N-terminal truncated glycogen debranching enzyme for the treatment of glycogen storage disease III

Abstract

This invention relates to a functional N-terminal truncated GDE polypeptide for the treatment of glycogen storage disease III.

Description

[Technical Field]

[0001] This invention relates to the treatment of glycogen storage disease (GSDIII). [Background technology]

[0002] Mutations in the AGL gene result in a deficiency of glycogen debranching enzyme (GDE), also known as "amylo-alpha-1,6-glucosidase, 4-alpha-glucanotransferase," which is involved in glycogenolysis. GDE has two independent catalytic activities that occur at different sites on the protein: 4-alpha-glucotransferase activity and amylo-1,6-glucosidase activity. Genetic deficiency of GDE leads to incomplete glycogenolysis in glycogen storage disease III (GSDIII), resulting in the accumulation of abnormal glycogen with short outer chains in various organs, mainly the liver and muscles. This disease is characterized by hepatomegaly, hypoglycemia, short stature, variable myopathy, and cardiomyopathy. Most patients have GSDIII (type IIIa), which involves both the liver and muscles, but some patients (about 15 percent) have liver lesions only (type IIIb). Liver symptoms usually occur 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, pp. 446-463). Muscle weakness may be present in childhood. It manifests in the 20s or 30s and is more common in adulthood. The incidence of progressive muscle weakness is significant, and patients may become wheelchair-bound in later stages. Patients may also develop cardiomyopathy. There is considerable clinical variability in the severity of symptoms that these patients develop. Progressive myopathy and / or cardiomyopathy are the leading causes of morbidity in adults (Kishnani et al., 2010, Genet Med 12, pp. 446-463; Cornelio et al., 1984, Arch Neurol 41, pp. 1027-1032; Coleman et al., 1992, Ann Intern Med 116, pp. 896-900). Reports of potentially disease-related neurological symptoms have come from clinicians caring for GSDIII patients, who have reported fluctuations in attention, executive function deficits, and emotional skill impairments (Michon et al., 2015, J Inherit Metab Dis, 38(3):573-580).Therefore, Agl is a disease. - / - In mouse models, extensive glycogen accumulation has been described in the liver, skeletal muscle, and heart, as well as, to a relatively smaller extent, in the central nervous system (Pagliarani et al., 2014, Biochim Biophys Acta, 1842(11):2318-2328; Liu et al., 2014, Mol Genet Metab, 111(4):467-476), but careful characterization of the neurocognitive phenotype associated with glycogen accumulation is still lacking. Current treatments are symptomatic, and there is no effective treatment for the disease. Hypoglycemia can be managed with frequent or nocturnal intragastric tube feeding containing cornstarch supplements, as well as nocturnal enteral infusion in addition to a daytime protein-rich diet. While temporary improvement in muscle symptoms after the use of a high-protein diet has been described in some patients, there are no systematic studies or long-term data demonstrating that a high-protein diet can prevent or treat progressive myopathy (Kishnani et al., 2010, Genet Med 12, pp. 446-463). These approaches have little effect on the long-term course and morbidity of these diseases.

[0003] Therefore, long-term treatment for GSDIII remains necessary. Gene therapy aimed at stably replacing the GDE protein in affected tissue appears to be a promising therapeutic approach. However, the large size of the GDE transgene is a major obstacle as it cannot fit within the size limits of most gene therapy vectors. In fact, the human AGL gene is 85 kb long and consists of 35 exons encoding 7.4 kb of mRNA, including a 4599-bp coding region and a 2371-bp 3' untranslated sequence for expressing a 175 kDa GDE protein (Bao Y et al., 1996, Genomics., 38(2):155-65). This is a serious problem because the minimum size of a GDE expression cassette (e.g., for an AAV vector, including at least the promoter, GDE coding sequence, poly(A) signaling and two ITRs) is larger than the 5 kb genome size limit that can be packaged into an AAV vector used for in vivo gene delivery.

[0004] The inventors previously proposed the use of dual AAV vectors to overcome this size limitation (WO2018 / 162748). Following this approach, two vectors, each containing half of the expression cassette, are used to transduce the same cell. While the use of dual AAV vectors is promising, it would be preferable to provide gene therapy strategies that implement only a single viral vector for both economic and practical reasons.

