene glgX, and isoamylase (EC 3.2.1.68).

[0117] Pullanase (EC 3.2.1.41) enzymes can be found in a variety of microbial species, including, but not limited to, Anaerobranca gottschalkii, Anoxybacillus sp., Anoxybacillus sp. SK3-4, Aureobasidium pullulans, Avena sativa, Bacillus acidopullulyticus, Bacillus cereus, Bacillus cereus FDTA 13, Bacillus circulans, Bacillus deramificans, Bacillus subtilis, Bacillus subtilis strain 168, thermophilic Bacillus sp. AN-7, Bacteroides thetaiotaomicron, Beta vulgaris, Desulfurococcus mucosus, Exiguobacterium acetylicum, Exiguobacterium acetylicum YH5, Exiguobacterium sp., Exiguobacterium sp. Sh3, Fervidobacterium pennivorans, Fervidobacterium pennivorans Ven5, Geobacillus stearothermophilus,

Geobacillus thermoleovorans, Geobacillus thermoleovorans US105, Halorubrum sp. Ha25, Hordeum vulgare, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella planticola, Klebsiella pneumonia, Klebsiella pneumoniae UNF5023, Laceyella sacchari, Lactococcus lactis, Lactococcus lactis IBB500, Micrococcus sp., Nostoc punctiforme, Oryctolagus cuniculus, Oryza sativa, Paenibacillus macerans, Pullulanibacillus naganoensis, Pyrococcus furiosus, Pyrococcus woesei, Raoultella planticola, Rhodothermus marinus, Saccharomyces cerevisiae, Spinacia oleracea, Streptococcus pyogenes, Streptococcus sp., Sulfolobus acidocaldarius, Sulfolobus acidocaldarius DSM 639, Thermoanaerobacter acetoethylicus, Thermoanaerobacter brockii subsp. Finnii, Thermoanaerobacter ethanolicus,

Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium saccharolyticum NTOU1 , Thermoanaerobacterium

thermosaccharolyticum, Thermoanaerobium sp., Thermococcus kodakarensis,

Thermococcus litoralis, Thermococcus litoralis DSM 5473, Thermococcus siculi,

Thermococcus siculi HJ21 , Thermotoga neapolitana, Thermus aquaticus, Thermus caldophilus, Thermus thermophiles, and Thermus thermophilus HB8 / ATCC 27634 / DSM 579. In some embodiments, pullanase is derived from Bacillus subtilis, Bacillus subtilis strain 168.

[0118] Limit dextrin alpha-1 , 6-hydrolase (GlgX) (E.C.3.2.1.-) enzymes can be found in a variety of microbial species, including, but not limited to Corynebacterium glutamicum, Corynebacterium glutamicum ATCC 13032, Escherichia coli, Escherichia coli BW25113, Escherichia coli K-12, Rhizobium tropici, Rhizobium tropic i PRF 81 , Synechococcus elongates, and Synechococcus elongatus PCC 6803. In some embodiments, limit dextrin alpha-1 , 6-hydrolase is derived from Escherichia coli K-12.

[0119] Isoamylase (EC 3.2.1.68) enzymes can be found in a variety of microbial species, including, but not limited to Amaranthus hybridus subsp. cruentus, Arthrobactersp., Arthrobacter sp. Q36, Bacillus sp., Chlamydomonas reinhardtii, Chlamydomonas reinhardtii 330, Cytophaga sp., Dickeya chrysanthemi, Dickeya chrysanthemi PY35, Flavo bacterium sp., Lipomyces kononenkoae, Musa acuminate, Myroides odoratus, Pseudomonas amyloderamosa, Pseudomonas amyloderamosa JD210, Pseudomonas amyloderamosa MI- 414, Pseudomonas amyloderamosa SB-15, Pseudomonas amyloderamosa SMP1 ,

Pseudomonas amyloderamosa WU-5315, Pseudomonas amyloderamosa WU7211-2, and Pseudomonas sp. In other embodiments, the isoamylase can be derived from Pseudomonas amyloderamosa SB- 15.

[0120] The gene therapy methods described herein can be applied to any protein or enzyme from a microbial species that is smaller (e.g., contains fewer nucleobases) than its human counterpart protein or enzyme such that it is more suited for packaging in vectors that have the capacity to carry only smaller genes.

[0121] Furthermore, the gene therapy compositions and methods described herein that utilize a microbial polypeptide for degrading glycogen can be used to treat not only GSDs, but also other disorders caused by a large defective gene.

[0122] Isolated Nucleic Acids

[0123] The present disclosure provides, in part, an isolated nucleic acid molecule, comprising a nucleic acid sequence encoding a microbial polypeptide for degrading glycogen, wherein the nucleic acid is codon-optimized for expression in a mammalian or a human cell.

[0124] As used herein, the term“isolated” nucleic acid molecule (e.g., an isolated DNA, isolated cDNA, or an isolated vector genome) means a nucleic acid molecule separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid.

[0125] Likewise, an“isolated” polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

[0126] The term“nucleotide” refers to sequences with conventional nucleotide bases, sugar residues and internucleotide phosphate linkages, but also to those that contain modifications of any or all of these moieties. The term“nucleotide” as used herein includes those moieties that contain not only the natively found purine and pyrimidine bases adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U), but also modified or analogous forms thereof. Polynucleotides include RNA and DNA sequences of more than one nucleotide in a single chain. Modified RNA or modified DNA, as used herein, refers to a nucleic acid molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occurs in nature.

[0127] The term“nucleic acid” or“nucleic acid molecule” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated, for example, by a polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any one or more of ligation, scission, endonuclease action, or exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally-occurring nucleotides), or a combination thereof. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include

phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, morpholino, or the like. Nucleic acid molecules can be either single stranded or double stranded.

[0128] The term“microbial polypeptide” refers to a polypeptide that is from a microorganism species (e.g., a bacterial species).

[0129] In some embodiments, the microbial polypeptide is a therapeutic glycogen debranching enzyme (GDE) from bacteria or another microorganism. In other embodiments, the microbial polypeptide has debranching enzyme activity that can cleave the a-1 , 6- glycosidic bonds in glycogen and/or limit dextrin (e.g., type I pullulanase, limit dextrin alpha- 1 , 6-hydrolase (GlgX), isoamylase, and derivatives thereof). In other embodiments, the microbial polypeptide can cleave both the a-1 , 6-glycosidic bonds and a-1 , 4-glycosidic bonds in glycogen (e.g., type II pullulanase or type III pullulan hydrolase, and derivatives thereof).

[0130] In some embodiments, the microbial polypeptide has an amino acid sequence as set forth in SEQ ID NO:01 or SEQ ID NO:04 or SEQ ID NO:07, or has at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:01 or SEQ ID NO:04 or SEQ ID NO:07. In other embodiments, the microbial polypeptide has at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:01 or SEQ ID NO:04 or SEQ ID NO:07.

[0131] In some embodiments, the nucleic acid sequence has a sequence as set forth in SEQ ID NO:03, SEQ ID NO:06, or SEQ ID NOS:08-25, or has at least 50%, 60%, 70%,

80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:03, SEQ ID NO:06, or SEQ ID NOS:08-25.

[0132] The term“sequence identity” refers to the number of identical or similar nucleotide bases on a comparison between a test and reference oligonucleotide or nucleotide sequence. Sequence identity can be determined by sequence alignment of nucleic acid to identify regions of similarity or identity. As described herein, sequence identity is generally determined by alignment to identify identical residues. Matches, mismatches, and gaps can be identified between compared sequences. Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions/length of the total aligned sequence x 100. In one non-limiting embodiment, the term“at least 90% sequence identity to” refers to percent identities from 90 to 100%, relative to the reference nucleotide sequence. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplary purposes a test and reference oligonucleotide length of 100 nucleotides are compared, no more than 10% (i.e., 10 out of 100) of the nucleotides in the test oligonucleotide differ from those of the reference oligonucleotide. Differences are defined as nucleic acid substitutions, insertions, or deletions.

[0133] The term“codon optimized” relates to the alteration of codons in nucleic acid molecules to reflect the typical codon usage of the host organism (e.g., mammals such as humans) without altering the polypeptide encoded by the DNA, to improve expression. Many methods and software tools for codon optimization have been reported previously. See, for example, genomes.urv.es/OPTIMIZER/; Puigbo et aL, Nucleic Acids Res. (2007) (Web Server issue): W126-W131 ; Chin et al. (2014) Bioinformatics, 30(15):2210-2; Fuglsang, (2003) Protein Expr Purit, 31(2):247-9; Narum et al., (2001) Infect. Immun., 69(12):7250- 7253, Outchkourov et al., (2002) Protein Expr. Purif, 24(1): 18-24, Feng et al., (200) Biochemistry , 39(50):15399-15409, Humphreys et ai, (2000) Protein Expr. Purif., 20(2):252- 64.

[0134] Table 1 below provides exemplary codon optimization for gene expression in humans.

[0135] In some embodiments, the nucleic acid sequence encoding a microbial polypeptide has a coding sequence that is less than about 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, or 5.5 kilobases (kb).

[0136] Table 2. Potential sequences related to this disclosure.

[0137] Nucleic Acid Vectors

[0138] Another aspect of the present disclosure provides a vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide for degrading glycogen, wherein the nucleic acid is codon optimized for expression in mammalian cells or mammals such as humans.

[0139] As described herein, an AAV vector containing a universally active CMV enhanced chicken beta-actin hybrid (CB) promoter (AAV-CB-Pull) or a liver-specific promoter (AAV- LSP-Pull) and a 2.2 kb coding sequence codon optimized for expressing a type I Pullulanase (derived from Bacillus subtilis strain 168) in human cells were constructed. The results showed that a ten-week treatment by intravenous administration of the AAV-CB-Pull vector in infant GSD Ilia mice (at two weeks of age) effectively reduced glycogen contents in skeletal muscle and heart without provoking Pullulanase-induced immune responses (immune response is impaired in infant mice during the first few weeks after birth). When the GSD Ilia mice were treated at an adult age (ten weeks), the AAV-LSP-Pull treatment markedly reduced liver glycogen storage and effectively prevented hepatic fibrosis. In contrast, the AAV-CB-Pull treatment in adult mice induced strong cytotoxic T cell responses and eventually lost treatment efficacy 7 weeks post treatment.

[0140] As further described herein, a combination treatment with the AAV-CB-Pull at two weeks of age and the AAV-LSP-Pull at 3 months of age (CB+LSP) effectively reduced glycogen accumulation in both liver and muscle and recovered liver and muscle functions in GSD Ilia mice. Furthermore, as described herein, an AAV vector having an immunotolerant LSP-CB dual promoter (consisting of the liver-specific promoter and the ubiquitous CB promoter) encoding a microbial polypeptide or a non-microbial polypeptide prevented cytotoxic T cell responses in both GSD Ilia and GSD IV mouse models, indicating that this immunotolerant dual vector system can be used to treat diseases having multisystem involvement (e.g., Pompe disease).

[0141] It will be apparent to those skilled in the art that any suitable vector can be used to deliver the isolated nucleic acids of the disclosure to the target cell(s) or subject of interest. The choice of delivery vector can be made based on a number of factors known in the art, including age and species of the target host, in vitro vs. in vivo delivery, level and

persistence of expression desired, intended purpose (e.g., for therapy or enzyme

production), the target cell or organ, route of delivery, size of the isolated nucleic acid, safety concerns, and the like.

[0142] Any suitable vector known in the art can be used to deliver, and optionally, express the isolated nucleic acids of the disclosure (e.g., viral and non-viral vectors), including, virus vectors (e.g., retrovirus, adenovirus, AAV, or herpes simplex virus), lipid vectors, poly-lysine vectors, synthetic polyamino polymer vectors that are used with nucleic acid molecules, such as a plasmid, and the like. In some embodiments, the non-viral vector can be a polymer based vector (e.g., polyethyleimine (PEI), chitosan, poly (DL-Lactide) (PLA), or poly (DL- lactidie-co-glycoside) (PLGA), dendrimers, polymethacrylate) a peptide based vector, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid based vector.

[0143] Any viral vector that is known in the art can be used in the present disclosure.

