N-terminally truncated GDE for treatment of glycogen storage disease III

Abstract

The present invention relates to functional N-terminally truncated GDE polypeptides for use in the treatment of glycogen storage disease III.

Description

[0001] The present invention relates to the treatment of glycogen storage disease III (GSDIII). Background Art

[0002] Mutations in the AGL gene result in a genetic deficiency of glycogen debranching enzyme (GDE) or "amyloid-α-1,6-glucosidase, 4-α-glucanotransferase", an enzyme involved in glycogen degradation. GDE has two independent catalytic activities, occurring at different sites on the protein: 4-α-glucanotransferase activity and amylo-1,6-glucosidase activity. Genetic deficiency of GDE in glycogen storage disease III (GSDIII) causes incomplete glycogenolysis, resulting in the accumulation of abnormal glycogen with short external chains in various organs, mainly the liver and muscle. The disease is characterized by hepatomegaly, hypoglycemia, short stature, variable myopathy and cardiomyopathy. Most patients have GSDIII involving both liver and muscle (type IIIa), while some patients (~15%) have only liver involvement (type IIIb). Liver symptoms usually appear in childhood. Cirrhosis and hepatocellular carcinoma have been reported in some cases (Chen et al., 2009, Scriver's Online Metabolic & Molecular Bases of Inherited Disease, New York: McGraw-Hill; Kishnani et al., 2010, Genet Med 12, 446-463). Muscle weakness may appear in childhood. It becomes more common in adults, with onset in the 20s or 30s. There is a significant incidence of progressive muscle weakness, and patients in the advanced stages may become wheelchair-bound. Patients may also develop cardiomyopathy. There is significant clinical variability in the severity of symptoms that occur in these patients. Progressive myopathy and / or cardiomyopathy and / or peripheral neuropathy are the main causes of morbidity in adults (Kishnani et al., 2010, Genet Med 12, 446-463; Cornelio et al., 1984, Arch Neurol 41, 1027-1032; Coleman et al., 1992, Ann Intern Med 116, 896-900). Reports of possible neurological manifestations associated with the disease have come from clinicians working with GSDIII patients, who reported fluctuations in attention, executive function deficits, and impaired emotional skills (Michon et al., 2015, J Inherit Metab Dis, 38(3):573-580).Thus, in the GDE- / - mouse model of the disease, a massive accumulation of glycogen throughout the nervous system has been documented (Pagliarani et al., 2014, Biochim Biophys Acta, 1842(11): 2318-2328; Liu et al., 2014, Mol Genet Metab, 111(4): 467-476), although a careful characterization of the phenotype associated with glycogen accumulation is still missing. Current treatments are symptomatic, and there is no effective therapy for the disease. Hypoglycemia can be controlled by frequent high-carbohydrate meals with corn starch supplements or nighttime gastric tube feedings. Patients with myopathy have been treated with a daytime high-protein diet plus nighttime enteral infusions. Temporary improvement of symptoms has been documented in some patients, but there are no systematic studies or long-term data demonstrating that a high-protein diet prevents or treats progressive myopathy (Kishnani et al., 2010, Genet Med 12, 446-463). These approaches have little effect on altering the long-term course and morbidity of these diseases.

[0003] Therefore, there is still a need for long-term treatment of GSDIII. Gene therapy aimed at stably replacing GDE protein in affected tissues seems to be a potential treatment approach. However, the large size of the GDE transgene constitutes a major obstacle because it cannot meet the size restrictions of most gene therapy vectors. In fact, the human AGL gene is 85 kb long, composed of 35 exons, encoding 7.4 kb of mRNA, which includes 4599 bp of coding region and 2371 bp of 3′ untranslated sequence to express 175 kDa GDE protein (Bao Y et al., 1996, Genomics., 38(2):155–65). This poses a real problem because the minimum size of the GDE expression cassette (including, for example, at least one promoter, GDE coding sequence, polyA signal and two ITRs for AAV vectors) would be greater than 5 kb, which is the limit of the genome size that can be packaged into AAV gene therapy vectors for in vivo gene delivery.

[0004] The inventors have previously proposed the use of dual AAV vectors to overcome this size limitation. According to this approach, two vectors are used to transduce the same cell, each vector containing a portion of the large transgenic gene coding sequence. Although the use of dual AAV vectors is promising, for economic and practical reasons, it is best to provide a gene therapy strategy that implements only one viral vector.

[0005] In patent applications WO2020 / 030661 and PCT / EP2021 / 073309, the inventors describe another method based on the use of truncated GDE polypeptides suitable for a single viral vector. However, alternatives to the truncated GDE polypeptides previously described in WO2020 / 030661 and PCT / EP2021 / 073309 are desirable. Summary of the invention

[0006] The present invention relates to a functional truncated GDE polypeptide, wherein the functional truncated GDE polypeptide comprises a deletion relative to a reference functional full-length human GDE sequence, and wherein the deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are:

[0007] - MGSFQY (SEQ ID NO: 7);

[0008] - MEKSGG (SEQ ID NO: 8);

[0009] - MILRVG (SEQ ID NO: 9);

[0010] - MGADNH (SEQ ID NO: 10); or

[0011] - MLDCVT (SEQ ID NO: 11).

[0012] In a specific embodiment, the deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8) or MILRVG (SEQ ID NO: 9), preferably wherein the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8).

[0013] In a specific embodiment, the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In a specific embodiment, the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO: 1, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO: 1.

[0014] In a specific embodiment, the functional truncated GDE polypeptide further comprises a deletion or a combination of deletions relative to the reference functional full-length human GDE sequence, such as a deletion or a combination of deletions in the C-terminal portion of the GDE sequence or a deletion or a combination of deletions in the central domain of the GDE sequence. In a specific embodiment, the functional truncated GDE polypeptide further comprises a deletion or a combination of deletions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and wherein the deletion is selected from any of the deletions referred to as ∆1, ∆2, ∆3, ∆4, ∆5, ∆6 and ∆7 in Table 2 below.

[0015] In a specific embodiment, the functional truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NO: 12-16, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 12-16. In a specific embodiment, the functional truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NO: 13 or 14, preferably SEQ ID NO: 13, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 13 or 14, preferably SEQ ID NO: 13.

[0016] The present invention also relates to a nucleic acid molecule encoding a functional truncated GDE polypeptide of the present invention.

[0017] The present invention also relates to an expression cassette, which preferably comprises in the following order:

[0018] - Promoter;

[0019] - optional introns;

[0020] - a nucleic acid molecule as claimed in the present invention; and

[0021] - Polyadenylation signal.

[0022] Another aspect of the present invention relates to a vector, in particular a viral vector, comprising the nucleic acid molecule or expression cassette of the present invention. In a specific embodiment, the vector is an AAV vector.

[0023] The present invention also relates to an isolated cell transformed with a nucleic acid molecule, an expression cassette or a vector according to the present invention, wherein the cell is in particular a hepatocyte, a myocyte, a cardiac cell or a CNS cell.

[0024] The present invention also relates to a functional truncated GDE polypeptide, nucleic acid molecule, expression cassette, vector or cell as defined above, which is used as a medicine. The present invention also relates to a functional truncated GDE polypeptide, nucleic acid molecule, expression cassette, vector or cell as defined above, which is used in a method for treating a disease caused by a mutation in the AGL gene encoding GDE. In another specific embodiment, the present invention relates to a functional truncated GDE polypeptide, nucleic acid molecule, expression cassette, vector or cell as defined above, which is used in a method for treating GSDIII (Corey's disease). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 ： (A) N-terminal regions of GDE enzymes in Candida glabrata (top) and humans (bottom). Truncation sites for the "∆1b2" and "∆1b3" proteins as well as for the novel truncated proteins ("∆1b4" to "∆1b8") are shown. (B) Experimental design for intramuscular injection of rAAV vectors containing truncated forms of GDE into GSDIII mice. Truncated "∆1b3" protein and full-length GDE (FS) were used as controls.

[0026] Figure 2 ： Glycogen quantification in the left tibialis anterior (TA) muscle 1 month after intramuscular injection of rAAV vectors containing the truncated GDE peptide (∆1b5). Truncated ∆1b3 peptide and full-length GDE were used as positive controls. KO and WT mice were injected with saline (PBS) as a negative control.

[0027] Figure 3 ：Western blot analysis of GDE expression from left tibialis anterior (TA) muscle lysates 1 month after intramuscular injection of rAAV vectors containing a truncated GDE polypeptide (∆1B5). Truncated ∆1B3 polypeptide and full-length GDE were used as positive controls. KO and WT mice were injected with saline (PBS) as a negative control.

[0028] Figure 4 ： based on Figure 3 Quantification of GDE protein expression by Western blot.

[0029] Figure 5 ： Quantification of vector genome copy number (VGCN) by quantitative PCR in the tibialis anterior (TA) muscle of mice injected with saline (PBS) or rAAV vectors expressing truncated GDE polypeptides (∆1b3 or ∆1b5) or full-length GDE protein (GDE full-size).

[0030] Figure 6 ： After injection of rAAV-∆1b3-GDE vector, Agl - / - Partial correction of muscle and heart damage in mouse models. (A) 4-month-old male AgL - / - Mice were injected in the tail vein with rAAV vector encoding ∆1b3-GDE at a dose of 1 x10 14 vg / kg. PBS injected Agl + / + and Agl - / - Mice were used as controls. (B) Glycogen content in the heart and triceps measured at the time of euthanasia. (C) Wire hanging test performed 3 months after vehicle injection, expressed as the number of falls per minute. Statistical analysis was performed by one-way ANOVA (vs. PBS-injected AgL - / - Compared with mice p<0.05, p<0.01 or p<0.001; compared with PBS-injected Agl + / + Compared with mice # p<0.05, ## p<0.01 or ### p<0.001; n=4 mice per group). All data are shown as mean ± SEM.

[0031] Figure 7 ： After injection of rAAV-∆1b5-GDE vector, Agl - / - Complete correction of muscle and cardiac damage in the mouse model. (A) 4-month-old male Ag1 - / - Mice were injected in the tail vein with rAAV vector encoding ∆1b5-GDE at a dose of 1 x10 14vg / kg. PBS injected Agl + / + and Agl - / - Mice were used as controls. (B) Glycogen content in the heart and triceps measured at the time of euthanasia. (C) Wire hanging test performed 3 months after vehicle injection, expressed as the number of falls per minute. Statistical analysis was performed by one-way ANOVA (vs. PBS-injected AgL - / - Compared with mice p<0.05, p<0.01 or p<0.001; compared with PBS-injected Agl + / + Compared with mice # p<0.05, ## p<0.01 or ### p<0.001; n = 7-9 mice per group from two independent experiments). All data are shown as mean ± SEM.

[0032] Figure 8 ： After injection of rAAV-∆1b5-GDE vector, Agl - / - Correction of muscle and heart damage in the rat model. (A) Six-week-old male Ag1 - / - Rats were injected in the tail vein with rAAV vector encoding ∆1b5-GDE at a dose of 1 x 10 14 vg / kg. PBS injected Agl - / - and Agl + / + Rats were used as controls. (B, D) Glycogen content in the heart (B) and triceps (D) measured at the time of euthanasia. (C, E) Histological analysis of the heart (C) and triceps (E) using HPS and PAS staining. Representative images are shown. Statistical analysis was performed by one-way ANOVA (vs. PBS-injected AgL - / - Compared with rats p<0.05, p<0.01 or p<0.001; compared with PBS-injected Agl + / + Compared with rats # p<0.05, ## p<0.01 or ### p<0.001; n=5 rats per group). All data are shown as mean ± SEM.

[0033] Fig. 9 ：Glycogen clearance in human induced pluripotent stem cell (hiPSC)-derived skeletal muscle cells treated with rAAV-∆1b5-GDE vectors. (A) AGL knockout (GSDIII) cells were transduced with rAAV vectors expressing GFP or ∆1b5-GDE at an MOI of 15,000 or 75,000. CRISPR ) Scheme of hiPSC-derived skeletal myoblasts and isogenic controls. (B) In GSDIII CRISPR Expression of specific myogenic markers (desmin, myosin heavy chain (MHC), and titin) and GFP in hiPSC-derived skeletal myotubes after rAAV transduction at MOI 75,000. Representative images are shown. (C) GSDIII CRISPR Glycogen content normalized to viable cell number measured in hiPSC-derived skeletal myotubes and isogenic controls (CTRL1). (D) GSDIII transduced as described above CRISPR PAS staining on hiPSC-derived skeletal myotubes and isogenic control (CTRL1). Representative images are shown. (E) Quantification of PAS staining. Statistical analysis was performed by one-way ANOVA (vs. GSDIII transduced with rAAV-GFP). CRISPR Cells compared p<0.05, p<0.01 or p<0.001; compared with CTRL1 cells # p<0.05, ## p<0.01 or ### p<0.001, experiments in Figures BC had three replicates, and experiments in Figures DE had five replicates). All data are shown as mean ± SEM.

[0034] Fig.10 ： Improved productivity and quality of vectors encoding truncated ∆1b5-GDE. (A) Schematic representation of the two expression cassettes evaluated and their corresponding sizes. (B) Triplicate small-scale rAAV production (50 mL culture) was performed for each vector, and for each replicate, viral titers were measured before (stock) and after (final product) purification. (C) rAAV vector DNA was extracted and loaded on a 1% agarose gel to assess genome integrity. Expected genome sizes range from 5.0 to 5.3 kb. (D) Analytical ultracentrifugation analysis of the proportion of complete particles (right peak in the curve) and empty particles (left peak in the curve). Statistical analysis was performed by one-way ANOVA (compared to miniCMV GDE full-length p<0.05, p<0.01 or p<0.001, experiments were performed in triplicate). All data are shown as mean ± SEM. DETAILED DESCRIPTION

[0035] In patent applications WO2020 / 030661 and PCT / EP2021 / 073309, the inventors have demonstrated that it is possible to use truncated GDE polypeptides whose size is compatible with encapsulation in AAV vectors while maintaining their enzymatic activity.

[0036] Despite the lack of knowledge about the three-dimensional structure of the GDE protein, the present inventors have identified novel N-terminally truncated GDE polypeptides that have high protein expression levels and excellent efficacy in glycogen reduction in GSDIII mice.

[0037] Therefore, the present invention relates to a functional N-terminally truncated GDE polypeptide. This polypeptide can be advantageously used in a method for treating a disease caused by a mutation in the AGL gene encoding GDE, in particular in a method for treating GSDIII (Corey's disease).

[0038] 1- N-terminally truncated GDE polypeptide

[0039] The truncated GDE polypeptide according to the present invention is a functional GDE polypeptide whose coding sequence is small enough to be efficiently packaged in a gene therapy vector, especially a single AAV vector.