[0005] In patent applications WO2020 / 030661 and WO2022 / 043280, the inventors described an alternative approach based on the use of a truncated GDE polypeptide that fits into a single viral vector. However, an alternative to the truncated GDE polypeptide previously described in WO2020 / 030661 and WO2022 / 043280 is desirable. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2018 / 162748 [Patent Document 2] WO2020 / 030661 [Patent Document 3] WO2022 / 043280 [Patent Document 4] U.S. Patent Application Publication No. 2003 / 0157064 [Patent Document 5] WO2020 / 219949 [Patent Document 6] WO2015110449 [Patent Document 7] WO2009130208 [Patent Document 8] WO2015 / 162302 [Patent Document 9] WO2021 / 219762 [Patent Document 10] WO2015013313 [Patent Document 11] WO2019 / 193119 [Patent Document 12] WO2020 / 200499 [Patent Document 13] WO2022 / 053630 [Patent Document 14] WO2020 / 216861 [Patent Document 15] WO2022 / 003211 [Patent Document 16] WO 2005 / 118792 [Non-patent literature]

[0007] [Non-Patent Document 1] Chen et al., 2009, Scriver's Online Metabolic & Molecular Bases of inherited Disease, New York: McGraw-Hill. [Non-Patent Document 2] Kishnaniら, 2010, Genet Med 12, pages 446~463 [Non-licensed Document 3] Cornelio, 1984, Arch Neurol 41, pp. 1027-1032 [Non-licensed Document 4] Coleman, 1992, Ann Intern Med 116, pages 896~900 [Non-licensed Document 5] Michon, 2015, J Inherit Metab Dis, 38(3): 573-580 pages [Non-licensed Document 6] Pagliaraniら, 2014, Biochim Biophys Acta, 1842(11):2318~2328 pages [Non-licensed Document 7] Liuら, 2014, Mol Genet Metab, 111(4):467~476 pages [Non-licensed Document 8] Bao Yら, 1996, Genomics., 38(2):155-65 [Non-licensed Document 9] Bao and colleagues (Genomics, 1997, 38, pages 155~165) [Non-licensed Document 10] Wang Bら, Construction and analysis of compact muscle-selective promoters for AAV vectors. Gene Ther. 2008 Nov;15(22):1489~99 pages [Non-licensed Document 11] Wangら, Gene Therapy, Volume 15, Pages 1489~1499 (2008) [Non-licensed Document 12] Salvaら、Mol Ther. Feb. 2007;15(2):320-9 [Non-licensed Document 13] Weintraub, Science, 251, 761 (1991) [Non-licensed Document 14] Wang et al., 2008 doi: 10.1038 / gt.2008.104 [Non-Patent Document 15] 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 [Non-Patent Document 16] Liver Specific Gene Promoter Database collected by Cold Spring Harbor Laboratory (http: / / rulai.cshl.edu / LSPD / ) [Non-Patent Document 17] Robert H. Costa et al., 1986 (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) [Non-Patent Document 18] Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993) [Non-Patent Document 19] Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991) [Non-Patent Document 20] Piccioli et al., Neuron, 15:373- 84 (1995) [Non-Patent Document 21] Boshart et al., Cell, 41:521-530 (1985) [Non-Patent Document 22] Rincon et al., Mol Ther. January 2015; 23(1): pp. 43-52. [Non-licensed Document 23] Chuahら, Mol Ther. 2014 September;22(9):1605-13 [Non-licensed Document 24] Nair, Blood. May 15, 2014;123(20):3195-9 [Non-licensed Document 25] Wuら, 2008, Mol Ther, 16(2): pages 280~289 [Non-licensed Document 26] Kurachi, 1995, J Biol Chem., 270(10): 5276~5281 pages [Non-licensed Document 27] Wongら, 1985, Chromosoma, 92(2): pages 124~135; [Non-licensed Document 28] Yewら, 1997, Hum Gene Ther, 8(5): pages 575~584; [Non-licensed Document 29] Choi T., 1991, Mol Cell Biol, 11(6): 3070-3074; [Non-licensed Document 30] Huang, 1990, Mol Cell Biol., 10(4):1805~1810 pages [Non-licensed Document 31] Wu Z.ら, Mol Ther., 2010, 18(1): pages 80~86 [Non-licensed Document 32] Lai Y.ら, Mol Ther., 2010, 18(1): pages 75~79 [Non-licensed Document 33] Wang Y., Hum Gene Ther Methods, 2012, 23(4): 225-33 [Non-licensed Document 34] Grieger, 2005, J Virol., 79(15): 9933~9944 pages [Non-licensed Document 35] Lingら, July 18, 2016, Hum Gene Ther Methods. [Non-licensed Document 36] Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042. [Non-Patent Document 37] Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, page 16026 [Non-Patent Document 38] Shen et al., Molecular Therapy, 2007. [Non-Patent Document 39] Tenney et al., Virology, 2014. [Non-Patent Document 40] 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., May 19, 2023. [Non-Patent Document 41] Weinmann, Jonas, et al., "Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants," Nature Communications, Vol. 11, pp. 15432, October 28, 2020. [Non-Patent Document 42] Tabebordbar, Mohammadsharif, et al., "Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species," Cell, Vol. 184, pp. 4919-4938, September 16, 2021. [Non-Patent Document 43] McCarty et al., Gene Therapy, 2003. [Non-Patent Document 44] "Remington's Pharmaceutical Sciences" by E.W. Martin [Overview of the project] [Means for solving the problem]