Examples of such viral vectors include, but are not limited to vectors derived from:

Adenoviridae; Birnaviridae; Bunyaviridae; Caliciviridae, Capillovirus group; Carlavirus group; Carmovirus virus group; Group Caulimovirus; Closterovirus Group; Commelina yellow mottle virus group; Comovirus virus group; Coronaviridae; PM2 phage group; Corcicoviridae; Group Cryptic virus; group Cryptovirus; Cucumovirus virus group family ([PHgr]6 phage group; Cysioviridae; Group Carnation ringspot; Dianthovirus virus group; Group Broad bean wilt; Fabavirus virus group; Filoviridae; Flaviviridae; Furovirus group; Group Germinivirus; Group Giardiavirus; Hepadnaviridae; Herpesviridae; Hordeivirus virus group; lllarvirus virus group; Inoviridae; Iridoviridae; Leviviridae; Lipothrixviridae; Luteovirus group; Marafivirus virus group; Maize chlorotic dwarf virus group; icroviridae; Myoviridae; Necrovirus group;

Nepovirus virus group; Nodaviridae; Orthomyxoviridae; Papovaviridae; Paramyxoviridae; Parsnip yellow fleck virus group; Partitiviridae; Parvoviridae; Pea enation mosaic virus group; Phycodnaviridae; Picornaviridae; Plasmaviridae; Prodoviridae; Polydnaviridae; Potexvirus group; Potyvirus; Poxyiridae; Reoviridae; Retroviridae; Rhabdoviridae; Group Rhizidiovirus; Siphoviridae; Sobemovirus group; SSV 1-Type Phages; Tectiviridae; Tenuivirus;

Tetraviridae; Group Tobamovirus; Group Tobravirus; Togaviridae; Group Tombusvirus; Group Torovirus; Totiviridae; Group Tymovirus; and plant virus satellites.

[0144] Protocols for producing recombinant viral vectors and for using viral vectors for nucleic acid delivery can be found in Current Protocols in Molecular Biology Ausubel, F. M. et al. (eds.) Greene Publishing Associates; (1989) and other standard laboratory manuals (e.g., Vectors for Gene Therapy, In: Current Protocols in Human Genetics, John Wiley and Sons, Inc.; 1997).

[0145] In some embodiments, the viral vector is selected from the group consisting of an adenovirus vector, an AAV vector (e.g., AAV serotypes and genetically modified AAV variants), a herpes simplex virus vector (e.g., HSV-1 , HSV), a retrovirus vector (e.g., MMSV, MSCV), a lentivirus vector (HIV-1 , HIV-2), and alphavirus vector (e.g., SFV, SIN, VEE, M1), a flavivirus vector (e.g., Kunjin, West Nile, Dengue virus), a rhabdovirus vector (e.g., Rabies, VSV), a measles virus vector (e.g., MV-Edm), a Newcastle disease virus vector, a poxvirus vector (VV), or a picornavirus vector (e.g., Coxsackievirus).

[0146] Protocols for producing recombinant viral vectors and for using viral vectors for nucleic acid delivery can be found in Current Protocols in Molecular Biology Ausubel, F. M. et al. (eds.) Greene Publishing Associates; (1989) and other standard laboratory manuals (e.g., Vectors for Gene Therapy, In: Current Protocols in Human Genetics, John Wley and Sons, Inc.; 1997). [0147] Viral vectors can be those previously employed for the delivery of transgenes including, for example, retrovirus, adenovirus, AAV, herpes virus, hybrid adenovirus-AAV, and poxvirus vectors. In some embodiments, the vector is an adenovirus vector, AAV vector or hybrid adenovirus-AAV vector.

[0148] In some embodiments, the delivery vector is an adenovirus vector. The term “adenovirus” as used herein encompasses all adenoviruses, including the

Mastadenovirus and Aviadenovirus genera.

[0149] The various regions of the adenovirus genome have been mapped and are understood by those skilled in the art. The genomic sequences of the various Ad serotypes, as well as the nucleotide sequence of the particular coding regions of the Ad genome, are known in the art and may be accessed, e.g., from GenBank and NCBI (see, e.g., GenBank Accession Nos. J0917, M73260, X73487, AF108105, L19443, NC 003266 and NCBI Accession Nos. NC 001405, NC 001460, NC 002067, NC 00454).

[0150] A recombinant adenovirus (rAd) vector genome can comprise the adenovirus terminal repeat sequences and packaging signal. An“adenovirus particle” or“recombinant adenovirus particle” comprises an adenovirus vector genome or recombinant adenovirus vector genome, respectively, packaged within an adenovirus capsid. Generally, the adenovirus vector genome is most stable at sizes of about 28 kb to 38 kb (approximately 75% to 105% of the native genome size). In the case of an adenovirus vector containing large deletions and a relatively small transgene,“stutter DNA” can be used to maintain the total size of the vector within the desired range by methods known in the art.

[0151] The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d 1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells. Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large relative to other nucleic acid delivery vectors.

[0152] In particular embodiments, the adenovirus genome contains a deletion therein, so that at least one of the adenovirus gene regions does not encode a functional protein. For example, first-generation adenovirus vectors are typically deleted for the E1 genes and packaged using a cell that expresses the E1 proteins (e.g., 293 cells). The E3 region is also frequently deleted as well, as there is no need for complementation of this deletion. In addition, deletions in the E4, E2a, protein IX, and fiber protein regions have been described. The deletions can be selected to avoid toxicity to the packaging cell.

[0153] AAV have also been employed as nucleic acid delivery vectors. AAV are

parvoviruses and have small icosahedral virions, 18-26 nanometers in diameter and contain a single stranded DNA molecule about 4.7 kb in size. The viruses contain either the sense or antisense strand of the DNA molecule and either strand is incorporated into the virion. Two open reading frames encode a series of Rep and Cap polypeptides. Rep polypeptides (Rep50, Rep52, Rep68 and Rep78) are involved in replication, rescue and integration of the AAV genome, although significant activity may be observed in the absence of all four Rep polypeptides. The Cap proteins (VP1 , VP2, VP3) form the virion capsid. Flanking the rep and cap open reading frames at the 5' and 3' ends of the genome are 145 basepair inverted terminal repeats (ITRs), the first 125 basepairs of which are capable of forming Y- or T- shaped duplex structures. It has been shown that the ITRs represent the minimal cis sequences required for replication, rescue, packaging and integration of the AAV genome. Typically, in recombinant AAV (rAAV) vectors, the entire rep and cap coding regions are excised and replaced with a transgene of interest.

[0154] Wild-type AAV can integrate their DNA into non-dividing cells, and exhibit a high frequency of stable integration into human chromosome 19. A rAAV vector genome will typically comprise the AAV terminal repeat sequences and packaging signal. An“AAV particle” or“rAAV particle” comprises an AAV vector genome or rAAV vector genome, respectively, packaged within an AAV capsid. The rAAV vector itself need not contain AAV genes encoding the capsid and Rep proteins. In particular embodiments of the disclosure, the rep and/or cap genes are deleted from the AAV genome. In a representative

embodiment, the rAAV vector retains only the terminal AAV sequences (ITRs) necessary for integration, excision, and replication.

[0155] Sources for the AAV capsid genes can include naturally isolated serotypes, including but not limited to, AAV1 , AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhIO, AAV11 , AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7 as well as bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, non-primate AAV, and any other virus classified by the International Committee on Taxonomy of Viruses (ICTV) as an AAV. In particular embodiments, the AAV capsids are chimeras either created by capsid evolution or by rational capsid engineering from the naturally isolated AAV variants to capture desirable serotype features such as enhanced or specific tissue tropism and host immune response escape, including but not limited to AAV-DJ, AAV-HAE1 , AAV-HAE2, AAVM41 , AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61 , AAV-B1 , AAV-AS, AAV9.45A-String (e.g., AAV9.45- AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS., AAV-PHP.B, AAV-PHP.eB, and AAV-PHP.S.

[0156] Because of packaging limitations, the total size of the rAAV genome can be less than about 5.2, 5.0, 4.8, 4.6 or 4.5 kb in size. The rAAV genome refers to the two inverted terminal repeats (ITRs) from the same AAV serotype or from two different AAV serotypes as well as the gene expression cassette comprising one or more promoters, a codon optimized nucleic acid sequence encoding a microbial polypeptide, and a poly A tail.

[0157] Any suitable method known in the art can be used to produce AAV vectors. In one particular method, AAV stocks can be produced by co-transfection of a rep/cap vector plasmid encoding AAV packaging functions and the vector plasmid containing the recombinant AAV genome into human cells infected with the helper adenovirus.

[0158] In other particular embodiments, the adenovirus helper virus is a hybrid helper virus that encodes AAV Rep and/or capsid proteins. Hybrid helper Ad/AAV vectors expressing AAV rep and/or cap genes and methods of producing AAV stocks using these reagents are known in the art. The hybrid Ad of the disclosure can express the AAV capsid proteins (i.e., VP1 , VP2, and VP3). Alternatively, or additionally, the hybrid adenovirus can express one or more of AAV Rep proteins (i.e., Rep40, Rep52, Rep68 and/or Rep78). The AAV sequences can be operatively associated with a tissue-specific promoter, a ubiquitous promoter, a constitutive or an inducible promoter, or combinations thereof.

[0159] The AAV rep and/or cap genes can alternatively be provided by a packaging cell that stably expresses the genes. In still further embodiments, the delivery vector is a hybrid Ad- AAV delivery vector, for example, as described in the working Examples and in U.S. Patent No. 7,858,367 (incorporated by reference herein in its entirety for its teaching of how to make and use hybrid Ad-AAV delivery vectors). Briefly, the hybrid Ad-AAV vector comprises an adenovirus vector genome comprising adenovirus (i) 5' and 3' cis-elements for viral replication and encapsidation and, further, (ii) a recombinant AAV vector genome comprising the AAV 5' and 3' inverted terminal repeats (ITRs), an AAV packaging sequence, and a heterologous sequence(s) flanked by the AAV ITRs, where the recombinant AAV vector genome is flanked by the adenovirus 5' and 3' cis-elements. The adenovirus vector genome can further be deleted, as described above.

[0160] Another vector for use in the present disclosure comprises Herpes Simplex Virus (HSV). Herpes simplex virions have an overall diameter of 150 to 200 nm and a genome consisting of one double-stranded DNA molecule that is 120 to 200 kilobases in length. Glycoprotein D (gD) is a structural component of the HSV envelope that mediates virus entry into host cells. The initial interaction of HSV with cell surface heparin sulfate proteoglycans is mediated by another glycoprotein, glycoprotein C (gC) and/or glycoprotein B (gB). This is followed by interaction with one or more of the viral glycoproteins with cellular receptors. Glycoprotein D of HSV binds directly to Herpes virus entry mediator (HVEM) of host cells. HVEM is a member of the tumor necrosis factor receptor superfamily. Finally, gD, gB and the complex of gH and gl_ act individually or in combination to trigger pH-independent fusion of the viral envelope with the host cell plasma membrane.

[0161] HSV can be modified for the delivery of transgenes to cells by producing a vector that exhibits only the latent function for long-term gene maintenance. HSV vectors are useful for nucleic acid delivery because they allow for a large DNA insert of up to or greater than 20 kilobases; they can be produced with extremely high titers; and they have been shown to express transgenes for a long period of time in the central nervous system as long as the lytic cycle does not occur.

[0162] In other embodiments of the present disclosure, the delivery vector of interest is a retrovirus. Retroviruses normally bind to a species specific cell surface receptor, e.g., CD4 (for HIV); CAT (for MLV-E; ecotropic Murine leukemic virus E); RAM1/GLVR2 (for murine leukemic virus-A; MLV-A); GLVR1 (for Gibbon Ape leukemia virus (GALV) and Feline leukemia virus B (FeLV-B)). The development of specialized cell lines (termed“packaging cells”) which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes. A replication-defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques.

[0163] Yet another suitable vector is a poxvirus vector. These viruses contain more than 100 proteins. Extracellular forms of the virus have two membranes while intracellular particles only have an inner membrane. The outer surface of the virus is made up of lipids and proteins that surround the biconcave core. Poxviruses are very complex antigenically, inducing both specific and cross- reacting antibodies after infection. Poxvirus can infect a wide range of cells. Poxvirus gene expression is well studied due to the interest in using vaccinia virus as a vector for expression of transgenes.