[0040] A "functional" GDE polypeptide means a polypeptide that at least partially retains at least one enzymatic activity of a GDE protein, preferably all enzymatic activities of a GDE protein. Therefore, the functional GDE polypeptide implemented in the present invention is able to rescue glycogen accumulation and muscle strength in vivo. As defined herein, GDE enzyme activity is 4-α-glucanotransferase activity and amylo-1,6-glucosidase activity that are involved in glycogen degradation. The transferase activity of GDE relocates three glucose units of glycogen from one chain to another. This leaves a glucose unit at the branch point, which is subsequently released as glucose by glucosidase activity. In a specific embodiment, the functional GDE polypeptide of the present invention has the same function as a full-length GDE polypeptide, particularly a full-length human GDE polypeptide. For example, the functional GDE polypeptide of the present invention may have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or at least 99% activity, or at least 100% activity, relative to one, preferably both, of the above enzymatic activities, compared to the full-length human GDE protein, in particular the full-length human GDE protein of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. The activity of the GDE protein of the present invention may even be more than 100% of the activity of the full-length human GDE protein, in particular the full-length human GDE protein of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, for example, 110%, 120%, 130%, 140%, 150%, 200%, 500%, 700% or even more than 1000%. In a specific embodiment, the functional GDE polypeptide of the present invention has the same function as the full-length GDE polypeptide, in particular with the full-length human GDE polypeptide in muscle tissue, such as the heart or quadriceps. For example, compared to the full-length human GDE protein, in particular the full-length human GDE protein of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, the activity of the functional GDE polypeptide of the present invention in muscle tissue, such as the heart or quadriceps, can be at least 50%, 60%, 70%, 80%, 90%, 95% or at least 99%, or at least 100% relative to one, preferably two, of the above enzyme activities.The activity of the GDE protein of the present invention in muscle tissue, such as the heart or quadriceps, can even be more than 100% of the activity of the full-length human GDE protein, especially the full-length human GDE protein of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, for example, 110%, 120%, 130%, 140%, 150%, 200%, 500%, 700% or even more than 1000%.

[0041] "Functional" GDE polypeptide means a non-pathological GDE polypeptide. In particular, the functional GDE polypeptide of the present invention is not a GDE polypeptide found in patients with GSDIII (Corey's disease), such as GSDIIIa or GSDIIIb.

[0042] Those skilled in the art can easily determine whether a polypeptide is a functional GDE polypeptide. Suitable methods are obvious to those skilled in the art. For example, a suitable in vitro method involves inserting a nucleic acid encoding a polypeptide into a vector such as a plasmid or a viral vector, transfecting or transducing a host cell such as a 293T or HeLa cell or other cells such as Huh7 with the vector, and determining GDE activity. Suitable methods are described in more detail in the experimental section below. For example, GDE activity can be determined by measuring the glucose produced after incubation of homogenized tissue or cell extracts transfected with a vector expressing a functional GDE polypeptide with limit dextrin (glycogen digested by glycogen phosphorylase). Other methods include assessing the muscle strength of treated GDE-KO animals by wire suspension after applying the vector, such as one month, two months or three months after application, assessing the rescue of glycogen accumulation in muscle and / or heart tissue and / or assessing the normalization of blood sugar in treated GDC-KO animals after applying the vector, such as one month, two months or three months after application, to test the efficacy of GDE. Furthermore, GDE expression in tissues of GDE KO animals can be assessed by western blotting after administration of the vector, for example, one month, two months, or three months after administration.

[0043] In the context of the present invention, "reference full-length human GDE sequence" encompasses all natural isoforms of human GDE. Bao and colleagues (Genomics, 1997, 38, 155-165) identified the existence of six transcript variants encoding three GDE protein isoforms. Transcript variants 1-4 encode the same protein, i.e., GDE isoform 1. Transcript variants 5 and 6 encode GDE isoforms 5 and 6, respectively.

[0044] In the context of the present invention, "reference full-length human GDE sequence" does not encode pathological GDE polypeptides. Specifically, the reference full-length human GDE sequence does not encode pathological variants found in GSDIII patients that contain mutations, deletions or insertions compared to wild-type non-pathological full-length human GDE sequences.

[0045] Therefore, the term "reference full-length human GDE polypeptide" covers all natural isoforms of human GDE, including precursor forms, as well as GDE proteins or fragments thereof modified or mutated by insertion, deletion and / or substitution as functional derivatives of GDE. Specifically, the reference full-length human GDE sequence is selected from SEQ ID NO: 1 (corresponding to wild-type GDE isoform 1, UniProtKB identification number: P35573-1), SEQ ID NO: 4 (corresponding to a variant of GDE isoform 1), SEQ ID NO: 2 (corresponding to wild-type GDE isoform 5, UniProtKB identification number: P35573-2), SEQ ID NO: 5 (corresponding to a variant of GDE isoform 5), SEQ ID NO: 3 (corresponding to wild-type GDE isoform 6, UniProtKB identification number: P35573-3) and SEQ ID NO: 6 (corresponding to a variant of GDE isoform 6).

[0046] In a specific embodiment, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, in particular to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In a specific embodiment, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4 and has the same length in terms of number of amino acids as SEQ ID NO: 1 or SEQ ID NO: 4. In a specific embodiment, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 5, and has the same length in terms of the number of amino acids as SEQ ID NO: 2 or SEQ ID NO: 5. In a specific embodiment, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 6, and has the same length in terms of the number of amino acids as SEQ ID NO: 3 or SEQ ID NO: 6.

[0047] The term "identical" and its deviations refer to the sequence identity between two nucleic acid molecules or between two polypeptide molecules. When a position in two compared sequences is occupied by the same base or the same amino acid, the molecules are identical at that position. The identity percentage between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in the two sequences are matched, the two sequences have 60% identity. Usually, the comparison is carried out when the two sequences are aligned to provide maximum identity. Various bioinformatics tools known to those skilled in the art can be used for comparing nucleic acid sequences, such as BLAST or FASTA.

[0048] In a specific embodiment, the reference full-length human GDE sequence has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, particularly SEQ ID NO: 1, corresponding to GDE isoform 1.

[0049] In a specific embodiment, the truncated GDE polypeptide of the present invention that is truncated relative to a reference full-length human GDE sequence may comprise one or more additional amino acid modifications relative to the reference full-length human GDE sequence. Specifically, in addition to the deletions further described below, the functional truncated GDE polypeptide may comprise one or more amino acid modifications such as amino acid insertions, deletions and / or substitutions compared to the reference full-length human GDE sequence. For example, the functional truncated GDE polypeptide may comprise 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) additional amino acid modifications, in particular 1 to 5 (e.g., 1, 2, 3, 4 or 5) additional amino acid modifications, as long as the function of the truncated GDE polypeptide is retained.

[0050] The functional truncated GDE polypeptide of the present invention is an N-terminal truncated GDE polypeptide. "N-terminal truncated GDE polypeptide" means a GDE polypeptide comprising a deletion relative to a reference functional full-length human GDE sequence, wherein the deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence. The "N-terminal portion" of the reference functional full-length GDE sequence refers to a region consisting of the first 280 amino acid residues (i.e., the most N-terminal 280 amino acid residues of the reference full-length GDE sequence), the first 200 amino acid residues (i.e., the most N-terminal 200 amino acid residues of the reference full-length GDE sequence), the first 150 amino acid residues (i.e., the most N-terminal 150 amino acid residues of the reference full-length GDE sequence), preferably the first 125 amino acid residues (i.e., the most N-terminal 125 amino acid residues of the reference full-length GDE sequence), and most preferably the first 123 amino acid residues (i.e., the most N-terminal 123 amino acid residues of the reference full-length GDE sequence).

[0051] As described in detail below, the N-terminally truncated GDE polypeptides of the present invention retain methionine as the first residue at the N-terminal end.

[0052] The functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are:

[0053] - MGSFQY (SEQ ID NO: 7);

[0054] - MEKSGG (SEQ ID NO: 8);

[0055] - MILRVG (SEQ ID NO: 9);

[0056] - MGADNH (SEQ ID NO: 10); or

[0057] - MLDCVT (SEQ ID NO: 11).

[0058] Preferably, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8).

[0059] In other words, the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE results in a truncated GDE polypeptide, wherein the first six amino acids at the N-terminal end are different from the six amino acids at the N-terminal end of the reference functional full-length human GDE sequence, and wherein the first six amino acids at the N-terminal end of the truncated polypeptide are SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, preferably SEQ ID NO: 8.

[0060] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0061] - wherein the reference functional full-length human GDE sequence has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

[0062] - and wherein the deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are:

[0063] - MGSFQY (SEQ ID NO: 7);

[0064] - MEKSGG (SEQ ID NO: 8);

[0065] - MILRVG (SEQ ID NO: 9);

[0066] - MGADNH (SEQ ID NO: 10); or

[0067] - MLDCVT (SEQ ID NO: 11).

[0068] In a preferred embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0069] - wherein the reference functional full-length human GDE sequence has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

[0070] - and wherein the deletion consists of deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8).

[0071] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0072] - wherein the reference functional full-length human GDE sequence has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

[0073] - and the deletion consists of:

[0074] (i) deletion of any amino acid between the first methionine and the sequence "GSFQY" (SEQ ID NO: 54);

[0075] (ii) deletion of any amino acid between the first methionine and the sequence "EKSGG" (SEQ ID NO: 55);

[0076] (iii) deletion of any amino acid between the first methionine and the sequence "ILRVG" (SEQ ID NO: 56);

[0077] (iv) deletion of any amino acid between the first methionine and the sequence "GADNH" (SEQ ID NO: 57); or

[0078] (v) Deletion of any amino acid between the first methionine and the sequence "LDCVT" (SEQ ID NO: 58).

[0079] According to this embodiment, Depend on Deletion of any amino acid between the first methionine and the sequence "GSFQY" compositionThe deletion means that all consecutive amino acids between the first methionine at the N-terminal end and the sequence "GSFQY" are deleted, while the first methionine and the sequence "GSFQY" (SEQ ID NO: 54) are not deleted.

[0080] According to this embodiment, Depend on Deletion of any amino acid between the first methionine and the sequence "EKSGG" composition The deletion means that all consecutive amino acids between the first methionine at the N-terminal end and the sequence "EKSGG" are deleted, while the first methionine and the sequence "EKSGG" (SEQ ID NO: 55) are not deleted.

[0081] According to this embodiment, Depend on Deletion of any amino acid between the first methionine and the sequence "ILRVG" composition The deletion means that all consecutive amino acids between the first methionine at the N-terminal end and the sequence "ILRVG" are deleted, while the first methionine and the sequence "ILRVG" (SEQ ID NO: 56) are not deleted.

[0082] According to this embodiment, Depend on Deletion of any amino acid between the first methionine and the sequence "GADNH" composition The deletion means that all consecutive amino acids between the first methionine at the N-terminal end and the sequence "GADNH" are deleted, while the first methionine and the sequence "GADNH" (SEQ ID NO: 57) are not deleted.

[0083] According to this embodiment, Depend on Deletion of any amino acid between the first methionine and the sequence "LDCVT" composition The deletion means that all consecutive amino acids between the first methionine at the N-terminal end and the sequence "LDCVT" are deleted, while the first methionine and the sequence "LDCVT" (SEQ ID NO: 58) are not deleted.

[0084] In a preferred embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0085] - wherein the reference functional full-length human GDE sequence has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6;

[0086] - and wherein the deletion consists of the deletion of any amino acid between the first methionine and the sequence "EKSGG".

[0087] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0088] - wherein the reference functional full-length human GDE sequence has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0089] - wherein the deletion consists of:

[0090] (i) a deletion of amino acids at positions 2 to 88, wherein the amino acids at positions 1 and 89 are not deleted;

[0091] (ii) a deletion of amino acids at positions 2 to 99, wherein the amino acids at positions 1 and 100 are not deleted;

[0092] (iii) a deletion of amino acids at positions 2 to 111, wherein the amino acids at positions 1 and 112 are not deleted;

[0093] (iv) a deletion of amino acids at positions 2 to 115, wherein the amino acids at positions 1 and 116 are not deleted; or

[0094] (v) a deletion of amino acids at positions 2 to 123, wherein the amino acids at positions 1 and 124 are not deleted.

[0095] In a preferred embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0096] - wherein the reference functional full-length human GDE sequence has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0097] - wherein the deletion consists of the deletion of amino acids at positions 2-99, and the amino acids at positions 1 and 100 are not deleted.

[0098] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises one and only one deletion in the N-terminal portion of the reference functional full-length human GDE sequence,

[0099] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0100] - wherein said N-terminal portion corresponds to a region consisting of the first 280 amino acid residues of said reference functional full-length human GDE sequence,

[0101] - and wherein said deletion in the N-terminal part of said reference functional full-length human GDE sequence consists of the deletion of:

[0102] (i) deletion of amino acids at positions 2-88 of the reference functional full-length human GDE sequence;

[0103] (ii) deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence;

[0104] (iii) deletion of amino acids at positions 2-111 of the reference functional full-length human GDE sequence;

[0105] (iv) deletion of amino acids at positions 2-115 of the reference functional full-length human GDE sequence;

[0106] (v) deletion of amino acids 2-123 of the reference functional full-length human GDE sequence.

[0107] In a preferred embodiment, the functional truncated GDE polypeptide of the present invention comprises one and only one deletion in the N-terminal portion of the reference functional full-length human GDE sequence,

[0108] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0109] - wherein said N-terminal portion corresponds to a region consisting of the first 280 amino acid residues of said reference functional full-length human GDE sequence,

[0110] - and wherein said deletion in the N-terminal part of said reference functional full-length human GDE sequence consists of deletion of amino acids at positions 2-99 of said reference functional full-length human GDE sequence.

[0111] "One and only one deletion" means that there is a deletion in the N-terminal portion corresponding to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence (i.e., the most N-terminal 280 amino acid residues of the reference full-length GDE sequence). For clarity, a functional truncated GDE comprising one and only one deletion of amino acids at positions 2-88 of the N-terminal portion of the reference functional full-length human GDE sequence corresponds to the following truncated polypeptide:

[0112] - wherein amino acids 2 to 88 of the reference functional full-length sequence are deleted, and

[0113] - wherein the amino acids at positions 1 and 89-280 are not deleted.

[0114] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises one and only one deletion relative to the reference functional full-length human GDE sequence,

[0115] - wherein the reference functional full-length human GDE sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 6,

[0116] - and wherein the deletion is referred to as ∆1b4, ∆1b5, ∆1b6, ∆1b7, ∆1b8, preferably ∆1b5 in Table 1:

[0117] For clarity, Table 1 should be understood as follows: When the reference full-length GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, the functional "∆1b4" truncated GDE polypeptide corresponds to a functional truncated GDE polypeptide derived from SEQ ID NO: 1 or SEQ ID NO: 4, which lacks all consecutive amino acids from positions 2-88 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0118] "∆1b4" truncated peptide

[0119] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MGSFQY (SEQ ID NO: 7).

[0120] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0121] - wherein the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and

[0122] - wherein the deletion consists of the deletion of amino acids in the N-terminal part of the reference functional full-length human GDE sequence such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are "MGSFQY" (SEQ ID NO: 7).

[0123] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0124] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0125] - wherein the deletion consists of a deletion of amino acids at positions 2-88 of the reference functional full-length GDE sequence, and

[0126] - wherein the amino acid at position 1 and the amino acid at position 89 of the reference functional full-length GDE sequence are not deleted.

[0127] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0128] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0129] - wherein the deletion consists of the deletion of amino acids at positions 2-88 of the reference functional full-length human GDE sequence,

[0130] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0131] - wherein amino acids 89 to 110, 89 to 130, 89 to 150, 89 to 170, 89 to 190, 89 to 210, 89 to 230, 89 to 250, 89 to 270 or 89 to 280 from the reference functional full-length GDE sequence are not deleted.