[0008] The present invention relates to a functional truncated GDE polypeptide comprising a deletion relative to a reference functional full-length human GDE sequence, wherein the deletion is such that the first six amino acids of the N-terminus of the functional truncated GDE polypeptide are - MQYYFL (array 7); - MFLQGN (sequence number 8); - MQGNEK (Sequence ID 9); - MGNEKS (Sequence ID 10); - MNEKSG (sequence number 11); - MKSGGG (sequence number 12); or - MSGGGY (Sequence ID 13) This invention relates to a functionally shortened GDE polypeptide, which consists of an amino acid deletion at the N-terminus of a reference functional full-length human GDE sequence in such a manner.

[0009] In a preferred embodiment, the deletion consists of an amino acid deletion in the N-terminal region of a reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally shortened GDE polypeptide become MNEKSG (SEQ ID NO: 11).

[0010] In certain embodiments, the reference functional full-length human GDE has the 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 percent 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. In certain embodiments, the reference functional full-length human GDE has the 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 percent sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO: 1.

[0011] In certain embodiments, the functionally truncated GDE polypeptide further comprises a deletion or combination of deletions relative to a reference functional full-length human GDE sequence, for example, a deletion or combination of deletions in the C-terminal region of the GDE sequence, or a deletion or combination of deletions in the central domain of the GDE sequence. In certain embodiments, the functionally truncated GDE polypeptide further comprises a deletion or 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, the deletion being selected from any of the deletions referred to as Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, and Δ7 in Table 2 below.

[0012] In certain embodiments, the functionally abbreviated GDE polypeptide has the amino acid sequence as shown in SEQ ID NOs. 14-20, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity with SEQ ID NOs. 14-20. In certain embodiments, the functionally abbreviated GDE polypeptide has the amino acid sequence as shown in SEQ ID NOs. 14-20. Preferably, the functionally abbreviated GDE polypeptide has the amino acid sequence as shown in SEQ ID NOs. 18, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity with SEQ ID NOs. 18.

[0013] In a particular embodiment, the functionally shortened GDE polypeptide described in any one of the claims is - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 14, and containing the sequence of SEQ ID NO: 21; - Having an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 15, and containing the sequence of SEQ ID NO: 22; - Having an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 16, and containing the sequence of SEQ ID NO: 23; - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 17, and containing the sequence of SEQ ID NO: 24; - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 18, and containing the sequence of SEQ ID NO: 25; - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 19, and containing the sequence of SEQ ID NO: 26; or - It has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 20, and includes the sequence of SEQ ID NO: 27.

[0014] The present invention also relates to nucleic acid molecules encoding the functionally shortened GDE polypeptide of the present invention.

