Acid alpha-glucosidase variants and uses thereof

Abstract

Acid alpha-glucosidase variant polypeptides and vectors comprising nucleic acid molecules or nucleic acid constructs encoding the variant polypeptides are provided. [Solution] A truncated acid alpha-glucosidase (GAA) polypeptide comprising the deletion of at least one amino acid from the N-terminus of the GAA polypeptide, wherein the parent polypeptide corresponds to a precursor form of the GAA polypeptide lacking its signal peptide, and the truncated GAA polypeptide has 1 to 75 consecutive amino acids deleted at its N-terminus compared to the parent GAA polypeptide, and wherein the truncated GAA polypeptide further comprises a signal peptide fused to its N-terminus.

Description

[Technical field]

[0001] The present invention relates to acid alpha-glucosidase variants and uses thereof.

[0002] Pompe disease, also known as glycogen storage disease (GSD) type II and acid maltase deficiency, is an autosomal recessive metabolic muscle disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is an exo-1,4 and 1,6-alpha-glucosidase that hydrolyzes glycogen to glucose in the lysosomes. Deficiency of GAA leads to accumulation of glycogen in the lysosomes, causing progressive damage to respiratory, cardiac and skeletal muscles. The disease ranges from a rapidly progressive infantile course that is usually fatal by 1-2 years of age to a slowly progressive heterogeneous course that causes significant morbidity and early mortality in children and adults. Hirschhorn RR, The Metabolic and Molecular Bases of Inherited Disease, 3: 3389-3420 (2001, McGraw-Hill); Van der Ploeg and Reuser, Lancet 372: 1342-1351 (2008).

[0003] Current therapies in humans to treat Pompe disease include the administration of recombinant human GAA, otherwise known as enzyme replacement therapy (ERT). ERT has demonstrated efficacy for severe infantile glycogen storage disease type II. However, the benefits of enzyme therapy are limited by the need for frequent infusions and the development of inhibitory antibodies against recombinant hGAA (Amalfitano, A., et al. (2001) Genet. In Med. 3:132-138). Furthermore, ERT does not effectively repair the entire body. This is likely due to a combination of poor biodistribution of the protein after delivery via a peripheral vein, lack of uptake from some tissues, and high immunogenicity.

[0004] The feasibility of gene therapy approaches to treat glycogen storage disease type II, as an alternative or adjunct to ERT, has been investigated (Amalfitano, A., et al. (1999) Proc. Natl. Acad. Sci. USA 96:8861-8866, Ding, E., et al. (2002) Mol. Ther. 5:436-446, Fraites, TJ, et al. (2002) Mol. Ther. 5:571-578, Tsujino, S., et al. (1998) Hum. Gene Ther. 9:1609-1616). However, muscle-directed gene transfer to correct genetic defects must face the limitations of the systemic nature of the disease and the fact that transgene expression in muscle tends to be more immunogenic than in other tissues.

[0005] Doerfler et al., 2016, described the combined administration of two constructs encoding human codon-optimized GAA, one under the control of a liver-specific promoter and the other under the control of a muscle-specific promoter. Expression of GAA driven by the liver-specific promoter was found to be consistent with Gaa - / - It has been used to promote immune tolerance to GAA in mouse models, while expression of GAA driven by a muscle-specific promoter provides expression of therapeutic proteins in tissue portions targeted for therapy. However, this strategy is not entirely satisfactory in that it requires the use of multiple constructs, which does not result in systemic expression of GAA.

[0006] Modified GAA proteins have been proposed in the past to improve the treatment of lysosomal storage diseases. In particular, WO2004064750 application and Sun et al. 2006 disclose chimeric GAA polypeptides that include a signal peptide operably linked to GAA as a method for enhancing targeting of proteins to the secretory pathway.

[0007] However, the treatments available to patients are not entirely satisfactory, and there remains a need in the art for improved GAA polypeptides and GAA production. In particular, there remains a need for long-term efficacy of treatment with GAA, high levels of GAA production, improved immune tolerance to the produced GAA polypeptides, and improved uptake of GAA by cells and tissues in need thereof. Furthermore, in WO2004064750 and Sun et al. 2006, the tissue distribution of the chimeric GAA polypeptides disclosed therein is not entirely satisfactory. Therefore, there remains a need for GAA polypeptides that will completely cure by allowing the restoration of glycogen accumulation in most, if not all, tissues of interest.

[0008] Summary of the Invention The present invention relates to GAA mutants that are expressed and secreted at higher levels compared to wild-type GAA protein and that cause improved repair of pathological accumulations of glycogen throughout the body, thereby inducing immune tolerance to GAA.

[0009] According to one embodiment, the present invention relates to a truncated GAA polypeptide comprising at least one amino acid deletion from the N-terminus of a parent GAA polypeptide, where the parent polypeptide corresponds to a precursor form of the GAA polypeptide lacking its signal peptide. In a particular embodiment, the truncated GAA polypeptide has at least 2, particularly at least 2, particularly at least 3, particularly at least 4, particularly at least 5, particularly at least 6, particularly at least 7, particularly at least 8 consecutive amino acids deleted at its N-terminus compared to the parent GAA polypeptide. In another embodiment, the truncated GAA polypeptide has at most 75, particularly at most 70, particularly at most 60, particularly at most 55, particularly at most 50, particularly at most 47, particularly at most 46, particularly at most 45, particularly at most 44, particularly at most 43 consecutive amino acids deleted at its N-terminus compared to the parent GAA polypeptide. In a further particular embodiment, the truncated GAA polypeptide has at most 47, particularly at most 46, particularly at most 45, particularly at most 44, particularly at most 43 consecutive amino acids deleted at its N-terminus compared to the parent GAA polypeptide. In another particular embodiment, the truncated GAA polypeptide has from 1 to 75, particularly from 1 to 47, particularly from 1 to 46, particularly from 1 to 45, particularly from 1 to 44, particularly from 1 to 43 consecutive amino acids deleted at its N-terminus compared to the parent GAA polypeptide. In another embodiment, the truncated GAA polypeptide has from 2 to 43, particularly from 3 to 43, particularly from 4 to 43, particularly from 5 to 43, particularly from 6 to 43, particularly from 7 to 43, particularly from 8 to 43 consecutive amino acids deleted at its N-terminus compared to the parent GAA polypeptide. In more particular embodiments, the truncated GAA polypeptide lacks 6, 7, 8, 9, 10, 27, 28, 29, 30, 31, 40, 41, 42, 43, 44, 45, 46 or 47 consecutive amino acids at its N-terminus compared to the parent GAA polypeptide, in particular is truncated by 7, 8, 9, 28, 29, 30, 41, 42, 43 or 44, more particularly 8, 29, 42 or 43 consecutive amino acids at its N-terminus compared to the parent GAA polypeptide. In a further particular embodiment, the parent polypeptide is human GAA (hGAA), in particular hGAA having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

[0010] In certain embodiments, the truncated GAA polypeptides of the invention have the sequences shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, and SEQ ID NO:35.

[0011] Furthermore, the truncated GAA polypeptide of the present invention may further comprise a signal peptide fused to its N-terminus, particularly a signal peptide selected from the group consisting of SEQ ID NOs: 3 to 7, particularly the signal peptide of SEQ ID NO: 3.

[0012] In another aspect, the present invention relates to a nucleic acid molecule encoding a truncated GAA polypeptide as described above, optionally fused via its N-terminus to a signal peptide. In some embodiments, the nucleic acid molecule relates to a nucleotide sequence optimized for improving expression of the truncated GAA polypeptide in vivo, particularly in a human subject, and / or for improving immune tolerance to the truncated GAA polypeptide.

[0013] In yet another aspect, the present invention relates to a nucleic acid construct comprising a nucleic acid molecule of the present invention operably linked to one or more regulatory sequences, such as a promoter, an intron, a polyadenylation signal and / or an enhancer (e.g., a cis-regulatory module, or CRM). In a particular embodiment, the promoter is a liver-specific promoter, preferably selected from the group consisting of alpha-1 antitrypsin promoter (hAAT), transthyretin promoter, albumin promoter, and thyroxine-binding globulin (TBG) promoter. In another particular embodiment, the promoter is a muscle-specific promoter, such as Spc5-12, MCK and desmin promoter. In another embodiment, the promoter is a ubiquitous promoter, such as CMV, CAG and PGK promoter. The nucleic acid construct may further optionally comprise an intron, particularly an intron selected from the group consisting of the human beta globin b2 (i.e. HBB2) intron, the FIX intron, the chicken beta-globin intron, and the SV40 intron, wherein the intron is optionally a modified intron, such as the modified HBB2 intron of SEQ ID NO: 17, the modified FIX intron of SEQ ID NO: 19, or the modified chicken beta-globin intron of SEQ ID NO: 21. In a particular embodiment of the nucleic acid construct of the invention, the construct comprises, preferably in this order, an enhancer; an intron; a promoter, particularly a liver-specific promoter; a nucleic acid sequence encoding a GAA protein; and a polyadenylation signal, wherein the construct comprises, preferably in this order, an ApoE regulatory region; an HBB2 intron, particularly a modified HBB2 intron; a hAAT promoter; a nucleic acid sequence encoding a truncated GAA polypeptide; and a bovine growth hormone polyadenylation signal. In a specific embodiment, the nucleic acid construct more particularly comprises the nucleotide sequence of any one of SEQ ID NOs: 22-26.

[0014] In another aspect, the present invention relates to a vector comprising the nucleic acid molecule or nucleic acid construct disclosed herein.The vector of the present invention can be a viral vector, preferably a retroviral vector, such as a lentiviral vector, or an AAV vector.Preferably, the vector is a single-stranded or double-stranded self-complementary AAV vector, preferably a capsid derived from AAV, such as AAV1, AAV2, mutant AAV2, AAV3, mutant AAV3, AAV3B, mutant AAV3B, AAV4, AAV5, AAV6, mutant AAV6, AAV7, AAV8, AAV9, AAV10, such as AAVcy10, and AAVrh10, AAVrh74, AAVdj, AAV-Anc80, AAV-LK03, AAV2i8, and an AAV vector with a porcine AAV capsid, such as AAVpo4 and AAVpo6 capsid, or a chimeric capsid. In specific embodiments, the vector is an AAV vector having an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly an AAV8, AAV9, or AAVrh74 capsid, more particularly an AAV8 capsid.

[0015] In yet another aspect, the present invention provides a cell transformed with a nucleic acid molecule, a nucleic acid construct or a vector of the invention, more particularly said cell being a liver cell or a muscle cell.

[0016] In certain aspects, the present invention provides pharmaceutical compositions comprising a truncated GAA polypeptide, nucleic acid molecule, nucleic acid construct, vector, or cell of the present invention in a pharma- ceutically acceptable carrier.

[0017] The present invention further relates to a truncated GAA polypeptide, a nucleic acid molecule, a nucleic acid construct, a vector or a cell of the invention for use as a medicament.

[0018] The present invention further provides a truncated GAA polypeptide, nucleic acid molecule, nucleic acid construct, vector, or cell of the present invention for use in a method for treating glycogen storage disease. In particular embodiments, the glycogen storage disease is glycogen storage disease type I, glycogen storage disease type II, glycogen storage disease type III, glycogen storage disease type IV, glycogen storage disease type V, glycogen storage disease type VI, glycogen storage disease type VII, glycogen storage disease type VIII, or fatal congenital glycogen storage disease of the heart. In more particular embodiments, the glycogen storage disease is selected from the group consisting of glycogen storage disease type I, glycogen storage disease type II, and glycogen storage disease type III, more particularly selected from the group consisting of glycogen storage disease type II and glycogen storage disease type III. In an even more particular embodiment, the glycogen storage disease is glycogen storage disease type II. [Brief description of the drawings]