[0164] In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed. Many non-viral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In particular embodiments, non-viral delivery systems rely on endocytic pathways for the uptake of the nucleic acid molecule by the targeted cell. Exemplary nucleic acid delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. [0165] In particular embodiments, plasmid vectors are used in the practice of the present disclosure. Naked plasmids can be introduced into muscle cells by injection into the tissue. Expression can extend over many months, although the number of positive cells is typically low (Wolff et al., (1989) Science 247:247). Cationic lipids have been demonstrated to aid in introduction of DNA into some cells in culture (Feigner and Ringold, (1989) Nature 337:387). Injection of cationic lipid plasmid DNA complexes into the circulation of mice has been shown to result in expression of the DNA in lung (Brigham et al., (1989) Am. J. Med.

Sci. 298:278). One advantage of plasmid DNA is that it can be introduced into non replicating cells.

[0166] In a representative embodiment, a nucleic acid molecule (e.g., a plasmid) can be entrapped in a lipid particle bearing positive changes on its surface and, optionally, tagged with antibodies against cell surface antigens of the target tissue.

[0167] Liposomes that consist of amphiphilic cationic molecules are useful non-viral vectors for nucleic acid delivery in vitro and in vivo. The positively charged liposomes are believed to complex with negatively charged nucleic acids via electrostatic interactions to form lipidmucleic acid complexes. The lipid ucleic acid complexes have several advantages as gene transfer vectors. Unlike viral vectors, the lipidmucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size. Since the complexes lack proteins, they can evoke fewer immunogenic and inflammatory responses. Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency.

[0168] Amphiphilic cationic lipidmucleic acid complexes can be used for in vivo transfection both in animals and in humans and can be prepared to have a long shelf-life.

[0169] Administering an immunosuppressive agent in combination with gene therapy could be another approach to help prevent or reduce cytotoxic T cell and/or antibody mediated immune responses towards gene therapy. Methods of delivering a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide for degrading glycogen for therapeutic methods are described in more detail below.

[0170] An isolated nucleic acid molecule can be carried by a delivery vector as described in the preceding section. Those skilled in the art will appreciate that the isolated nucleic acid encoding the microbial polypeptides for degrading glycogen can be operably associated with appropriate expression control sequences, e.g., transcription/translation control signals or secretory signal sequences, which can be included in the isolated nucleic acid or by a vector backbone. For example, specific initiation signals can be required for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which can include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic. [0171] A variety of promoter/enhancer elements can be used depending on the level and tissue-specific expression desired. The promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the therapeutic gene expression desired. The promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0172] The promoter can be chosen so that it will function in the target cell(s) of interest. Tissue-specific promoters refer to promoters that have activity in only certain cell types. The use of a tissue-specific promoter in a nucleic acid expression cassette can restrict unwanted transgene expression in the unaffected tissues as well as facilitate persistent transgene expression by escaping from transgene induced host immune responses.

[0173] Ubiquitous promoters refer to promoters that are strongly active in a wide range of cells and tissues, including, but not limited to, the liver, kidney, skeletal muscle, cardiac muscle, smooth muscle, diaphragm muscle, brain, spinal cord, endothelial cells, intestinal cells, pulmonary cells (e.g., smooth muscle or epithelium), peritoneal epithelial cells and fibroblasts.

[0174] Ubiquitous promoters include, but are not limited to, a CMV major immediate-early enhancer/chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EF1-a) promoter, a simian vacuolating virus 40 (SV40) promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a b-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous promoters.

[0175] Constitutive promoters refer to unregulated promoters that allow for continual transcription of its associate gene. In some embodiments, a constitutive promoter can also be a ubiquitous promoter.

[0176] Inducible promoters refer to promoters that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

[0177] Liver-specific promoters include, but are not limited to, the a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone-binding globulin promoter, the a-1 -anti-trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human a1 -antitrypsin (hAAT) promoter, the ApoEhAAT promoter composed of the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC172 promoter consisting of the hAAT promoter and the a1-microglobulin enhancer, the DC190 promoter containing the human albumin promoter and the prothrombin enhancer, and other natural and synthetic liver-specific promoters.

[0178] Muscle specific promoters include, but are not limited to, the MHCK7 promoter, the muscle creatine kinase (MCK) promoter/enhancer, the slow isoform of troponin I (TnIS) promoter, the MYODI promoter, the MYLK2 promoter, the SPc5-12 promoter, the desmin (Des) promoter, the unc45b promoter, and other natual and synthetic muscle-specific promoters.

[0179] Skeletal muscle-specific promoters include, but are not limited to, the HSA promoter, the human a-skeletal actin promoter.

[0180] Heart-specific promoters include, but art not limited to, the MYH6 promoter, the TNNI3 promoter, the cardiac troponin C (cTnC) promoter, the alpha-myosin heavy chain (a- MHC) promoter, and the MYBPC3 promoter.

[0181] Neuron-specific promoters include, but are not limited to the synapsin I (SYN) promoter, the calcium/calmodulin-dependent protein kinase II promoter, the tubulin alpha I promoter, the enolase promoter, and the platelet-derived growth factor beta chain promoter.

[0182] The tissue-specific promoters can be operably linked to one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) enhancer elements or combined to form a tandem promoter (e.g., liver- specific/muscle-specific tandem promoter, liver-specific/neuron-specific tandem promoter, or muscle-specific/ neuron-specific tandem promoter). When two or more tissue-specific promoters are present, the isolated nucleic acid can be targeted to two or more different tissues at the same time.

[0183] In some embodiments, the vector can comprise one or more immunotolerant promoters (e.g., an immunotolerant liver-specific promoter or an immunotolerant dual promoter). As used herein, the term“immunotolerant” refers to unresponsiveness to an antigen (e.g., a vector, a therapeutic protein, a bacterial GDE, etc.). An immunotolerant promoter can reduce, ameliorate, or prevent transgene-induced immune responses that can be associated with gene therapy. Assays known in the art to measure immune responses, such as immunohistochemical detection of cytotoxic T cell responses, can be used to determine whether one or more promoters can confer immunotolerant properties.

[0184] In some embodiments, the vector can comprise an immunotolerant dual promoter having a liver-specific promoter and a ubiquitous promoter. In some embodiments, the liver- specific promoters that can be used in the immunotolerant dual promoter system include, but are not limited to, the a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter, the human albumin (hALB) promoter, the thyroid hormone-binding globulin promoter, the a-1-anti-trypsin promoter, the bovine albumin (bAlb) promoter, the murine albumin (mAlb) promoter, the human a1-antitrypsin (hAAT) promoter, the ApoEhAAT promoter composed of the ApoE enhancer and the hAAT promoter, the transthyretin (TTR) promoter, the liver fatty acid binding protein promoter, the hepatitis B virus (HBV) promoter, the DC172 promoter consisting of the hAAT promoter and the a1-microglobulin enhancer, the DC190 promoter containing the human albumin promoter and the prothrombin enhancer, and other natural and synthetic liver-specific promoters.

[0185] In some embodiments, the ubiquitous promoters that can be used in the

immunotolerant dual promoter system include, but are not limited to, a CMV major immediate-early enhancer/chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EF1-a) promoter, a simian vacuolating virus 40 (SV40) promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a b- kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natual or synthetic ubiquitous promoters.

[0186] In some embodiments, the immunotolerant dual promoter comprises a a1- microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter and a CMV enhancer/beta-actin (CB) promoter. In some embodiments, the immunotolerant dual promoter comprises a a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter and a CMV enhancer/beta-actin (CB) promoter. The a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter can have the nucleic acid sequence as set forth in SEQ ID NO: 31 , or can have at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:31. The CMV enhancer/chicken b-actin promoter can have the nucleic acid sequence as set forth in SEQ ID NO: 32, or can have at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:32.

[0187] In some embodiments, the immunotolerant dual promoter has a nucleic acid sequence as set forth in SEQ ID NO:30, or has at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:30. The nucleic acid sequence set forth in SEQ ID NO:30 is shown in Table 2, and the un-bolded portion of the sequence represents the a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter and the bolded portion of the sequence represents the CMV enhancer/chicken b-actin promoter.

[0188] The dual promoters can be engineered into the gene expression cassette such that the 3’ end of the liver-specific promoter is operably linked to the 5’ end of the ubiquitous promoter or the 3’ end of the ubiquitous promoter is operably linked to the 5’ end of the liver- specific promoter. Delivering a therapeutic gene under the control of the dual promoters described herein has the surprising advantage of preventing a transgene-induced T cell response of a therapeutic transgene product for gene therapy of human genetic diseases that affect multiple tissues (e.g., GSD III or GSD IV).

[0189] Thus, the disclosure relates to methods and compositions of the use of an immunotolerant dual promoter system consisting of a liver-specific promoter and a ubiquitous promoter to prevent host immune responses against a therapeutic transgene product for gene therapy of human genetic diseases that affect multiple tissues. The disclosure also relates to gene replacement therapy approaches to deliver a functional therapeutic gene under the control of the said immunotolerant dual promoter with a viral or non-viral vector.

[0190] The transgene under control of the immunotolerant dual promoter system described herein can encode a variety of therapeutic proteins/enzymes and polypeptides, including microbial polypeptides for degrading glycogen and non-microbial proteins to treat human genetic diseases that affect multiple tissues. Therapeutic proteins and polypeptides that can be used with the immunotolerant dual promoter system described herein include, but are not limited to, cluster of differentiation 39 (CD39) protein, cluster of differentiation 73 (CD73) protein, Recombinant Anti-Inflammation fusion protein (RAIN) (CD73-39 fusion), interleukin- 1 inhibitor, tumor necrosis factor-a inhibitor, interleukin-12 inhibitor, interleukin-1 receptor antagonist, interleukin-18 binding protein, soluble tumor necrosis factor-a receptor p55 or soluble tumor necrosis factor-a protein 75, dominant negative I KB kinase-b, inter leukin-4, interleukin-10, interleukin-13, interferon-b, vasoactive intestinal polypeptide, cystic fibrosis transmembrane regulator protein (CFTR), dystrophin, utrophin, blood coagulation (clotting) factor (e.g., Factor XIII, Factor IX, Factor X, Factor VIII, Factor Vila, protein C, Factor VII, B domain-deleted Factor VIII, or a longer half-life variant of a coagulation factor), retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, b- globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, lysosomal acid a-glucosidase (GAA; also known as acid maltase), a-galactosidase A, b-galactosidase, b-hexosaminidase A, b-hexosaminidase B, GM ₂ activator protein, glucocerebrosidase, arylsulfatase A, galactosylceramidase, acid sphingomyelinase, acid ceramidase, acid lipase, a-L-iduronidase, iduronate sulfatase, heparan N-sulfatase, a-N-acetylglucosaminidase, glucosaminide acetyltransferase, N-acetylglucosamine-6-sulfatase, arylsulfatase B, b- glucuronidase, a-mannosidase, b-mannosidase, a-L-fucosidase, N-aspartyl-b- glucosaminidase, N-acetylgalactosamine 4-sulfatase, a-neuraminidase, lysosomal protective protein, a-N-acetyl-galactosaminidase, N-acetylglucosamine-1 -phosphotransferase, glycogen branching enzyme (GDE), a microbial polypeptide for degrading glycogen, glycogen debranching enzyme (GDE), cystine transport protein, sialic acid transport protein, the CLN3 gene product, palmitoyl-protein thioesterase, saposin A, saposin B, saposin C, and saposin D, hypoxanthine guanine phosphoribosyl transferase, P-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched chain keto acid dehydrogenase, a hormone, a growth factor, insulin-like growth factor 1 or 2, platelet derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor a and b, a cytokine, interferon-a, interferon-g, inter leukin-2, interleukin-12, granulocyte-macrophage colony stimulating factor, lymphotoxin, a suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, a drug resistance protein, a tumor suppressor protein (e.g., p53, Rb, Wt-1 , NFI , Von Hippel-Lindau (VHL), SERCA2a, adenomatous polyposis coli (APC)), VEGF, microdystrophin, lysosomal acid lipase, arylsulfatase A and B, ATP7A and B, a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDRI, insulin, glucokinase, guanylate cyclase 2D (LCA- GUCY2D),

Rab escort protein 1 (Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid

aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked Retinoschisis), USH1C

(Usher's Syndrome IC), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4

(Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1 , CLN2, or a gene product implicated in lysosomal storage diseases (e.g., sulfatases, N-acetylglucosamine-1 - phosphate transferase, cathepsin A, GM2-AP, NPC1 , VPC2, a sphingo lipid activator protein), and any other peptide or protein that has a therapeutic effect in a subject in need thereof.