[0015] The present invention - Promoter; - Introns as needed; - Nucleic acid molecules according to the claims of the present invention; and - Polyadenylation signal The present invention also relates to an expression cassette that preferably contains these in this order.

[0016] Another aspect of the present invention relates to a vector comprising a nucleic acid molecule or expression cassette of the present invention, particularly a viral vector. In a particular embodiment, the vector is an AAV vector.

[0017] The present invention also relates to isolated cells transformed with the nucleic acid molecule, expression cassette, or vector of the present invention, wherein the cells are, in particular, liver cells, muscle cells, cardiac cells, or CNS cells.

[0018] The present invention also relates to functional truncated GDE polypeptides, nucleic acid molecules, expression cassettes, vectors, or cells as defined above for use as pharmaceuticals. The present invention also relates to functional truncated GDE polypeptides, nucleic acid molecules, expression cassettes, vectors, or cells as defined above for use in methods for treating diseases caused by mutations in the AGL gene encoding GDE. In a further specific embodiment, the present invention relates to functional truncated GDE polypeptides, nucleic acid molecules, expression cassettes, vectors, or cells as defined above for use in methods for treating GSDIII (German disease of Coli). [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows the N-terminal region of the GDE enzyme in Candida glabrata (top) and human (bottom). The shortened sites of the "Δ1b2" and "Δ1b3" proteins, as well as the shortened sites of the shortened proteins ("Δ1b9" to "Δ1b15"), are shown. [Figure 2] (A) This figure shows the experimental design for intramuscular injection of an rAAV vector containing shortened GDE ("Δ1b9" to "Δ1b15") into GSDIII mice. The shortened "Δ1b3" protein and full-length GDE (FS) were used as controls. Tissue analysis was performed one month after intramuscular injection. KO (Agl- / -) and WT (Agl+ / +) mice were injected with saline (PBS) as a negative control. (B) This figure shows the Western blot analysis of GDE and vinculin expression from left tibialis anterior muscle (TA) lysate one month after intramuscular injection of an rAAV vector containing shortened GDE polypeptide. (C) This figure shows the quantification of GDE protein expression based on the Western blot in Figure 2(B). [Figure 3] (A) This figure shows the experimental design for intravenous injection of rAAV vectors containing shortened forms of GDE ("Δ1b3" and "Δ1b13") into GSDIII mice. Tissue analysis was performed 2 months after intravenous injection. KO (Agl- / -) and WT (Agl+ / +) mice were injected with saline (PBS) as a negative control. (B) This figure shows the glycogen content in quadriceps muscle lysate 2 months after intravenous injection of rAAV vectors containing shortened GDE polypeptides. [Modes for carrying out the invention]

[0020] In patent applications WO2020 / 030661 and WO2022 / 043280, the inventors demonstrated that a shortened GDE polypeptide having a size suitable for encapsulation in a capsid in an AAV vector while retaining its enzymatic activity can be used.

[0021] Despite lacking knowledge of the three-dimensional structure of GDE proteins, the inventors identified a novel N-terminal truncated GDE polypeptide with high protein expression levels and therefore potentially good efficacy in vivo.

[0022] Therefore, the present invention relates to a functional N-terminal truncated GDE polypeptide. This polypeptide can be advantageously used in methods for treating diseases caused by mutations in the AGL gene encoding GDE, particularly in methods for treating GSDIII (German disease of Coli).

[0023] 1 - N-terminal truncated GDE polypeptide The truncated GDE polypeptide according to the present invention is a functional GDE polypeptide whose coding sequence is small enough to be efficiently packaged into a gene therapy vector, particularly into a single AAV vector.