[0019] [Figure 1]Deletion of a portion of hGAA increases its secretion in vitro. Panel A. Human hepatoma cells (Huh7) were transfected using Lipofectamine™ with a control plasmid expressing green fluorescent protein (GFP) or with plasmids expressing wild-type hGAA (hGAA) or hGAA sequences optimized according to two distinct algorithms (hGAAco1 and co2, respectively). The different hGAA constructs contained the wild-type signal peptide or the human alpha-1-antitrypsin signal peptide (sp2). A truncated hGAA was obtained by deletion of 8 amino acids (Δ8) after the signal peptide. 48 hours after transfection, hGAA activity in the culture medium was measured by a fluorogenic enzyme assay and GAA activity was evaluated against a standard curve of the reaction products as indicated in Materials and Methods. The histogram plots show the mean ± standard error of the levels of secreted hGAA derived from three different experiments. Statistical analysis was performed by paired t-test and the resulting p-values ​​are reported in the histograms (*=p<0.05 as indicated). Panel B. Human hepatoma cells (Huh7) were transfected using lipofectamine with a control plasmid expressing GFP or with a plasmid expressing hGAAco1 with wild-type signal peptide or chymotrypsinogen B1 signal peptide (sp7). The hGAA protein was truncated by removing 8 or 42 amino acids after the signal peptide (Δ8 and Δ42, respectively). 48 hours after transfection, hGAA activity in the culture medium was measured by fluorogenic enzyme assay as indicated above. The histogram plots show the mean ± standard error of the levels of secreted hGAA derived from three different experiments. Statistical analysis was performed by analysis of variance (*=p<0.05 as indicated). [Diagram 2]Deletion of a portion of hGAA increases its secretion into the bloodstream in a mouse model of Pompe disease. Three-month-old GAA- / - mice (n=4-5 mice per group) were intravenously injected with PBS or 2x1012vg / kg of an AAV8 vector expressing an optimized version of hGAA (hGAAco1) under the transcriptional control of a liver-specific promoter. The wild-type signal peptide of hGAA was replaced with the chymotrypsinogen B1 signal peptide (sp7), and the hGAA sequence was used either as the full-length native sequence or as a truncated sequence by removing 8 or 42 amino acids after the signal peptide (Δ8 and Δ42, respectively). One month after injection, the mice were bled and hGAA activity in the serum was measured using a fluorogenic assay. Statistical analysis was performed by analysis of variance (*=p<0.05 as indicated). [Diagram 3] Signal peptide enhances secretion of hGAA. Human hepatoma cells (Huh7) were transfected by Lipofectamine™ with a control plasmid (GFP), a plasmid expressing wild-type hGAA (designated as sp1), or a plasmid expressing the optimized Δ8 hGAA (hGAAco) sequence fused to signal peptides 6-8 (sp6-8). 48 hours after transfection, the activity of hGAA in the culture medium was measured by a fluorogenic enzyme assay and GAA activity was evaluated against a standard curve of 4-methylumbelliferone. Histogram plots show the mean ± standard error of the levels of secreted hGAA derived from three different experiments. Statistical analysis was performed by analysis of variance (*=p<0.05 vs. mock-transfected cells). [Figure 4]Truncated Δ8hGAA effectively restores glycogen accumulation in a mouse model of Pompe disease. Four-month-old wild-type (WT) and GAA- / - mice (n=4–5 mice per group) were intravenously injected with PBS or 6×1011vg / kg of AAV8 vector expressing sequence-optimized Δ8hGAA (hGAAco) under the transcriptional control of the human α-1-antitrypsin promoter and fused to signal peptides 1, 2, 7, and 8 (sp1, 2, 7, 8). Panel A. Histograms show hGAA activity measured by fluorogenic assay in blood 3 months after vector injection. Statistical analysis was performed by analysis of variance, and the histograms report the p-values ​​obtained relative to GAA- / - animals treated with PBS (*=p<0.05). Panels B–D. Biochemical restoration of glycogen content in heart, diaphragm, and quadriceps. Four-month-old GAA- / - mice were treated as described above. Three months after injection, mice were sacrificed and glycogen content was assessed. Histograms show glycogen content expressed as glucose released after enzymatic digestion of glycogen measured in the heart (panel B), diaphragm (panel C) and quadriceps (panel D). Statistical analysis was performed by analysis of variance (*=p<0.05 vs. PBS-injected GAA- / - mice). [Diagram 5] Highly secreted hGAA reduces humoral responses in a mouse model of Pompe disease. Four-month-old GAA- / - mice were intravenously injected with PBS or two different doses (5x1011 or 2x1012 vg / kg) of AAV8 vectors containing an optimized sequence under the transcriptional control of the human α-1-antitrypsin promoter and encoding Δ8hGAA fused to signal peptide 1 (co), signal peptide 2 (sp2-Δ8-co), signal peptide 7 (sp7-Δ8-co) or signal peptide 8 (sp8-Δ8-co). One month after injection, serum was analyzed for the presence of anti-hGAA antibodies by ELISA. Quantification was performed using purified mouse IgG as a standard. Statistical analysis was performed by analysis of variance with Dunnett's post-hoc test (*=p<0.01). [Figure 6]Injection of AAV8-hAAT-sp7-Δ8-hGAAco1 results in efficient secretion of hGAA into the blood and uptake into muscle in non-human primates. Two cynomolgus monkeys were injected with 2×1012 vg / kg AAV8-hAAT-sp7-Δ8-hGAAco1 on day 0. Panel A. Western blots of hGAA performed on serum from two monkeys obtained 12 days before and 30 days after vector administration. The band positions of molecular weight markers run in parallel with the samples are shown on the left. Panel B. Three months after vector injection, the monkeys were sacrificed and tissues were collected for biochemical evaluation of hGAA uptake. Western blots of hGAA were performed on tissue extracts obtained from biceps and diaphragm. Anti-tubulin antibody was used as a loading control. The band positions of molecular weight markers run in parallel with the samples are shown on the left. [Figure 7] Biochemical restoration of glycogen content in livers of GDE- / - animals injected with vectors expressing hGAA. Three-month-old wild-type (WT) or GDE- / - mice were intravenously injected with PBS or an AAV8 vector expressing codon-optimized hGAA under the transcriptional control of the human α-1-antitrypsin promoter and fused to signal peptide 7 (AAV8-hAAT-sp7-Δ8-hGAAco1) at a dose of 1×1011 or 1×1012 vg / mouse. Histogram plots show glycogen content, expressed as glucose released after enzymatic digestion of glycogen measured in liver. Statistical analysis was performed by analysis of variance (*=p<0.05 vs. PBS-injected GDE- / - mice, §=p<0.05 vs. PBS-injected WT animals). [Figure 8]GAA activity in the medium of cells transfected with plasmids encoding various GAA mutants. GAA activity was measured in the medium of HuH7 cells 24 hours (panel A) and 48 hours (panel B) after transfection of plasmids containing optimized sequences encoding native GAA (co) combined with the native GAAsp1 signal peptide or an engineered GAA (sp7-co) containing native GAA combined with a heterologous sp7 signal peptide. The effect of various deletions in the GAA coding sequence after the sp7 signal peptide was evaluated (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7-Δ47-co, sp7-Δ62-co). A plasmid encoding eGFP was used as a negative control. Statistical analysis was performed by one-way ANOVA with Tukey's post-hoc test. Hash symbols (#) in the bar graphs indicate statistically significant differences with respect to co; tau symbols (τ) indicate statistically significant differences with respect to sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, and sp7-Δ43-co. Data are means ± standard deviations of two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, unless different symbols are used. [Figure 9]Intracellular GAA activity of various GAA mutants. GAA activity was measured in lysates of HuH7 cells 48 hours after transfection of plasmids containing optimized sequences encoding native GAA combined with the native GAAsp1 signal peptide (co) or engineered GAA (sp7-co) containing native GAA combined with a heterologous sp7 signal peptide. The effect of various deletions in the GAA coding sequence after the signal peptide was evaluated (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7-Δ47-co, sp7-Δ62-co). A plasmid encoding eGFP was used as a negative control. Statistical analysis was performed by one-way ANOVA with Tukey post-hoc test. Tau symbols (τ) indicate statistically significant differences relative to sp7-co, sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co. Data are means ± standard deviations from two independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 unless different symbols are used. [Figure 10]Increased GAA activity in cell culture medium using the Δ8 deletion in combination with sp6 or sp8 signal peptide. GAA activity was measured in culture medium (panel A) and lysates (panel B) of HuH7 cells 48 hours after transfection of plasmids containing optimized sequences encoding native GAA in combination with the native GAAsp1 signal peptide (co) or engineered GAA (sp6-co or sp8-co) containing native GAA in combination with a heterologous sp6 or sp8 signal peptide. The effect of deleting 8 amino acids in the GAA coding sequence after the signal peptide is evaluated (sp6-Δ8-co, or sp8-Δ8-co). A plasmid encoding eGFP was used as a negative control. Statistical analysis was performed by one-way ANOVA with Tukey post-hoc test. Asterisks in the bar graphs indicate statistically significant differences relative to co. Data are the mean ± standard deviation of two independent experiments. Unless different symbols are used, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0020] Detailed Description of the Invention The present invention relates to truncated GAA polypeptides, nucleic acid molecules encoding such truncated GAA polypeptides, nucleic acid constructs comprising said nucleic acids, vectors comprising said nucleic acid constructs, cells comprising said nucleic acid molecules or constructs or vectors, and pharmaceutical compositions comprising a polypeptide, nucleic acid molecule, nucleic acid construct, vector or cell according to the invention. The inventors have surprisingly shown that the truncated forms of GAA according to the invention greatly improve the secretion of GAA while reducing its immunogenicity.

[0021] Lysosomal acid α-glucosidase or "GAA" (EC 3.2.1.20) (1,4-α-D-glucan glucohydrolase) is an exo-1,4-α-D-glucosidase that releases glucose by hydrolyzing both α-1,4 and α-1,6 linkages in oligosaccharides. Deficiency of GAA causes glycogen storage disease type II (GSDII), also called Pompe disease (the term formally refers to the infantile-onset form of the disease). It catalyzes the complete breakdown of glycogen and is slow at branching. The 28 kb human acid α-glucosidase gene on chromosome 17 encodes a 3.6 kb mRNA that produces a 951 amino acid polypeptide (Hoefsloot et al., (1988) EMBO J. 7: 1697; Martiniuk et al., (1990) DNA and Cell Biology 9: 85). The enzyme undergoes cotranslational N-linked glycosylation in the endoplasmic reticulum. It is synthesized as a 110 kDa precursor form and matures by extensive glycosylation modification, phosphorylation, and proteolytic processing through an approximately 90 kDa endosomal intermediate to the final lysosomal 76 and 67 kDa forms (Hoefsloot, (1988) EMBO J. 7: 1697; Hoefsloot et al., (1990) Biochem. J. 272: 485; Wisselaar et al., (1993) J. Biol. Chem. 268: 2223; Hermans et al., (1993) Biochem. J. 289: 681).

[0022] Acid α-glucosidase deficiency in patients with GSD type II causes massive accumulation of glycogen in lysosomes, disrupting cellular function (Hirschhorn, R. and Reuser, AJ (2001), in The Metabolic and Molecular Basis for Inherited Disease, (eds, Scriver, CR et al.) pages 3389-3419 (McGraw-Hill, New York). In the most common infantile form, patients show progressive muscle degeneration and cardiomyopathy and die before the age of 2 years. Severe wasting is present in juvenile-onset and adult-onset forms.

[0023] In addition, patients with other glycogen storage diseases may benefit from the administration of optimized forms of GAA. For example, administration of GAA has been shown to reduce glycogen in primary myoblasts from patients with glycogen storage disease type III (GSD III) (Sun et al. (2013) Mol Genet Metab 108(2): 145; WO 2010 / 005565).

[0024] In particular, in the context of the present invention, a "precursor form of GAA" is a polypeptide form of GAA that includes its native signal peptide. For example, the sequence of SEQ ID NO: 2 is the precursor form of human GAA (hGAA). Amino acid residues 1-27 in SEQ ID NO: 2 correspond to the signal peptide of the hGAA polypeptide. This sequence of the signal peptide of hGAA is also shown in SEQ ID NO: 4.

[0025] In the context of the present invention, the truncated GAA polypeptides of the present invention are derived from parent GAA polypeptides. According to the present invention, a "parent GAA polypeptide" is a functional precursor GAA sequence as defined above, but lacking its signal peptide. For example, referring to a typical wild-type human GAA polypeptide, the complete wild-type GAA polypeptide (i.e. the precursor form of GAA) is shown in SEQ ID NO: 2 or SEQ ID NO: 30 and has a signal peptide (corresponding to amino acids 1-27 of SEQ ID NO: 2 or SEQ ID NO: 30), whereas the parent GAA polypeptides serving as the basis for the truncated GAA forms of these wild-type human GAA polypeptides are shown in SEQ ID NO: 1 and SEQ ID NO: 33, respectively, and do not have any signal peptide. In this example, amino acids 28-952 of SEQ ID NO: 2 and the latter part corresponding to amino acids 28-952 of SEQ ID NO: 30 are referred to as parent GAA polypeptides.

[0026] According to the present invention, the truncated GAA polypeptide of the present invention is a functional GAA polypeptide, i.e. it has the function of a wild-type GAA polypeptide. As defined above, the function of wild-type GAA is to release glucose by hydrolyzing both α-1,4 and α-1,6 bonds of oligosaccharides and polysaccharides, more particularly glycogen. A functional GAA polypeptide encoded by a nucleic acid of the present invention may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least 100% hydrolysis activity on glycogen compared to the wild-type GAA polypeptide of SEQ ID NO: 1 or SEQ ID NO: 33. The activity of the GAA protein encoded by a nucleic acid of the present invention may even be more than 100%, for example more than 110%, 120%, 130%, 140% or even more than 150% relative to the activity of the wild-type GAA protein of SEQ ID NO: 1 or SEQ ID NO: 33.

[0027] The amino acid sequence of the parent GAA polypeptide or its coding sequence can be obtained from any source, including avian and mammalian species. As used herein, the term "avian" includes, but is not limited to, chicken, duck, goose, quail, turkey, and pheasant. As used herein, the term "mammal" includes, but is not limited to, humans, monkeys, and other non-human primates, cows, sheep, goats, horses, cats, dogs, lagomorphs, and the like. In an embodiment of the invention, the parent GAA polypeptide is a human, mouse, or quail, particularly a human GAA polypeptide.

[0028] Furthermore, the parent GAA polypeptide may be a functional variant of a GAA polypeptide, comprising one or more amino acid modifications, such as insertions, deletions, and / or substitutions of amino acids compared to the GAA polypeptide. For example, the parent polypeptide may be a functional derivative of a human GAA polypeptide, such as a polypeptide of SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1, having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity to this human GAA polypeptide. For example, in addition to the truncation as defined above, the functional variant of a GAA polypeptide may have 0-50, 0-30, 0-20, 0-15, 0-10, or 0-5 amino acid changes relative to the parent GAA polypeptide, such as the parent GAA polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1. In particular, the parent GAA polypeptide may consist of a human GAA polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1.

[0029] The term "identical" and its derivatives, when referring to a polypeptide, means that if a position in two compared polypeptide sequences is occupied by the same amino acid (e.g., if a position in each of the two polypeptides is occupied by leucine), the polypeptides are identical at that position. The percentage of identity between two polypeptides is a function of the number of matching positions shared by the two sequences, divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two polypeptides are identical, the two sequences are 60% identical. Generally, two sequences are aligned and compared to obtain the maximum percentage of identity. Nucleic acid sequences can be aligned using various bioinformatics tools known to those skilled in the art, such as BLAST or FASTA.