[0132] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0133] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0134] - wherein the deletion consists of the deletion of amino acids at positions 2-88 of the reference functional full-length human GDE sequence,

[0135] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0136] - wherein amino acids 89 to 280 from said reference functional full-length GDE sequence are not deleted.

[0137] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0138] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0139] - wherein the deletion consists of the deletion of amino acids at positions 2-88 of the reference functional full-length human GDE sequence,

[0140] - and wherein the functional truncated GDE polypeptide comprises the sequence "MGSFQY" (SEQ ID NO: 7).

[0141] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0142] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0143] - wherein the deletion consists of the deletion of amino acids at positions 2-88 of the reference functional full-length human GDE sequence,

[0144] - wherein the functional truncated GDE polypeptide comprises the sequence "MGSFQY" (SEQ ID NO: 7),

[0145] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0146] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0147] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0148] - wherein the deletion consists of the deletion of amino acids at positions 2-88 of the reference functional full-length human GDE sequence,

[0149] - and wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO:17.

[0150] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0151] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0152] - wherein the deletion consists of the deletion of amino acids at positions 2-88 of the reference functional full-length human GDE sequence,

[0153] - wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 17,

[0154] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0155] In a specific embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 12 or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 12, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 12, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.

[0156] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 12, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 12, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has substantially the same function or enzymatic activity as the GDE polypeptide of SEQ ID NO: 12, in particular the same enzymatic activity involved in glycogen degradation in muscle tissue, such as the heart or quadriceps. In a specific embodiment, the functional variant of SEQ ID NO: 12 has substantially the same ability to rescue glycogen accumulation and muscle strength in vivo as the GDE polypeptide of SEQ ID NO: 12. In a specific embodiment, the functional variant of SEQ ID NO: 12 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 12.

[0157] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 12, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 12, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has the same N-terminal portion as SEQ ID NO: 12. In a specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 12 is "MGSFQY" (SEQ ID NO: 7). In another specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 12 corresponds to the sequence of SEQ ID NO: 17.

[0158] In a specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 12 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, and 1-200 of SEQ ID NO: 12. In other words, according to this embodiment, the functional variant of SEQ ID NO: 12 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 12. In a specific embodiment, the N-terminal end of the functional variant of SEQ ID NO: 12 consists of amino acids 1-50, 1-100 or 1-150 of SEQ ID NO: 12. In other words, according to this embodiment, the functional variant of SEQ ID NO: 12 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-50, 1-100 or 1-150.

[0159] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO:12.

[0160] "∆1b5" truncated peptide

[0161] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are "MEKSGG" (SEQ ID NO: 8).

[0162] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0163] - wherein the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and

[0164] - wherein said deletion consists of the deletion of amino acids in the N-terminal portion of said reference functional full-length human GDE sequence such that the first six amino acids at the N-terminus of said functional truncated GDE polypeptide are "MEKSGG" (SEQ ID NO: 8).

[0165] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0166] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0167] - wherein the deletion consists of a deletion of amino acids at positions 2-99 of the reference functional full-length GDE sequence, and

[0168] - wherein the amino acid at position 1 and the amino acid at position 100 of the reference functional full-length GDE sequence are not deleted.

[0169] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0170] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0171] - wherein the deletion consists of the deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence,

[0172] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0173] - wherein amino acids at positions 100-110, 100 to 130, 100 to 150, 100 to 170, 100 to 190, 100 to 210, 100 to 230, 100 to 250, 100 to 270 or amino acids 100 to 280 from said reference functional full-length GDE sequence are not deleted.

[0174] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0175] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0176] - wherein the deletion consists of the deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence,

[0177] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0178] - wherein amino acids from positions 100 to 280 from said reference functional full-length GDE sequence are not deleted.

[0179] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0180] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0181] - wherein the deletion consists of the deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence,

[0182] - and wherein the functional truncated GDE polypeptide comprises the sequence "MEKSGG" (SEQ ID NO: 8).

[0183] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0184] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0185] - wherein the deletion consists of the deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence,

[0186] - wherein the functional truncated GDE polypeptide comprises the sequence "MEKSGG" (SEQ ID NO: 8),

[0187] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0188] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0189] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0190] - wherein the deletion consists of the deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence,

[0191] - and wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO:18.

[0192] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0193] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0194] - wherein the deletion consists of the deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence,

[0195] - wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 18,

[0196] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0197] In a specific embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 13 or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 13, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 13, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.

[0198] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 13, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 13, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has substantially the same function or enzymatic activity as the GDE polypeptide of SEQ ID NO: 13, in particular the same enzymatic activity involved in glycogen degradation in muscle tissue, such as the heart or quadriceps. In a specific embodiment, the functional variant of SEQ ID NO: 13 has substantially the same ability to rescue glycogen accumulation and muscle strength in vivo as the GDE polypeptide of SEQ ID NO: 13. In a specific embodiment, the functional variant of SEQ ID NO: 13 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 13.

[0199] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 13, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 13, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has the same N-terminal portion as SEQ ID NO: 13. In a specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 13 is "MEKSGG" (SEQ ID NO: 8). In another specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 13 corresponds to the sequence of SEQ ID NO: 18.

[0200] In a specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 13 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, and 1-200 of SEQ ID NO: 13. In other words, according to this embodiment, the functional variant of SEQ ID NO: 13 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 13. In a specific embodiment, the N-terminal end of the functional variant of SEQ ID NO: 13 consists of amino acids 1-50, 1-100 or 1-150 of SEQ ID NO: 13. In other words, according to this embodiment, the functional variant of SEQ ID NO: 13 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-50, 1-100 or 1-150.

[0201] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO:13.

[0202] "∆1b6" truncated peptide

[0203] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are "MILRVG" (SEQ ID NO: 9).

[0204] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0205] - wherein the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and

[0206] - wherein the deletion consists of the deletion of amino acids in the N-terminal part of the reference functional full-length human GDE sequence such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are "MILRVG" (SEQ ID NO: 9).

[0207] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0208] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0209] - wherein the deletion consists of the deletion of amino acids at positions 2-111 of the reference functional full-length GDE sequence, and

[0210] - wherein the amino acid at position 1 and the amino acid at position 112 of the reference functional full-length GDE sequence are not deleted.

[0211] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0212] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0213] - wherein the deletion consists of the deletion of amino acids at positions 2-111 of the reference functional full-length human GDE sequence,

[0214] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0215] - wherein amino acids 112 to 130, 112 to 150, 112 to 170, 112 to 190, 112 to 210, 112 to 230, 112 to 250, 112 to 270 or amino acids 112 to 280 from the reference functional full-length GDE sequence are not deleted.

[0216] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0217] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0218] - wherein the deletion consists of the deletion of amino acids at positions 2-111 of the reference functional full-length human GDE sequence,

[0219] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0220] - wherein amino acids 112 to 280 from said reference functional full-length GDE sequence are not deleted.

[0221] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0222] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0223] - wherein the deletion consists of the deletion of amino acids at positions 2-111 of the reference functional full-length human GDE sequence,

[0224] - and wherein the functional truncated GDE polypeptide comprises the sequence "MILRVG" (SEQ ID NO: 9).

[0225] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0226] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0227] - wherein the deletion consists of the deletion of amino acids at positions 2-111 of the reference functional full-length human GDE sequence,

[0228] - wherein the functional truncated GDE polypeptide comprises the sequence "MILRVG" (SEQ ID NO: 9),

[0229] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0230] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0231] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0232] - wherein the deletion consists of the deletion of amino acids at positions 2-111 of the reference functional full-length human GDE sequence,

[0233] - and wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO:19.

[0234] In a specific embodiment, a functional truncated GDE polypeptide of the invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0235] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0236] - wherein the deletion consists of the deletion of amino acids at positions 2-111 of the reference functional full-length human GDE sequence,

[0237] - wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 19,

[0238] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0239] In a specific embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 14 or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 14, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 14, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.

[0240] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 14, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 14, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has substantially the same function or enzymatic activity as the GDE polypeptide of SEQ ID NO: 14, in particular the same enzymatic activity involved in glycogen degradation in muscle tissue, such as the heart or quadriceps. In a specific embodiment, the functional variant of SEQ ID NO: 14 has substantially the same ability to rescue glycogen accumulation and muscle strength in vivo as the GDE polypeptide of SEQ ID NO: 14. In a specific embodiment, the functional variant of SEQ ID NO: 14 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 14.

[0241] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 14, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 14, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has the same N-terminal portion as SEQ ID NO: 14. In a specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 14 is "MILRVG" (SEQ ID NO: 9). In another specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 14 corresponds to the sequence of SEQ ID NO: 19.

[0242] In another embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 14 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, and 1-200 of SEQ ID NO: 14. In other words, according to this embodiment, the functional variant of SEQ ID NO: 14 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 14. In a specific embodiment, the N-terminal end of the functional variant of SEQ ID NO: 14 consists of amino acids 1-50, 1-100 or 1-150 of SEQ ID NO: 14. In other words, according to this embodiment, the functional variant of SEQ ID NO: 14 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-50, 1-100 or 1-150.

[0243] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO:14.

[0244] "∆1b7" truncated peptide

[0245] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are "MGADNH" (SEQ ID NO: 10).

[0246] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0247] - wherein the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and

[0248] - wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are "MGADNH" (SEQ ID NO: 10).

[0249] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0250] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0251] - wherein the deletion consists of a deletion of amino acids at positions 2-115 of the reference functional full-length GDE sequence, and

[0252] - wherein the amino acid at position 1 and the amino acid at position 116 of the reference functional full-length GDE sequence are not deleted.

[0253] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0254] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0255] - wherein the deletion consists of the deletion of amino acids at positions 2-115 of the reference functional full-length human GDE sequence,

[0256] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0257] - wherein amino acids 116 to 130, 116 to 150, 116 to 170, 116 to 190, 116 to 210, 116 to 230, 116 to 250, 116 to 270 or amino acids 116 to 280 from said reference functional full-length GDE sequence are not deleted.

[0258] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0259] - wherein the functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0260] - wherein the deletion consists of the deletion of amino acids at positions 2-115 of the reference functional full-length human GDE sequence,

[0261] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0262] - wherein amino acids 116 to 280 from said reference functional full-length GDE sequence are not deleted.

[0263] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0264] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0265] - wherein the deletion consists of the deletion of amino acids at positions 2-115 of the reference functional full-length human GDE sequence,

[0266] - and wherein the functional truncated GDE polypeptide comprises the sequence "MGADNH" (SEQ ID NO: 10).

[0267] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0268] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0269] - wherein the deletion consists of the deletion of amino acids at positions 2-115 of the reference functional full-length human GDE sequence,

[0270] - wherein the functional truncated GDE polypeptide comprises the sequence "MGADNH" (SEQ ID NO: 10),

[0271] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0272] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0273] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0274] - wherein the deletion consists of the deletion of amino acids at positions 2-115 of the reference functional full-length human GDE sequence,

[0275] - and wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO:20.

[0276] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0277] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0278] - wherein the deletion consists of the deletion of amino acids at positions 2-115 of the reference functional full-length human GDE sequence,

[0279] - wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 20,

[0280] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0281] In a specific embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 15 or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 15, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 15, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.

[0282] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 15, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 15, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has substantially the same function or enzymatic activity as the GDE polypeptide of SEQ ID NO: 15, in particular the same enzymatic activity involved in glycogen degradation in muscle tissue, such as the heart or quadriceps. In a specific embodiment, the functional variant of SEQ ID NO: 15 has substantially the same ability to rescue glycogen accumulation and muscle strength in vivo as the GDE polypeptide of SEQ ID NO: 15. In a specific embodiment, the functional variant of SEQ ID NO: 15 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 15.

[0283] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 15, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 15, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has the same N-terminal portion as SEQ ID NO: 15. In a specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 15 is "MGADNH" (SEQ ID NO: 10). In another specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 15 corresponds to the sequence of SEQ ID NO: 20.

[0284] In another embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 15 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, and 1-200 of SEQ ID NO: 15. In other words, according to this embodiment, the functional variant of SEQ ID NO: 15 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 15. In a specific embodiment, the N-terminal end of the functional variant of SEQ ID NO: 15 consists of amino acids 1-50, 1-100 or 1-150 of SEQ ID NO: 15. In other words, according to this embodiment, the functional variant of SEQ ID NO: 15 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-50, 1-100 or 1-150.

[0285] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO:15.

[0286] "∆1b8" truncated peptide

[0287] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are "MLDCVT" (SEQ ID NO: 11).

[0288] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0289] - wherein the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and

[0290] - wherein the deletion consists of the deletion of amino acids in the N-terminal part of the reference functional full-length human GDE sequence such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are "MLDCVT" (SEQ ID NO: 11).

[0291] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0292] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0293] - wherein the deletion consists of a deletion of amino acids at positions 2-123 of the reference functional full-length GDE sequence, and

[0294] - wherein the amino acid at position 1 and the amino acid at position 124 of the reference functional full-length GDE sequence are not deleted.

[0295] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0296] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0297] - wherein the deletion consists of the deletion of amino acids at positions 2-123 of the reference functional full-length human GDE sequence,

[0298] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0299] - wherein amino acids 124 to 130, 124 to 150, 124 to 170, 124 to 190, 124 to 210, 124 to 230, 124 to 250, 124 to 270 or amino acids 124 to 280 from said reference functional full-length GDE sequence are not deleted.

[0300] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence,

[0301] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0302] - wherein the deletion consists of the deletion of amino acids at positions 2-123 of the reference functional full-length human GDE sequence,

[0303] - wherein the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, and

[0304] - wherein amino acids 124 to 280 from said reference functional full-length GDE sequence are not deleted.

[0305] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0306] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0307] - wherein the deletion consists of the deletion of amino acids at positions 2-123 of the reference functional full-length human GDE sequence,

[0308] - and wherein the functional truncated GDE polypeptide comprises the sequence "MLDCVT" (SEQ ID NO: 11).

[0309] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0310] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0311] - wherein the deletion consists of the deletion of amino acids at positions 2-123 of the reference functional full-length human GDE sequence,

[0312] - wherein the functional truncated GDE polypeptide comprises the sequence "MLDCVT" (SEQ ID NO: 11),

[0313] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0314] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0315] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0316] - wherein the deletion consists of the deletion of amino acids at positions 2-123 of the reference functional full-length human GDE sequence,

[0317] - and wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO:21.

[0318] In a specific embodiment, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence,

[0319] - wherein the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length,

[0320] - wherein the deletion consists of the deletion of amino acids at positions 2-123 of the reference functional full-length human GDE sequence,

[0321] - wherein the functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 21,

[0322] - and wherein the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0323] In a specific embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 16 or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 16, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 16, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.

[0324] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 16, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 16, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has substantially the same function or enzymatic activity as the GDE polypeptide of SEQ ID NO: 16, in particular the same enzymatic activity involved in glycogen degradation in muscle tissue, such as the heart or quadriceps. In a specific embodiment, the functional variant of SEQ ID NO: 16 has substantially the same ability to rescue glycogen accumulation and muscle strength in vivo as the GDE polypeptide of SEQ ID NO: 16. In a specific embodiment, the functional variant of SEQ ID NO: 16 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 16.