[0024] A "functional" GDE polypeptide means a polypeptide that retains at least one, preferably all, of the enzymatic activities of the GDE protein. As a result, the functional GDE polypeptides realized by the present invention can restore glycogen storage and muscle strength in vivo. As defined herein, GDE enzymatic activity is 4-alpha-glucotransferase activity and amylo-1,6-glucosidase activity, which are involved in glycogenolysis. The transferase activity of GDE transfers three glucose units of glycogen from one chain to another. This leaves one glucose unit at the branching point, which is then released as glucose by glucosidase activity. In certain embodiments, the functional GDE polypeptides of the present invention have the same functionality as full-length GDE polypeptides, and in particular, full-length human GDE polypeptides. 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% of the enzyme activity of one, preferably both, of the above-mentioned proteins, or at least 100% of the activity of the full-length human GDE protein, particularly 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 exceed 100% of the activity of the full-length human GDE protein, particularly 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, exceeding 110%, 120%, 130%, 140%, 150%, 200%, 500%, 700%, or even exceeding 1000%. In certain embodiments, the functional GDE polypeptide of the present invention has the same functionality as a full-length GDE polypeptide, and in particular, as a full-length human GDE polypeptide in muscle tissue such as the heart or quadriceps muscle.For example, the functional GDE polypeptide of the present invention may have at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the enzyme activity of one, preferably both, of the above-mentioned proteins in muscle tissue such as the heart or quadriceps, or at least 100% of the activity of full-length human GDE protein, particularly 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 in muscle tissue such as the heart or quadriceps may exceed 100% of the activity of full-length human GDE protein, particularly 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, exceeding 110%, 120%, 130%, 140%, 150%, 200%, 500%, 700%, or even exceeding 1000%.

[0025] The term "functional" GDE polypeptide also refers to non-pathological GDE polypeptides. In particular, the functional GDE polypeptide of the present invention is not the GDE polypeptide found in patients with GSDIII (GSDIII b), such as GSDIIIa or GSDIIIb.

[0026] Those skilled in the art can easily determine whether a polypeptide is a functional GDE polypeptide. Preferred methods will be obvious to those skilled in the art. For example, one preferred in vitro method involves inserting a nucleic acid encoding the polypeptide into a vector, e.g., a plasmid or viral vector; transfecting or transducing the vector into a host cell, e.g., 293T or HeLa cells, or other cells, e.g., Huh7; and assaying the GDE activity. Alternatively, GDE activity can be determined by measuring the glucose produced after incubating a homogenized tissue or cell extract pre-transfected with a vector expressing a functional GDE polypeptide with limiting dextrin (glycogen digested by glycogen phosphorylase). Other methods include testing the efficacy of GDE by evaluating the recovery of glycogen storage in muscle and / or cardiac tissue by assessing the muscle strength of treated GDE-KO animals by wire hang after vector administration, for example, 1, 2, or 3 months after administration, and / or by evaluating the normalization of blood glucose in treated GDE-KO animals after vector administration, for example, 1, 2, or 3 months after administration. Furthermore, GDE expression can be evaluated by Western blotting in the tissues of GDE-KO animals after vector administration, for example, 1, 2, or 3 months after administration. Some preferred methods are described in more detail in the experimental section below.

[0027] In relation to the present invention, the "reference full-length human GDE sequence" encompasses all native isoforms of human GDE. Bao and colleagues (Genomics, 1997, 38, pp. 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.

[0028] With respect to the present invention, the "reference full-length human GDE sequence" does not encode a pathogenic GDE polypeptide. In particular, the reference full-length human GDE sequence does not encode pathogenic variants, including mutations, deletions, or insertions, that are found in patients with GSDIII, compared to a wild-type non-pathological full-length GDE sequence.

[0029] Therefore, the term "reference full-length human GDE polypeptide" includes not only all native isoforms of human GDE, including precursor forms, but also functional derivatives of GDE, such as GDE proteins or fragments thereof that have been modified or mutated by insertion, deletion, and / or substitution. In particular, the reference full-length human GDE sequence is selected from the group consisting of 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).

[0030] In certain embodiments, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent 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, and in particular with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In certain embodiments, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4, and has the same length as SEQ ID NO: 1 or SEQ ID NO: 4 in terms of the number of amino acids. In certain embodiments, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity with SEQ ID NO: 2 or SEQ ID NO: 5, and has the same length as SEQ ID NO: 2 or SEQ ID NO: 5 in terms of the number of amino acids. In certain embodiments, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity with SEQ ID NO: 3 or SEQ ID NO: 6, and has the same length as SEQ ID NO: 3 or SEQ ID NO: 6 in terms of the number of amino acids.