[0030] The parent GAA polypeptide may also be a GAA variant such as GAA II as described by Kunita et al., (1997) Biochemica et Biophysica Acta 1362: 269; GAA polymorphisms and SNPs are described by Hirschhorn, R. and Reuser, AJ (2001) In The Metabolic and Molecular Basis for Inherited Disease (see Scriver, CR, Beaudet, AL, Sly, WS & Valle, D. Eds., pp. 3389- 3419. McGraw-Hill, New York, pages 3403-3405). Any variant GAA polypeptide known in the art may be used as a basis for defining a parent GAA polypeptide. Exemplary mutant GAA polypeptides include SEQ ID NO:2 (NCBI Reference Sequence NP_000143.2); SEQ ID NO:29 (GenBank AAA52506.1); SEQ ID NO:30 (GenBank CAA68763.1); SEQ ID NO:31 (GenBank:EAW89583.1) and SEQ ID NO:32 (GenBank ABI53718.1). Other useful variants include those described in Hoefsloot et al., (1988) EMBO J. 7: 1697; and Van Hove et al., (1996) Proc. Natl. Acad. Sci. USA 93: 65 (human), and GenBank Accession No. NM_008064 (mouse). Other mutant GAA polypeptides include those described in WO 2012 / 145644, WO 00 / 34451, and U.S. Patent No. 6,858,425. In certain embodiments, the parent GAA polypeptide is obtained from the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:30.

[0031] A truncated form of GAA according to the present invention is an N-terminal truncated form of a parent GAA polypeptide, in which at least one amino acid is deleted from the N-terminus of the parent GAA polypeptide.

[0032] By "truncated" is meant a GAA polypeptide in which one or several consecutive amino acids have been deleted from the N-terminal portion of the parent GAA polypeptide. For example, the GAA portion may be truncated by 1 to 75 consecutive amino acids, or by more than 75 consecutive amino acids, from its N-terminus, as compared to the parent GAA polypeptide. Specifically, a truncated GAA polypeptide has from its N-terminus 1 to 75 consecutive amino acids, or more than 75 consecutive amino acids, as compared to the parent GAA protein (particularly a truncated version of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1). [ka] Consecutive amino acids may be truncated. Using alternative nomenclature, a GAA polypeptide resulting from truncation of one amino acid in a parent GAA polypeptide is referred to as a Δ1 GAA truncated form, a GAA polypeptide resulting from truncation of two consecutive amino acids from the N-terminus is referred to as a Δ2 GAA truncated form, a GAA polypeptide resulting from truncation of three consecutive amino acids in a parent GAA polypeptide is referred to as a Δ3 GAA truncated form, etc. In certain embodiments, the truncated GAA polypeptides of the present invention are [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0033] In another specific embodiment, the truncated GAA polypeptide of the invention comprises [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0034] In another specific embodiment, the truncated GAA polypeptide of the invention comprises [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0035] In another specific embodiment, the truncated GAA polypeptide of the invention comprises [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0036] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0037] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0038] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0039] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0040] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0041] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0042] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0043] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0044] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0045] In a further specific embodiment, the truncated GAA polypeptide of the invention comprises: [ka] It is a truncated form of GAA (particularly a truncated form of the parent hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0046] In a further particular embodiment, the truncated GAA polypeptide of the present invention is a Δ6, Δ7, Δ8, Δ9 or Δ10 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1), particularly a Δ7, Δ8 or Δ9 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1), more particularly a Δ8 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0047] In a further particular embodiment, the truncated GAA polypeptide of the present invention is a Δ27, Δ28, Δ29, Δ30 or Δ31 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1), particularly a Δ28, Δ29 or Δ30 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1), more particularly a Δ29 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0048] In another particular embodiment, the truncated GAA polypeptide of the present invention is a Δ40, Δ41, Δ42, Δ43 or Δ44 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1), particularly a Δ41, Δ42 or Δ43 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1), more particularly a Δ42 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0049] In a further particular embodiment, the truncated GAA polypeptide of the present invention is a Δ41, Δ42, Δ43, Δ44 or Δ45 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1), particularly a Δ42, Δ43 or Δ44 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1), more particularly a Δ43 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1).

[0050] In another embodiment, the truncated GAA polypeptide of the present invention is a Δ6, Δ7, Δ8, Δ9, Δ10, Δ27, Δ28, Δ29, Δ30, Δ31, Δ40, Δ41, Δ42, Δ43, Δ44, Δ45, Δ46, or Δ47 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0051] In another embodiment, the truncated GAA polypeptide of the present invention is a Δ7, Δ8, Δ9, Δ28, Δ29, Δ30, Δ41, Δ42, Δ43, or Δ44 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0052] In another embodiment, the truncated GAA polypeptide of the present invention is a Δ6, Δ7, Δ8, Δ9, Δ10, Δ40, Δ41, Δ42, Δ43, or Δ44 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0053] In another embodiment, the truncated GAA polypeptide of the present invention is a Δ8, Δ29, Δ42, Δ43, or Δ47 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0054] In another embodiment, the truncated GAA polypeptide of the present invention is a Δ8, Δ29, Δ42, or Δ43 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0055] In another embodiment, the truncated GAA polypeptide of the invention is a Δ8 or Δ42 truncated form of GAA (particularly the hGAA protein shown in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1).

[0056] In a particular embodiment of the invention, the truncated GAA polypeptide of the invention is a truncated version of a functional human GAA polypeptide. In a further particular embodiment, the parent hGAA polypeptide is the hGAA polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1. In a variation of this embodiment, the truncated GAA polypeptide of the invention is a hGAA polypeptide, more particularly the hGAA polypeptide set forth in SEQ ID NO: 1 or SEQ ID NO: 33, even more particularly the hGAA polypeptide set forth in SEQ ID NO: 1, or a functional variant thereof which comprises amino acid substitutions in the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1 and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 1 or SEQ ID NO: 33, in particular SEQ ID NO: 1. [ka] It is a GAA truncated form.

[0057] In a variant of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0058] In a variant of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0059] In a variant of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0060] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0061] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0062] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0063] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0064] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0065] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0066] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0067] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0068] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0069] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0070] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0071] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0072] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0073] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0074] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0075] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0076] In another variation of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, even more particularly as set forth in SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] It is a GAA truncated form.

[0077] In another variation of this embodiment, the truncated GAA polypeptide of the present invention is a Δ6, Δ7, Δ8, Δ9 or Δ10, particularly a Δ7, Δ8 or Δ9, more particularly a Δ8 truncated form of an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, or a functional variant thereof which comprises an amino acid substitution in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, and which has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1.

[0078] In another variation of this embodiment, the truncated GAA polypeptide of the present invention is a Δ27, Δ28, Δ29, Δ30 or Δ31, particularly a Δ28, Δ29 or Δ30, more particularly a Δ29 truncated form of an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, or a functional variant thereof which comprises an amino acid substitution in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, and which has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1.

[0079] In another variation of this embodiment, the truncated GAA polypeptide of the present invention is a Δ40, Δ41, Δ42, Δ43 or Δ44, particularly a Δ41, Δ42 or Δ43, more particularly a Δ42 truncated form of an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, or a functional variant thereof which comprises an amino acid substitution in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, and has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1.

[0080] In another variation of this embodiment, the truncated GAA polypeptide of the present invention is a Δ41, Δ42, Δ43, Δ44 or Δ45, particularly a Δ42, Δ43 or Δ44, more particularly a Δ43 truncated form of an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, or a functional variant thereof which comprises an amino acid substitution in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, and has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1.

[0081] In another variant of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] especially [ka] In particular, Δ8, Δ29, Δ42, or Δ43 truncations.

[0082] In another variant of this embodiment, the truncated GAA polypeptide of the invention is an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, or a functional variant thereof which comprises amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1, and has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, in particular SEQ ID NO:1. [ka] In particular, the Δ8 or Δ42 truncated forms.

[0083] In another variation of this embodiment, the truncated GAA polypeptide of the present invention is a Δ8, Δ29, Δ42, Δ43, or Δ47 truncated form of an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, or a functional variant thereof which contains amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, and has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1.

[0084] In another variation of this embodiment, the truncated GAA polypeptide of the present invention is a Δ8, Δ29, Δ42, or Δ43 truncated form of an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, or a functional variant thereof which contains amino acid substitutions in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, and has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1.

[0085] In another variation of this embodiment, the truncated GAA polypeptide of the present invention is a Δ8 or Δ42 truncated form of an hGAA polypeptide, more particularly an hGAA polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, or a functional variant thereof which comprises an amino acid substitution in the sequence as set forth in SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1, and has at least 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO:1 or SEQ ID NO:33, particularly SEQ ID NO:1.

[0086] In a specific embodiment, the truncated hGAA polypeptide of the present invention has an amino acid sequence consisting of the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36, or a functional variant thereof comprising 1 to 5, particularly 1 to 4, particularly 1 to 3, more particularly 1 to 2, particularly 1 amino acid substitution compared to the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36. In another specific embodiment, the truncated hGAA polypeptide of the present invention has an amino acid sequence consisting of the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34 or SEQ ID NO: 35, or a functional variant thereof comprising 1 to 5 amino acid substitutions compared to the sequence shown in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34 or SEQ ID NO: 35. In a specific embodiment, the truncated hGAA polypeptide of the present invention has an amino acid sequence consisting of the sequence shown in SEQ ID NO:27 or SEQ ID NO:28, or a functional variant thereof comprising 1 to 5, particularly 1 to 4, particularly 1 to 3, more particularly 1 to 2, particularly 1 amino acid substitution compared to the sequence shown in SEQ ID NO:27 or SEQ ID NO:28.

[0087] The truncated GAA polypeptide according to the invention may further comprise a signal peptide, for example the native signal peptide of GAA or an alternative signal peptide derived from another secreted protein. Non-limiting examples of such signal peptides include those shown in SEQ ID NOs: 3-7. The inventors have surprisingly shown that fusing the truncated GAA polypeptide of the invention to an alternative signal peptide further enhances its secretion. The invention hereby provides a chimeric GAA polypeptide comprising a signal portion and a truncated GAA polypeptide portion, the truncated GAA polypeptide portion being a truncated GAA polypeptide as defined above. In a particular embodiment, the signal peptide is the native signal peptide of GAA, for example the signal peptide of hGAA as shown in SEQ ID NO: 4. In another embodiment, the signal peptide is an exogenous (i.e. alternative) signal peptide derived from a protein different from GAA. In a particular embodiment, the alternative signal peptide is selected from the group consisting of SEQ ID NOs: 3, 5, 6, and 7, or functional derivatives thereof as defined below.

[0088] The present inventors have shown that an exogenous signal peptide fused to the remainder of the GAA protein increases secretion of the resulting chimeric GAA polypeptide compared to a corresponding GAA polypeptide containing its native signal peptide. Furthermore, the truncated GAA polypeptide portion also increases secretion of the chimeric GAA polypeptide (containing both the signal peptide and the truncated GAA polypeptide) compared to a chimeric GAA polypeptide containing the same signal peptide fused to the parent GAA polypeptide.

[0089] Particular exogenous signal peptides operable in the present invention include amino acids 1-20 from chymotrypsinogen B2 (SEQ ID NO:3), the signal peptide of human alpha-1-antitrypsin (SEQ ID NO:5), amino acids 1-25 from iduronate-2-sulfatase (SEQ ID NO:6), and amino acids 1-23 from protease C1 inhibitor (SEQ ID NO:7). The signal peptides of SEQ ID NO:3 and SEQ ID NO:5 through SEQ ID NO:7 allow for higher secretion of the chimeric GAA protein both in vitro and in vivo compared to GAA containing its native signal peptide. In a particular embodiment, the signal peptide has the sequence as set forth in SEQ ID NO: 3 to 7 or is a functional derivative thereof, i.e. a sequence which comprises deletion(s), insertion(s) or substitution(s) of 1 to 5, in particular 1 to 4, in particular 1 to 3, more in particular 1 to 2, in particular 1 amino acid compared to the sequence as set forth in SEQ ID NO: 3 to 7, insofar as the resulting sequence corresponds to a functional signal peptide, i.e. a signal peptide enabling secretion of the GAA protein.