[0325] In a specific embodiment, the functional variant having at least 70% sequence identity to SEQ ID NO: 16, such as at least 75% or at least 80% sequence identity to SEQ ID NO: 16, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity, has the same N-terminal portion as SEQ ID NO: 16. In a specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 16 is "MLDCVT" (SEQ ID NO: 11). In another specific embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 16 corresponds to the sequence of SEQ ID NO: 21.

[0326] In another embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 16 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, and 1-200 of SEQ ID NO: 16. In other words, according to this embodiment, the functional variant of SEQ ID NO: 16 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 16. In a specific embodiment, the N-terminal end of the functional variant of SEQ ID NO: 16 consists of amino acids 1-50, 1-100 or 1-150 of SEQ ID NO: 16. In other words, according to this embodiment, the functional variant of SEQ ID NO: 16 does not comprise any mutation, insertion or deletion in the region corresponding to amino acids 1-50, 1-100 or 1-150.

[0327] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO:16.

[0328] Combination with other missing

[0329] In a specific embodiment, the functional truncated GDE polypeptide of the invention comprising a deletion in the N-terminal portion of a reference functional full-length human GDE sequence as described above may further comprise a deletion or a combination of deletions in other portions of the reference functional full-length human GDE sequence.

[0330] In a specific embodiment, the functional truncated GDE polypeptide comprises:

[0331] - a deletion of one and only one consecutive amino acid in the N-terminal part of the reference functional full-length human GDE sequence, wherein the N-terminal part corresponds to the first 280 amino acid residues of the reference functional full-length human GDE sequence,

[0332] - another deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence.

[0333] In a specific embodiment, the functional truncated GDE polypeptide comprises:

[0334] - a deletion of one and only one consecutive amino acid in the N-terminal portion of the reference functional full-length human GDE sequence, wherein the N-terminal portion corresponds to the first 280 amino acid residues of the reference functional full-length human GDE sequence, and

[0335] - another deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence,

[0336] wherein the reference functional full-length human GDE sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 6,

[0337] And the deletions described therein are referred to as ∆1b4, ∆1b5, ∆1b6, ∆1b7, ∆1b8 in Table 1.

[0338] In specific embodiments, a functional truncated GDE polypeptide of the invention comprises at least the amino acid residues at positions 429-666, 866-892, 1088-1194, 1235-1420 relative to SEQ ID NO:1 or SEQ ID NO:4.

[0339] In a specific embodiment, a functional truncated GDE polypeptide of the present invention comprising a deletion in the N-terminal portion of a reference functional full-length human GDE sequence may further comprise a deletion or a combination of deletions in the C-terminal portion of the reference full-length GDE sequence and / or in the central domain of the reference full-length GDE sequence.

[0340] In a specific embodiment, the C-terminal portion of the reference full-length GDE sequence corresponds to the region consisting of the last 112 amino acids of the reference full-length human GDE sequence, ie, the most C-terminal 112 amino acids.

[0341] In a specific embodiment, the central domain of the reference full-length GDE sequence of SEQ ID NO: 1 or SEQ ID NO: 4 corresponds to the region consisting of amino acids at positions 710 to 865 of SEQ ID NO: 1 or SEQ ID NO: 4.

[0342] In a specific embodiment, the functional truncated GDE polypeptide comprises:

[0343] - one and only one deletion in the N-terminal portion of the reference functional full-length human GDE sequence, wherein the N-terminal portion corresponds to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence, and

[0344] - another deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence,

[0345] wherein the reference functional full-length human GDE sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 6,

[0346] and wherein the deletions in the N-terminal portion are referred to as ∆1b4, ∆1b5, ∆1b6, ∆1b7, ∆1b8 in Table 1 ;

[0347] And wherein the additional deletion or combination of deletions in the other part of the reference functional full-length human GDE sequence is selected from any of the deletions referred to as ∆1, ∆2, ∆3, ∆4, ∆5, ∆6 and ∆7 in Table 2:

[0348] 2- Nucleic acid molecules encoding the N-terminally truncated GDE polypeptide

[0349] In another aspect, the present invention relates to a nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above.

[0350] The term "nucleic acid molecule" (or nucleic acid sequence) refers to a DNA or RNA molecule in single-stranded or double-stranded form, in particular a DNA encoding a functional truncated GDE polypeptide according to the present invention.

[0351] In a preferred embodiment, the nucleic acid molecule encoding the functional truncated GDE polypeptide is small enough to be packaged in a gene therapy vector such as an AAV vector in combination with a suitable regulatory sequence. The size of the nucleic acid molecule encoding the functional truncated GDE polypeptide is preferably less than about 4.5 kb, preferably less than about 4.4 kb.

[0352] "Gene therapy vector" means any vector suitable for gene therapy. In particular, the gene therapy vector can be a plasmid or a recombinant virus, such as a viral vector derived from a retrovirus or a lentivirus. Preferably, the viral vector is an AAV vector, such as an AAV vector suitable for transducing liver tissue or muscle cells. Extensive experience in clinical trials and preclinical models of muscle diseases has shown that adeno-associated virus (AAV) is the preferred vector for in vivo gene therapy of GSDIII. These vectors efficiently transduce the liver and muscle, their production is scalable, and they have relatively low immunogenic properties compared to other gene therapy vectors. However, one of the biggest limitations of using AAV for gene replacement is their limited encapsidation size limit (about 5kb). In fact, during the production of recombinant AAV, the efficiency of encapsidation of genomes larger than 5 kb is low, and the resulting AAV may contain fragmented genomes, reducing the efficiency of gene transfer.

[0353] The sequence of the nucleic acid molecule encoding the functional truncated GDE polypeptide of the present invention can be optimized for expressing the GDE polypeptide in vivo. Sequence optimization can include a large number of changes in the nucleic acid sequence, including codon optimization, increasing GC content, reducing the number of CpG islands, reducing the number of optional open reading frames (ARFs) and / or reducing the number of splice donors and splice acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules may encode the same protein. In addition, it is well known that the genetic codes of different organisms often tend to use one of several codons encoding the same amino acid over other codons. Changes are introduced in the nucleotide sequence by codon optimization, which utilizes the codon bias present in a given cell background, so that the codon-optimized nucleotide sequence obtained is more likely to be expressed at a relatively higher level than the non-codon-optimized sequence in such a given cell background. In a preferred embodiment of the present invention, such an optimized nucleotide sequence encoding a functional truncated GDE polypeptide is codon-optimized to improve its expression in human cells compared to a non-codon-optimized nucleotide sequence encoding the same functional truncated GDE polypeptide, for example, by utilizing human-specific codon usage bias. The nucleic acid sequence encoding the full-length human GDE isoform 1 is shown in SEQ ID NO: 32. An example of the corresponding codon-optimized sequence is shown in SEQ ID NO: 33.

[0354] In a particular embodiment, the nucleic acid molecule of the invention encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 12. In a particular embodiment, the nucleic acid molecule of the invention comprises or consists of the sequence shown in SEQ ID NO: 22 or SEQ ID NO: 23, which encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 12. In a particular embodiment, the nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90 or at least 95% identity with the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 23, preferably SEQ ID NO: 22. In a particular embodiment, the nucleic acid molecule of the invention has at least 95% identity, such as at least 96, 97, 98, 99 or 100% identity with the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 23, preferably SEQ ID NO: 22.

[0355] In a particular embodiment, the nucleic acid molecule of the invention encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 13. In a particular embodiment, the nucleic acid molecule of the invention comprises or consists of the sequence shown in SEQ ID NO: 24 or SEQ ID NO: 25, which encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 13. In a particular embodiment, the nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90 or at least 95% identity with the nucleotide sequence of SEQ ID NO: 24 or SEQ ID NO: 25, preferably SEQ ID NO: 24. In a particular embodiment, the nucleic acid molecule of the invention has at least 95% identity, such as at least 96, 97, 98, 99 or 100% identity with the nucleotide sequence of SEQ ID NO: 24 or SEQ ID NO: 25, preferably SEQ ID NO: 24.

[0356] In a specific embodiment, the nucleic acid molecule of the invention encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 14. In a specific embodiment, the nucleic acid molecule of the invention comprises or consists of the sequence shown in SEQ ID NO: 26 or SEQ ID NO: 27, which encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 14. In a specific embodiment, the nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90 or at least 95% identity with the nucleotide sequence of SEQ ID NO: 26 or SEQ ID NO: 27, preferably SEQ ID NO: 26. In a specific embodiment, the nucleic acid molecule of the invention has at least 95% identity, such as at least 96, 97, 98, 99 or 100% identity with the nucleotide sequence of SEQ ID NO: 26 or SEQ ID NO: 27, preferably SEQ ID NO: 26.

[0357] In a particular embodiment, the nucleic acid molecule of the invention encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the nucleic acid molecule of the invention comprises or consists of the sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29, which encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 15. In a particular embodiment, the nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90 or at least 95% identity with the nucleotide sequence of SEQ ID NO: 28 or SEQ ID NO: 29, preferably SEQ ID NO: 28. In a particular embodiment, the nucleic acid molecule of the invention has at least 95% identity, such as at least 96, 97, 98, 99 or 100% identity with the nucleotide sequence of SEQ ID NO: 28 or SEQ ID NO: 29, preferably SEQ ID NO: 28.

[0358] In a specific embodiment, the nucleic acid molecule of the invention encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 16. In a specific embodiment, the nucleic acid molecule of the invention comprises or consists of the sequence shown in SEQ ID NO: 30 or SEQ ID NO: 31, which encodes a functional truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 16. In a specific embodiment, the nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90 or at least 95% identity with the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31, preferably SEQ ID NO: 30. In a specific embodiment, the nucleic acid molecule of the invention has at least 95% identity, such as at least 96, 97, 98, 99 or 100% identity with the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31, preferably SEQ ID NO: 30.

[0359] The nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above may have at least 80, at least 85, at least 90 or at least 95% identity with any of the nucleotide sequences in SEQ ID NOs: 22 to 31. In a specific embodiment, the nucleic acid molecule of the invention has at least 95% identity with any of the nucleotide sequences in SEQ ID NOs: 22 to 31, such as at least 96, 97, 98, 99 or 100% identity.

[0360] Preferably, the nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above may have at least 80, at least 85, at least 90 or at least 95% identity with any of the nucleotide sequences of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30. In a specific embodiment, the nucleic acid molecule of the invention has at least 95% identity, such as at least 96, 97, 98, 99 or 100% identity with any of the nucleotide sequences of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30.

[0361] 3- Nucleic acid construct

[0362] The present invention further relates to a nucleic acid construct comprising a nucleic acid molecule of the present invention as described above. The nucleic acid construct may correspond to an expression cassette comprising a nucleic acid sequence of the present invention that is operably connected to one or more expression control sequences and / or other sequences that improve expression. The term "operably connected" as used herein refers to a polynucleotide element that is connected in a functional relationship. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, it is "operably connected". For example, if a promoter or another transcriptional regulatory sequence affects the transcription of a coding sequence, it is operably connected to the coding sequence. Such expression control sequences are known in the art, such as promoters, enhancers (such as cis-regulatory modules (CRMs)), introns, polyA signals, etc.

[0363] In a specific embodiment, the expression cassette may comprise a promoter. The promoter may be a ubiquitous or tissue-specific promoter, particularly a promoter that promotes expression in cells or tissues where GDE expression is desired, such as cells or tissues where GDE expression is desired in GDE-deficient patients.

[0364] In a first specific embodiment, the promoter is a muscle-specific promoter. Non-limiting examples of muscle-specific promoters include muscle creatine kinase (MCK) promoter. Non-limiting examples of suitable muscle creatine kinase promoters are human muscle creatine kinase promoter and truncated mouse muscle creatine kinase (tMCK) promoter (Wang B et al., Construction and analysis of compact muscle-selective promoters for AAV vectors, Gene Ther. 2008 Nov; 15 (22): 1489-99) (representative GenBank accession number AF188002). Human muscle creatine kinase has gene ID number 1158 (representative GenBank accession number NC_000019.9, registered on December 26, 2012). Other examples of muscle-specific promoters include the synthetic promoter C5.12 (spC5.12, also referred to herein as "C5.12"), such as spC5.12 or spC5.12 promoter (disclosed in Wang et al., Gene Therapy, Vol. 15, pp. 1489-1499 (2008)), the MHCK7 promoter (Salva et al., Mol Ther. 2007 Feb; 15(2):320-9), myosin light chain (MLC) promoters such as MLC2 (Gene ID No. 4633; representative GenBank accession No. NG_007554.1, accessed December 26, 2012), myosin heavy chain (MHC) promoters such as α-MHC (Gene ID No. 4624; representative GenBank accession No. NG_023444.1, accessed December 26, 2012), the desmin promoter (Gene ID No. 1674; representative GenBank accession No. NG_023444.1, accessed December 26, 2012). Representative GenBank accession number NG_008043.1, accessed on December 26, 2012), cardiac troponin C promoter (Gene ID No. 7134; Representative GenBank accession number NG_008963.1, accessed on December 26, 2012), troponin I promoter (Gene ID Nos. 7135, 7136, and 7137; Representative GenBank accession numbers NG_016649.1, NG_011621.1, and NG_007866.2, accessioned on December 26, 2012), myoD gene family promoters (Weintraub et al., Science, 251, 761 (1991); gene ID number 4654; representative GenBank accession number NM_002478, accessioned on December 26, 2012), α-actin promoters (gene ID numbers 58, 59, and 70; representative GenBank accession numbers NG_006672.1, NG_011541.1, and NG_007553.1, accessioned on December 26, 2012), β-actin promoters (gene ID number 60; representative GenBank accession number NG_007992.1, accessioned on December 26, 2012), 1433.1 and NM_001199893, accessioned on December 26, 2012), the gamma-actin promoter (Gene ID Nos. 71 and 72; representative GenBank accession Nos. NG_011433.1 and NM_001199893, accessioned on December 26, 2012), the muscle-specific promoter located in intron 1 of the ocular Pitx3 (Gene ID No. 5309) (Coulon et al., the muscle-selective promoter corresponds to residues 11219-11527 of representative GenBank accession No. NG_008147, accessioned on December 26, 2012), as well as the promoter described in U.S. Patent Publication No. US 2003 / 0157064 and the CK6 promoter (Wang et al., 2008 doi: 10.1038 / gt.2008.104). In another specific embodiment, the muscle-specific promoter is the E-Syn promoter described in Wang et al., Gene Therapy, Vol. 15, pp. 1489-1499 (2008), which comprises a combination of an MCK-derived enhancer and a spC5.12 promoter. In a specific embodiment of the present invention, the muscle-specific promoter is selected from the group consisting of a spC5.12 promoter, a MHCK7 promoter, an E-syn promoter, a muscle creatine kinase promoter, a myosin light chain (MLC) promoter, a myosin heavy chain (MHC) promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an α-actin promoter, a β-actin promoter, a γ-actin promoter, a muscle-specific promoter located in intron 1 of eye-type Pitx3, a CK6 promoter, a CK8 promoter, and an Acta1 promoter. In another embodiment, the muscle-specific promoter is selected from the group consisting of a spC5.12 promoter, a desmin promoter, and a MCK promoter. In another embodiment, the muscle-specific promoter is selected from the group consisting of spC5.12 and MCK promoters. In a preferred embodiment, the muscle-specific promoter is the spC5.12 promoter.