[0031] The term "identical" and its variations refer to sequence identity between two nucleic acid molecules or between two polypeptide molecules. If the same base or amino acid occupies the same position in both of the two sequences being compared, then those molecules are identical at that position. The percentage of identity between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared × 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences are 60% identical. Generally, comparison is performed by aligning the two sequences to obtain the greatest possible identity. Various bioinformatics tools known to those skilled in the art, such as BLAST or FASTA, can be used to align nucleic acid sequences.

[0032] In certain embodiments, the reference full-length human GDE sequence has the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, in particular SEQ ID NO: 1, which corresponds to GDE isoform 1.

[0033] In certain embodiments, the shortened GDE polypeptide of the present invention, which is shortened relative to a reference full-length human GDE sequence, may include one or more additional amino acid modifications relative to the reference full-length human GDE sequence. In particular, in addition to the deletions described further below, the functional shortened GDE polypeptide may include one or more amino acid modifications, e.g., amino acid insertions, deletions, and / or substitutions, relative to the reference full-length human GDE sequence. For example, the functional shortened GDE polypeptide may include 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, provided that the functionality of the shortened GDE polypeptide is preserved.

[0034] The functionally shortened GDE polypeptide of the present invention is an N-terminally shortened GDE polypeptide. An "N-terminally shortened GDE polypeptide" means a GDE polypeptide that contains a deletion relative to a reference functional full-length human GDE sequence, wherein the deletion consists of an amino acid deletion at the N-terminus of the reference functional full-length human GDE sequence. The "N-terminus" of the reference functional full-length human GDE sequence refers to a region consisting of the first 280 amino acid residues (i.e., the 280 amino acid residues at the very N-terminus of the reference full-length GDE sequence), the first 200 amino acid residues (i.e., the 200 amino acid residues at the very N-terminus of the reference full-length GDE sequence), the first 150 amino acid residues (i.e., the 150 amino acid residues at the very N-terminus of the reference full-length GDE sequence), preferably the first 125 amino acid residues (i.e., the 125 amino acid residues at the very N-terminus of the reference full-length GDE sequence), and most preferably the first 123 amino acid residues (i.e., the 123 amino acid residues at the very N-terminus of the reference full-length GDE sequence).

[0035] As detailed below, the N-terminal truncated GDE polypeptide of the present invention retains methionine as the first residue at the N-terminal end.

[0036] The functionally truncated GDE polypeptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence, wherein the deletion is in the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide. - MQYYFL (array 7); - MFLQGN (sequence number 8); - MQGNEK (Sequence ID 9); - MGNEKS (Sequence ID 10); - MNEKSG (sequence number 11); - MKSGGG (sequence number 12); or - MSGGGY (Sequence ID 13) This consists of an amino acid deletion at the N-terminus of the reference functional full-length human GDE sequence.

[0037] Preferably, the functionally shortened GDE polypeptide of the present invention contains a deletion from a reference functional full-length human GDE sequence, wherein the deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence such that the first six amino acids of the N-terminus of the functionally shortened GDE polypeptide become MNEKSG (SEQ ID NO: 11).

[0038] In other words, a deletion of amino acids at the N-terminus of a reference functional full-length human GDE results in a shortened GDE polypeptide in which the first six amino acids at the N-terminus are different from the first six amino acids at the N-terminus of the reference functional full-length human GDE sequence, and the first six amino acids at the N-terminus of the shortened polypeptide are sequence numbers 7, 8, 9, 10, 11, 12, or 13.

[0039] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence has an amino acid sequence selected from the group consisting 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 deletion is due to the first six amino acids at the N-terminus of the functionally truncated GDE polypeptide. - MQYYFL (array 7); - MFLQGN (sequence number 8); - MQGNEK (Sequence ID 9); - MGNEKS (Sequence ID 10); - MNEKSG (sequence number 11); - MKSGGG (sequence number 12); or - MSGGGY (Sequence ID 13) This consists of an amino acid deletion at the N-terminus of the reference functional full-length human GDE sequence.

[0040] In preferred embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence has an amino acid sequence selected from the group consisting 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 deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally shortened GDE polypeptide become MNEKSG (SEQ ID NO: 11).