[0090] In certain embodiments, the GAA polypeptide of the present invention comprises: - A8 truncated forms of GAA, for example a combination of a Δ8 truncated form of hGAA as shown in SEQ ID NO: 27 and SEQ ID NO: 3; - A8 truncated forms of GAA, for example a combination of a Δ8 truncated form of hGAA as shown in SEQ ID NO: 27 and SEQ ID NO: 4; - A8 truncated forms of GAA, for example a combination of a Δ8 truncated form of hGAA as shown in SEQ ID NO: 27 and SEQ ID NO: 5; - A8 truncated forms of GAA, for example a combination of a A8 truncated form of hGAA as shown in SEQ ID NO: 27 and SEQ ID NO: 6; - A8 truncated forms of GAA, for example a combination of a Δ8 truncated form of hGAA as shown in SEQ ID NO: 27 and SEQ ID NO: 7; - A29 truncated form of GAA, for example a combination of a Δ29 truncated form of hGAA as shown in SEQ ID NO: 34 and SEQ ID NO: 3; - A29 truncated form of GAA, for example a combination of a Δ29 truncated form of hGAA as shown in SEQ ID NO: 34 and SEQ ID NO: 4; - A29 truncated form of GAA, for example a combination of a Δ29 truncated form of hGAA as shown in SEQ ID NO: 34 and SEQ ID NO: 5; - A29 truncated form of GAA, for example a combination of a Δ29 truncated form of hGAA as shown in SEQ ID NO: 34 and SEQ ID NO: 6; - A29 truncated form of GAA, for example a combination of a A29 truncated form of hGAA as shown in SEQ ID NO: 34 and SEQ ID NO: 7; - A42 truncated form of GAA, for example a combination of a Δ42 truncated form of hGAA as shown in SEQ ID NO: 28 and SEQ ID NO: 3; - A42 truncated form of GAA, for example a combination of a Δ42 truncated form of hGAA as shown in SEQ ID NO: 28 and SEQ ID NO: 4; - A42 truncated form of GAA, for example a combination of A42 truncated form of hGAA as shown in SEQ ID NO: 28 and SEQ ID NO: 5; - A42 truncated form of GAA, for example a combination of a Δ42 truncated form of hGAA as shown in SEQ ID NO: 28 and SEQ ID NO: 6; - A42 truncated form of GAA, for example a combination of a Δ42 truncated form of hGAA as shown in SEQ ID NO: 28 and SEQ ID NO: 7; - A43 truncated form of GAA, for example a combination of a A43 truncated form of hGAA as shown in SEQ ID NO: 35 and SEQ ID NO: 3; - A43 truncated form of GAA, for example a combination of a Δ43 truncated form of hGAA as shown in SEQ ID NO: 35 and SEQ ID NO: 4; - A43 truncated form of GAA, for example a combination of A43 truncated form of hGAA as shown in SEQ ID NO: 35 and SEQ ID NO: 5; - A43 truncated form of GAA, for example a combination of A43 truncated form of hGAA as shown in SEQ ID NO: 35 and SEQ ID NO: 6; - A43 truncated forms of GAA, for example the A43 truncated form of hGAA as shown in SEQ ID NO: 35 in combination with SEQ ID NO: 7; - A Δ47 truncated form of GAA, for example a combination of a Δ47 truncated form of hGAA as shown in SEQ ID NO: 36 and SEQ ID NO: 3; - A Δ47 truncated form of GAA, for example a combination of a Δ47 truncated form of hGAA as shown in SEQ ID NO: 36 and SEQ ID NO: 4; - A47 truncated form of GAA, for example a combination of the A47 truncated form of hGAA shown in SEQ ID NO: 36 and SEQ ID NO: 5; - A47 truncated form of GAA, for example a combination of the A47 truncated form of hGAA shown in SEQ ID NO: 36 and SEQ ID NO: 6; - a Δ47 truncated form of GAA, for example a combination of a Δ47 truncated form of hGAA as shown in SEQ ID NO: 36 and SEQ ID NO: 7; or or functional derivatives thereof having at least 90% identity, in particular at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the resulting sequence combination. In these embodiments, as described above, the signal peptide portion may be a sequence which comprises 1 to 5, in particular 1 to 4, in particular 1 to 3, more particularly 1 to 2, in particular 1 amino acid deletion(s), insertion(s) or substitution(s) compared to the sequences shown in SEQ ID NOs: 3 to 7, insofar as the resulting sequence corresponds to a functional signal peptide, i.e. a signal peptide which allows secretion of the resulting truncated chimeric GAA protein.

[0091] The relative proportion of newly synthesized GAA that is secreted from cells can be routinely determined by methods known in the art and described in the Examples. Secreted protein can be detected by direct measurement of the protein itself (e.g., by Western blot) or by protein activity assays (e.g., enzyme assays) in cell culture medium, serum, milk, etc.

[0092] One of skill in the art will further understand that a truncated or chimeric GAA polypeptide can contain additional amino acids, for example as a result of manipulation of the nucleic acid construct, such as the addition of restriction enzyme sites, so long as these additional amino acids do not render the signal peptide or GAA polypeptide non-functional. The additional amino acids may be truncated or may be retained by the mature polypeptide so long as such retention does not result in a non-functional polypeptide.

[0093] In another aspect, the present invention relates to a nucleic acid molecule encoding a truncated GAA polypeptide of the present invention or a chimeric GAA polypeptide of the present invention.

[0094] The sequence of the nucleic acid molecule of the present invention encoding truncated GAA is optimized for the expression of GAA polypeptide in vivo. Sequence optimization can include many changes in the nucleic acid sequence, including codon optimization, increasing GC content, reducing the number of CpG islands, reducing the number of alternative open reading frames (ARFs), and reducing the number of splice donor sites and splice acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules can code for the same protein. It is also well known that the genetic code of various organisms is often biased towards using one of several codons that code for the same amino acid over other amino acids. Through codon optimization, changes are introduced into the nucleotide sequence that take advantage of the codon bias present in a given cellular context, making the resulting codon-optimized nucleotide sequence more likely to be expressed at a relatively high level in such a given cellular context compared to a sequence that is not codon-optimized. In a preferred embodiment of the present invention, such an optimized nucleotide sequence encoding a truncated GAA protein has improved expression in human cells compared to a non-codon-optimized nucleotide sequence encoding the same truncated GAA protein, e.g., by optimizing the codons by taking advantage of human-specific codon usage bias.

[0095] In certain embodiments, the optimized GAA coding sequence is codon optimized and / or has an increased GC content and / or has a reduced number of alternative open reading frames and / or has a reduced number of splice donor and / or splice acceptor sites compared to nucleotides 82-2859 of the wild-type hGAA coding sequence of SEQ ID NO: 8. For example, the nucleic acid sequences of the invention have at least a 2, 3, 4, 5, or 10% increase in GC content in the GAA sequence compared to the sequence of the wild-type GAA sequence. In certain embodiments, the nucleic acid sequences of the invention have a 2, 3, 4, or more particularly a 5% or 10% (particularly 5%) increase in GC content in the GAA sequence compared to the sequence of the wild-type GAA nucleotide sequence. In certain embodiments, the nucleic acid sequence of the invention encoding a functional GAA polypeptide is "substantially identical" to nucleotides 82-2859 of the sequence shown in SEQ ID NO:8, i.e., about 70% identical, more preferably about 80% identical, even more preferably about 90% identical, even more preferably about 95% identical, even more preferably about 97%, 98%, or even 99% identical. As noted above, in addition to the GC content and / or number of ARFs, sequence optimization may also include reducing the number of CpG islands and / or reducing the number of splice donor and acceptor sites within the sequence. Of course, as is well known to those skilled in the art, sequence optimization is a balance between all these parameters, which means that a sequence may be considered optimized if at least one of the above parameters is improved, even if one or more of the other parameters are not, as long as the optimized sequence results in improved transgene expression in vivo, such as improved expression and / or reduced immune response to the transgene.

[0096] Furthermore, the adaptability of a nucleotide sequence encoding a functional GAA to the codon usage of human cells can be expressed as a codon adaptation index (CAI). The codon adaptation index is defined herein as a measure of the relative adaptability of a gene's codon usage to the codon usage of highly expressed human genes. The relative adaptability (w) of each codon is the ratio of the usage of each codon to the usage of the most abundant codon for the same amino acid. The CAI is defined as the geometric mean of these relative adaptability values. Nonsynonymous codons and stop codons (depending on the genetic code) are excluded. CAI values ​​range from 0 to 1, with higher numbers indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; see also Kim et al, Gene. 1997, 199:293-301; zur Megede et al, Journal of Virology, 2000, 74: 2628-2635). Preferably, a nucleic acid molecule encoding GAA has a CAI of at least 0.75 (especially 0.77), 0.8, 0.85, 0.90, 0.92, or 0.94.

[0097] The term "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or RNA molecule, particularly DNA, in single- or double-stranded form, that encodes a GAA protein according to the present invention.

[0098] The inventors have discovered that the truncated GAA polypeptides, when expressed from the nucleic acid molecules encoding them, result in surprisingly high levels of functional GAA protein expression both in vitro and in vivo, as compared to wild-type GAA cDNA. Moreover, as also shown by the inventors, the truncated GAA protein produced from liver and muscle cells expressing the nucleic acid molecules of the invention does not induce any immune response. This means that the nucleic acid molecules can be used to produce high levels of GAA protein, providing therapeutic advantages such as avoiding reliance on immunosuppressant treatment, allowing treatment with low doses of immunosuppressants, and allowing repeated administration of the nucleic acid molecules of the invention to subjects in need thereof. Therefore, the truncated GAA polypeptides of the invention and the nucleic acid molecules of the invention are of particular interest in situations where GAA expression and / or activity is deficient, or where high expression levels of GAA can ameliorate diseases such as glycogen storage diseases. In particular, the glycogen storage disease may be glycogen storage disease type I (von Gierke's disease), glycogen storage disease type II (Pompe's disease), glycogen storage disease type III (Cohri's disease), glycogen storage disease type IV, glycogen storage disease type V, glycogen storage disease type VI, glycogen storage disease type VII, glycogen storage disease type VIII, or fatal congenital glycogen storage disease of the heart. More particularly, the glycogen storage disease is selected from the group consisting of glycogen storage disease type I, glycogen storage disease type II, and glycogen storage disease type III, and even more particularly, from the group consisting of glycogen storage disease type II and glycogen storage disease type III. In an even more particular embodiment, the glycogen storage disease is glycogen storage disease type II. In particular, the nucleic acid molecules of the invention may be useful in gene therapy for treating GAA deficiency conditions or other conditions involving glycogen accumulation, such as glycogen storage disease type I (von Gierke's disease), glycogen storage disease type II (Pompe's disease), glycogen storage disease type III (Cori's disease), glycogen storage disease type IV, glycogen storage disease type V, glycogen storage disease type VI, glycogen storage disease type VII, glycogen storage disease type VIII, and fatal congenital glycogen storage diseases of the heart, more particularly glycogen storage disease type I, glycogen storage disease type II or glycogen storage disease type III, and even more particularly glycogen storage disease type II and glycogen storage disease type III. In an even more particular embodiment, the nucleic acid molecules of the invention may be useful in gene therapy for treating glycogen storage disease type II.

[0099] In another embodiment of the invention, a portion of a nucleic acid molecule of the invention encoding a truncated GAA polypeptide portion has at least 75% (e.g., 77.7%), or at least 80%, or at least 82% (e.g., 83.1%) identity to the corresponding portion of the nucleotide sequence encoding SEQ ID NO:1 or SEQ ID NO:33, which is the wild-type hGAA polypeptide sequence lacking the signal peptide, particularly SEQ ID NO:1.

[0100] The truncated GAA portion of the nucleic acid molecule of the present invention preferably has an identity of at least 85%, more preferably at least 90%, even more preferably at least 92%, particularly at least 95%, for example at least 98, 99 or 100%, to the nucleotide sequence of SEQ ID NO: 10 or 11, which is the optimized sequence of the sequence.

[0101] The term "identical" and its derivatives refer to the sequence identity between two nucleic acid molecules. If a position in both of the two compared sequences is occupied by the same base, for example, if the position in each of the two DNA molecules is occupied by adenine, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in the two sequences are identical, the two sequences are 60% identical. Generally, two sequences are aligned and compared to obtain the maximum percentage of identity. Nucleic acid sequences can be aligned using various bioinformatics tools known to those skilled in the art, such as BLAST or FASTA.

[0102] Furthermore, the nucleic acid molecule of the present invention encodes a functional GAA protein, i.e., it encodes a human GAA protein that has the function of a wild-type GAA protein when expressed. As defined above, the function of wild-type GAA is to release glucose by hydrolyzing both α-1,4 and α-1,6 bonds of oligosaccharides and polysaccharides, more particularly glycogen. A functional GAA protein encoded by a nucleic acid of the present invention may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% hydrolysis activity against glycogen compared to the wild-type GAA protein of SEQ ID NO: 1, 2, 30, or 33. The activity of the GAA protein encoded by a nucleic acid of the present invention may even be more than 100%, for example more than 110%, 120%, 130%, 140%, or even more than 150%, relative to the activity of the wild-type GAA protein of SEQ ID NO: 1, 2, 30, or 33.

[0103] Those skilled in the art can easily determine whether the nucleic acid described in the present invention expresses a functional GAA protein. Suitable methods will be clear to those skilled in the art. For example, one suitable in vitro method includes inserting the nucleic acid into a vector, such as a plasmid or viral vector, transfecting or transducing a host cell, such as 293T cells or HeLa cells, or other cells, such as Huh7, with the vector, and assaying for GAA activity. Alternatively, a suitable in vivo method includes transducing a vector containing the nucleic acid into a mouse model of Pompe disease or another glycogen storage disease, and assaying for the presence of functional GAA in the plasma and GAA in the tissues of the mouse. Suitable methods are described in more detail in the experimental section below.

[0104] In particular embodiments, the nucleic acid molecule of the invention comprises a sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 13, which encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 27; a sequence as set forth in SEQ ID NO: 48 or SEQ ID NO: 49, which encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 28; a sequence as set forth in SEQ ID NO: 50 or SEQ ID NO: 51, which encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 35; or a sequence as set forth in SEQ ID NO: 52 or SEQ ID NO: 53, which encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 36. In further embodiments, the nucleic acid molecule of the invention comprises a sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 13, which encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 27; a sequence as set forth in SEQ ID NO: 48 or SEQ ID NO: 49, which encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 28; or a sequence as set forth in SEQ ID NO: 50 or SEQ ID NO: 51, which encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 35. In particular embodiments, the nucleic acid molecule of the invention comprises a sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 13, which encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 27.

[0105] The present invention also relates to a nucleic acid construct comprising the nucleic acid molecule of the present invention. The nucleic acid construct may correspond to an expression cassette comprising the nucleic acid sequence of the present invention operably linked to one or more expression control sequences and / or other sequences that improve the expression of the transgene and / or sequences that enhance the secretion of the encoded protein and / or sequences that enhance the uptake of the encoded protein. As used herein, the term "operably linked" refers to the linkage of polynucleotide sequences in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter, or another transcriptional regulatory sequence, is operably linked to a coding sequence when it affects the transcription of the coding sequence. Such expression control sequences, such as promoters, enhancers (e.g., cis-regulatory modules (CRMs)), introns, polyA signals, etc., are known in the art.