[0191] In some embodiments, the nucleic acid sequence encoding a therapeutic protein under the control of a dual promoter has a coding sequence that is less than about 1.0, 1.1 ,

1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2,

3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3,

5.4, or 5.5 kilobases (kb).

[0192] The promoter can further be“specific” for these cells and tissues, in that it can only show significant activity in the specific cell or tissue type. In some embodiments, the tissue- specific promoter is a liver-specific promoter, a muscle-specific promoter, a neuron-specific promoter, or a combination thereof.

[0193] The isolated nucleic acid can be operatively associated with an ubiquitous promoter, for example, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EF1-a) promoter, a simian vacuolating virus 40 (SV40) promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a b-kin promoter, a murine

phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natual or synthetic ubiquitous promoters. A hybrid promoter containing the CMV major immediate- early enhancer and chicken beta-actin (CB) promoter is also suitable. Driving heterologous nucleotide transcription with the CMV promoter can result in down-regulation of expression in immunocompetent animals. Accordingly, it can be advantageous to operably associate the isolated nucleic acid molecule with a modified CMV promoter that does not result in this down-regulation of transgene expression. In some embodiments, the ubiquitous promoter is a CMV enhancer/chicken b-actin promoter.

[0194] The vector can comprise a ubiquitous promoter and/or a tissue-specific promoter operably linked to the nucleic acid molecule. The term“operably linked” means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. A nucleic acid molecule that is operably linked to a promoter can be under transcriptional initiation regulation of the promoter.

[0195] The isolated nucleic acids of the disclosure can comprise two or more coding sequences. In embodiments wherein there is more than one coding sequence, the coding sequences can be operatively associated with separate promoters or, alternatively, with a single upstream promoter and one or more downstream internal ribosome entry site (IRES) sequences (e.g., the picornavirus EMC IRES sequence).

[0196] In some embodiments, the isolated nucleic acid encoding the microbial polypeptides for degrading glycogen can be operably associated with or fused to a secretory signal sequence (e.g., for secreting the microbial polypeptide to the blood circulation that subsequently can be taken up by other tissues).

[0197] The secretory signal sequence can be derived in whole or in part from the secretory signal of a secreted polypeptide (e.g., from the precursor) and/or can be in whole or in part synthetic. The secretory signal sequence can be from any species of origin, including mammals, plants, yeast, bacteria, protozoa or fungi. The length of the secretory signal sequence can be from about 10-15 to 50-60 amino acids in length. Further, known secretory signals from secreted polypeptides can be altered or modified (e.g., by substitution, deletion, truncation or insertion of amino acids) as long as the resulting secretory signal sequence functions to enhance secretion of an operably linked lysosomal polypeptide.

[0198] Exemplary secreted proteins (and their secretory signals) include but are not limited to: erythropoietin, coagulation Factor IX, cystatin, lactotransferrin, plasma protease C1 inhibitor, apolipoproteins (e.g., APO A, C, E), MCP-1 , a-2-HS-glycoprotein, a-1- microgolubilin, complement (e.g., C1Q, C3), vitronectin, lymphotoxin-a, azurocidin, VIP, metalloproteinase inhibitor 2, glypican-1 , pancreatic hormone, clusterin, hepatocyte growth factor, insulin, a-1-antichymotrypsin, growth hormone, type IV collagenase, guanylin, properdin, proenkephalin A, inhibin b (e.g., A chain), prealbumin, angiocenin, lutropin (e.g., b chain), insulin-like growth factor binding protein 1 and 2, proactivator polypeptide, fibrinogen (e.g., b chain), gastric triacylglycerol lipase, midkine, neutrophil defensins 1 , 2, and 3, a-1- antitrypsin, matrix gla-protein, a-tryptase, bile-salt-activated lipase, chymotrypsinogen B, elastin, IG lambda chain V region, platelet factor 4 variant, chromogranin A, WNT-1 proto oncogene protein, oncostatin M, b-neoendorphin-dynorphin, von Willebrand factor, plasma serine protease inhibitor, serum amyloid A protein, nidogen, fibronectin, rennin, osteonectin, histatin 3, phospholipase A2, cartilage matrix Protein, GM-CSF, matrilysin, neuroendocrine protein 7B2, placental protein 11 , gelsolin, IGF 1 and 2, M-CSF, transcobalamin I, lactase- phlorizin hydrolase, elastase 2B, pepsinogen A, MIP 1-b, prolactin, trypsinogen II, gastrin releasing peptide II, atrial natriuretic factor, secreted alkaline phosphatase, pancreatic a- amylase, secretogranin I, b-casein, serotransferrin, tissue factor pathway inhibitor, follitropin b-chain, coagulation factor XII, growth hormone-releasing factor, prostate seminal plasma protein, interleukins (e.g., 2, 3, 4, 5, 9, 11), inhibin (e.g., alpha chain), angiotensinogen, thyroglobulin, IG heavy or light chains, plasminogen activator inhibitor-1 , lysozyme C, plasminogen activator, antileukoproteinase 1 , statherin, fibulin-1 , isoform B, uromodulin, thyroxine-binding globulin, axonin-1 , endometrial a-2 globulin, interferon (e.g., alpha, beta, gamma), b-2-microglobulin, procholecystokinin, progastricsin, prostatic acid phosphatase, bone sialoprotein II, colipase, Alzheimer's amyloid A4 protein, PDGF (e.g., A or B chain), coagulation factor V, triacylglycerol lipase, haptoglobuin-2, corticosteroid-binding globulin, triacylglycerol lipase, prorelaxin H2, follistatin 1 and 2, platelet glycoprotein IX, GCSF,

VEGF, heparin cofactor II, antithrombin-lll, leukemia inhibitory factor, interstitial collagenase, pleiotrophin, small inducible cytokine A1 , melanin-concentrating hormone, angiotensin converting enzyme, pancreatic trypsin inhibitor, coagulation factor VIII, a-fetoprotein, a- lactalbumin, senogelin II, kappa casein, glucagon, thyrotropin beta chain, transcobalamin II, thrombospondin 1 , parathyroid hormone, vasopressin copeptin, tissue factor, motilin, MPIF- 1 , kininogen, neuroendocrine convertase 2, stem cell factor procollagen a1 chain, plasma kallikrein keratinocyte growth factor, as well as any other secreted hormone, growth factor, cytokine, enzyme, coagulation factor, milk protein, immunoglobulin chain, and the like. [0199] In other particular embodiments, the secretory signal sequence is derived in part or in whole from a secreted polypeptide that is produced by liver cells.

[0200] In some embodiments of the disclosure, the total size of the gene expression cassette comprising the isolated nucleic acid molecule is less than about 5.0, 4.8, 4.7, 4.6, 4.5, 4.3, 4.2, 4.0, 3.8, 3.7, 3.6, 3.5, 3.2, 3.0, 2.8, 2.5, 2.2, 2.0, or 1.8 kb in length. A gene expression cassette can comprise, for example, one or more promoters, an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide, and a polyadenylation sequence (poly A sequence). Relatively small gene expression cassettes can be particularly advantageous for delivery by, e.g., AAV vectors.

[0201] In some embodiments, the vector comprising an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a microbial polypeptide having fewer than about 4.0 kilobases (e.g., about 0.5, 1.0, 1.5, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 3.9 kbs), wherein a counterpart human nucleic acid sequence has a coding sequence that is greater than about 4.0 kilobases (e.g., 4.1 , 4.2, 4.4, 4.6, 4.8, 5.0. , 5.5., 6.0, 6.5, or 7.0 kbs), and wherein the nucleic acid sequence encoding the microbial polypeptide is codon- optimized for expression in a human cell. A counterpart human nucleic acid sequence of a microbial nucleic acid sequence refers to a human nucleic acid sequence that encodes a polypeptide that has the same or similar catalytic function as a polypeptide encoded by a microbial nucleic acid sequence (e.g., a human nucleic acid sequence encoding a GDE capable of degrading glycogen is a counterpart of a microbial nucleic acid sequence encoding an enzyme capable of degrading glycogen).

[0202] Another aspect of the present disclosure provides an isolated cell comprising a vector comprising a codon-optimized microbial polypeptide for degrading glycogen.

[0203] Gene Therapy Methods. Pharmaceutical Formulations, and Modes of

Administration

[0204] Another aspect of the present disclosure provides a pharmaceutical formulation comprising the microbial polypeptide for degrading glycogen. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an isolated nucleic acid or vector of the disclosure in a pharmaceutically-acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like. For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL® (BASF, Parsippany, N.J.). For other methods of administration, the carrier can be either solid or liquid.

[0205] By“pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the isolated nucleic acid or vector without causing any undesirable biological effects such as toxicity. Thus, such a pharmaceutical composition can be used, for example, in transfection of a cell ex vivo or in administering an isolated nucleic acid or vector directly to a subject.

[0206] In the case of a viral vector, virus particles can be contacted with the cells at the appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cells. Titers of virus to administer can vary, depending upon the target cell type and the particular virus vector, and can be determined by those of skill in the art. Typically, at least about 10 ³ virus particles, at least about 10 ⁵ particles, at least about 10 ⁷ particles, at least about 10 ⁹ particles, at least about 10 ¹¹ particles, or at least about

10 ¹² particles are administered to the cell. In exemplary embodiments, about 10 ⁷ to about 10 ¹⁵ particles, about 10 ⁷ to about 10 ¹³ particles, about 10 ⁸ to about 10 ¹² particles, about 10 ¹ ° to about 10 ¹⁵ particles, about 10 ¹¹ to about 10 ¹⁵ particles, about 10 ¹² to about 10 ¹⁴ particles, or about 10 ¹² to about 10 ¹³ particles are administered.

[0207] The cell to be administered the vectors of the disclosure can be of any type, including but not limited to neuronal cells (including cells of the peripheral and central nervous systems), retinal cells, epithelial cells (including dermal, gut, respiratory, bladder, pulmonary, peritoneal and breast tissue epithelium), muscle (including cardiac, smooth muscle, including pulmonary smooth muscle cells, skeletal muscle, and diaphragm muscle), pancreatic cells (including islet cells), kidney cells, hepatic cells (including parenchyma), cells of the intestine, fibroblasts (e.g., skin fibroblasts such as human skin fibroblasts), fibroblast-derived cells, endothelial cells, intestinal cells, germ cells, lung cells (including bronchial cells and alveolar cells), prostate cells, stem cells, progenitor cells, dendritic cells, and the like. Alternatively, the cell is a cancer cell (including tumor cells). Moreover, the cells can be from any species of origin, as indicated above.

[0208] Another aspect of the present disclosure provides a method of treating a deficiency of a polypeptide for degrading glycogen in a subject, comprising administering to the subject a therapeutically effective amount of an isolated nucleic acid encoding a microbial polypeptide for degrading glycogen, a vector comprising an isolated nucleic acid encoding a microbial polypeptide for degrading glycogen or pharmaceutical composition comprising an isolated nucleic acid encoding a microbial polypeptide for degrading glycogen. The subject can be suffering from a GSD (e.g., GSD III).

[0209] Administration of the nucleic acid or delivery vectors of the present disclosure to a human subject or an animal can be by any means known in the art. The subject can be a mammalian subject, more particularly a human subject. In other embodiments, the subject is in need of treatment, for example, has been diagnosed with or is suspected of having a deficiency of a polypeptide for degrading glycogen.

[0210] Dosages will depend upon the mode of administration, the severity of the disease or condition to be treated, the individual subject's condition, the particular vector, and the gene to be delivered, and can be determined in a routine manner. In some embodiments, the isolated nucleic acid molecule or vector is administered to the subject in a therapeutically effective amount, as that term is defined above.