[0365] In another specific embodiment, the promoter is a liver-specific promoter. Non-limiting examples of liver-specific promoters include HSE promoter (liver-specific promoter), alpha-1 antitrypsin promoter (hAAT), transthyretin promoter, albumin promoter, thyroxine-binding globulin (TBG) promoter, LSP promoter (comprising thyroid hormone-binding globulin promoter sequence, two copies of alpha 1-microglobulin / bikunin enhancer sequence and leader sequence - 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.), etc. Other useful liver-specific promoters are 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 liver-specific promoter in the context of the present invention is a promoter comprising an H1 enhancer and a TTR promoter. An example of such a liver-specific promoter is the HSE promoter. The HSE promoter contains the mouse TTR promoter and the H1 enhancer described in SEQ NO: 51 and is derived from the original 1986 paper by Robert H. Costa et al. (Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific, Mol Cell Biol. 1986; 6(12): 4697-4708). In a preferred embodiment, the liver-specific promoter is a HSE promoter having the sequence of SEQ ID NO: 53, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 53, such as at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 53.

[0366] In another specific embodiment, the promoter is a neuron-specific promoter. Non-limiting examples of neuron-specific promoters include, but are not limited to, the following promoters: synapsin-1 (Syn) promoter, neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)) and neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373- 84 (1995)), and other neuron-specific promoters apparent to professionals. In a specific embodiment, the neuron-specific promoter is the Syn promoter. Other neuron-specific promoters include, but are not limited to, the synapsin-2 promoter, the tyrosine hydroxylase promoter, the dopamine β-hydroxylase promoter, the hypoxanthine phosphoribosyltransferase promoter, the low affinity NGF receptor promoter, and the choline acetyltransferase promoter (Bejanin et al., 1992; Carroll et al., 1995; Chin and Greengard, 1994; Foss-Petter et al., 1990; Harrington et al., 1987; Mercer et al., 1991; Patei et al., 1986). Representative motor neuron-specific promoters include, but are not limited to, the promoter for calcitonin gene-related peptide (CGRP), a known motor neuron-derived factor. Other promoters that function in motor neurons include the promoters for choline acetyltransferase (ChAT), neuron-specific enolase (NSE), synapsin, and Hb9. Other neuron-specific promoters useful in the present invention include, but are not limited to, GFAP (for astrocytes), calbindin 2 (for interneurons), Mnx1 (motor neurons), nestin (neurons), parvalbumin, somatostatin, and Plp1 (oligodendrocytes and Schwann cells).

[0367] In a preferred embodiment, the promoter is a ubiquitous promoter. Representative ubiquitous promoters include cytomegalovirus enhancer / chicken beta actin (CAG) promoter, cytomegalovirus enhancer / promoter (CMV) (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)] or a short version of the CMV promoter, PGK promoter, SV40 early promoter, retrovirus Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerol kinase (PGK) promoter, EF1 α promoter or a short version of the EF1a promoter and Ins84 promoter (as described in WO2020 / 219949). In a preferred embodiment, the promoter is a truncated version of the CMV promoter.

[0368] Furthermore, the promoter may also be an endogenous promoter such as an albumin promoter or a GDE promoter.

[0369] Short promoters are of particular interest in the present invention, such as shorter versions of known promoters. In a preferred embodiment, the promoter is less than about 500 pb, preferably less than about 450 pb, preferably less than about 400 pb.

[0370] In a preferred embodiment, the promoter is a shorter version of any promoter described herein, such as a shorter version of the CMV promoter or a shorter version of the EF1a promoter or a promoter comprising the H1 enhancer and the TTR promoter.

[0371] In a preferred embodiment, the promoter is a short version of the CMV promoter. More preferably, the promoter is a short version of the CMV promoter having the sequence SEQ ID NO: 43, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 43, such as at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 43.

[0372] In another specific embodiment, the promoter is a short version of the EF1a promoter. Preferably, the promoter is a short version of the EF1a promoter having the sequence shown in SEQ ID NO: 52, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 52, such as at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 52.

[0373] In another specific embodiment, the promoter is the Ins84 promoter as described in WO2020 / 219949.

[0374] The expression cassette comprising the nucleic acid molecule of the present invention can be adjusted according to the target population of GSDIII patients, the clinical manifestations of the GSDIII disease and / or the target tissue. For example, for patients with clinical manifestations of the GSDIII disease mainly in the muscle (e.g. adolescent or adult GSDIII patients), the expression cassette preferably comprises a muscle-specific promoter or any promoter capable of inducing strong expression of the nucleic acid molecule of the present invention in the muscle, such as the above-mentioned miniCMV promoter, in particular the miniCMV promoter of SEQ ID NO: 43. For patients with clinical manifestations of the GSDIII disease in the liver, the expression cassette preferably comprises a liver-specific promoter or any promoter capable of inducing strong expression of the nucleic acid molecule of the present invention in the liver, such as the above-mentioned HSE promoter, in particular the HSE promoter of SEQ ID NO: 53. For patients with clinical manifestations of the GSDIII disease in the muscle and liver, a promoter capable of inducing expression of the nucleic acid molecule of the present invention in both tissues is preferably used.

[0375] In a particular embodiment, the promoter is associated with an enhancer sequence such as a cis-regulatory module (CRM) or an artificial enhancer sequence. CRMs useful in the practice 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. Other regulatory elements capable of particularly enhancing the muscle-specific expression of a gene, particularly in cardiac muscle and / or skeletal muscle, are those described in WO2015110449. Specific examples of nucleic acid regulatory elements comprising artificial sequences include regulatory elements obtained by rearranging the transcription factor binding sites (TFBS) present in the sequences disclosed in WO2015110449. The rearrangement may encompass changing the order of the TFBS and / or changing the position of one or more TFBS relative to other TFBS and / or changing the number of copies of one or more TFBS. For example, nucleic acid regulatory elements for enhancing muscle-specific gene expression, particularly cardiac and skeletal muscle-specific gene expression, may comprise binding sites for E2A, HNH1, NF1, C / EBP, LRF, MyoD and SREBP, or binding sites for E2A, NF1, p53, C / EBP, LRF and SREBP, or binding sites for E2A, HNH1, NF1, C / EBP, LRF and SREBP. 1. Binding sites for HNF3a, HNF3b, NF1, C / EBP, LRF, MyoD and SREBP, or binding sites for E2A, HNF3a, NF1, C / EBP, LRF, MyoD and SREBP, or binding sites for E2A, HNF3a, NF1, CEBP, LRF, MyoD and SREBP, or binding sites for HNF4, NF1, RSRFC4, C / EBP, LRF and MyoD, or binding sites for NF1, PPAR, p53, C / EBP, LRF and MyoD.For example, nucleic acid regulatory elements for enhancing muscle-specific gene expression, particularly skeletal muscle-specific gene expression, may also comprise binding sites for E2A, NF1, SRFC, p53, C / EBP, LRF and MyoD, or binding sites for E2A, NF1, C / EBP, LRF, MyoD and SREBP, or binding sites for E2A, HNF3a, C / EBP, LRF, MyoD, SEREBP and Tal1-b, or binding sites for E2A, SRF, p53, C / EBP, LRF, MyoD and SREBP, or binding sites for HNF4, NF1, RSRFC4, C / EBP, LRF and SREBP, or binding sites for E2A, HNF3a, HNF3b, NF1, SRF, C / EBP, LRF, MyoD and SREBP, or binding sites for E2A, CEBP and MyoD. In other examples, the nucleic acid regulatory elements comprise at least 2, such as 2, 3, 4 or more copies of one or more of the above-described TFBS. Other regulatory elements capable of enhancing in particular the liver-specific expression of a gene are those disclosed in WO2009130208.

[0376] In a specific embodiment, the enhancer is a short-sized enhancer. Specifically, the enhancer used in the present invention can be composed of 10 to 175 nucleotides, such as 40 to 100 nucleotides, and particularly 50 to 80 nucleotides. In a specific embodiment, the enhancer is a 72-nucleotide HS-CRM8 enhancer consisting of SEQ ID NO: 51, or a functional variant of SEQ ID NO: 51 having enhancer activity. In another embodiment, the enhancer is a functional variant of the 72-nucleotide HS-CRM8 enhancer, which has at least 80% identity with SEQ ID NO: 51, such as at least 85% identity with SEQ ID NO: 51, particularly at least 90% identity, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% identity.

[0377] In another specific embodiment, the nucleic acid construct comprises an intron, particularly an intron placed between the promoter and the GDE coding sequence. Introns can be introduced to improve mRNA stability and protein production. In another embodiment, the intron is a human β-globin b2 (or HBB2) intron, a coagulation factor IX (FIX) intron, an SV40 intron, hCMV intron A (hCMVI), a TPL intron (TPLI), a CHEF1 gene intron 1 (CHEFI), an MVM intron (Wu et al., 2008), a FIX truncated intron 1 (Wu et al., 2008, Mol Ther, 16(2):280–289; Kurachi et al., 1995, J Biol Chem., 270(10):5276–5281), a β-globin / immunoglobulin heavy chain hybrid intron (the 5´-donor site is from a human β-globin intron and the 3´-acceptor site is from an immunoglobulin heavy chain variable region intron, Wu et al., 2008, Mol Ther, 16(2):280–289; Kurachi et al., 1995, J Biol Chem., 270(10):5276–5281), a hybrid intron consisting of an adenovirus splice donor and an immunoglobulin G splice (Wong et al., 1985, Chromosoma, 92(2):124–135; Yew et al., 1997, Hum Gene Ther, 8(5):575–584; Choi T. et al., 1991, Mol Cell Biol, 11(6):3070–3074; Huang et al., 1990, Mol Cell Biol.,10(4):1805–1810), a hybrid 19S / 16S SV40 intron (the 5´-donor site is from the 19S intron and the 3´-acceptor site is from the 16S intron, Yew et al., 1997, Hum Gene Ther, 8(5):575–584), Ther, 8(5):575–584) or chicken β-globin intron. In another embodiment, the intron is a modified intron (particularly a modified HBB2 or FIX intron) designed to reduce the number of alternative open reading frames (ARFs) present in the intron or even completely remove them. Preferably, ARFs with a length span of more than 50 bp and having a stop codon in frame with the start codon are removed. ARFs can be removed by modifying the sequence of the intron. For example, the modification can be performed by nucleotide substitution, insertion or deletion, preferably by nucleotide substitution. As an example, one or more nucleotides, particularly one nucleotide, in the ATG or GTG start codon present in the sequence of the intron of interest can be replaced to produce a non-start codon.For example, ATG or GTG within the sequence of an intron of interest can be replaced with CTG, which is not a start codon.

[0378] The classical HBB2 intron is shown in SEQ ID NO: 34. For example, the HBB2 intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In a specific embodiment, the modified HBB2 intron has the sequence shown in SEQ ID NO: 35. The classical FIX intron is derived from the first intron of human FIX and is shown in SEQ ID NO: 36. The FIX intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In a specific embodiment, the modified FIX intron has the sequence shown in SEQ ID NO: 37. The classical chicken beta globin intron used in the nucleic acid construct is shown in SEQ ID NO: 38. The chicken beta globin intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In a specific embodiment, the modified chicken beta globin intron has the sequence shown in SEQ ID NO: 39.

[0379] The inventors have previously shown in WO 2015 / 162302 that such modified introns, in particular modified HBB2 or FIX introns, have advantageous properties and can significantly improve the expression of the introduced gene.

[0380] In a specific embodiment, the nucleic acid construct of the present invention is an expression cassette, which comprises, in a 5' to 3' direction, a promoter optionally preceded by an enhancer, a coding sequence of the present invention (i.e., a nucleic acid molecule encoding a functional truncated GDE polypeptide) and a polyadenylation signal, such as a pA58 polyadenylation signal (pA58 polyA), a bovine growth hormone polyadenylation signal (bGH polyA), an SV40 polyadenylation signal or another naturally occurring or artificial polyadenylation signal. Preferably, the polyadenylation signal is bGH polyA or pA58 polyA, more preferably pA58 polyA. In a specific embodiment, the polyadenylation signal is bGH poly A as shown in SEQ ID NO: 41. In a specific embodiment, a very short polyA signal is used. For example, a very short polyA signal comprising less than 20 nucleotides is used. In a specific embodiment, the polyadenylation signal is the human soluble neuropilin-1 (sNRP) polyadenylation signal (sNRP polyA; SEQ ID NO: 40). In a preferred embodiment, the polyadenylation signal is the pA58 polyadenylation signal as shown in SEQ ID NO: 42.

[0381] In a specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in a 5' to 3' direction, a promoter optionally preceded by an enhancer, an intron, a coding sequence of the present invention, and a polyadenylation signal. In another embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in a 5' to 3' direction, a promoter, a coding sequence of the present invention, and a polyadenylation signal. In another embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in a 5' to 3' direction, an enhancer, a promoter, a coding sequence of the present invention, and a polyadenylation signal. In another specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in a 5' to 3' direction, an enhancer, a promoter, an intron, a coding sequence of the present invention, and a polyadenylation signal. In another specific embodiment of the present invention, the expression cassette comprises, in a 5' to 3' direction, a promoter, an optional intron, a coding sequence of the present invention, and a polyA signal.

[0382] In another embodiment, the nucleic acid construct of the invention is an expression cassette comprising, in 5' to 3' direction, a SpC5-12 promoter or a CMV promoter, such as a mini-CMV promoter, a coding sequence of the invention and a polyadenylation signal (e.g., bGH polyA or pA58 polyA, particularly pA58 polyA). In another embodiment, the nucleic acid construct of the invention is an expression cassette comprising, in 5' to 3' direction, an enhancer, a SpC5-12 promoter or a CMV promoter, such as a mini-CMV promoter, a coding sequence of the invention and a polyadenylation signal (e.g., bGH polyA or pA58 polyA, particularly pA58 polyA).

[0383] In another specific embodiment, the expression cassette comprises, in 5' to 3' direction, the SpC5-12 promoter, a sequence encoding the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, and bGH polyA or pA58 polyA, in particular pA58 polyA. In another embodiment, the expression cassette comprises, in 5' to 3' direction, the CMV promoter, a sequence encoding the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, and bGH polyA or pA58 polyA, in particular pA58 polyA.

[0384] In a preferred embodiment, the expression cassette comprises, in 5' to 3' direction: a CMV promoter, e.g. a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide as defined above, e.g. a sequence encoding the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16; and bGH polyA or pA58 polyA, in particular pA58 polyA.

[0385] In a specific embodiment, the expression cassette comprises, in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide as defined above, for example a sequence selected from SEQ ID NO: 22 to SEQ ID NO: 31; and bGH polyA or pA58 polyA, in particular pA58 polyA.

[0386] In a specific embodiment, the expression cassette comprises, in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 12; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises, in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 22 or SEQ ID NO: 23 encoding a functional truncated GDE polypeptide of SEQ ID NO: 12; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises in 5' to 3' direction: the mini-CMV promoter of SEQ ID NO:43; a sequence of SEQ ID NO:22 or SEQ ID NO:23 encoding a functionally truncated GDE polypeptide of SEQ ID NO:12; and the pA58 polyA of SEQ ID NO:42.