[0106] In particular, the expression cassette may comprise a promoter. The promoter may be a ubiquitous or tissue-specific promoter, particularly a promoter capable of promoting expression in cells or tissues in which expression of GAA is desired, such as in GAA-deficient patients. In a particular embodiment, the promoter is a liver-specific promoter, such as alpha-1 antitrypsin promoter (hAAT) (SEQ ID NO: 14), transthyretin promoter, albumin promoter, thyroxine-binding globulin (TBG) promoter, LSP promoter (containing thyroid hormone-binding globulin promoter sequence, two copies of alpha-1-microglobulin / bikunin enhancer sequence, and leader sequence, 34.Ill, CR, et al. (1997). Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A. Blood Coag. Fibrinol. 8: S23-S30). Other useful liver-specific promoters are also known in the art, such as those listed in the liver-specific gene promoter database compiled by Cold Spring Harbor Laboratory (http: / / rulai.cshl.edu / LSPD / ).A preferred promoter in the context of the present invention is the hAAT promoter.In another embodiment, the promoter is a promoter that directs expression in tissue or cell of interest (e.g., muscle cell) and liver cell.For example, to a certain extent, promoters specific to muscle cells, such as desmin, Spc5-12 and MCK promoters, may exhibit some leakage of expression into liver cells, which may be beneficial for inducing immune tolerance in subjects to the GAA polypeptide expressed from the nucleic acid of the present invention.

[0107] Other tissue-specific or non-tissue-specific promoters may also be useful in the practice of the present invention. For example, the expression cassette may include a tissue-specific promoter that is a promoter different from the liver-specific promoter. For example, the promoter may be a muscle-specific promoter, such as the desmin promoter (and desmin promoter variants, such as the desmin promoter with natural or artificial enhancers), SPc5-12 or MCK promoter. In another embodiment, the promoter is a promoter specific to other cell lineages, such as the erythropoietin promoter for the expression of GAA polypeptides from cells of the erythroid lineage.

[0108] In another embodiment, the promoter is a ubiquitous promoter. Representative ubiquitous promoters include the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, the cytomegalovirus enhancer / promoter (CMV), the PGK promoter, and the SV40 early promoter.

[0109] Furthermore, the promoter may also be an endogenous promoter, such as the albumin promoter or the GAA promoter.

[0110] In certain embodiments, the promoter is linked to an enhancer sequence, such as a cis-regulatory module (CRM) or an artificial enhancer sequence. For example, the promoter may be linked to an enhancer sequence, such as the human ApoE regulatory region (or human apolipoprotein E / CI locus, hepatic regulatory region HCR-1-Genbank accession number U32510, as shown in SEQ ID NO: 15). In certain embodiments, the enhancer sequence, such as the ApoE sequence, is linked to a liver-specific promoter, such as the promoters listed above, in particular the hAAT promoter. Other CRMs useful in the implementation of the present invention include those described in Rincon et al., Mol Ther. 2015 Jan;23(1):43-52, Chuah et al., Mol Ther. 2014 Sep;22(9):1605-13 or Nair et al., Blood. 2014 May 15;123(20):3195-9.

[0111] In another particular embodiment, the nucleic acid construct comprises an intron, particularly an intron located between the promoter and the GAA coding sequence. Introns can be introduced to increase mRNA stability and protein production. In a further embodiment, the nucleic acid construct comprises a human beta globin b2 (i.e. HBB2) intron, a coagulation factor IX (FIX) intron, an SV40 intron, or a chicken beta-globin intron. In another further embodiment, the nucleic acid construct of the invention contains a modified intron, particularly a modified HBB2 or FIX intron, designed to reduce or even completely remove the number of alternative open reading frames (ARFs) found in the intron. Preferably, ARFs that span 50 bp in length and have initiation and termination codons in the reading frame are removed. ARFs may be removed by modifying the sequence of the intron. For example, modifications can be made using nucleotide substitutions, insertions, or deletions, preferably by nucleotide substitutions. As an example, a non-start codon can be created by replacing one or more nucleotides, particularly one nucleotide, in the ATG or GTG start codon present in the intron sequence of interest. For example, the ATG or GTG can be replaced by CTG, which is not a start codon, in the sequence of the intron of interest.

[0112] The classical HBB2 intron used in the nucleic acid construct is shown in SEQ ID NO: 16. For example, this HBB2 intron may be modified by eliminating the start codons in the intron (ATG and GTG codons). In a particular embodiment, the modified HBB2 intron included in the construct has the sequence shown in SEQ ID NO: 17. The classical FIX intron used in the nucleic acid construct is derived from the first intron of human FIX and is shown in SEQ ID NO: 18. The FIX intron may be modified by eliminating the start codons in the intron (ATG and GTG codons). In a particular embodiment, the modified FIX intron included in the construct of the invention has the sequence shown in SEQ ID NO: 19. The classical chicken-β globin intron used in the nucleic acid construct is shown in SEQ ID NO: 20. The chicken-β globin intron may be modified by eliminating the start codons in the intron (ATG and GTG codons). In a particular embodiment, the modified chicken-β globin intron included in the construct of the invention has the sequence shown in SEQ ID NO: 21.

[0113] The inventors have previously shown in WO 2015 / 162302 that such modified introns, in particular modified HBB2 or FIX introns, have beneficial properties and can significantly improve transgene expression.

[0114] In certain embodiments, the nucleic acid construct of the invention is an expression cassette comprising, in a 5' to 3' direction, a promoter, optionally preceded by an enhancer, a coding sequence of the invention (i.e., an optimized truncated GAA coding sequence of the invention, a chimeric GAA coding sequence of the invention, or a chimeric and sequence optimized GAA coding sequence of the invention), and a polyadenylation signal (e.g., a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or another naturally occurring or artificial polyadenylation signal). In certain embodiments, the nucleic acid construct of the invention is an expression cassette comprising, in a 5' to 3' direction, a promoter, optionally preceded by an enhancer (e.g., an ApoE regulatory region), an intron (particularly an intron as defined above), a coding sequence of the invention, and a polyadenylation signal. In a further particular embodiment, the nucleic acid construct of the invention is an expression cassette comprising, in a 5' to 3' direction, an enhancer such as the ApoE regulatory region, a promoter, an intron (particularly an intron as defined above), a coding sequence of the invention, and a polyadenylation signal. In a further particular embodiment of the invention, the expression cassette comprises, in a 5' to 3' direction, an ApoE regulatory region, a hAAT liver-specific promoter, an HBB2 intron (particularly a modified HBB2 intron as defined above), a coding sequence of the invention, and a bovine growth hormone polyadenylation signal, e.g. - SEQ ID NO: 22, which contains a non-optimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 5; - SEQ ID NO: 23, which encodes a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and contains an optimized sequence encoding the signal peptide of SEQ ID NO: 5; - SEQ ID NO: 24, which encodes a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and which contains another optimized sequence encoding the signal peptide of SEQ ID NO: 5; - SEQ ID NO: 25, which comprises an optimized sequence encoding a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and a signal peptide of SEQ ID NO: 3; - SEQ ID NO: 26, which comprises an optimized sequence encoding a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and a signal peptide of SEQ ID NO: 3; - SEQ ID NO: 37, which comprises a Δ29 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the non-optimized sequence of SEQ ID NO: 9) and a non-optimized sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 38, which comprises an optimized sequence encoding a Δ29 truncated form of GAA (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) derived from the parent hGAA of SEQ ID NO: 1 and a signal peptide of SEQ ID NO: 3; - SEQ ID NO: 39, which comprises a Δ29 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and another optimized sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 40, which comprises a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the non-optimized sequence of SEQ ID NO: 9) and a non-optimized sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 41, which comprises a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and another optimized sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 42, which comprises a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the non-optimized sequence of SEQ ID NO: 9) and a non-optimized sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 43, which comprises an optimized sequence encoding a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and a signal peptide of SEQ ID NO: 3; - SEQ ID NO: 44, which comprises a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and another optimized sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 45, which comprises a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the non-optimized sequence of SEQ ID NO: 9) and a non-optimized sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 46, which comprises a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and an optimized sequence encoding the signal peptide of SEQ ID NO: 3; and SEQ ID NO: 47, which comprises a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and another optimized sequence encoding the signal peptide of SEQ ID NO: 3; The construct is shown in.

[0115] Other expression cassettes of the invention can include the following nucleic acid sequences: - a non-optimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - a non-optimized nucleotide sequence encoding a Δ29 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - a non-optimized nucleotide sequence encoding a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - a non-optimized nucleotide sequence encoding a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - a non-optimized nucleotide sequence encoding a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ29 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ29 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6, or 7.

[0116] In an alternative embodiment of these particular constructs, the sequence encoding SEQ ID NO:1 is replaced by a sequence encoding SEQ ID NO:33.

[0117] In a particular embodiment, the expression cassette comprises an ApoE regulatory region, a hAAT liver-specific promoter, a codon-optimized HBB2 intron, a coding sequence of the invention, and a bovine growth hormone polyadenylation signal.

[0118] When designing the nucleic acid construct of the present invention, those skilled in the art will be careful to consider the size limit of the vector used to deliver the construct to cells or organs. In particular, those skilled in the art believe that the main limitation of AAV vectors is their loading capacity, which may vary depending on the AAV serotype, but is approximately limited by the size of the parental viral genome. For example, 5kb is the maximum size that is generally believed to be packaged into AAV8 capsid. (Wu Z. et al., Mol Ther., 2010, 18(1): 80-86; Lai Y. et al., Mol Ther., 2010, 18(1): 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4): 225-33). Thus, in practicing the present invention, one skilled in the art will take care to select the components of the nucleic acid construct of the present invention such that the resulting nucleic acid sequence, including sequences encoding the AAV 5'- and 3'-ITRs, preferably does not exceed 110% of the carrying capacity of the AAV vector to which it is attached, and particularly preferably does not exceed 5.5 kb.

[0119] The present invention also relates to a vector comprising a nucleic acid molecule or construct as disclosed herein. In particular, the vector of the present invention is a vector suitable for expression of a protein, preferably for use in gene therapy. In one embodiment, the vector is a plasmid vector. In another embodiment, the vector is a nanoparticle containing a messenger RNA encoding a nucleic acid molecule of the present invention, in particular a GAA polypeptide of the present invention. In another embodiment, the vector is a transposon-based system that allows the integration of a nucleic acid molecule or construct of the present invention into the genome of a target cell, such as the hyperactive Sleeping Beauty (SB100X) transposon system (Mates et al. 2009). In another embodiment, the vector is a viral vector suitable for gene therapy, targeting any cell of interest, such as liver tissue or liver cells, muscle cells, CNS cells (e.g. brain cells), or hematopoietic stem cells, such as cells of the erythroid lineage (e.g. red blood cells). In this case, the nucleic acid construct of the present invention also contains sequences suitable for generating an effective viral vector, as is well known in the art. In a particular embodiment, the viral vector is derived from an integrating virus. In particular, the viral vector may be derived from a retrovirus or a lentivirus.In further particular embodiments, the viral vector is an AAV vector, such as an AAV vector suitable for transducing liver tissue or liver cells, more particularly AAV-1, -2 and AAV-2 variants (e.g., the quadruple mutant capsid optimized AAV-2, including the engineered capsid with Y44+500+730F+T491V changes disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods [article published online ahead of print]), AAV-3 and AAV-3 variants (e.g., the AAV vectors described in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042), AAV-3B and AAV-3B mutants, AAV-4, -5, -6, and AAV-6 mutants (e.g., AAV6 mutants comprising the triple mutant AAV6 capsid Y731F / Y705F / T492V form disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p.16026), AAV-7, -8, -9, -10, such as AAV-cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV serotypes, such as AAVpo4 and AAVpo6, or retroviral vectors, such as lentiviral vectors and alpha-retroviruses. As known in the art, depending on the specific viral vector to be used, additional suitable sequences will be inserted into the nucleic acid construct of the present invention to obtain a functional viral vector.Suitable sequences include AAV ITRs for AAV vectors, or LTRs for lentiviral vectors.Therefore, the present invention also relates to such expression cassettes flanked by ITRs or LTRs on each side.

[0120] The advantages of viral vectors are discussed in the following parts of this disclosure. Viral vectors are preferred for delivering the nucleic acid molecules or constructs of the invention, for example retroviral vectors, such as lentiviral vectors, or non-pathogenic parvoviruses, more preferably AAV vectors. The human parvovirus adeno-associated virus (AAV) is a naturally replication-deficient dependant virus that can establish a latent infection by integrating into the genome of infected cells. The last property appears to be unique among mammalian viruses, since integration occurs at a specific site in the human genome, called AAVS1, located on chromosome 19 (19q13.3-qter).

[0121] Therefore, AAV vectors have attracted considerable interest as potential vectors for human gene therapy. Favorable properties of the virus include its lack of any human disease, its ability to infect both dividing and non-dividing cells, and its ability to infect a wide variety of cell lines derived from various tissues.

[0122] Among the well-characterized AAV serotypes isolated from humans or non-human primates (NHPs), human serotype 2 was the first AAV to be developed as a gene transfer vector. Other currently used AAV serotypes include AAV-1, AAV-2 variants (e.g., the quadruple mutant capsid optimized AAV-2, including an engineered capsid with the Y44+500+730F+T491V changes, as disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods [article published online ahead of print]), AAV-3 and AAV-3 variants (e.g., the AAV3-ST variant, including an engineered AAV3 capsid with two amino acid changes, S663V+T492V, as disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042), AAV-3B and AAV-3B variants, AAV-4, -5, -6, and AAV-6 variants (e.g., the AAV-2 variant, including an engineered AAV3 capsid with the Y44+500+730F+T491V changes, as disclosed in Rosario et al., 2016, Mol Ther Methods Clin. Dev. 3, p.16026), AAV-7, -8, -9, -10, such as AAV-cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV serotypes, such as AAVpo4 and AAVpo6, and AAV serotypes of tyrosine, lysine, and serine capsid mutants. In addition, other non-natural engineered mutants and chimeric AAVs may be useful.

[0123] AAV viruses can be engineered using routine molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, for minimal immunogenicity, for tuning the stability and longevity of the particle, for efficient degradation, and for precise delivery to the nucleus.

[0124] Desirable AAV fragments for assembly into vectors include the cap proteins (including vp1, vp2, vp3) and hypervariable regions, the rep proteins (including rep78, rep68, rep52, and rep40) and sequences encoding these proteins. These fragments can be readily utilized in a variety of vector systems and host cells.