[0211] With respect to viral vectors, at least about 10 ³ virus particles, at least about 10 ⁵ virus particles, at least about 10 ⁷ virus particles, at least about 10 ⁹ , at least about 10 ¹¹ virus particles, or at least about 10 ¹² virus particles are administered to the subject per treatment. Exemplary doses are virus titers of about 10 ⁷ to about 10 ¹⁵ particles, about 10 ⁷ to about 10 ¹⁴ particles, about 10 ⁸ to about 10 ¹³ particles, about 10 ¹⁰ to about 10 ¹⁵ particles, about 10 ¹¹ to about 10 ¹⁵ particles, about 10 ¹² to about 10 ¹⁴ particles, or about 10 ¹² to about 10 ¹³ particles.

[0212] In some embodiments, more than one administration (e.g., two, three, four, or more administrations) can be employed to achieve therapeutic levels of nucleic acid expression. In some embodiments, more than one administration of the same vector can be employed to achieve the durability of therapeutic levels of nucleic acid expression (e.g., two, three, or four administrations of an AAV-LSP-Pull vector).

[0213] In other embodiments, more than one administration of a different vector can be employed to achieve therapeutic levels of nucleic acid expression in different affected tissues (e.g., administration of an AAV-CB-Pull vector and administration of an AAV-LSP- Pull vector). Administration of more than one different types of vectors can include, for example, vectors of the same serotype carrying different elements (e.g., administration of an AAV9-CB-Pull vector and administration of an AAV9-LSP-Pull vector) or vectors of different serotypes carrying different elements (e.g., administration of an AAV9-CB-Pull vector and administration of an AAV8-LSP-Pull vector). Administration of more than one different vector can also refer to administering, for example, and AAV vector and an Ad vector carrying similar or different elements.

[0214] More than one administration can be co-administering more than one vector at the same time or administering one vector and then subsequently administering two, three, four, or more vectors at a time period of minutes, hours, days, weeks, or months after the initial administration.

[0215] In some embodiments, tissue-restricted gene expression using a single or a tandem tissue-specific promoter can prevent, reduce, or suppress bacterial enzyme induced immune responses towards gene therapy. The term“immune response” as used herein refers to host immunity (cytotoxic T cell and/or antibody mediated immune responses) during gene therapy against transgene expression and/or viral vectors (e.g., AAV). In other embodiments, an immune response to the gene therapy approaches described herein can be prevented, reduced, or suppressed by administering an immunosuppressive agent instead of relying on tissue-restricted gene expression. An immunosuppressive agent can include, but is not limited to, agents acting on B cells, T cells, plasma cells. Examples are proteasome inhibitors (e.g., bortezomib, carfilzomib, Ixazomib), corticosteroids (e.g., hydrocortisone, cortisone, ethamethasoneb, prednisone, prednisolone, triamcinolone, dexamethasone, fludrocortisone, bethamethasone, triamcinolone, methylprednisolone), Janus kinase inhibitors (e.g., ruxolitinib, tofacitinib, oclacitinib, baricitinib), calcinerurin inhibitors (e.g., cyclosporine, tacrolimus), mTOR inhibitors (e.g., rapamycin sirolimus, temsirolimus, everolimus, ridaforolimus), inosine monophosphate dehydrogenase (IMDH) inhibitors (e.g., mycophenolic acid), methotrexate, biologic drugs (e.g., etanercept), or monoclonal antibodies (e.g., rituximab, adalimumab, infliximab, efalizumab). In some embodiments, more than one immunosuppressive agent can be administerd in combination with gene therapy.

[0216] In other embodiments, an immune response to the gene therapy approaches described herein can be prevented, reduced, or suppressed by performing plasmapheresis in combination with or as an alternative to administering one or more immunosuppressive agents. Plasmapheresis is a procedure that can remove harmful antibodies from the blood.

[0217] In other embodiments, an immune response to the gene therapy approaches described herein can be prevented, reduced, or suppressed by utilizing vectors that express a therapeutic enzyme under the control of an immunotolerant dual promoter (e.g., the LSP- CB dual promoter).

[0218] In some embodiments, the gene therapy methods described herein can be enhanced by administering an inhibitor of glycogen synthase in combination with a nucleic acid or delivery vector of the present disclosure. In humans, glycogen synthase 1 (encoded by the GYS1 gene) regulates glycogen/glucose levels in the muscle and other tissues and glycogen synthase 2 (encoded by the GYS2 gene) regulates glycogen/glucose levels in the liver. Glycogen synthase inhibitors include, but are not limited to, small molecule drugs or RNA interference (RNAi) polynucleotides (e.g., double stranded RNA (dsRNA), antisense oligonucleotides (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNAs) oligonucleotides, and aptamers, and the like, as described by Pursell,

N. et al. (2018) Molecular Therapy 26(7): 1771 -1782)).

[0219] Modes of administration include oral, rectal, transmucosal, topical, transdermal, inhalation, parenteral, e.g., intravenous, subcutaneous, intradermal, intramuscular (i.e. , administration to cardiac, skeletal, diaphragm and/or smooth muscle), and intraarticular administration, and the like, as well as direct tissue (e.g., muscle) or organ injection (e.g., into the liver, into the brain for delivery to the central nervous system), alternatively, intrathecal, direct intramuscular (e.g., into cardiac, skeletal, or diaphragm muscle), intraventricular, intravenous, intraperitoneal, intranasal, or intraocular. Additional modes of administration can include intraarterially, intraperitoneally, or directly into utero. Administration to the liver (discussed below) is another representative mode of administration.

[0220] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. An injection medium will typically be an aqueous liquid that contains the additives usual for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.

[0221] In some embodiments, the isolated nucleic acid molecule or vector is delivered to the liver of the subject. Administration to the liver can be achieved by any method known in the art, including, but not limited to intravenous administration, intraportal administration, intrabiliary administration, intra-arterial administration, injection into the liver parenchyma, and intrasplenic injection.

[0222] Intramuscular delivery and intracardiac delivery to skeletal muscle or cardiac muscle, respectively, or direct injection into diaphragm muscle can be used. In other particular embodiments, intraperitoneal administration is used to deliver the isolated nucleic acid or vector to diaphragm muscle.

[0223] In particular embodiments, the isolated nucleic acid molecule (e.g., carried by an Ad, AAV or hybrid Ad/AAV vector) encoding a microbial polypeptide for degrading glycogen is introduced into a depot organ or tissue (e.g., liver, skeletal muscle, lung) and the polypeptide is expressed therein and secreted into the circulatory system, where it is optionally delivered to other target tissues, in a therapeutically effective amount. Intramuscular delivery to skeletal muscle or delivery to the liver is illustrative for the practice of this embodiment of the disclosure. Alternatively, the isolated nucleic acid or vector can be administered to the brain (e.g., to treat MPS disorders such as Sly disease), where the polypeptide can be expressed and secreted by transformed or transduced cells (e.g., neurons, glial cells) and taken up by other brain cells.

[0224] Certain aspects of the disclosure are now explained further via the following non limiting examples.

[0225] EXAMPLES

[0226] Materials and Methods For Examples 1-6

[0227] AAV vector construction and AAV viral vector preparation:

[0228] A 2.2kb codon optimized coding sequence (SEQ ID NO:03) for Pullulanase was synthesized at GenScript (Piscataway, NJ) and cloned into an AAV vector (AAV-CBhGAA) (Sun, B.D. et al. (2003) Mol. Ther. 7(2): 193-201 ) containing a CMV enhanced chicken beta- actin hybrid (CB) promoter to replace the human GAA cDNA, to generate the AAV-CB-Pull vector. The Scal-Kpnl fragment containing the liver-specific promoter (LSP) from the AAV- LSPhGAA vector (Franco, L.M. et al., (2005) Mol. Ther. 12(5):876-84) was cloned into the AAV-CB-Pull vector to replace the CB promoter, to generate the AAV-LSP-Pull vector. To generate the pAAV-MHCK7-Pull vector plasmid, the MHCK7 promoter was amplified from the AAV-MHCK7hGAApA vector (Sun, B., et al. (2008) Mol Ther, 16(8): 1366-71) using primers: Xbal-MHCK7-F (5'-ccccttaagagctgcatgtctaagctagaccc-3') (SEQ ID NO: 33) and Kpnl-MHCK7-R (5'-cggggtacccgctggctggctcctgagt-3') (SEQ ID NO: 34). The amplified PCR fragment was digested with Xbal and Kpnl and then cloned into the pAAV-LSP-Pull vector to replace the LSP promoter. To generate the pAAV-LSP-CB-Pull vector plasmid containing the LSP-CB dual promoter, the LSP promoter was amplified from the pAAV-LSP-hGAA vector (Franco, L.M., et al. (2005) Mol Ther., 12(5):876-84) using primers: Xbal-LSP-F (5'- AGTTCTAGAGCGGCCGCCAG-3') (SEQ ID NO: 35) and Aflll-LSP-R (5'- CCCCTT AAGCCATTTTT AT AGCAT GTCCT GT ATTGCAAAACT A-3') (SEQ ID NO: 36); the CB promoter was amplified from the pAAV-CB-Pull vector using primers Aflll-CB-F (5'- CCCCTT AAGGTTCCGCGTT ACAT AACTT ACGGT AAAT -3') (SEQ ID NO: 37) and Kpnl-CB- R (5'-GTCGACGGTACCGCGCAG-3') (SEQ ID NO: 38). The amplified Xbal-LSP-Aflll and Aflll-CB-Kpnl fragments were ligated through the Aflll site and amplified again using primers: Xbal-LSP-F and Kpnl-CB-R (see above). The amplified Xbal-LSP-CB-Kpnl fragment was purified and cloned into the pAAV-LSP-Pull vector at Xbal and Kpnl sites to replace the LSP promoter region. The LSP-CB dual promoter (SEQ ID NO:30) described in the Examples is a a1-microglobulin/bikunin enhancer/thyroid hormone-binding globulin promoter (SEQ ID NO:31) and a CMV enhancer/beta-actin (CB) promoter (SEQ ID NO:32) .

[0229] These AAV vectors were packaged as AAV8 or AAV9 in HEK 293T cells using standard phosphate-mediated transfection and purified using iodixanol gradient

ultracentrifugation (Sun, B., et al. (2008) Mol. Ther., 16(8): 1366-71 ; Hermens, W.T., et al. (1999) Hum Gene Ther, 10(11): 1885-91 ; Lock, M. et al., (2010) Human Gene Therapy, 21 (10):1259-1271). The titer of the viral stock was determined using purified viral DNA and southern blotting with a biotin-labeled probe generated with Prime-A-Gene labeling kit (Promega, Madison, Wl). All viral vector stocks were handled according to the Biohazard Safety Level 2 guidelines published by the National Institutes of Health.

[0230] Animals and virus administration:

[0231] Animal care and experiments were conducted in accordance with Duke University Institutional Animal Care and Use Committee-approved guidelines.

[0232] AAV copy number determination in mouse tissues by real-time PCR:

[0233] DNA was extracted from frozen tissues using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wl). Real-time PCR was performed using SYBR Green (Roche, Basel, Switzerland) and gene-specific primer pairs for Pullulanase (primers 5’- GCCACT GGATGCCT ACAACT -3’ (SEQ ID NO:26) and 5’- CGTGCTGGTGCAGTGT ATT G- 3’ (SEQ ID NO:27); and for mouse beta-actin (internal control, primers: 5’- AGAGGGAAATCGTGCGT GAC-3’ (SEQ ID NO:28) and 5’-

CAAT AGT GAT GACCT GGCCGT -3’ (SEQ ID NO:29)).The AAV-CB-Pull or AAV-LSP-Pull plasmid DNA was used to generate a standard curve for viral vector copy number calculation (Yi, H. et al. , (2017) Hum Gene Ther, 28(3):286-294; Lim, J.A., et ai, (2018) Molecular Therapy, 26(5): 382-83).