[0387] In a preferred embodiment, the expression cassette comprises, in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular a mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 13; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises, in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular a mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 24 or SEQ ID NO: 25 encoding a functional truncated GDE polypeptide of SEQ ID NO: 13; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises in 5' to 3' direction: the mini-CMV promoter of SEQ ID NO:43; the sequence of SEQ ID NO:24 or SEQ ID NO:25 encoding a functionally truncated GDE polypeptide of SEQ ID NO:13; and the pA58 polyA of SEQ ID NO:42.

[0388] In another embodiment, the expression cassette comprises in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular a mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 14; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular a mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 26 or SEQ ID NO: 27 encoding a functional truncated GDE polypeptide of SEQ ID NO: 14; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises in 5' to 3' direction: the mini-CMV promoter of SEQ ID NO:43; the sequence of SEQ ID NO:26 or SEQ ID NO:27 encoding a functionally truncated GDE polypeptide of SEQ ID NO:14; and the pA58 polyA of SEQ ID NO:42.

[0389] In another embodiment, the expression cassette comprises in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular a mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 15; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular a mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 28 or SEQ ID NO: 29 encoding a functional truncated GDE polypeptide of SEQ ID NO: 15; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises in 5' to 3' direction: the mini-CMV promoter of SEQ ID NO:43; the sequence of SEQ ID NO:28 or SEQ ID NO:29 encoding a functionally truncated GDE polypeptide of SEQ ID NO:15; and the pA58 polyA of SEQ ID NO:42.

[0390] In another embodiment, the expression cassette comprises in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 16; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises in 5' to 3' direction: a CMV promoter, such as a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 30 or SEQ ID NO: 31 encoding a functional truncated GDE polypeptide of SEQ ID NO: 16; and bGH polyA or pA58 polyA, in particular pA58 polyA. In a specific embodiment, the expression cassette comprises in 5' to 3' direction: the mini-CMV promoter of SEQ ID NO:43; the sequence of SEQ ID NO:30 or SEQ ID NO:31 encoding a functional truncated GDE polypeptide of SEQ ID NO:16; and the pA58 polyA of SEQ ID NO:42.

[0391] In a specific embodiment, the expression cassette comprises or consists of a sequence as shown in SEQ ID NO: 44 to SEQ ID NO: 48. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 44 to SEQ ID NO: 48.

[0392] In a specific embodiment, the expression cassette comprises or consists of a sequence as shown in SEQ ID NO: 44. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 44.

[0393] In a preferred embodiment, the expression cassette comprises or consists of a sequence as shown in SEQ ID NO: 45. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 45.

[0394] In another embodiment, the expression cassette comprises or consists of a sequence as shown in SEQ ID NO: 46. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 46.

[0395] In another embodiment, the expression cassette comprises or consists of a sequence as shown in SEQ ID NO: 47. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 47.

[0396] In another embodiment, the expression cassette comprises or consists of a sequence as shown in SEQ ID NO: 48. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even at least 99% sequence identity to SEQ ID NO: 48.

[0397] In designing the nucleic acid constructs of the present invention, those skilled in the art will take into account the size limits of the vectors used to deliver the constructs to cells or organs. Specifically, those skilled in the art understand that the main limitation of the AAV vector is its carrying capacity, which can vary from one AAV serotype to another, but is believed to be limited to the size of the parental viral genome. For example, 5 kb is generally considered to be the maximum size packaged in the 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). In addition, during the production of recombinant AAV, the efficiency of encapsidation of genomes greater than 5 kb is low, and the resulting AAV may contain fragmented genomes, reducing the efficiency of gene transfer. Therefore, those skilled in the art will carefully select the components of the nucleic acid construct of the present invention in practicing the present invention so that the resulting nucleic acid sequence (including the sequence encoding AAV 5'- and 3'-ITR) preferably does not exceed 110% of the carrying capacity of the AAV vector used, and in particular preferably does not exceed 5 kb. AAV vectors with a larger carrying capacity can also be used in the context of the present invention. For example, AAV particles lacking the Vp2 subunit have been shown to successfully package a larger genome (i.e., 6 kb) while maintaining the integrity of the encapsidated genome (Grieger et al., 2005, J Virol., 79(15):9933–9944).

[0398] 4-Carrier

[0399] The present invention also relates to a vector comprising a nucleic acid molecule or construct disclosed herein. In a specific embodiment, the vector comprises a nucleic acid molecule or construct encoding a functional truncated GDE polypeptide defined herein.

[0400] Specifically, the vector of the present invention is suitable for protein expression, preferably for gene therapy. In one embodiment, the vector is a plasmid vector. In another embodiment, the vector is a nanoparticle containing a nucleic acid molecule of the present invention, particularly a messenger RNA encoding a functional truncated GDE polypeptide of the present invention. In another embodiment, the vector is a transposon-based system that allows the nucleic acid molecule or construct of the present invention to be integrated into the genome of a target cell, such as the extremely active 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 cell, muscle cell, CNS cell (such as brain cell) or hematopoietic stem cell such as erythroid cell (such as erythrocyte). In this case, the nucleic acid construct of the present invention also contains a sequence suitable for producing an efficient viral vector, as is well known in the art.

[0401] Viral vectors are preferably used to deliver the nucleic acid molecules or constructs of the invention, such as retroviral vectors, such as lentiviral vectors, or non-pathogenic parvoviruses, more preferably AAV vectors. Human parvovirus Adeno-associated virus (AAV) is a naturally replication-defective dependent virus that is able to integrate into the genome of infected cells to establish a latent infection. This last property appears to be unique among mammalian viruses, as integration occurs at a specific site in the human genome called AAVS1, which is located on chromosome 19 (19q13.3-qter).

[0402] Therefore, AAV vectors have attracted great interest as potential vectors for human gene therapy. Advantageous properties of the virus include that it has not been associated with any human disease, that it can infect both dividing and non-dividing cells, and that it can infect a wide range of cell lines derived from different tissues.

[0403] Among the AAV serotypes isolated from humans or nonhuman primates (NHPs) and well characterized, 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., a quadruple-mutated capsid-optimized AAV-2 comprising an engineered capsid with changes Y44+500+730F+T491V, disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods.), -3 and AAV-3 variants (e.g., an AAV3-ST variant comprising an engineered AAV3 capsid with two amino acid changes S663V+T492V, disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (e.g., an AAV6 variant comprising a triple-mutated AAV6 capsid of the form Y731F / Y705F / T492V, disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p.16026), -7, -8, -9, -2G9, -10 such as cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV serotypes such as AAVpo4 and AAVpo6, and tyrosine, lysine and serine capsid mutants of AAV serotypes, etc. In addition, other non-natural engineered variants and chimeric AAVs may also be useful.

[0404] AAV viruses can be engineered using conventional molecular biology techniques, such that these particles can be optimized for cell-specific delivery of nucleic acid sequences, for minimizing immunogenicity, for regulating stability and particle lifespan, for efficient degradation, and for accurate delivery to the nucleus.

[0405] To assemble into vectors, ideal AAV fragments include cap protein (including vp1, vp2, vp3 and hypervariable regions), rep protein (including rep 78, rep 68, rep 52 and rep 40) and sequences encoding these proteins. These fragments can be easily used in various vector systems and host cells.

[0406] AAV-based recombinant vectors lacking the Rep protein integrate into the host genome with low efficiency and exist primarily as stable circular episomes that can persist in target cells for years.

[0407] As an alternative to using natural serotypes of AAV, artificial AAV serotypes may be used in the context of the present invention, including but not limited to AAV with non-naturally occurring capsid proteins. Such artificial capsids may be produced by any suitable technique using selected AAV sequences (e.g., fragments of the vp1 capsid protein) in combination with heterologous sequences, which may be obtained from different selected AAV serotypes, non-contiguous portions of the same AAV serotype, non-AAV viral sources, or non-viral sources. The artificial AAV serotype may be, but is not limited to, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid.

[0408] In the context of the present invention, the AAV vector comprises an AAV capsid capable of transducing target cells of interest, i.e., cells of tolerogenic tissues (e.g., hepatocytes) and cells of therapeutic target tissues, such as muscle cells, CNS cells, or cardiac cells. In a specific embodiment, the AAV vector comprises an AAV capsid capable of transducing muscle cells or cardiac cells.

[0409] According to certain embodiments, the AAV vector is AAV-1, -2, an AAV-2 variant (e.g., a quadruple-mutated capsid-optimized AAV-2 comprising an engineered capsid with changes Y44+500+730F+T491V, disclosed in Ling et al., 2016 Jul18, Hum Gene Ther Methods. [Epub ahead of print]), -3, and an AAV-3 variant (e.g., an AAV3-ST variant comprising an engineered AAV3 capsid with two amino acid changes S663V+T492V, disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (e.g., AAV6 variants comprising an AAV6 capsid of the triple mutation Y731F / Y705F / T492V form, disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p. 16026), -7, -8, -9, -9P1, -2G9, -10 such as -cy10 and -rh10, -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP, AAV2i8, porcine AAV such as AAVpo1 (as described in WO2021 / 219762), AAVpo4 and AAVpo6, and tyrosine, lysine and serine capsid mutants of AAV serotypes. In a specific embodiment, the AAV vector is of the AAV6, AAV8, AAV9, AAV9P1, AAVrh74 or AAV2i8 serotype (i.e., the AAV vector has a capsid of the AAV6, AAV8, AAV9, AAV9P1, AAVrh74 or AAV2i8 serotype). In another specific embodiment, the AAV vector is a pseudotype vector, i.e., its genome and capsid are derived from AAVs of different serotypes. For example, the pseudotyped AAV vector may be a vector whose genome is derived from one of the above-mentioned AAV serotypes and whose capsid is derived from another serotype. For example, the pseudotyped vector may have a capsid derived from the AAV6, AAV8, AAV9, AAV9P1, AAVrh74 or AAV2i8 serotype, and its genome may be derived from a different serotype. In a specific embodiment, the AAV vector has a capsid of the AAV6, AAV8, AAV9 or AAVrh74 serotype, particularly the AAV6, AAV8, AAV9 or AAV9P1 serotype, more particularly the AAV6, AAV9 or AAV9P1 serotype.

[0410] In a specific embodiment in which the vector is used to deliver a therapeutic transgene to muscle cells, the AAV vector may be particularly selected from AAV8, AAV9 and AAVrh74.

[0411] In another specific embodiment in which the vector is used to deliver the transgene to hepatocytes, the AAV vector can be particularly selected from AAV1, AAV5, AAV8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAVrh74, AAV-LK03, AAV2G9, AAV.PHP, AAV-Anc80 and AAV3B.

[0412] In another specific embodiment, in which the vector is used to deliver a transgene to the CNS, the AAV vector may be particularly selected from AAV9, AAV9P1, AAV10 and AAV2G9.

[0413] In another embodiment, the capsid is a modified capsid. In the context of the present invention, a "modified capsid" may be a chimeric capsid or a capsid comprising one or more variant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0414] In a specific embodiment, the AAV vector is a chimeric vector, i.e., its capsid comprises VP capsid proteins derived from at least two different AAV serotypes, or comprises at least one chimeric VP protein that combines VP protein regions or domains derived 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 may be derived from a combination of AAV8 capsid sequences and sequences of an AAV serotype other than the AAV8 serotype, such as any of the serotypes specifically mentioned above. In another embodiment, the capsid of the AAV vector comprises one or more variant VP capsid proteins, such as those described in WO2015013313, in particular RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid variants that exhibit high liver tropism.

[0415] In another embodiment, the modified capsid may also be derived from a capsid modification inserted by error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). In a specific embodiment, the capsid comprises a P1 insertion, as described in WO2019 / 193119 or WO2020 / 200499 or WO2022 / 053630. In addition, capsid variants may include single amino acid changes, such as tyrosine mutants (e.g., as described in Zhong et al., 2008). In a specific embodiment, the vector is AAV9rh74 comprising a P1 insertion (e.g., as described in Sellier, P et al., "Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompe phenotype in adult and neonate Gaa- / - mice", Journal of inherited metabolic disease, 10.1002 / jimd.12625. 19 May. 2023). In another specific embodiment, the vector is a P1-displaying AAV9 mutant referred to as "AAVMYO", as described in Weinmann et al. (Weinmann, Jonas et al., "Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants", Nature communications vol. 11, 1 5432. 28 Oct. 2020).

[0416] In a specific embodiment, the AAV vector is an AAV vector described in WO2019 / 193119 or an AAV vector described in WO2020 / 200499.

[0417] In another embodiment, the AAV vector is an AAV vector described in WO2020 / 216861 or an AAV vector described in WO2022 / 003211. Specifically, the AAV vector may have a hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, such as a hybrid capsid between AAV8 and AAV2 / 13.

[0418] In addition, the genome of the AAV vector can be a single-stranded or self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting the terminal melting site from one of the AAV terminal repeat sequences. These replicating genomes are modified vectors that are half the length of the wild-type AAV genome and have a tendency to package DNA dimers. In a preferred embodiment, the AAV vector used in the practice of the present invention has a single-stranded genome and also preferably comprises an AAV8, AAV9, AAVrh74, AAVrh74-P1, AAV9rh74-P1, AAV2i8 capsid or a hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, in particular AAV8, AAV9, AAV9rh74-P1 or a hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, more particularly an AAV9 capsid.

[0419] The AAV vectors used to package the GDE sequences of the present invention can also be modified to increase their carrying capacity. For example, AAV vectors lacking the Vp2 subunit have been shown to successfully package larger genomes (i.e., 6 kb) while maintaining the integrity of the encapsidated genome (Grieger et al., 2005).

[0420] As known in the art, other suitable sequences can be introduced into the nucleic acid construct of the present invention to obtain a functional viral vector. Suitable sequences include AAV ITR.

[0421] In a specific embodiment, the AAV vector comprises a muscle-specific promoter as described above, particularly a muscle-specific promoter that exhibits some leakiness of expression in hepatocytes.

[0422] In another specific embodiment of the present invention, the AAV vector comprises a liver-specific promoter as described above. Since the present embodiment develops an efficient and optimized vector to express GDE in hepatocytes and induce immune tolerance to the protein, the tolerogenic and metabolic properties of the liver are advantageously achieved.

[0423] In a specific embodiment, a double recombinant AAV vector system comprising two AAV vectors as described in WO 2018 / 162748 is used to deliver a nucleic acid molecule or construct encoding a functional truncated GDE polypeptide as defined above. Specifically, the double AAV vector system comprises:

[0424] - a first AAV vector comprising, between the 5' and 3' AAV ITRs, a first nucleic acid sequence encoding an N-terminal portion of a truncated GDE polypeptide as defined above, and

[0425] - a second AAV vector comprising between the 5' and 3' AAV ITRs a second nucleic acid sequence encoding a portion of a truncated GDE polypeptide as defined above,

[0426] and wherein the first and second nucleic acid sequences encoding said GDE comprise polynucleotide regions allowing for the production of a full-length truncated GDE polypeptide as defined above.

[0427] In another specific embodiment, the AAV vector is a single AAV vector comprising a nucleic acid molecule or construct encoding a functionally truncated GDE polypeptide as defined above.