[0125] AAV-based recombinant vectors lacking Rep proteins integrate into the host genome with low efficiency and exist primarily as stable circular episomes that can persist in target cells for years. Instead of using AAV natural serotypes, artificial AAV serotypes, including but not limited to AAVs with non-naturally occurring capsid proteins, can be used in the context of the present invention. Such artificial capsids can be made by any suitable technique using selected AAV sequences (e.g., fragments of vp1 capsid protein) in combination with heterologous sequences that can be obtained from selected different AAV serotypes, from non-contiguous parts of the same AAV serotype, from non-AAV viral sources, or from non-viral sources. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids.

[0126] Thus, the present invention relates to an AAV vector comprising a nucleic acid molecule or construct of the present invention. In the context of the present invention, an AAV vector comprises an AAV capsid capable of transducing a target cell of interest, in particular a hepatoma cell. According to certain embodiments, the AAV vectors include AAV-1, -2, AAV-2 variants (e.g., the quadruple mutant capsid optimized AAV-2, including an engineered capsid with Y44+500+730F+T491V changes, as disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods [article published online ahead of print]), AAV-3 and AAV-3 variants (e.g., the AAV3-ST variant, including an engineered AAV3 capsid with two amino acid changes, S663V+T492V, as disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042), AAV-3B and AAV-3B variants, AAV-4, -5, -6, and AAV-6 variants (e.g., the AAV-4 variant, including an engineered AAV3 capsid with Y44+500+730F+T491V changes, as disclosed in Rosario et al., 2016, Mol. Ther. Vol. 24(6), p. 1042). and AAV6 mutants including the triple mutant AAV6 capsid Y731F / Y705F / T492V type disclosed in Methods Clin Dev. 3, p.16026), AAV-7, -8, -9, -10, such as AAV-cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV, such as AAVpo4 and AAVpo6, and AAV serotypes of tyrosine, lysine, and serine capsid mutants. In certain embodiments, the AAV vector is of the AAV8, AAV9, AAVrh74, or AAV2i8 serotype (i.e., the AAV vector has a capsid of the AAV8, AAV9, AAVrh74, or AAV2i8 serotype). In further specific embodiments, the AAV vector is a pseudotyped vector, i.e., its genome and capsid are derived from different serotypes of AAV. For example, a pseudotyped AAV vector can be a vector whose genome is derived from one AAV serotype as described above and whose capsid is derived from another serotype.For example, the genome of a pseudotype vector can have a capsid derived from an AAV8, AAV9, AAVrh74, or AAV2i8 serotype, or the genome can be derived from a different serotype. In certain embodiments, the AAV vector has a capsid of an AAV8, AAV9, or AAVrh74 serotype, particularly an AAV8 or AAV9 serotype, more particularly an AAV8 serotype.

[0127] In a specific embodiment where the vector is for use in delivering a transgene to muscle cells, the AAV vector may be selected from the group consisting of AAV8, AAV9, and AAVrh74, among others. In another specific embodiment where the vector is for use in delivering a transgene to liver cells, the AAV vector may be selected from the group consisting of AAV5, AAV8, AAV9, AAV-LK03, AAV-Anc80, and AAV3B, among others.

[0128] In another embodiment, the capsid is a modified capsid. In the context of the present invention, a "modified capsid" can be a chimeric capsid or a capsid that includes one or more mutant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0129] In certain embodiments, the AAV vector is a chimeric vector, i.e., its capsid comprises VP capsid proteins from at least two different AAV serotypes, or at least one chimeric VP protein combined with VP protein regions or domains from at least two AAV serotypes. Examples of such chimeric AAV vectors useful for transducing hepatocytes are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, a chimeric AAV vector can be obtained from a combination of AAV8 capsid sequences and sequences of an AAV serotype different from the AAV8 serotype, such as any of those specifically described above. In another embodiment, the capsid of the AAV vector comprises one or more mutant VP capsid proteins such as those described in WO2015013313, in particular the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6 capsid mutants, which exhibit enhanced liver tropism.

[0130] In another embodiment, modified capsids can also result from capsid modifications inserted by error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). Additionally, capsid variants can contain single amino acid changes such as tyrosine mutations (e.g., as described in Zhong et al., 2008).

[0131] Furthermore, the genome of the AAV vector can be either a single-stranded or a self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). A self-complementary double-stranded AAV vector is generated by deleting the terminal release site (trs) from one of the AAV terminal repeat sequences. These modified vectors, whose replicative genomes are half the length of the wild-type AAV genome, have a tendency to package DNA dimers. In a preferred embodiment, the AAV vectors employed in the practice of the present invention have a single-stranded genome, and more preferably comprise an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly an AAV8, AAV9, or AAVrh74 capsid, such as an AAV8 or AAV9 capsid, more particularly an AAV8 capsid.

[0132] In a particularly preferred embodiment, the present invention relates to an AAV vector, comprising a nucleic acid construct of the present invention in a single-stranded or double-stranded self-complementary genome (e.g., single-stranded genome). In one embodiment, the AAV vector comprises an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, in particular an AAV8, AAV9 or AAVrh74 capsid, such as an AAV8 or AAV9 capsid, more particularly an AAV8 capsid. In a further particular embodiment, the nucleic acid is operably linked to a promoter, in particular a ubiquitous promoter or a liver-specific promoter. According to a particular variant embodiment, the promoter is a ubiquitous promoter, such as a cytomegalovirus enhancer / chicken β-actin (CAG) promoter, a cytomegalovirus enhancer / promoter (CMV), a PGK promoter, and an SV40 early promoter. In a particular variant, the ubiquitous promoter is a CAG promoter. According to another variant, the promoter is a liver-specific promoter, such as alpha-1 antitrypsin promoter (hAAT), transthyretin promoter, albumin promoter, and thyroxine-binding globulin (TBG) promoter. In a particular variant, the liver-specific promoter is the hAAT liver-specific promoter of SEQ ID NO: 14. In a further particular embodiment, the nucleic acid construct contained in the genome of the AAV vector of the present invention further comprises an intron as described above, such as an intron located between the promoter and the nucleic acid sequence encoding the GAA coding sequence (i.e., the optimized GAA coding sequence of the present invention, the chimeric GAA coding sequence of the present invention, or the chimeric and optimized GAA coding sequence of the present invention). Representative introns that may be included in the nucleic acid construct introduced into the AAV vector genome include, but are not limited to, the human beta globin b2 (i.e., HBB2) intron, the FIX intron, and the chicken beta-globin intron. The introns in the genome of the AAV vector may be classical (i.e., unmodified) introns, or may be modified introns designed to reduce, or even completely eliminate, the number of alternative open reading frames (ARFs) within the intron.The modified and unmodified introns that can be used to implement this embodiment, in which the nucleic acid of the present invention is introduced into the AAV vector, are described in detail above. In a particular embodiment, the AAV vector of the present invention, particularly the AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly the AAV8, AAV9, or AAVrh74 capsid, such as the AAV8 or AAV9 capsid, more particularly the AAV8 capsid, comprises a modified (i.e., optimized) intron in its genome, such as the modified HBB2 intron of SEQ ID NO: 17, the modified FIX intron of SEQ ID NO: 19, and the modified chicken β-globin intron of SEQ ID NO: 21. In a further particular embodiment, the vector of the invention comprises an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, in particular an AAV8, AAV9, or AAVrh74 capsid, such as an AAV8 or AAV9 capsid, more particularly an AAV8 capsid, and has a genome that contains, from 5' to 3', an AAV 5'-ITR (e.g., the AAV2 5'-ITR); an ApoE regulatory region; a hAAT liver-specific promoter; an HBB2 intron (in particular a modified HBB2 intron as defined above); a GAA coding sequence of the invention; a bovine growth hormone polyadenylation signal; and an AAV 3'-ITR (e.g., the AAV2 3'-ITR). and an AAV vector comprising a genome comprising the nucleic acid constructs set forth in SEQ ID NOs: 22-26 and 37-47 flanked by 3'-ITRs. Other nucleic acid constructs useful in the practice of the invention include those described above, such as: - a non-optimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - a non-optimized nucleotide sequence encoding a Δ29 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - a non-optimized nucleotide sequence encoding a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - a non-optimized nucleotide sequence encoding a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - a non-optimized nucleotide sequence encoding a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ8 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ29 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ29 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4 or 6; - an optimized nucleotide sequence encoding a Δ42 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ43 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12) and encoding a signal peptide of SEQ ID NO: 4, 6 or 7; - an optimized nucleotide sequence encoding a Δ47 truncated form of GAA derived from the parent hGAA of SEQ ID NO: 1 (nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13) and encoding a signal peptide of SEQ ID NO: 4, 6, or 7.

[0133] In an alternative embodiment of these particular constructs, the sequence encoding SEQ ID NO:1 is replaced by a sequence encoding SEQ ID NO:33.

[0134] In a particular embodiment of the present invention, the nucleic acid construct of the present invention comprises a liver-specific promoter as described above, and the vector is a viral vector capable of transducing liver tissue or liver cells as described above.The inventors present data below showing that, thanks to this embodiment, the immune tolerance-inducing and metabolic properties of the liver are advantageously utilized in developing a highly effective and optimized vector for expressing a secretable form of GAA in hepatoma cells and inducing immune tolerance to the protein.

[0135] Furthermore, in a further specific embodiment, the present invention provides a combination of two vectors, such as two viral vectors, in particular two AAV vectors, for improving gene delivery and treatment efficacy in cells of interest.For example, the two vectors may have the nucleic acid molecule of the present invention encoding the GAA protein of the present invention, which is under the control of one different promoter, in each of these two vectors.In a specific embodiment, one vector comprises a promoter (such as one of those described above) that is a liver-specific promoter, and the other vector comprises a promoter that is specific to another tissue of interest for the treatment of glycogen storage disease, such as a muscle-specific promoter, such as the desmin promoter.In a specific variant of this embodiment, this combination of vectors corresponds to multiple co-packaged AAV vectors produced as described in WO2015196179.

[0136] The present invention also relates to cells, such as hepatocytes, transformed with the nucleic acid molecule or construct of the present invention, as in the case of ex vivo gene therapy. The cells of the present invention can be delivered to a subject in need thereof, such as a GAA-deficient patient, by any suitable route of administration, for example via injection into the liver or bloodstream of the subject. In a particular embodiment, the present invention comprises the step of introducing the nucleic acid molecule, nucleic acid construct or vector, particularly a lentiviral vector, of the present invention into hepatocytes, particularly hepatocytes of a subject to be treated, and administering to the subject the transformed hepatocytes into which the nucleic acid has been introduced. Advantageously, this embodiment is useful for secreting GAA from the cells. In a particular embodiment, the hepatocytes are hepatocytes derived from the patient to be treated, or hepatic stem cells that have been further transformed and differentiated in vitro into hepatocytes for subsequent administration to the patient.

[0137] The present invention further relates to a transgenic non-human animal comprising within its genome a nucleic acid molecule or construct encoding a GAA protein according to the invention. In a particular embodiment, the animal is a mouse.

[0138] Aside from the specific delivery systems embodied in the examples below, a variety of delivery systems are known and can be used to administer the nucleic acid molecules or constructs of the invention, including, for example, encapsulation of recombinant cells capable of expressing the coding sequences of the invention within liposomes, microparticles, microcapsules, receptor-mediated endocytosis, construction of a therapeutic nucleic acid as part of a retrovirus or other vector, etc.

[0139] According to one embodiment, it may be desirable to introduce the GAA polypeptide, nucleic acid molecule, nucleic acid construct or cell of the present invention into the liver of a subject by any suitable route.In addition, naked DNA such as minicircle and transposon can be used for delivery or lentiviral vector.In addition, gene editing technology such as zinc finger nuclease, meganuclease, TALEN and CRISPR can also be used to deliver the coding sequence of the present invention.

[0140] The present invention also provides pharmaceutical compositions comprising the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides, or cells of the present invention. Such compositions comprise a therapeutically effective amount of a therapeutic agent (the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides, or cells of the present invention) and a pharmaceutically acceptable carrier. In specific embodiments, the term "pharmaceutically acceptable" means approved by a U.S. or state government regulatory agency for use in animals and humans, or listed in the United States Pharmacopoeia or the European Pharmacopoeia or other generally recognized pharmacopoeias. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.

[0141] If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. Such compositions will contain a therapeutically effective amount of a therapeutic agent, preferably in purified form, together with an appropriate amount of carrier to provide the dosage form for proper administration to a subject. In a specific embodiment, the nucleic acid, vector, or cell of the present invention is formulated into a composition containing phosphate buffered saline and supplemented with 0.25% human serum albumin. In another particular embodiment, the nucleic acid, vector or cell of the invention is formulated in a composition comprising lactated Ringer's solution and a non-ionic surfactant, such as Pluronic F68, at a final concentration of 0.01-0.0001%, for example at a concentration of 0.001%, by weight of the total composition. The formulation may further comprise serum albumin, in particular human serum albumin, for example 0.25% human serum albumin. Other suitable formulations for either storage or administration are known in the art, in particular from WO 2005 / 118792 or Allay et al., 2011.

[0142] In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to humans.Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer solution.If necessary, the composition can also contain a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the injection site.

[0143] In one embodiment, the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the invention can be delivered in vesicles, in particular liposomes, hi yet another embodiment, the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the invention can be delivered in a controlled release system.

[0144] Methods of administration of the nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the present invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. In certain embodiments, administration is via intravenous or intramuscular routes. The nucleic acid molecules, nucleic acid constructs, vectors, GAA polypeptides or cells of the present invention (whether vectorized or not) can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucosal linings (such as oral, rectal, and intestinal mucosa), and may be administered together with other biologically active agents. Administration may be systemic or local.

[0145] In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the present invention locally to the area in need of treatment, e.g., the liver. This can be accomplished, for example, by using an implant, which is a porous, non-porous, or gel-like material, such as a membrane, e.g., a silicone rubber membrane, or a fiber.