[0234] Pullulanase activity and glycogen content assay

[0235] Frozen tissues were homogenized in distilled water (1 mg/20 pl_ of water) using a homogenizer, followed by sonication for 15 sec and centrifugation at 18,000 g at 4 °C for 15 min. Pullulanase activity was assayed with the tissue homogenates using the

Pullulanase/Limit-Dextrinase Assay Kit (PullG6 Method) (Megazyme, Chicago, IL) following manufacturer's instructions.

[0236] For measuring glycogen content, the 1 :5 diluted tissue lysates were boiled for 3 min (to inactivate endogenous enzymes) and incubated with 0.175 U/mL (final concentration in the reaction) of amyloglucosidase (Sigma-Aldrich Co., St. Louis, MO) for 90 min at 37 °C. The reaction mixtures were then boiled again for 3 min to stop the reaction. 30 pL of the mixtures were incubated with 1 mL of Pointe Scientific Glucose (Hexokinase) Liquid

Reagents (Fisher, Hampton, NH) for at least 10 min at RT. The absorbance at 340 nm was read on a Shimadzu UV-1700 PharmaSpec UV-VIS Spectrophotometer. Protein

concentration was determined by BCA assay and used to normalize the data (Yi, H. et al., (2017) Hum Gene Ther, 28(3):286-294).

[0237] Histology:

[0238] Fresh tissues were fixed in 10% neutral-buffered formalin (NBF) for 48 h. After primary immersion fixation, the samples were post-fixed with 1 % periodic acid (PA) in 10% NBF for 48 h at 4 °C. The samples were then washed with PBS, dehydrated with ascending grades of alcohol, cleared with xylene, and infiltrated with paraffin. For Periodic acid-Schiff (PAS) staining, sections of paraffin-embedded tissues were processed and stained using Schiff reagent as described (Lim, J.A., et al. (2018) Mol Ther., 26(5): 382-83). Briefly, the slides were oxidized with freshly made 0.5% PA for 5 min and rinsed with distilled water for 1 min. The slides were then stained with Schiff reagent for 15 min and washed with tap water for 10 min. The slides were counterstained with Hematoxylin and rinsed with tap water, incubated with bluing reagent for 1 min, dehydrated, and mounted. For trichrome staining, the paraffin-embedded liver sections were processed and stained using Masson's trichrome staining kit (Sigma-Aldrich Co., St. Louis, MO) following the manufacturer's protocol. The images were taken on a BZ-X710 microscope (Keyence America, Itasca, IL).

[0239] Immunohistochemistry for detection of cytotoxic T cells:

[0240] Sections of the paraffin-embedded liver were deparaffinized and rehydrated. The antigen retrieval was by heat mediation using citrate buffer (pH 6.0). Then, the slides were incubated in 10% normal goat serum with 1 % BSA in TBS for 2 hours at room temperature. The anti-CD4 or anti-CD8a monoclonal antibody (Abeam, Cambridge, MA) was diluted in TBS with 1 % BSA and incubated overnight at 4 °C. And then, the samples were washed with TBS containing 0.025% Triton X-100 and incubated in 0.3% H ₂ 0 ₂ in TBS for 15 minutes. The HRP-conjugated secondary antibody was diluted in TBS with 1% BSA and incubated for 1 hour at room temperature. The samples were washed and developed using SignalStain DAB substrate kit (Cell Signaling Technology, Danvers, MA) (Sun, B. et ai, (2005) Mol Ther, 11 (6): 889-98).

[0241] Western blot:

[0242] Frozen tissues were homogenized on ice in RIPA buffer [PBS containing 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA)] using a glass homogenizer. Lysates were cleared by centrifugation at 18,000 g at 4 °C for 15 minutes. The protein concentration of the supernatants was measured using the BCA assay. Equal amounts of protein were run on SDS-PAGE gels and transferred to nitrocellulose membrane. The membranes were blocked in 3% BSA/PBST, incubated with primary antibodies overnight at 4 °C, washed, incubated with secondary antibodies, washed again, and developed using ECL kit (Bio-rad, Hercules, CA). The images were obtained by the image analyzer (Bio-rad, Hercules, CA). The following primary antibodies were used: rabbit anti AGL (Abeam, ab71423) and mouse anti b-actin-HRP (Sigma-Aldrich, A3854).

[0243] Plasma enzyme activity measurement:

[0244] Plasma ALT, AST, and CK enzyme activities were assessed using the Liquid ALT Reagent Set, Liquid AST Reagent Set, and Creatinine Reagent Set (Pointe Scientific) per the manufacturer's protocol.

[0245] Determination of Urinary Glc4 concentration:

[0246] Urinary Glc4 concentration was determined by stable isotope-dilution electrospray tandem mass spectrometry as previously described (Young, S.P. et ai (2003) Biochem,

316(2): 175-80). [0247] Accelerating Rotarod test:

[0248] Mice were trained on a rotarod (ENV-577M, Med Associates Inc, Fairfax, VT) by first allowing them to stay for 3 minutes on the drum which was rotating at a constant speed of four rotations per minute (waiting mode). Mice were then trained twice on a gradually accelerating rotarod (4.0 - 40 rpm). Trained mice were then tested during three sessions using accelerating rotarod protocol, and the latency to fall was recorded. This routine provided at least 5 minutes of rest between the sessions (Lim, J.A., et al. (2018) Mol Ther., 26(5): 382-83).

[0249] Wire-hang test:

[0250] Mice are placed on a 1/4" mesh wire screen with either 2 or 4 limbs. Then the screen is inverted 8" above a mouse cage cushioned with bedding, and the time until the mouse drops off is recorded (Zhang, P., et al., (2012) Hum Gene Ther, 23(5):460-72).

[0251] Treadmill fatigue testing:

[0252] Treadmill testing will be performed every 1 to 2 months on a treadmill device (model LE 8709, Panlab, Spain) according to manufacturer's instructions. Running speed can be adjusted from ~5 to 150 m/min, and the running surface can be inclined from -25° to +25° above horizontal in 5° increments depending on the mouse strain. A stimulus can be created using the electrical shock grids, and grids can be enabled or disabled individually for each lane. The testing protocol include 3 steps: Acclimation, Warm-up, and Exhaustion exercise. The test will be ended when the mouse sits on the shock grid consecutive 5 seconds on the shock grid without attempting to reengage the treadmill (Knab, A.M., et al. (2009) Physiol Behav, 98(4):433-40).

[0253] Statistics:

[0254] Statistical significance was determined by unpaired two-tailed Student’s t-test using Prism software (GraphPad, La Jolla, CA); data are presented as mean ± standard deviation (SD). *P < 0.05 was considered statistically significant. * indicate P-values < 0.05; ** indicate P-values < 0.01 ; *** indicate P-values < 0.001 ; **** indicate P-values < 0.0001.

Example 1 : Generation of a mouse model of GSD Ilia

[0255] Heterozygous Agl mutant mice (Agl ^Tm1a ) in the C57BL/6N background that carry a mutant Agl allele (FIG. 1A) were purchased form the European Mouse Mutant Archive (EMMA) and crossed with the CMV-Cre mice (the Jackson Laboratory), to convert the mutant Agl allele into a knockout allele by deleting the exons 6~10 and the neo expression cassette (FIG. 1B). The resulting Agl ^+/ mice were interbred to generate homozygous Agl ^/_ mice. Agl ^/_ mice were used as breeders to produce Agl-KO mice (Ag ^/_ or GSD Ilia mice in this disclosure). No GDE protein was detected in liver, heart, and skeletal tissue of the GSD Ilia mice (FIG. 2). PAS-stained tissue sections from 3-month-old GSD Ilia mice showed massive glycogen accumulation in the liver, heart, skeletal muscles (quadriceps,

gastrocnemius, soleus muscle, tongue, and diaphragm) and smooth muscle (bladder), and significant amounts in the kidney and the entire region of the brain (data not shown). FIG. 3 shows the pattern of glycogen accumulation in liver, heart, and skeletal muscles of GSD Ilia mice with age. Masson Trichrome stained liver and kidney sections from 3-month-old GSD Ilia mice revealed mild fibrosis in both tissues (data not shown). Increased liver/body weight ratio (hepatomegaly), lowered blood glucose concentration (hypoglycemia), and elevated serum AST, ALT, ALP, and CK activities were consistently observed in the affected mice at different ages, when compared to the age-matched wild-type (WT) controls (data not shown). The levels of urinary Glc4, a glucose tetrasaccharide and a biomarker being used for GSD II and GSD III (Kumlien, J. et al. (1988) Clin. Chim. Acta, 176(1):39-48; Young, S.P. et al. (2003) Anal Biochem, 316(2):175-80; Pompe Disease Diagnostic Working, (2008) Mol. Genet. Metab., 93(3):275-81 ; Manwaring, V. et al. (2012) J Inherit. Metab Dis. 35(2):311-6), were also markedly elevated in these GSD Ilia mice compared to WT controls.

[0256] The following behavioral tests were performed in WT and GSD Ilia mice at 3 and 6 months of age, to assess the functional deficits in GSD Ilia mice. Treadmill fatigue test was used to evaluate cardiac function and exercise tolerance (Knab, A.M. et al. (2009) Physiol Behav. 98(4):433-440) (Knab, A.M., et al. (2009) Physiol Behav, 98(4):433-40); four limb wire-hang test for muscle strength (Zhang, P. et al. (2012) Hum. Gene Ther, 23(5):460-72; Miniarikova, J. et al. (2017) Gene Ther. 24(10):630-639); Rota-rod test for motor

coordination and balance (Sun, B. et al. (2008) Mol Ther 16(8): 1366-71 ; Sun, B. et al (2005) Mol Ther, 11(6): 889-98). a

[0257] The affected GSD Ilia mice showed a remarkable reduction in treadmill and wire- hang performance and a moderate but significant (p<0.05) decrease in Rota-rod test at 6 months of age, compared with the age-matched WT (FIG. 4). No significant difference was observed in other tests between the GSD Ilia and WT mice (data not shown). Similar results were observed when mice were tested at 3 months of age (data not shown). These data indicate that the neuromuscular dysfunction in the GSD Ilia mice is likely a result of muscle damage, rather than neurological impairment despite the widespread glycogen accumulation in the brain. At a minimum, neuromuscular dysfunction in the GSD Ilia mice can be due to peripheral nerve involvement.

[0258] Notably, based on the results described above, the GSD Ilia mouse model showed symptoms of liver and skeletal muscle defects similar to human GSD Ilia patients, such a liver fibrosis, hepatomegaly, increased plasma alanine aminotransferase (ALT) activity, elevated disease urinary Glc4, and impaired muscle functions.

Example 2: Correction of glycogen accumulation in liver of GSD Ilia mice and GSD Ilia dogs with the liver-targeted AAV-LSP-Pull vector.

[0259] In this experiment, the feasibility of using pullulanase-based gene therapy to reduce glycogen accumulation and the effectiveness of using a tissue- restricted gene expression approach to suppress pullulanase-induced cellular immune responses in GSD Ilia mice was tested. Ten-week-old GSD Ilia mice were intravenously injected with the AAV-CB-Pull or the AAV-LSP-Pull vector (both packaged as AAV9) at a dose of 5x10 ¹² vg/kg and euthanized two and seven weeks later to collect tissues and blood. Age-matched untreated (UT) GSD Ilia mice and WT mice were included as controls.

[0260] At two weeks after AAV injection, the AAV-LSP-Pull treatment resulted in a significantly (2-fold) higher Pullulanase activity in the liver than the AAV-CB-Pull treatment (FIG. 5A), but both treatments dramatically reduced glycogen content in the liver by >90% (FIG. 5B). Light microscopy of PAS-stained liver sections confirmed the clearance of glycogen accumulation and the restoration of normal hepatic morphology by the AAV treatments (FIG. 6). Consistent with the correction of liver glycogen, the liver/body weight ratios were reduced to the WT value by either AAV treatment (FIG. 7A) but the plasma ALT activities were decreased only in the AAV- LSP- Pull-treated mice (FIG. 7B). The lower Pullulanase activity in the liver (FIG. 5A) and the lack of the efficacy in the heart and skeletal muscle (data not shown) by the AAC-CB-Pull treatment are likely caused by the Pullulanase- induced cellular immune responses in these tissues. As expected, immunohistochemical staining of tissue sections showed multiple CD4+ and CD8+ lymphocytic infiltrates present in the AAV-CB-Pull treated liver, but barely detectable in the AAV-LSP-Pull treated liver two weeks after the AAV treatment (FIG. 8), a similar phenomenon was also observed in the heart and skeletal muscle (data not shown).