[0428] 5-Cell

[0429] The present invention also relates to cells transformed or transduced with nucleic acid molecules, constructs or vectors of the present invention, in particular isolated cells, such as hepatocytes, heart cells, CNS cells or myocytes. In a specific embodiment, the cell is an isolated human cell. In another specific embodiment, the cell is not a human embryonic stem cell. The cell of the present invention expresses a functional truncated GDE polypeptide as described above. The cell of the present invention can be delivered to the subject by any suitable route of administration, for example, by injection in the liver, CNS, heart, muscle or bloodstream of a subject in need, such as a GDE defective patient. In a specific embodiment, the present invention includes transducing liver or myocytes, in particular liver or myocytes of a subject to be treated, and administering the transduced liver and / or myocytes into which the nucleic acid has been introduced to the subject. In a specific embodiment, the hepatocyte is a hepatocyte from a patient to be treated, or a liver stem cell, which is further transformed and differentiated into a hepatocyte in vitro for subsequent administration to the patient. In another embodiment, the cell is a myocyte from a patient to be treated, or a muscle stem cell, which is further transformed and optionally differentiated into a myocyte in vitro for subsequent administration to the patient.

[0430] 6- Pharmaceutical Composition

[0431] The present invention also provides a pharmaceutical composition comprising a nucleic acid molecule, a nucleic acid construct, a vector, a functional truncated GDE polypeptide or a cell of the present invention. Such compositions may comprise a therapeutically effective amount of the therapeutic agent (a nucleic acid molecule, a nucleic acid construct, a vector, a functional truncated GDE polypeptide or a cell of the present invention) and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the U.S. or European Pharmacopoeia or other generally recognized pharmacopoeias, and can be used for animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient or solvent administered with a therapeutic agent. Such pharmaceutical carriers may be sterile liquids such as water and oils, including oils of petroleum, animal, plant or synthetic origin such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol and the like.

[0432] If necessary, the composition may also contain a small amount of a wetting agent or emulsifier or a pH buffer. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may include standard carriers such as pharmaceutical grade 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 contain a therapeutically effective amount of a therapeutic agent, preferably in purified form, and a suitable amount of a carrier, so as to provide a form suitable for administration to a subject. In a specific embodiment, the nucleic acid, vector, or cell of the present invention is formulated in a composition comprising phosphate buffered saline supplemented with 0.25% human serum albumin. In another specific embodiment, the nucleic acid, vector or cell of the invention is formulated in a composition comprising lactated Ringer's solution and a nonionic surfactant such as pluronic F68, the final concentration of the surfactant being 0.01-0.0001% by weight of the total weight of the composition, such as a concentration of 0.001%. The formulation may also comprise serum albumin, particularly human serum albumin, such as 0.25% human serum albumin. Other formulations suitable for storage or administration are known in the art, particularly from WO 2005 / 118792 or Allay et al., 2011.

[0433] In a preferred embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site.

[0434] In an embodiment, nucleic acid molecules of the present invention, nucleic acid constructs, carriers, functional truncated GDE polypeptides or cells can be delivered in vesicles, particularly liposomes. In another embodiment, nucleic acid molecules of the present invention, nucleic acid constructs, carriers, functional truncated GDE polypeptides or cells can be delivered in a controlled release system.

[0435] In a specific embodiment, the nucleic acid molecule is delivered as mRNA, which corresponds to a transcript encoding a functional truncated GDE polypeptide of the invention. Specifically, the mRNA of the invention can be delivered using liposomes such as lipid nanoparticles (LNP).

[0436] Methods of administration of nucleic acid molecules, nucleic acid constructs, vectors, functional truncated polypeptides or cells of the present invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. In a specific embodiment, the administration is by intravenous or intramuscular routes. The nucleic acid molecules, nucleic acid constructs, vectors, functional truncated polypeptides or cells of the present invention, whether vectorized or not, can be administered by any convenient route, such as by infusion or bolus injection, by administration through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered with other biologically active agents. Administration can be systemic or local.

[0437] In certain embodiments, it may be desirable to administer the pharmaceutical compositions of the invention locally to an area in need of treatment, such as the liver or muscle. This can be achieved, for example, using an implant that is a porous, non-porous or gel-like material, including membranes such as silicone rubber membranes or fibers.

[0438] In a specific embodiment, the functional truncated GDE polypeptides of the present invention are used for enzyme replacement therapy (ERT), in particular for the treatment of GSDIII. The term "enzyme replacement therapy" or "ERT" generally refers to the introduction of a purified enzyme into an individual lacking such an enzyme. The administered polypeptides of the present invention can be obtained from natural sources, obtained by recombinant expression, produced in vitro, or purified from isolated tissues or fluids. In particular, when used for ERT, the polypeptides of the present invention can be administered parenterally, for example, by intraperitoneal, intramuscular, intravascular (i.e., intravenous or intraarterial) administration. In particular, the polypeptides are administered by intravenous injection. The administration can be repeated frequently, for example, daily, weekly, biweekly or monthly, in particular weekly or biweekly.

[0439] The amount of the therapeutic agent of the present invention (i.e., the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide or cell of the present invention) that is effective in the treatment of GSDIII can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays can be optionally used to help predict the optimal dosage range. The precise dosage used in the formulation also depends on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and the circumstances of each patient. The dosage of the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide or cell of the present invention administered to a subject in need will vary with 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 necessary to obtain a therapeutic effect. A professional and technical person can easily determine the desired dosage range based on these and other factors based on his knowledge in the field. In the case where the treatment includes administering a viral vector, such as an AAV vector, to the subject, a typical dose of the vector is at least 1x10 8 vector genomes / kg body weight (vg / kg), e.g. at least 1x10 9 vg / kg, at least 1x10 10 vg / kg, at least 1x10 11 vg / kg, at least 1x10 12 vg / kg, at least 1x10 13 vg / kg or at least 1x10 14 vg / kg.

[0440] 7- Treatment methods

[0441] The present invention also relates to a method for treating GSDIII, comprising the step of delivering a therapeutically effective amount of the nucleic acid molecule, nucleic acid construct, vector, functionally truncated GDE polypeptide, pharmaceutical composition or cell of the present invention to a subject in need thereof.

[0442] Cirrhosis and hepatocellular carcinoma may also occur in GSDIII patients. Therefore, the present invention also relates to a method for treating cirrhosis and hepatocellular carcinoma in GSDIII patients, the method comprising delivering a therapeutically effective amount of nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides, pharmaceutical compositions or cells of the present invention to a subject in need thereof.

[0443] The present invention also relates to a method for treating GSDIII, which does not induce an immune response to the transgenic gene (i.e., to the functional truncated GDE polypeptide encoded by the nucleic acid molecule), or induces a reduced immune response to the transgenic gene, the method comprising delivering a therapeutically effective amount of the nucleic acid, vector, functional truncated GDE polypeptide, pharmaceutical composition or cell of the present invention to a subject in need. The present invention also relates to a method for treating GSDIII, the method comprising repeatedly administering a therapeutically effective amount of the nucleic acid, vector, functional truncated GDE polypeptide, pharmaceutical composition or cell of the present invention to a subject in need. In this regard, the nucleic acid molecule, nucleic acid construct or vector of the present invention comprises a promoter functional in hepatocytes, thereby allowing immune tolerance to the expressed functional truncated GDE polypeptide produced therefrom. Similarly, in this regard, the pharmaceutical composition used in this aspect comprises a nucleic acid molecule, a nucleic acid construct or a vector, which comprises a promoter functional in hepatocytes. In the case of delivering cells, particularly liver, heart, CNS or muscle cells, the cells can be previously collected from the subject in need of treatment and engineered by introducing nucleic acid molecules, nucleic acid constructs or vectors of the present invention therein so that they can produce the cells of the functional truncated GDE polypeptide. According to an embodiment, in the case of including repeated administration, the administration can be repeated at least once or many times, and it can even be considered to be carried out according to a regular schedule, such as weekly, monthly or annually. The regular schedule can also include administration once every 2, 3, 4, 5, 6, 7, 8, 9 or 10 years or more than 10 years. In another specific embodiment, each administration of the viral vector of the present invention is carried out using different viruses for each subsequent administration, thereby avoiding the reduction in efficacy caused by the possible immune response of the viral vector used for the previous administration. For example, the first administration can be carried out using the AAV vector comprising the AAV8 capsid, and the subsequent administration uses the vector comprising the AAV9 capsid.

[0444] According to the present invention, treatment may include a curative, alleviating or preventive effect. Thus, therapeutic and prophylactic treatments include amelioration or prevention of symptoms of GSDIII or otherwise reducing the risk of developing a specific glycogen storage disease. The term "prophylactic" may be considered to reduce the severity of a specific condition or reduce its onset. "Prophylactic" also includes preventing the re-emergence of the condition in a patient previously diagnosed with a specific condition. "Therapeutic" may also reduce the severity of an existing condition. The term "treatment" is used herein to refer to any regimen that may be beneficial to an animal, particularly a mammal, and more particularly a human subject.

[0445] The present invention also relates to an ex vivo gene therapy method for treating GSDIII, which comprises introducing the nucleic acid molecule, nucleic acid construct or vector of the present invention into isolated cells of a patient in need, such as isolated hematopoietic stem cells, and introducing the cells into the patient in need.

[0446] The present invention also relates to the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, cell or pharmaceutical composition of the present invention, which is used as a drug.

[0447] The present invention also relates to the nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides, cells or pharmaceutical compositions of the present invention, which are used in methods for treating diseases caused by mutations in the AGL gene encoding GDE, in particular for treating GSDIII (Corey's disease) such as GSDIIIa and GSDIIIb, in particular GSDIIIa.

[0448] The present invention also relates to the use of the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, cell or pharmaceutical composition of the present invention for preparing a medicament for treating GSDIII (Corey's disease).

[0449] The present invention also relates to the nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides, cells or pharmaceutical compositions of the present invention, which are used in methods for delivering the GDE protein to affected tissues, especially muscle and liver tissues, especially muscle.

[0450] Example

[0451] The present invention is further described in detail with reference to the following experimental examples and the accompanying drawings. These examples are provided for illustrative purposes only and are not intended to be limiting.

[0452] In patent applications WO2020 / 030661 and PCT / EP2021 / 073309, it was shown that it is possible to produce truncated forms of GDE proteins while retaining enzymatic activity. In particular, the truncated proteins "∆1b2" and "∆1b3" were shown to be highly effective in reducing the amount of glycogen in different muscle tissues. "∆1b2" corresponds to the GDE polypeptide of SEQ ID NO: 1 in which amino acids 2-81 are deleted. "∆1b3" corresponds to the GDE polypeptide of SEQ ID NO: 1 in which amino acids 2-103 are deleted. However, both proteins were shown to be less expressed or less stable in vivo than the full-size GDE. Therefore, the aim was to modify the N-terminal truncation site to find more stable, better expressed truncated proteins, and ultimately to improve the ability of the proteins to reduce glycogen in vivo.

[0453] To evaluate the activity of truncated GDE, AAV vectors expressing truncated GDE (∆1b5 or ∆1b3) or full-size protein (AAV-GDE-full-size) were generated. AAV9rh74-P1 vector (i.e., an AAV vector with a hybrid capsid between AAV9 and AAVrh74 modified with peptide P1) was used throughout the experiments. The transgene was cloned into a transgene expression cassette optimized for muscle expression and composed of a mini CMV promoter and a pA58 polyadenylation signal. The expression cassettes for the ∆1b5, ∆1b3, and GDE-full-size constructs are shown in SEQ ID NO: 45, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.

[0454] Embodiment 1:

[0455] The carrier was placed at 3x10 11 vg / mouse was injected into the left tibialis anterior muscle of 4-6 month old GSDIII mice ( Figure 1 PBS-injected wild-type (WT, PBS) or knockout mice (KO, PBS) were used as controls. Mice were sacrificed 1 month after injection to measure the glycogen content in the left tibialis anterior muscle.

[0456] Measurement of glycogen content

[0457] Glycogen content in tissue homogenates was measured indirectly as glucose released after complete digestion with Aspergillus Niger amyloglucosidase (Sigma-Aldrich, ref A1602). Samples were incubated at 95°C for 10 min and then cooled at 4°C. After the addition of amyloglucosidase (final concentration 4 U / mL) and potassium acetate, pH 5.5 (final concentration 25 mM) for 90 min at 37°C, the reaction was terminated by incubating the samples at 95°C for 10 min. Control reactions without amyloglucosidase and incubated under the same conditions were prepared for each sample. The released glucose was determined using a glucose assay kit (Sigma-Aldrich) and the resulting absorbance was acquired at a wavelength of 540 nm on an EnSpire alpha plate reader (PerkinElmer). The glucose released after amyloglucosidase was then normalized by the total protein concentration.

[0458] Measurement of GDE expression

[0459] Mouse tissues were placed in a cOmplete TMHomogenize in phosphate-buffered saline (PBS, ThermoFisher Scientific) with protease inhibitor cocktail (Roche, ref 4693132001). TM Protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific). Fractions of 50 µg of total protein were loaded per well for PBS- and rAAV-injected Agl- / - mice, and 10 µg of total protein were loaded per well for PBS-injected Agl+ / + mice. TM SDS-PAGE electrophoresis was performed in 4% paraformaldehyde (4% paraformaldehyde, Invitrogen). After transfer, the membrane was blocked with Intercept blocking buffer (LI-CORBiosciences) and incubated with anti-GDE rabbit polyclonal antibody (16582-1-AP, Proteintech) and anti-vinculin mouse monoclonal antibody (V9131, Sigma). The membrane was washed, incubated with appropriate secondary antibodies (LI-COR Biosciences), and visualized by Odyssey imaging system (LI-COR Biosciences).

[0460] Quantification of vector genome copy number

[0461] DNA was extracted from tissue homogenates using the Nucleomag Pathogen Extraction Kit from Macherey-Nagel (#744210.4) following the manufacturer's instructions. The vector genome copy number was determined using a qPCR assay. The PCR primers used in the reaction were located in the glucosyltransferase domain of the full-length and truncated codon-optimized GDE (SEQ-33; forward: 5'-CTG AAG CTG TGG GAG TTC TT-3' (SEQ ID NO: 59) and reverse: 5'-CTC TTG GTC ACT CTTCTG TTC TC-3' (SEQ ID NO: 60)). As an internal control, primers located within the mouse titin gene were used (forward: 5'-AAA ACG AGC AGT GAC GTG AGC-3' (SEQ ID NO: 61) and reverse: 5'-TTC AGT CAT GCTGCT AGC GC-3' (SEQ ID NO: 62)).

[0462] result

[0463] The results showed that the glycogen content of mice injected with truncated GDE constructs was significantly reduced compared with the control group injected with PBS. In terms of glycogen depletion, GDE proteins "∆1b3", "∆1b5" and the full-length form of GDE (GDE full size) showed significant efficacy in injected muscles ( Figure 2 ).

[0464] Compared with the ∆1b3 GDE construct, the ∆1b5 GDE construct showed much better protein expression levels ( Figure 3 and 4 ), although the truncation sites are very close ( Figure 1 ). This experiment shows that it is not possible to predict the expression level of a newly truncated protein based solely on the location of the truncation site.