[0146] The amount of the therapeutic agent of the present invention (i.e., the nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the present invention) that will be effective in treating glycogen storage disease can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays can be optionally used to help predict optimal dosage ranges. The exact dose to be used in the formulation will also depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the physician and each patient's circumstances. The dose of the nucleic acid molecule, nucleic acid construct, vector, GAA polypeptide or cell of the present invention administered to a subject in need thereof will vary based on several factors, including, but not limited to, the route of administration, the specific disease being treated, the age of the subject, or the expression level required to achieve a therapeutic effect. One of skill in the art can easily determine the required dosage range based on these and other factors based on his or her knowledge in the art. For treatments involving administering a viral vector, such as an AAV vector, to a subject, a typical dose of the vector is at least 1×10 per kg of body weight. 8 vector genomes (vg / kg), e.g., at least 1 × 10 9 vg / kg, at least 1 × 10 10 vg / kg, at least 1 × 10 11 vg / kg, at least 1 × 10 12 vg / kg, at least 1 × 10 13 vg / kg, or at least 1 × 10 14 vg / kg.

[0147] The present invention also relates to a method for treating glycogen storage disease comprising delivering a therapeutically effective amount of a nucleic acid, vector, GAA polypeptide, pharmaceutical composition, or cell of the present invention to a subject in need thereof.

[0148] The present invention also relates to a method for treating glycogen storage disease, which elicits no immune response against the transgene (i.e. against the GAA polypeptide of the present invention) or elicits a reduced immune response against the transgene, comprising the step of delivering a therapeutically effective amount of a nucleic acid molecule, nucleic acid construct, vector, pharmaceutical composition, or cell of the present invention to a subject in need thereof. The present invention also relates to a method for treating glycogen storage disease, which comprises the repeated administration of a therapeutically effective amount of a nucleic acid molecule, nucleic acid construct, vector, pharmaceutical composition, or cell of the present invention to a subject in need thereof. In this embodiment, the nucleic acid molecule or nucleic acid construct of the present invention comprises a promoter that is functional in hepatocytes, thereby allowing immune tolerance to the expressed GAA polypeptide produced therefrom. Similarly, in this embodiment, the pharmaceutical composition used in this embodiment comprises a nucleic acid molecule or nucleic acid construct that comprises a promoter that is functional in hepatocytes. In the case of delivery of hepatocytes, the cells may be cells previously collected from a subject in need of treatment and engineered to enable them to produce the GAA polypeptide of the present invention by introducing therein a nucleic acid molecule or nucleic acid construct of the present invention. According to one embodiment of the aspect that includes repeated administration, the administration can be repeated at least once or more, and can be considered to be carried out according to a periodic schedule, such as once a week, once a month, or once a year. Periodic schedules can also include once every 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, or once every 10 years or more. In another specific embodiment, the administration of each administration of the viral vector of the present invention is carried out using a different virus for each successive administration, thereby avoiding the decrease in efficacy due to possible immune response against the viral vector that was previously administered. For example, the first administration of a viral vector that includes AAV8 capsid can be followed by the administration of a vector that includes AAV9 capsid, or even the administration of a virus that is not related to AAV, such as a retroviral or lentiviral vector.

[0149] According to the present invention, treatment may include a curative, ameliorative or prophylactic effect. Thus, therapeutic and prophylactic treatment includes amelioration of symptoms of a particular glycogen storage disease, or prevention or otherwise reducing the risk of developing a particular glycogen storage disease. The term "prevention" may be considered as reducing the severity or onset of a particular condition. "Prevention" also includes preventing the recurrence of a particular condition in a patient previously diagnosed with the condition. "Treatment" may also reduce the severity of an existing condition. The term "treatment" is used herein to refer to any regimen that may benefit an animal, particularly a mammal, and more particularly a human subject.

[0150] The present invention also relates to an ex vivo gene therapy for the treatment of glycogen storage disease, comprising the steps of introducing a nucleic acid molecule or a nucleic acid construct of the present invention into an isolated cell, such as an isolated hematopoietic stem cell, of a patient in need thereof, and introducing said cell into said patient in need thereof. In a particular embodiment of this aspect, the nucleic acid molecule or construct is introduced into the cell using a vector as defined above. In a particular embodiment, the vector is an integrative viral vector. In a further particular embodiment, the viral vector is a retroviral vector, such as a lentiviral vector. For example, lentiviral vectors as disclosed in van Til et al., 2010, Blood, 115(26), p. 5329, may be used to implement the method of the present invention.

[0151] The present invention also relates to a nucleic acid molecule, a nucleic acid construct, a vector, a GAA polypeptide, or a cell of the invention for use as a medicament.

[0152] The present invention also relates to a nucleic acid molecule, a nucleic acid construct, a vector, a GAA polypeptide, or a cell of the present invention for use in a method for treating a disease caused by a mutation in the GAA gene, in particular in a method for treating Pompe disease. The present invention further relates to a nucleic acid molecule, a nucleic acid construct, a vector, a GAA polypeptide, or a cell of the present invention for use in a method for treating a glycogen storage disease, such as glycogen storage disease type I (von Gierke disease), glycogen storage disease type II (Pompe disease), glycogen storage disease type III (Cohri disease), glycogen storage disease type IV, glycogen storage disease type V, glycogen storage disease type VI, glycogen storage disease type VII, glycogen storage disease type VIII, and fatal congenital glycogen storage diseases of the heart, more particularly glycogen storage disease type I, glycogen storage disease type II or glycogen storage disease type III, even more particularly glycogen storage disease type II and glycogen storage disease type III, most particularly glycogen storage disease type II. The truncated GAA polypeptides of the present invention may be administered to a patient in need thereof for use in enzyme replacement therapy (ERT), for example for glycogen storage diseases such as Glycogen Storage Disease Type I (von Gierke's Disease), Glycogen Storage Disease Type II (Pompe's Disease), Glycogen Storage Disease Type III (Cohri's Disease), Glycogen Storage Disease Type IV, Glycogen Storage Disease Type V, Glycogen Storage Disease Type VI, Glycogen Storage Disease Type VII, Glycogen Storage Disease Type VIII, and fatal congenital glycogen storage diseases of the heart, more particularly Glycogen Storage Disease Type I, Glycogen Storage Disease Type II or Glycogen Storage Disease Type III, even more particularly Glycogen Storage Disease Type II and Glycogen Storage Disease Type III, most particularly Glycogen Storage Disease Type II.

[0153] The present invention further relates to the use of a nucleic acid molecule, a nucleic acid construct, a vector, a GAA polypeptide, or a cell of the invention in the manufacture of a medicament useful for the treatment of a glycogen storage disease, such as Glycogen Storage Disease Type I (von Gierke's Disease), Glycogen Storage Disease Type II (Pompe's Disease), Glycogen Storage Disease Type III (Cohri's Disease), Glycogen Storage Disease Type IV, Glycogen Storage Disease Type V, Glycogen Storage Disease Type VI, Glycogen Storage Disease Type VII, Glycogen Storage Disease Type VIII, and fatal congenital glycogen storage diseases of the heart, more particularly Glycogen Storage Disease Type I, Glycogen Storage Disease Type II or Glycogen Storage Disease Type III, even more particularly Glycogen Storage Disease Type II and Glycogen Storage Disease Type III, most particularly Glycogen Storage Disease Type II.

[0154] Working Example The present invention is further described by reference to the following experimental examples and the accompanying drawings, which are provided by way of illustration only and are not intended to be limiting.

[0155] Materials and Methods GAA activity GAA activity was measured after homogenization of frozen tissue samples in distilled water. 50-100 mg of tissue was weighed, homogenized, and then centrifuged at 10000×g for 20 min. Reactions were assembled with 10 μl of supernatant and 20 μl of substrate (4MU α-D-glucoside) in a 96-well plate. The reaction mixtures were incubated at 37°C for 1 h and then stopped by the addition of 150 μl of sodium carbonate buffer (pH 10.5). Using a standard curve (0-2500 pmol / μl of 4MU), the fluorescent 4MU released from each reaction mixture was measured using an EnSpire Alpha Plate Reader (PerkinElmer) at 449 nm (emission) and 360 nm (excitation). Protein concentrations of clarified supernatants were quantified by BCA (Thermo Fisher Scientific). To calculate GAA activity, the released 4MU concentration was divided by the protein concentration of the sample and activity was reported as nmol / hr / mg protein.

[0156] Mouse studies Gaa- / - mice were generated by targeted disruption of exon 6 and maintained on a C57BL / 6J / 129X1 / SvJ background (Raben N. et al 1998). A volume of 0.2 ml of vector was delivered via the tail vein. Serum samples were collected monthly to monitor the levels of secreted hGAA. PBS-injected diseased animals and wild-type littermates were used as controls.

[0157] Non-human primate studies Male cynomolgus macaques were housed in stainless steel cages and maintained on a 12-hour light-dark cycle. All macaques demonstrated neutralizing antibody titers of less than 1:5 prior to the start of the study. 12AAV8-hAAT-sp7-Δ8-hGAAco1 at a dose of vg / kg was injected via the saphenous vein. Blood samples were taken 12 days before and 30 days after injection via the femoral vein. Whole blood was collected into EDTA-containing tubes and centrifuged to separate serum. All macaques were euthanized 3 months after vector administration. Animals were first anesthetized with a mixture of ketamine / dexmedetomidine and then euthanized using intravenous sodium pentobarbital. Tissues were immediately collected and frozen in liquid nitrogen.

[0158] Western blot analysis Complete homogenates were obtained from frozen muscles. Protein concentrations in the extracts were determined by Pierce BCA protein assay (Thermo Fisher Scientific) according to the manufacturer's instructions. Western blots were performed using anti-hGAA antibody (Abcam). Anti-tubulin antibody (Sigma-Aldrich) was used as a loading control.

[0159] result With the aim of designing a novel form of GAA with improved secretion and reduced immunogenicity, the inventors decided to generate truncated forms of GAA, optionally in combination with alternative signal peptides.

[0160] Human GAA, shown in SEQ ID NO:2, served as the basis for designing these novel forms. SEQ ID NO:1 corresponds to the sequence of SEQ ID NO:2, lacking the corresponding native signal peptide of GAA (amino acids 1-27 of SEQ ID NO:2). Nucleic acid constructs were designed to encode a GAA polypeptide derived from SEQ ID NO:1 truncated at its N-terminus. We started by designing a nucleic acid sequence based on a wild-type hGAA coding sequence (SEQ ID NO:9, corresponding to nucleotides 82-2859 of SEQ ID NO:8, the wild-type hGAA coding sequence including the signal peptide coding sequence) with the codons corresponding to the first 8 amino acids of SEQ ID NO:1 deleted (Δ8). In addition to the wild-type hGAA coding sequence, we designed optimized nucleic acid sequences (SEQ ID NO:10 and SEQ ID NO:11, corresponding to the optimized coding sequences of hGAAco1 and hGAAco2, respectively) encoding Δ8 truncated hGAA polypeptides to eliminate possible sequence-specific effects.

[0161] [Table 1]

[0162] Amino acids 1-27 of the hGAA sequence (corresponding to the native signal peptide of hGAA, defined here as sp1; its sequence is shown in SEQ ID NO:4) have been replaced by amino acids 1-24 of the human α-1-antitrypsin sequence (NP_000286.3), defined here as sp2 (sequence shown in SEQ ID NO:5). We transfected human hepatoma cells (Huh-7) with a construct encoding a truncated hGAA in parallel with its full-sized version, and we measured the amount of hGAA released into the medium after 48 hours (Figure 1A). The Δ8 deletion of the hGAA sequence resulted in a significant 50% increase in secretion levels for both the wild-type (hGAA) and codon-optimized (hGAAco2) sequences. The same truncation performed on a different codon-optimized sequence (hGAAco1) also improved the secretion of hGAA to a similar extent.

[0163] To confirm that changes in the sequence after the signal peptide could improve the secretion of hGAA, we further truncated the hGAA polypeptide. We eliminated the codons corresponding to the first 42 amino acids of hGAA from the hGAAco1 construct (Δ42), and we replaced them with the signal peptide (sp7; sequence shown in SEQ ID NO:3) derived from chymotrypsinogen B1. We then compared the secretion efficiency obtained with this new deletion construct with its Δ8 version fused to the sp7 signal peptide to that obtained with the full-sized hGAAco1 with sp1 or sp7. We transfected such constructs into Huh-7 cells, and we measured the hGAA activity in the medium after 48 hours. As expected, we were able to measure hGAA activity after transfection of full-sized hGAAco1 (p=0.055 vs. GFP), and its secretion was increased 2-fold by replacing the wild-type signal peptide with sp7 (p=0.006 vs. hGAAco1). Surprisingly, both the Δ8hGAA and Δ42hGAA sequences fused to the sp7 signal peptide showed a 2-fold increase in secretion of hGAA compared to the full-sized sequence (p=0.0002 and p=0.0003, respectively, for sp7-hGAAco1, FIG. 1B).

[0164] In conclusion, these data demonstrate that truncation of the hGAA sequence coupled with an effective signal peptide can increase protein secretion in vitro. Furthermore, truncation has one important advantage over mutagenesis of the native sequence, since truncation does not generate major neoantigens, which is an advantage in engineering therapeutic products.

[0165] We then confirmed these findings in vivo in a mouse model of Pompe disease. We injected GAA- / - mice (Raben et al J. Bio. Chem. 1998) with AAV8 vectors expressing full-size, Δ8, or Δ42 hGAAco1 fused to the sp7 signal peptide under the transcriptional control of a very strong liver-specific promoter resulting from the fusion of the apolipoprotein B enhancer and the human α-1-antitrypsin promoter (hAAT). 12 One month after injection of vg / kg of vector, mice were bled and hGAA activity was measured in the serum. Treatment of mice with vector expressing full-length hGAAco1 fused to sp7 showed elevated levels of hGAA in the bloodstream (p=0.115 vs. PBS). Surprisingly, both truncated hGAA, Δ8 and Δ42, significantly increased serum hGAA levels (p=0.014 and p=0.013, respectively).