[0041] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence has an amino acid sequence selected from the group consisting 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 aforementioned deletion is, (i) Deletion of any amino acid between the first methionine and the sequence "QYYFL" (SEQ ID NO: 66); (ii) Deletion of any amino acid between the first methionine and the sequence "FLQGN" (SEQ ID NO: 67); (iii) Deletion of any amino acid between the first methionine and the sequence "QGNEK" (SEQ ID NO: 68); (iv) Deletion of any amino acid between the first methionine and the sequence "GNEKS" (SEQ ID NO: 69); (v) Deletion of any amino acid between the first methionine and the sequence "NEKSG" (SEQ ID NO: 70); (vi) Deletion of any amino acid between the first methionine and the sequence "KSGGG" (SEQ ID NO: 71); (vii) Deletion of any amino acid between the first methionine and the sequence "SGGGY" (SEQ ID NO: 72) It consists of.

[0042] According to this embodiment, a deletion consisting of the deletion of any amino acid between the first methionine and the sequence "QYYFL" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "QYYFL" are deleted, but the first methionine and the sequence "QYYFL" (SEQ ID NO: 66) are not deleted.

[0043] According to this embodiment, a deletion consisting of the deletion of any amino acid between the first methionine and the sequence "FLQGN" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "FLQGN" are deleted, but the first methionine and the sequence "FLQGN" (SEQ ID NO: 67) are not deleted.

[0044] According to this embodiment, a deletion consisting of the deletion of any amino acid between the first methionine and the sequence "QGNEK" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "QGNEK" are deleted, but the first methionine and the sequence "QGNEK" (SEQ ID NO: 68) are not deleted.

[0045] According to this embodiment, a deletion consisting of the deletion of any amino acid between the first methionine and the sequence "GNEKS" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "GNEKS" are deleted, but the first methionine and the sequence "GNEKS" (SEQ ID NO: 69) are not deleted.

[0046] According to this embodiment, a deletion consisting of the deletion of any amino acid between the first methionine and the sequence "NEKSG" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "NEKSG" are deleted, but the first methionine and the sequence "NEKSG" (SEQ ID NO: 70) are not deleted.

[0047] According to this embodiment, a deletion consisting of the deletion of any amino acid between the first methionine and the sequence "KSGGG" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "KSGGG" are deleted, but the first methionine and the sequence "KSGGG" (SEQ ID NO: 71) are not deleted.

[0048] According to this embodiment, a deletion consisting of the deletion of any amino acid between the first methionine and the sequence "SGGGY" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "SGGGY" are deleted, but the first methionine and the sequence "SGGGY" (SEQ ID NO: 72) are not deleted.

[0049] In preferred embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence has an amino acid sequence selected from the group consisting 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 deletion consists of a deletion of any amino acid between the first methionine and the sequence "NEKSG" (SEQ ID NO: 70).

[0050] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence has the amino acids 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. - The aforementioned deletion is, (i) The amino acid is missing from positions 2 to 91, and the amino acids at positions 1 and 92 are not missing; (ii) The amino acid is missing from positions 2 to 94, and the amino acids at positions 1 and 95 are not missing. (iii) The amino acid is missing from positions 2 to 96, and the amino acids at positions 1 and 97 are not missing; (iv) The amino acid deletion consists of positions 2-97, and the amino acids at positions 1 and 98 are not deleted; (v) The amino acid deletion consists of positions 2-98, and the amino acids at positions 1 and 99 are not deleted; (vi) Consists of amino acid deletions between positions 2 and 100, with amino acids at positions 1 and 101 being present or (vii) Consists of amino acid deletions from positions 2 to 101, with amino acids at positions 1 and 102 being present.

[0051] In preferred embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence has the amino acids 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. - The aforementioned deletions consist of amino acid deletions between positions 2 and 98, while amino acids at positions 1 and 99 are not deleted.