[0261] At seven weeks after AAV injection, the AAV-LSP-Pull treated livers still had very high Pullulanase activities and low glycogen contents; in contrast, Pullulanase activity became undetectable and glycogen content returned to the UT level in the AAV-CB-Pull treated liver (FIG. 5A, 5B). Consistent with the enzyme activity results, AAV copy numbers were significantly lower in the AAV-CB-Pull treated livers than that in the AAV-LSP-Pull treated liver (FIG. 9). The liver/body weight ratio and plasma ALT activity in the AAC-LSP- Pull treated mice were significantly lower than those in the AAV-CB-Pull-treated or untreated mice (FIG. 10A-10B), suggesting functional improvement of liver. In addition, Trichrome- stained liver sections showed that the AAV-LSP-Pull treatment effectively prevented progression of liver fibrosis seven weeks after AAV injection (FIG. 11). [0262] These results demonstrate that gene therapy with the liver-targeted AAV-LSP-Pull vector can effectively prevent Pullulanase-induced cytotoxic T cell immune responses and correct liver disease in GSD Ilia mice.

[0263] The efficacy of AAV-LSP-Pull treatment in correcting liver glycogen storage was further tested in a naturally occurring GSD Ilia dog model (Yi, H., et ai (2012) Dis Model Mech, 5(6):804-11 ; Yi, H., et ai (2014) J Mol Med (Berl), 92(6): 641-50; Brooks, E.D., et ai (2016) Comp Med, 66(1):41-51). Twelve-week-old GSD Ilia dogs (n=2) were intravenously injected with the AAV-LSP-Pull vector packaged as AAV9 at a dose of 3.8 x 10 ¹² vg/kg. Age- matched untreated (UT) GSD Ilia dogs were used as untreated controls (n=3). Two weeks after AAV treatment, liver and muscle biopsies were performed on both the AAV-treated and untreated GSD Ilia dogs following overnight fasting, to analyze the Pullulanase expression by Western blot and glycogen content in these tissues. Pre- and post-treatment radiographic imaging was done on the two treated dogs to measure the size of liver.

[0264] Two weeks after AAV-LSP-Pull treatment, Pullulanase expression was detected in the livers from both treated dogs (FIG. 12A); glycogen content was significantly reduced (- 35%) in the AAV-treated livers compared to the UT livers (FIG. 12B). No Pullulanase expression and glycogen reduction were observed in the skeletal muscle of the AAV-treated mice (not shown). Radiographic images of GSD Ilia dogs showed abnormal shape of the abdomen (yellow dot lines) with unusual curvatures before treatment due to hepatomegaly; two weeks after gene therapy, liver size was obviously reduced and the abdomen shape became less curvature (FIG. 13A). The liver size was significantly reduced (-35%) two weeks after AAV treatment compared to that at pre-treatment (FIG. 13B).

[0265] These data indicate that gene therapy with an AAV vector expressing Pullulanase is a feasible treatment approach for GSD III and the Pullulanase-induced cellular immune responses can be overcome by tissue-restricted gene expression. This liver-targeted gene therapy approach is suitable for treating GSD Nib patients (-15% of total GSD III patients) who have disease limited to the liver. For treatment of GSD Ilia patients, a supplementary therapy (muscle-directed gene therapy with the AAV-MHCK7-Pull vector) can be used to correct muscle specific symptoms along with the AAV-LSP-Pull treatment.

Example 3. Neonatal gene therapy with the AAV-CB-Pull vector achieved long-term systemic correction of glycogen storage in GSD Ilia mice

[0266] Immune response is impaired in neonatal mice during the first few weeks after birth (Ramachandran, P.S., et ai. (2017) Human Gene Then, 28(2): 154- 167) and GSD animals often accumulate very low levels of glycogen in muscle tissues at this stage. The AAV-CB- Pull vector packaged as AAV9 was intravenously injected into 14-day-old GSD Ilia mice at a dose of 5x10 ¹² vg/kg. Age-matched untreated mice were included as UT controls. All mice were euthanized 10 weeks after AAV injection following overnight fasting. The AAV treatment resulted in highly elevated Pullulanase activities in the heart and skeletal muscle (quadriceps) (FIG. 14A) and markedly reduced glycogen contents in these tissues (FIG. 14B). Consistent with measured glycogen contents, PAS stained tissue sections confirmed the clearance of glycogen accumulation in heart and skeletal muscle (FIG. 15). Plasma ALT and CK activities and urinary Glc4 levels were lowered by the AAV-CB-Pull treatment (FIG. 16A-16C). The failure of the AAV-CB-Pull treatment to correct liver in this experiment is likely caused by the rapid growth of liver during the 10-week experimental time. This study demonstrated a long-term benefit of neonatal gene therapy with the AAV-CB-Pull vector in GSD Ilia mice.

Example 4. Testing the ability of the AAV-MHCK7-Pull vector to achieve long-term efficacy in reversing muscular defects in GSD Ilia mice (for patients with GSD Ilia).

[0267] Systemic administration of the AAV-MHCK7-Pull vector can achieve long-term correction of cardiac and skeletal muscles in GSD Ilia mice and a high dose of vector administration is required for the correction of skeletal muscles. The AAV-MHCK7-Pull vector was packaged as AAV9 in this experiment. This experiment includes 2 groups starting with n=12 mice per group: Group 1 : no treatment; Group 2: AAV-MHCK7-Pull treatment. The AAV-MHCK7-Pull vector at 5x10 ¹³ vg/kg was intravenously injected into ten-week-old GSD Ilia mice. Mice from each group were euthanized after 12 weeks following overnight fasting to collect tissues and blood. Functional tests including treadmill and wire-hang were performed at 3, 4, and 5 months of age. Gender- and age-matched untreated GSD III mice were used as controls. Fresh tissue specimens were either immediately frozen on dry ice and stored at -80°C until used for biochemical analyses, or fixed immediately for histology.

[0268] Twelve weeks after AAV treatment, Pullulanase activity was significantly increased in the heart and skeletal muscle, but not in the liver, of the AAV-treated mice (FIG. 17A). Consistent with the enzyme activity results, AAV treatment significantly reduced glycogen levels in the heart and quadriceps while glycogen content in the liver was not significantly changes (FIG. 17B). Treadmill test was used for evaluating exercise intolerance. The running distance was dramatically increased for the AAV-treated mice compared to the untreated mice (FIG. 18A). Wire-hang test was performed to assess limb muscle strength by measuring the time of latency to fall. The AAV-treated mice showed significantly increased hanging time compared to the untreated mice throughout the treatment period (FIG. 18B). Example 5: Combination treatment with AAV9-CB-Pull and AAV8-LSP-Pull corrects glycogen storage in both liver and muscle tissues of GSD Ilia mice

[0269] Because GSD Ilia manifests mainly in liver, heart and skeletal muscles, the study focused on these tissues. The AAV vector plasmids pAAV-CB-Pull and pAAV-LSP-Pull (FIG. 19A) were packaged into AAV9 (AAV9-CB-Pull) and AAV8 (AAV8-LSP-Pull), respectively. Two-week-old GSD Ilia mice were intravenously injected with AAV9-CB-Pull at a dose of 1 x 10 ¹³ vg/kg. After 10 weeks, half of the mice were sacrificed to evaluate the efficacy of AAV9- CB-Pull treatment in different tissues at 3 months of age (FIG. 19B, 3 mo, gray arrow) and the other half were further treated with AAV8-LSP-Pull (1 x 10 ¹³ vg/kg) for another 10 weeks to correct liver abnormalities (FIG. 19B, 5 mo, black arrow). Behavioral tests were performed to evaluate muscle functions during the treatments at 2, 2.5, 3, 4, and 5 months old age (FIG. 19B, open arrows).

[0270] To evaluate the transduction efficiency of the AAV vectors in liver and muscles, we first checked AAV genome copy numbers using real-time-PCR and protein expression with Western blot. Ten weeks after the AAV9-CB-Pull (CB) treatment, AAV copy numbers were high in the heart (8.69±2.22 vg/genome) and low in the quadriceps (0.68±0.15) and liver (0.40±0.32) (FIG. 19C). Protein level was high in the heart, moderate in the quadriceps, and undetectable in the liver (FIG. 19D). Consistent with the Western blot results, Pullulanase enzyme activity was profoundly high in the heart (33.77±8.51 mU/mg) and readily detectable in the quadriceps (6.64±2.11 mU/mg) but undetectable in the liver at 3 months age (FIG. 19E). However, 10 weeks after the secondary treatment with AAV8-LSP-Pull (CB+LSP), the AAV genome copy number, protein expression, and enzyme activity were all significantly increased in the liver (FIG. 19C, FIG. 19D, FIG. 19E, black bars); enzyme activity was lowered in the heart (18.69±6.19 mU/mg) and still detectable in the skeletal muscle (FIG. 19E, black bars). Both the CB and CB+LSP combination treatments significantly decreased glycogen contents in the heart (-75-80%) and skeletal muscle (-80%) compared to those in the untreated mice (FIG. 20A). Glycogen level did not change significantly in the CB treated liver but decreased remarkably (-75%) in the CB+LSP treated liver (FIG. 20A).

[0271] PAS staining of tissue sections confirmed the glycogen content results. CB treatment effectively cleared glycogen accumulation in the heart and skeletal muscles (quadriceps, gastrocnemius, soleus, diaphragm, and tongue), but had no effect on the liver, smooth muscle (bladder), brain (cerebellum), or the spinal cord (FIG. 20B and FIG. 20C, CB). Additional treatment with the second AAV8-LSP-Pull vector cleared glycogen accumulation in the liver (FIG. 20B, CB+LSP).

[0272] Another hepatic disease manifestation in GSD III is progressive liver fibrosis. The liver fibrosis status was evaluated in the untreated and AAV treated GSD Ilia mice by trichrome staining. Untreated GSD Ilia liver showed similar early stage (stage 1-2) fibrosis with appearance of blue on staining at both 3 and 5 months of age (FIG. 20D, white arrows). The early (on postnatal day 14) treatment with AAV9-CB-Pull, which did not change the glycogen level in the liver, did not prevent liver fibrosis (FIG. 20D). There was no obvious fibrotic tissues in the CB+LSP treated liver (FIG. 20D), indicating that the secondary AAV8- LSP-Pull treatment can reverse and prevent early stage liver fibrosis in adult GSD Ilia mice.

[0273] Hepatomegaly is one of the most common symptoms of GSD III patients.

Therefore, the liver size using the liver-to-bodyweight ratio was measured. The liver-to- bodyweight ratio clearly increased in the untreated GSD Ilia mice (7~8%) compared to WT level (about 4%) (FIG. 21 A). Consistent with the Pullulanase activity and glycogen data, the liver size remained unchanged in the CB treated mice but was normalized to the WT level in the CB+LSP treated mice (FIG. 21A). Furthermore, the activity of ALT in the plasma, which is commonly used for monitoring liver damage, was reduced to the WT level in CB+LSP treated mice (FIG. 21 B). The CB treated mice showed a slightly decreased in plasma ALT activity compared with the untreated mice at 3 months of age (p=0.09, FIG. 21 B). A significant increase in plasma ALT level in the untreated GSD Ilia mice from 3 month of age (264.0±153.4 U/L) to 5 months of age (744.0±170.4 U/L), which indicated progressive liver damage during that time (FIG. 21 B).

[0274] Urinary Glc4, a known disease biomarker for Pompe disease (GSD II) that is often correlated with the levels of glycogen accumulation in skeletal muscle, has been indicated as a potential biomarker for GSD III. The concentration of urinary Glc4 was reduced to the WT level in the CB treated GSD Ilia mice at 3 months of age (FIG. 21 C).

[0275] The behavioral tests including treadmill, inverted wire hang, and Rota-rod have been broadly used for assessment of muscle function in mice. After AAV treatments, all the three tests showed significant improvement of muscle function (FIG. 21 D). Notably, the running distance improved dramatically in the treadmill test after AAV treatments (FIG. 21D).