[0166] These data indicate that deletion of the first amino acid of hGAA significantly improves the levels of hGAA secreted into the bloodstream.

[0167] Additionally, another signal peptide corresponding to amino acids 1-25 (sp6; SEQ ID NO: 6) derived from iduronate-2-sulfatase was fused to the Δ8 truncated form of hGAA. We transfected hepatoma cells (Huh-7) with plasmids expressing GFP or wild-type hGAA (hGAA; parent polypeptide corresponding to amino acid residues 28-952 of SEQ ID NO: 30) in parallel with plasmids expressing optimized hGAA (hGAAco1) fused to sp1, sp2, sp6, sp7, or sp8. 48 hours after transfection, growth media was analyzed for the presence of hGAA. Notably, these constructs led to secretion of significantly higher levels of hGAA than those observed in GFP-negative cells as indicated by transfected cells (Figure 3).

[0168] We then assessed glycogen content in the heart, diaphragm, and quadriceps of GAA- / - mice treated as described above with Δ8-hGAA. Of note, we observed high levels of hGAA in tissues after treatment with Δ8-hGAAco expression vector (data not shown), which correlated with a significant decrease in glycogen content in all tissues considered (Figure 4B-D). Notably, glycogen levels measured after treatment with vectors carrying the highly effective signal peptides sp7 and 8 in the heart (Figure 4B) were indistinguishable from those observed in unaffected animals (p=0.983 and 0.996 vs. wild type, respectively). Importantly, the levels observed after treatment with both sp7 and sp8 vectors were significantly reduced compared to GAA- / - animals injected with PBS or treated with hGAAco expression vector fused to sp1 signal peptide.

[0169] We also tested whether transduction of the liver with our vectors induces a humoral response to the transgene. Mice were intravenously injected with AAV8 vectors expressing hGAAco1(co) with the native sp1 signal peptide or Δ8-hGAAco1 fused to sp2, sp7, or sp8 under the transcriptional control of a liver-specific promoter. The results are presented in Figure 5. Gaa- / - mice injected intramuscularly with AAV expressing Δ8-hGAAco1 under the transcriptional control of a constitutive promoter showed very high levels of total IgG (about 150 μg / mL), whereas vectors expressing the same protein in the liver generally showed lower levels of humoral response. Interestingly, mice injected with sp1 hGAAco1(co) expressing vectors showed detectable levels of antibodies at both doses, whereas mice injected with the engineered highly secreted vector showed undetectable IgG levels. These data indicate that transgene expression in the liver is necessary for the induction of peripheral tolerance, and they also provide the perspective that high circulating levels of hGAA, achieved by fusion with an effective signal peptide, might induce a reduced humoral response to the protein itself.

[0170] The best performing vectors selected in the mouse study were injected into two non-human primates (NHPs, Macaca fascicularis) to confirm the efficacy of secretion and uptake into muscle of our vectors. We administered 2×10 12Two monkeys were injected with 1000 mg / kg of AAV8-hAAT-sp7-Δ8-hGAAco1. One month after injection, we measured the hGAA levels in the serum of two animals by Western blot using a specific anti-hGAA antibody. We observed a clear band with a size compatible with that of hGAA in two monkeys. This band was not present in serum samples obtained 12 days before injection of the vector, thus confirming the specificity of our detection method (FIG. 6A). Three months after injection, we sacrificed the animals and we obtained tissues to confirm whether the hGAA secreted from the liver into the bloodstream was effectively taken up by the muscle. We performed Western blots using an antibody specific for hGAA on total lysates obtained from the biceps and diaphragm of two monkeys. Interestingly, we could observe a clear band in animal number 2, which also showed the highest hGAA levels in the bloodstream (Figure 6B). Also, in animal number 1, we could observe a faint band with a molecular weight consistent with that of hGAA in both analyzed muscles. These data show that the AAV8-hAAT-sp7-Δ8-hGAAco1 vector effectively transduces the liver of non-human primates. They also demonstrate that the protein secreted into the bloodstream is effectively taken up into muscle, and that this uptake correlates with the levels of hGAA measured in the blood.

[0171] We also determined the effect of the best-behaved vector (AAV8-hAAT-sp7-Δ8-hGAAco1) selected in mouse studies in a GSD III mouse model. We developed a glycogen debranching enzyme (GDE) knockout mouse model. This model recapitulates the disease phenotype observed in humans with glycogen storage disease type III (GSDIII). Notably, GDE- / - mice, which completely lack GDE activity, have impaired muscle strength and accumulate glycogen in various tissues. Interestingly, they also accumulate glycogen in the liver, which is also seen in humans. Here, we tested whether overexpression of sp7-Δ8-hGAA in the liver rescues the glycogen accumulation observed in GDE- / - mice. We administered 1×10 11 Or 1×10 12 vg / mice were injected with AAV8-hAAT-sp7-Δ8-hGAAco1. As a control, we injected wild type (WT) and GDE− / − mice with PBS in parallel. Three months after the administration of the vector, the mice were sacrificed and glycogen levels were quantified in the liver. The results are reported in Figure 7. As previously reported (Pagliarani et al. and our model), GDE− / − mice showed a significant increase in glycogen accumulation in the liver (p=1.3×10 -7 ), which have 5 times more glycogen than wild-type animals. Remarkably, 1 × 10 11 and 1 x 1 x 10 12 Treatment of vg / mice with the AAV8-hAAT-sp7-Δ8-hGAAco1 vector induced a statistically significant decrease in glycogen content (p=4.5×10, respectively). -5 and 1.4 x 10 -6 Importantly, glycogen levels measured in the livers of mice injected with the AAV8-hAAT-sp7-Δ8-hGAAco1 vector were indistinguishable from those measured in wild-type animals, especially at the highest dose (1 × 10 11 p=0.053, 1×10 12 0.244 in the dose cohort).

[0172] We performed an analysis of GAA activity in media and lysates of HuH7 cells transfected with different GAA versions (all codon optimized): 1. native GAA (co) containing the native sp1 GAA signal peptide, 2. engineered GAA containing a heterologous sp7 signal peptide (sp7-co), and 3. engineered GAA containing a heterologous sp7 signal peptide followed by various amino acid deletions (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co, sp7-Δ47-co, and sp7-Δ62-co, in which 8, 29, 42, 47, and 62 first N-terminal amino acids of SEQ ID NO:1, respectively, are deleted). The analysis showed significantly higher GAA activity in the medium of cells transfected with Δ8, Δ29, Δ42 and Δ43 GAA versions compared to both engineered non-deleted GAA (sp7-co) and native GAA (co) (Figure 8). Instead, significantly lower GAA activity was observed in the medium of cells transfected with Δ47 and Δ62 GAA versions compared to other engineered GAA versions [with deletions (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co and without deletion (sp7-co)]. Interestingly, (Figure 9) intracellular GAA activity did not differ between the productive deleted (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co) and non-deleted versions (sp7-co), indicating that they are all efficiently produced and processed in the cells. Instead, intracellular GAA activity was very low in the sp7-Δ47-co and sp7-Δ62-co versions, showing significantly lower activity compared to all other engineered versions [with deletions (sp7-Δ8-co, sp7-Δ29-co, sp7-Δ42-co, sp7-Δ43-co) and without deletions (sp7-co)].

[0173] We also performed an analysis of GAA activity in the medium and lysates of HuH7 cells transfected with various GAA versions (all codons optimized): 1. native GAA (co) containing the native sp1 GAA signal peptide, 2. engineered GAA containing heterologous sp6 or sp8 signal peptides (sp6-co, sp8-co), and 3. engineered GAA containing heterologous sp6 or sp8 signal peptides followed by a deletion of 8 amino acids (sp6-Δ8-co, sp8-Δ8-co)). The analysis showed significantly higher GAA activity in the medium of cells transfected with the Δ8 versions compared to i. their respective engineered non-deleted GAA versions (sp6-co or sp8-co); and ii. native GAA (co) (Figure 10). Interestingly, intracellular GAA activity did not differ between all engineered GAA versions (both deleted and non-deleted), indicating that they are efficiently produced and processed intracellularly (cell lysate panels). Instead, intracellular GAA activity was significantly higher when native GAA (co) was used compared to the engineered versions, indicating that native GAA is primarily retained intracellularly.

Claims

1. A recombinant adeno-associated virus (AAV) vector comprising an expression cassette, the expression cassette comprising a nucleic acid molecule encoding a truncated acid alpha-glucosidase (GAA) polypeptide, the truncated GAA polypeptide comprising a deletion of 6 to 46 contiguous amino acids from its N-terminus compared to a parent GAA polypeptide, the parent polypeptide corresponding to a precursor form of a GAA polypeptide lacking its signal peptide, the truncated GAA polypeptide further comprising a signal peptide fused to its N-terminus, the signal peptide being selected from the group consisting of SEQ ID NOs: 3, 6 and 7; wherein the expression cassette comprises, in that order, an enhancer, a promoter, an intron, the nucleic acid molecule encoding the truncated GAA polypeptide, and a polyadenylation signal. Recombinant AAV vectors.

2. A recombinant AAV vector as described in claim 1, wherein the nucleic acid molecule encoding the truncated GAA polypeptide is optimized for improving expression of the truncated GAA polypeptide in vivo and / or for improving immune tolerance to the truncated GAA polypeptide.

3. A recombinant AAV vector described in claim 1 or 2, wherein the nucleic acid molecule has an array as shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO:

51.

4. A recombinant AAV vector described in any one of claims 1 to 3, wherein the parent polypeptide is human GAA (hGAA).

5. The recombinant AAV vector of claim 4, wherein the parent polypeptide is hGAA having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:33, or is hGAA that is a functional mutant of hGAA having the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:

33.

6. The recombinant AAV vector of claim 4, wherein the parent polypeptide is hGAA having the amino acid sequence shown in SEQ ID NO:1, or is hGAA that is a functional mutant of hGAA having the amino acid sequence shown in SEQ ID NO:

1.

7. A recombinant AAV vector described in any one of claims 1 to 6, wherein the truncated GAA polypeptide has the amino acid sequence shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34 or SEQ ID NO:

35.

8. A recombinant AAV vector described in any one of claims 1 to 7, wherein the promoter is a liver-specific promoter.

9. The recombinant AAV vector of claim 8, wherein the liver-specific promoter is selected from the group consisting of an alpha-1 antitrypsin promoter (hAAT), a transthyretin promoter, an albumin promoter, and a thyroxine-binding globulin (TBG) promoter.

10. The recombinant AAV vector of any one of claims 1 to 9, wherein the intron is selected from the group consisting of a human beta globin b2 (i.e., HBB2) intron, a FIX intron, a chicken beta-globin intron, and an SV40 intron, wherein the intron is optionally a modified intron.

11. The recombinant AAV vector of claim 10, wherein the modified intron is selected from a modified HBB2 intron of SEQ ID NO: 17, a modified FIX intron of SEQ ID NO: 19, and a modified chicken β-globin intron of SEQ ID NO:

21.

12. The recombinant AAV vector of any one of claims 1 to 11, wherein the expression cassette comprises, in this order: an ApoE regulatory region; a hAAT promoter; an HBB2 intron; a nucleic acid sequence encoding a truncated GAA polypeptide; and a bovine growth hormone polyadenylation signal.

13. The recombinant AAV vector described in claim 12, wherein the expression cassette comprises any one of the nucleotide sequences of SEQ ID NOs: 22 to 26.

14. The recombinant AAV vector according to any one of claims 1 to 13, which is a single-stranded or double-stranded self-complementary AAV vector.

15. The recombinant AAV vector described in claim 14, wherein the recombinant AAV vector is an AAV vector having a capsid derived from AAV.

16. The recombinant AAV vector of claim 15, wherein the capsid derived from AAV is selected from the group consisting of AAV1, AAV2, mutated AAV2, AAV3, mutated AAV3, AAV3B, mutated AAV3B, AAV4, AAV5, AAV6, mutated AAV6, AAV7, AAV8, AAV9, AAV10, AAVcy10, AAVrhlO, AAVrh74, AAVdj, AAV-Anc80, AAV-LK03, AAV2i8, porcine AAV, AAVpo4, AAVpo6 capsid and chimeric capsid.

17. The recombinant AAV vector of claim 14, wherein the AAV vector has an AAV8, AAV9, AAVrh74 or AAV2i8 capsid.

18. The recombinant AAV vector of claim 14, wherein the AAV vector has an AAV8, AAV9 or AAVrh74 capsid.

19. The recombinant AAV vector of claim 14, wherein the AAV vector has an AAV8 capsid.

20. A cell transformed with a recombinant AAV vector of any one of claims 1 to 19.

21. The cell of claim 20, wherein the cell is a liver cell or a muscle cell.

22. A pharmaceutical composition comprising the recombinant AAV vector of any one of claims 1 to 19, or the cell of claim 20 or 21, in a pharma- ceutically acceptable carrier.

23. Use of a recombinant AAV vector according to any one of claims 1 to 19, or a cell according to claim 20 or 21, in the manufacture of a medicament.

24. The pharmaceutical composition of claim 22 for treating glycogen storage disease.

25. The pharmaceutical composition of claim 24 for treating glycogen storage disease type I (von Gierke disease), glycogen storage disease type II (Pompe disease), glycogen storage disease type III (Cohri disease), glycogen storage disease type IV, glycogen storage disease type V, glycogen storage disease type VI, glycogen storage disease type VII, glycogen storage disease type VIII, or fatal congenital glycogen storage disease of the heart.

26. The pharmaceutical composition of claim 24 for treating glycogen storage disease type I, glycogen storage disease type II, or glycogen storage disease type III.

27. The pharmaceutical composition of claim 24 for treating glycogen storage disease II and glycogen storage disease III.

28. The pharmaceutical composition of claim 24 for treating glycogen storage disease II.

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