[0465] Furthermore, quantification of vector genome copy number (VGCN) in injected muscles showed that VGCN was lower in tibialis anterior muscles injected with the ∆1b5 GDE construct compared to the ∆1b3 GDE construct ( Figure 5 ), emphasizing that Figure 3-4 The higher expression observed with the ∆1b5 GDE construct was not due to a higher amount of rAAV vector injected.

[0466] Embodiment 2:

[0467] The expression of ∆1b3-GDE ( Figure 6 ) or ∆1b5-GDE ( Figure 7 ) of rAAV vector. - / - Mice were injected via the tail vein with a gene encoding ∆1b3-GDE ( Figure 6 ) or ∆1b5-GDE ( Figure 7 ) of rAAV vectors at a dose of 1 x 10 14 vg / kg. PBS injected Agl + / + and Agl - / - Mice were used as controls. Glycogen content in the heart and triceps was measured at euthanasia as described above. Wire hanging test was performed 3 months after vehicle injection and expressed as the number of falls per minute. The number of falls was recorded in a 3-min period using a 4-mm thick wire. The average number of falls per minute for each animal is reported.

[0468] result

[0469] The results showed that compared with the PBS-injected control group, the truncated ∆1b3-GDE construct ( picture 6B) and ∆1B5-GDE construct ( picture7B) showed a significant decrease in glycogen content in the heart and triceps of mice. The ∆1B5-GDE construct showed better efficacy than the ∆1B3-GDE construct in glycogen depletion in the heart and triceps.

[0470] Furthermore, treatment with rAAV-∆1b3-GDE and rAAV-∆1b5-GDE improved muscle strength assessed by wire suspension. Compared with the PBS-injected group, mice showed a lower frequency of falls at 3 months after injection (Fig. Figure 6 C and 7C).

[0471] Importantly, treatment with rAAV-∆1b5-GDE proved to be much more efficient in rescuing muscle strength compared to rAAV-∆1b3-GDE. Three months after injection, rAAV-∆1b5-GDE-injected mice showed a lower fall frequency compared to the PBS-injected group (9.8 falls / min ± 1.8 vs. 31.4 falls / min ± 1.4, corresponding to a 69% reduction), almost reaching wild-type levels ( Figure 7 C). In contrast, at 3 months post-injection, rAAV-∆1b3-GDE-injected mice showed approximately 17 falls / min ± 1.9 (compared to 23.8 ± 3.9 in the PBS-injected group, corresponding to only a 29% decrease).

[0472] These data clearly demonstrate that treatment with rAAV-∆1b5-GDE reverses muscle damage in adult symptomatic GSDIII mice at both the biochemical and functional levels.

[0473] Embodiment 3:

[0474] To confirm the efficacy of the rAAV-∆1b5-GDE construct in a larger animal model, the vector was administered to the GSDIII rat model. 14 Six-week-old Aglipidae were treated with rAAV-∆1b5-GDE via tail vein injection. - / - Rats were analyzed three months after injection ( Figure 8 A). PBS-injected Agl - / - and Agl + / + Rats were used as controls. Glycogen content in the heart and triceps was measured at the time of euthanasia as described above.

[0475] Histological analysis of the heart and triceps was performed with HPS (hematoxylin-fluorescein-saffron stain) and PAS (periodic acid-Schiff stain) staining. For muscle histology, the heart and triceps were snap-frozen in isopentane that had been previously frozen in liquid nitrogen. Serial 8-µm transverse sections were cut in a Leica CM3050 S cryostat (Leica Biosystems). To minimize sampling errors, two sections were obtained per specimen and stained with HPS or PAS according to standard procedures. Images were digitized using an Axioscan Z1 slide scanner (Zeiss, Germany) under a Zeiss Plan-Apochromat 10X / 0.45 M27 dry objective (ZEISS, Germany). Tiled scan images were reconstructed using ZEN software (ZEISS, Germany).

[0476] result

[0477] In rAAV-∆1b5-GDE-injected rats, a significant 50% reduction in glycogen accumulation in the heart was observed three months after injection ( Figure 8 B), which is also evident in PAS staining ( Figure 8 C). Almost complete glycogen clearance is also observed in skeletal muscles such as the triceps ( Figure 8 D), and histological analysis showed normalization of muscle structure and decreased glycogen accumulation ( Figure 8 E).

[0478] In conclusion, treatment of Agl with rAAV vector encoding ∆1b5-GDE - / - The data obtained in rats confirm those obtained in the mouse model of the disease and further support the clinical translation of rAAV-∆1b5-GDE for the treatment of muscle disease in GSDIII patients.

[0479] Embodiment 4:

[0480] The activity of the truncated ∆1b5-GDE was also evaluated in the context of human pathology using an in vitro human skeletal muscle model of GSDIII. Skeletal muscle myoblasts and myotubes were derived from GSDIII clones generated by CRISPR / Cas9 technology. CRISPR ) Human induced pluripotent stem cells (hiPSCs) edited to knock down the AGL gene.

[0481] Transduction of hiPSC-derived skeletal muscle cells

[0482] GSDIII CRISPRhiPSCs have been previously generated using CRISPR knockdown of the AGL gene (Rossiaud et al., Pathological modeling of Glycogen Storage Disease type III with CRISPR / Cas9 edited human pluripotent stem cells, Front. Cell Dev. Biol., 11 May 2023). Control hiPSCs were an isogenic cell line (CTRL1). CRISPR and CTRL1 hiPSCs were differentiated into skeletal myoblasts as previously described (Rossiaud et al.). After expansion in 96-well plates, hiPSC-derived skeletal myoblasts were transduced with rAAV vectors encoding GFP or ∆1b5-GDE under the control of the miniCMV promoter at an MOI of 75,000 or 15,000 for 72 h. hiPSC-derived skeletal myoblasts were then differentiated into myotubes as previously described (Rossiaud et al.).

[0483] Measurement of glycogen content in skeletal myotubes

[0484] As previously described, after 4 days of differentiation into myotubes, hiPSC-derived skeletal myotubes were starved for 3 days in glucose-free DMEM medium (ref 11966025, ThermoFisher Scientific) containing 10% fetal bovine serum to induce glycogen degradation in CTRL1 myotubes (Rossiaud et al.). Cells were lysed using HCl 0.3 M and Tris 450 mM pH 8.0. Glycogen-Glo TM Glycogen was quantified using a 24-well assay kit (ref CS1823B01, Promega) and using CellTiter-Glo TM Luminescent cell viability assay (ref G7570, Promega) was used for normalization.

[0485] Immunostaining assay

[0486] Skeletal myotubes derived from hiPSCs were fixed with 4% paraformaldehyde (Euromedex) for 10 min at room temperature. After washing twice in PBS, cells were permeabilized with 0.5% Triton X-100 for 5 min and blocked in PBS supplemented with 1% bovine serum albumin (BSA, Sigma) for 1 h at room temperature. Skeletal myotubes were stained for specific skeletal myogenic markers overnight at 4°C using primary antibodies (desmin, ref AF3844 R&D 1:200; MF20, ref 3ea DSHB 1:50; titin, ref T5650 US Biological 1:50). After washing 3 times in PBS, staining was revealed with appropriate Alexa Fluor secondary antibodies 1:1000 (donkey anti-goat AF488 ref A11055 Invitrogen 1:1000; donkey anti-mouse AF488 ref A21202 Invitrogen 1:1000) for 1 h at room temperature in the dark and nuclei were visualized with Hoechst solution 1:3000 (Invitrogen). Cells were imaged using an LSM-800 confocal microscope (Zeiss) associated with Zen Black software and a 20X objective.

[0487] Periodic acid-Schiff (PAS) staining of skeletal myotubes

[0488] PAS staining of hiPSC-derived skeletal myotubes was performed using the PAS staining kit (Sigma-Aldrich) following the manufacturer’s instructions. Briefly, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing twice in PBS, cells were treated with periodic acid for 5 min at room temperature. After washing three times in distilled water, cells were treated with Schiff’s reagent for 15 min at room temperature. Finally, after washing four times in tap water, staining was visualized using an EVOS XL Core microscope (Invitrogen). Images were processed and analyzed using a FIJI custom script. First, the colors were segmented and only the green channel was retained as it was the channel with the highest contrast. Then, images were manually thresholded into binary images where the PAS signal was black and the background was white. The threshold was set to maximize the difference between genotypes and once calculated, the same threshold was applied to all images for quantification. Quantification of PAS staining was obtained using the following formula: PAS staining area / total image area x 100, which then gives the percentage of PAS staining within the image.

[0489] result

[0490] GSDIII was transduced with rAAV vectors expressing ∆1b5-GDE or GFP according to previously reported protocols (Rossiaud et al. CRISPR and CTRL1 hiPSC-derived skeletal myoblasts ( Fig. 9 A). Skeletal myoblasts were differentiated into skeletal myotubes after transduction with rAAV. Transduction with rAAV expressing GFP or ∆1b5-GDE did not alter the differentiation of myoblasts into myotubes, and immunostaining analysis showed similar expression of skeletal myogenic markers ( Fig. 9 B) GSDIII transduced with a control vector CRISPR Compared with myotubes, GSDIII transduced with rAAV expressing ∆1b5-GDE CRISPR Skeletal myotubes showed a significant decrease in glycogen content ( Fig. 9 C). PAS staining of transduced skeletal myotubes confirmed these results ( Fig. 9 DE).

[0491] These data confirm that the truncated ∆1b5 GDE clears glycogen not only in mouse and rat muscle but also in the human skeletal muscle model of GSDIII without any specific toxicity.

[0492] Embodiment 5:

[0493] Oversized rAAV vector production has a significant impact on the yield and quality of the final product, which are critical parameters for translating rAAV-based gene therapies into the clinic. To evaluate the impact of using truncated ∆1b5-GDE on vector yield and quality, an oversized expression cassette (5.3 kb) expressing full-length human GDE was compared to a 5 kb expression cassette encoding truncated ∆1b5-GDE ( Fig.10 Small-scale rAAV production (50 mL culture) was performed in triplicate for each vector, and viral titer was measured for each replicate before purification (stock) and after purification (final product). rAAV vector DNA was extracted and loaded on a 1% agarose gel to assess genomic integrity. Analytical ultracentrifugation (AUC) was performed to analyze the proportion of intact and empty particles.

[0494] Viral genome analysis on agarose gel

[0495] DNA was extracted using the High Pure Viral Nucleic Acid Kit (Roche, ref 11858874001). The purified viral DNA was then loaded on a 1% agarose gel (EurobioScientific) stained with SybrSafe® gel stain (Invitrogen) to visualize the viral DNA. The expected genome size ranged from 5.0 to 5.3 kb.

[0496] Analytical Ultracentrifugation (AUC)

[0497] AUC measures the sedimentation coefficient of macromolecules by tracking the optical density of samples undergoing ultracentrifugation over time. The difference in sedimentation coefficients measured by Raleigh interferometry or absorbance at 260 nm depends on the amount of viral genome in the capsid. AUC analysis was performed using a Proteome Lab XL-I (Beckman Coulter, Indianapolis, IN). A 400 µL aliquot of rAAV vector and 400 µL formulation buffer were loaded into a dual-sector velocity cell. Sedimentation velocity centrifugation was performed at 20,000 rpm and 20°C. The radial concentration was recorded simultaneously over time using absorbance (260 nm) and Raleigh interferometry optics until the lightest sedimenting component passed through the optical window (approximately 1.5 h). The absorbance data were required to calculate the molar concentration and percentage values ​​of empty and genome-containing capsids using the extinction coefficient. The molar concentrations of both genome-containing and empty capsids were calculated using Beer's law, and the percentages of full genome-containing and empty capsids were calculated.

[0498] result

[0499] The use of ∆1b5-GDE cDNA allowed an approximately tenfold increase in rAAV production ( Fig.10 B), while allowing efficient encapsidation of non-truncated genomes ( Fig.10 C). As measured by analytical ultracentrifugation (AUC), the increase in yield and genome quality resulted in a dramatic increase in the ratio of full to empty particles, with up to 37% full particles measured in AAV-∆1b5-GDE compared to only 15% full particles measured in AAV-full-length GDE ( Fig.10 D).

[0500] Taken together, these data suggest that although ∆1b5-GDE has similar in vivo efficacy to the full-size enzyme, it allows for the production of rAAV vectors with higher yield and quality, thus providing a promising gene therapy candidate for the treatment of GSDIII.

Claims

1. A functional truncated GDE polypeptide, wherein the functional truncated GDE polypeptide comprises a deletion relative to a reference functional full-length human GDE sequence, and wherein the deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are: - MGSFQY (SEQ ID NO: 7); - MEKSGG (SEQ ID NO: 8); - MILRVG (SEQ ID NO: 9); - MGADNH (SEQ ID NO: 10); or - MLDCVT (SEQ ID NO: 11).

2. The functional truncated GDE polypeptide according to claim 1, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8) or MILRVG (SEQ ID NO: 9), preferably wherein the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8).

3. The functional truncated GDE polypeptide according to claim 1 or 2, wherein the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

4. The functional truncated GDE polypeptide according to claim 1, wherein the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO: 1, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4, preferably with SEQ ID NO:

1.

5. A functional truncated GDE polypeptide according to any one of the preceding claims, wherein the functional truncated GDE polypeptide further comprises a deletion or a combination of deletions relative to the reference functional full-length human GDE sequence, such as a deletion or a combination of deletions in the C-terminal portion of the GDE sequence or a deletion or a combination of deletions in the central domain of the GDE sequence; In particular, wherein the functional truncated GDE polypeptide further comprises a deletion or a combination of deletions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and wherein the deletion is selected from any of the deletions referred to as ∆1, ∆2, ∆3, ∆4, ∆5, ∆6 and ∆7 in Table 2.

6. The functional truncated GDE polypeptide according to any one of the preceding claims, wherein the functional truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NOs: 12-16, or an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NOs: 12-16.

7. The functional truncated GDE polypeptide according to any one of the preceding claims, wherein the functional truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NO: 13 or 14, preferably SEQ ID NO: 13, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 13 or 14, preferably SEQ ID NO:

13.

8. A nucleic acid molecule encoding a functional truncated GDE polypeptide according to any one of claims 1 to 7.

9. An expression cassette, preferably comprising in the following order: - Promoter; - optional introns; - a nucleic acid molecule according to claim 8; and - Polyadenylation signal.

10. A vector, in particular a viral vector, comprising the nucleic acid molecule according to claim 8 or the expression cassette according to claim 9.

11. The vector according to claim 10, which is an AAV vector.

12. An isolated cell transformed with the nucleic acid molecule according to claim 8, the expression cassette according to claim 9 or the vector according to claims 10-11, wherein the cell is in particular a hepatocyte, a myocyte, a heart cell or a CNS cell.

13. A functionally truncated GDE polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, an expression cassette according to claim 9, a vector according to claims 10-11 or a cell according to claim 12 for use as a medicament.

14. A functionally truncated GDE polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, an expression cassette according to claim 9, a vector according to claims 10-11 or a cell according to claim 12, for use in a method for treating a disease caused by a mutation in the AGL gene encoding GDE.

15. A functionally truncated GDE polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, an expression cassette according to claim 9, a vector according to claims 10-11 or a cell according to claim 12 for use in a method for treating GSDIII (Corey's disease).

Images