[0276] The improved performance in the Rota-rod test after Pullulanase treatments (FIG. 21 D) could be the result of skeletal muscle recovery because the treatment did not apparently affect the glycogen accumulation in the brain and spinal cord (FIG. 20C). Thus, the combination treatment with AAV9-CB-Pull at an infant age (2 weeks) and AAV8-LSP-Pull at an adult age (3 months) effectively reduced glycogen accumulation in both liver and muscle and recovered liver function and improved muscle strength.

In addition to the Pullulanase used in this study, other bacterial enzymes with a similar glycogen degrading activity can also possibly be used for GSD III gene therapy, such as type I Pullulanase derived from other bacteria species and strains38 and the bacterial GDE encoded by the glgX gene in Escherichia coli. See FIG. 22 - FIG. 28 (corresponding to SEQ ID NOs: 01-07, respectively) and Table 2. Example 6: Gene therapy with an AAV vector expressing Pullulanase under the control of an immunotolerant LSP-CB dual promoter in GSD Ilia mice

[0277] In this study, the effectiveness of the use of a dual promoter consisting of a liver- specific promoter and the ubiquitous CB promoter to prevent immune responses against Pullulanase and correct genetic defects in both liver and muscle tissues of GSD Ilia mice was investigated.

[0278] The AAV9-LSP-Pull, AAV9-CB-Pull, and AAV9-LSP-CB-Pull were all packaged as AAV9. Ten-week-old GSD Ilia mice were intravenously injected with AAV9-LSP-Pull, AAV9- CB-Pull, AAV9-LSP-Pull+AAV9-CB-Pull, or AAV9-LSP-CB-Pull at a dose of 5.0 x 10 ¹² vg/kg (FIG. 29A). After ten weeks, the mice were sacrificed to collect tissues and blood. The mice were examined for the treadmill test at 2, 3, 4, and 5 months of age. Gender- and age- matched untreated GSD Ilia mice were used as controls. Fresh tissue specimens were either immediately frozen on dry ice and stored at -80°C until used for biochemical analyses, or fixed immediately for histology.

[0279] AAV vector genome copy numbers were evaluated by real-time PCR in the liver, heart, and quadriceps ten weeks after AAV treatment. AAV copy numbers were markedly high in all the tested tissues of the AAV-LSP-CB-Pull (Dual) treated mice and moderate elevated in the liver of AAV-LSP-Pull (LSP) treated mice. Very low AAV genome copies were detected in the heart and skeletal muscle of the treated LSP treated mice and in all the tissues from mice treated with AAV-CB-Pull alone (CB) or co-treated with LSP and CB (Co) (FIG. 29B).

[0280] Pullulanase activities were highly elevated in all the tested tissues (liver, heart, and skeletal muscle) by the LSP-CB dual promoter treatment. Pullulanase activity was increased only in the liver of LSP treated mice. There is no significantly increase in Pullulanase activity in any tissues of the CB treated and LSP+CB co-treated mice (FIG. 30A). Consistent with the Pullulanase activity results, glycogen contents were significantly in all the tissues of the LSP-CB dual promoter treated mice, but only in the liver of LSP treated mice. No glycogen reduction was observed in any tissues of the CB treated and LSP+CB co-treated mice (FIG. 30B). PAS-staining of tissue sections confirmed the reduction of glycogen accumulation in those tissues by the dual promoter treatment (FIG. 31).

[0281] The ratio of liver to body weight was measured to determine liver size. The ratio reduced significantly in the LSP and LSP-CB dual promoter treated mice but showed no significant change in the CB treated to the untreated mice. Co-injected mice showed a decrease in liver size but to a lesser extent than the LSP or LSP-CB dual promoter treated mice (FIG. 32A). The activities of alanine aminotransferase (ALT) and aspartate

aminotransferase (AST) in plasma were measured to evaluate liver function. Both LSP and LSP-CB dual promoter treatments significantly decreased ALT and AST levels. Co-injected mice showed slightly decreased in ALT level but not in AST level, compared to the UT mice. CB treatment had no effect on the plasma ALT and AST activities (FIG. 32B and FIG. 32C). Trichrome staining was used to detect liver fibrosis. Significant fibrotic tissues were observed in the livers from UT, CB treated, and co-treated mice. In contrast, LSP and LSP-CB dual promoter treatments fully prevented liver fibrosis in the GSD Ilia mice (FIG. 32D).

[0282] Treadmill test was used for evaluating exercise intolerance in the untreated and AAV treated GSD Ilia mice. Only the LSP-CB dual promoter treated mice showed significantly improved running distance and other treatment groups showed no differences compared to the UT mice (FIG. 33).

[0283] Immunohistochemical detection of cytotoxic T cell responses showed that infiltration of CD4+ or CD8+ lymphocytes increased in the CB-treated and Co-treated livers but were barely visible in LSP treated and LSP-CB dual promoter treated livers (FIG. 34).

[0284] This study demonstrated that the LSP-CB dual promoter effectively prevented Pullulanase-induced immune responses in GSD Ilia mice, leading to persistent Pullulanase expression and glycogen correction of the genetic defects in all the affected tissues (e.g., the liver and muscle tissues). These data indicate that this immunotolerant LSP-CB dual promoter technology can be used for gene therapy for human inherited diseases with broad tissue involvement.

Example 7: Gene therapy with an AAV vector under the control of an immunotolerant LSP-CB dual promoter in a mouse model of GSD IV

[0285] The immunotolerant and therapeutic properties of an AAV vector having a dual promoter, as described above in Example 6, was further investigated in a mouse model for adult polyglucosan body disease APBD harboring the homozygous Y329S mutation in the Gbe1 gene. The affected mice are designated as Gbe1 ^ys/ys mice and they are phenotypically very similar to APBD patients with residual GBE activity and widespread, progressive increase of PG deposition in all tissues, including the CNS (Akman, H.O., et ai, (2015) Hum Mol Genet, 24(23) :6801 -10).

[0286] Glycogen branching enzyme (GBE) is the enzyme that introduces branches to the growing glycogen molecule during the synthesis of glycogen in animals. GBE deficiency causes glycogen storage disease type IV (GSD IV), which is characterized by the accumulation of a poorly soluble amylopectin-like glycogen, called polyglucosan bodies (PBs), in liver, skeletal muscle, heart, and the central nervous system (CNS). GSD IV is clinically variable. The classical form of GSD IV is characterized by failure to thrive, hepatosplenomegaly, and progressive liver cirrhosis, which normally lead to death by 5 years of age. In addition to the hepatic presentation, four neuromuscular forms can be distinguished based on the ages at onset: fatal perinatal neuromuscular type, congenital muscular type, childhood neuromuscular type, and adult neuromuscular type (Bruno, C. et al. (2004) Neurology, 63(6):1053-8; Moses, S.W. et ai, (2002) Curr Mol Med, 2(2):177-88; Bao, Y. et al. (1996) J Clin Invest, 97(4):941-8). Most early onset GSD IV patients die in infancy or early childhood due to severe hypotonia, respiratory distress, cardiomyopathy and/or liver dysfunction. Adult onset GSD IV constitutes the majority of adult polyglucosan body disease (APBD) (Bruno, C. et ai (2004) Neurology, 63(6): 1053-8; Moses, S.W. et al., (2002) Curr Mol Med, 2(2):177-88; Bao, Y. et ai. (1996) J Clin Invest, 97(4):941-8). Y329S is the most common mutation in the GBE1 gene in APBD patients of Ashkenazi Jewish ancestry (Lossos, A. et ai. (1998) Ann Neurol, 44(6): 867-72). APBD can present as an isolated myopathy or as a multisystem disorder with intracellular accumulation of PG in the central and peripheral nervous systems and in all muscles (Mochel, F., et al. (2012) Ann Neurol, 72(3):433-441 ; Klein, C.J., (1993) GeneReviews(R)). There is currently no definitive treatment for GSD IV, though liver transplantation has been successful in some cases to alleviate liver symptoms (Ban, H.R., et ai. (2009) Eur J Pediatr, 152:S71-S76; Selby, R., et al. (1993) Eur J Pediatr, 152:S71-S76; Davis, M.K. and Weinstein, D.A. (2008) Pediatr Transplant, 12(2): 137-45).

[0287] It has been shown that a single intravenous injection of 5x10 ¹³ vg/kg AAV-hGBE vector packaged as AAV serotype 9 (AAV9) into 2-week-old Gbe1 ^ys/ys mice almost completely prevented PB formation in skeletal muscles, and partially corrected glycogen accumulation in the brain for up to 9 months of age (Yi, H. et al. (2017) Hum Gene Ther, 28(3):286-294). However, when the same vector was administered into adult mice, no hGBE expression or glycogen reduction was observed in any tissues at 6 months post vector treatment (unpublished data). This discrepancy was likely caused by a stronger cellular immune response towards human protein (hGBE) in adult mice. Thus, a novel method using an immunotolerant dual promoter (LSP-CB) consisting of a liver-specific promoter and a universally active promoter to prevent hGBE-induced cytotoxic T cell response in Gbe1 ^ys/ys mice was investigated.

[0288] Materials and Methods

[0289] Preparation of AAV vector plasmids. The pAAV-CB-hGBE vector, which contains a CMV-enhanced chicken beta-actin hybrid promoter (CB), was described previously (Yi, H. et al. (2017) Hum Gene Ther, 28(3):286-294); to construct the pAAV-LSP- CB-hGBE vector, the Xba\-Kpn\ fragment containing the LSP-CB dual promoter from the AAV- LSP-CB- Pul I promoter was subcloned into the pAAV-CB-hGBE vector, to replace the CB promoter. Both AAV vectors had a human growth hormone polyA signal sequence (FIG. 35). [0290] Expression of GBE in HEK293 cells: Cells plated in 10-cm plates were transfected with 10pg of plasmid per plate. Cells were harvested 48 hours after transfection. For Western blotting, cells were lysed in RIPA buffer; for GBE activity assay, cells were homogenized in cold water (Yi, H. et al. (2017) Hum Gene Ther, 28(3):286-294).

[0291] AAV packaging, purification, and mouse injection: Both AAV vectors were packaged as AAV9 in HEK293T cells using phosphate triple transfection method and purified using iodixanol gradient ultracentrifugation (Yi, H. et ai (2017) Hum Gene Ther, 28(3):286-294). Two-month-old Gbe1 ^ys/ys mice were intravenously injected with the AAV-CB- hGBE or AAV-LSPCB-hGBE at a dose of 2x10 ¹³ vg/kg (n=3 each group), and liver, heart and muscle (quadriceps) tissues were collected 2 weeks later.

[0292] GBE activity assay and Western blotting: All procedures were described previously (Yi, H. et al. (2017) Hum Gene Ther, 28(3):286-294).

[0293] Immunohistochemical staining: Fresh liver specimens were fixed in 10% neutral formalin and the paraffin-embedded tissue sections were stained with an anti-CD4 or anti CD8a antibody (Abeam).

[0294] Results

[0295] Forty-eight hours after transfection of HEK293 cells, both AAV-CB-hGBE and AAV- LSPCB-hGBE vectors resulted in high-level hGBE expression as determined by the Western blot and GBE activity assays (FIG. 36A and FIG. 36B). This suggests that both CB promoter and LSPCB dual promoter are highly active in the non-liver cells.

[0296] Two weeks post AAV injection, the GBE activities were markedly higher in the liver, heart, and skeletal muscle of the AAV-LSPCB-hGBE treated Gbe1 ^ys/ys mice than those of the AAV-CB-hGBE treated mice or the untreated mice (FIG. 37).

[0297] Immunohistochemical stained liver sections revealed that multiple CD4+ and CD8+ lymphocytic infiltrates were present in the AAV-CB-hGBE treated liver but barely detectable in the AAV-LSPCB-hGBE treated liver (FIG. 38).

[0298] These results demonstrate that the cellular immunity against hGBE expressed from the CB promoter was associated with waning hGBE levels in the AAV-CB-hGBE treated mice. Our data indicate that the use of the LSPCB dual promoter can prevent transgene- induced immune responses and achieve long-term efficacy of gene therapy in the Gbe1 ^ys/ys mice.

[0299] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be incorporated within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated herein by reference for all purposes.