[0052] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises one and one deletion at the N-terminus of a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The N-terminus corresponds to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence. - The deletion at the N-terminus of the reference functional full-length human GDE sequence is, (i) Deletion of amino acids at positions 2-91 of the reference functional full-length human GDE sequence; (ii) Deletion of amino acids at positions 2-94 of the reference functional full-length human GDE sequence; (iii) Deletion of amino acids at positions 2-96 of the reference functional full-length human GDE sequence; (iv) Deletion of amino acids at positions 2-97 of the reference functional full-length human GDE sequence; (v) Deletion of amino acids at positions 2-98 of the reference functional full-length human GDE sequence; (vi) Deletion of an amino acid between positions 2 and 100 of the reference functional full-length human GDE sequence; or (vii) Deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence. It consists of.

[0053] In preferred embodiments, the functionally shortened GDE polypeptide of the present invention comprises one and one deletion at the N-terminus of a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The N-terminus corresponds to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence. - The deletion at the N-terminus of the reference functional full-length human GDE sequence consists of a deletion of amino acids between positions 2 and 98 of the reference functional full-length human GDE sequence.

[0054] "One and single deletions" means that there is a single deletion in the N-terminal region corresponding to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence (i.e., the 280 amino acid residues at the very N-terminus of the reference full-length GDE sequence). For clarity, functional truncated GDEs containing one and single deletions of amino acids between positions 2 and 91 in the N-terminal region of the reference functional full-length human GDE sequence are: - The amino acids at positions 2-91 of the reference functional full-length sequence are deleted. - The amino acids at position 1 and positions 92-280 are not deleted. Supports shortened polypeptides.

[0055] In certain embodiments, the functionally truncated GDE polypeptide of the present invention comprises one and one deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence was selected from the group consisting of SEQ ID NOs: 1 to 6. - The deletions are referred to as Δ1b9, Δ1b10, Δ1b11, Δ1b12, Δ1b13, Δ1b14, or Δ1b15 in Table 1:

[0056] [Table 1]

[0057] To clarify, 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 "Δ1b9" truncated GDE polypeptide corresponds to a functional truncated GDE polypeptide derived from SEQ ID NO: 1 or SEQ ID NO: 4, in which all consecutive amino acids from positions 2 to 91 are deleted compared to SEQ ID NO: 1 or SEQ ID NO: 4.

[0058] "Δ1b9" truncated polypeptide In certain embodiments, the functionally truncated GDE polypeptide of the present invention comprises a deletion from a reference functional full-length human GDE sequence, wherein the deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become MQYYFL (SEQ ID NO: 7).

[0059] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - The deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MQYYFL" (SEQ ID NO: 7).

[0060] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-91 of the reference functional full-length GDE sequence. - The amino acids at position 1 and 92 of the reference functional full-length GDE sequence are not deleted.

[0061] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-91 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 92-110, 92-130, 92-150, 92-170, 92-190, 92-210, 92-230, 92-250, 92-270, or 92-280 of the reference functional full-length GDE sequence are not deleted.

[0062] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-91 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 92-280 of the reference functional full-length GDE sequence are not deleted.

[0063] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-91 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence "MQYYFL" (SEQ ID NO: 7).

[0064] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-91 of the reference functional full-length human GDE sequence. - The aforementioned functionally shortened GDE polypeptide contains the sequence "MQYYFL" (SEQ ID NO: 7), - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0065] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-91 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence of SEQ ID NO: 21.

[0066] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-91 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide comprises the sequence of sequence number 21, - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0067] In certain embodiments, the functionally truncated GDE polypeptide includes or comprises SEQ ID NO: 14, or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 14, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 14.

[0068] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 14, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 14 has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 14, in particular, the same enzymatic activity involved in glycogenolysis in muscle tissue such as the heart or quadriceps. In certain embodiments, a functional variant of SEQ ID NO: 14 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 14 to restore glycogen storage and muscle strength in vivo. In certain embodiments, a functional variant of SEQ ID NO: 14 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 14.

[0069] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 14, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 14, has the same N-terminus as SEQ ID NO: 14. In certain embodiments, the N-terminal amino acid of the functional variant of SEQ ID NO: 14 is "MQYYFL" (SEQ ID NO: 7). In another particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 14 corresponds to the sequence of SEQ ID NO: 21.

[0070] In certain embodiments, the N-terminal amino acid of the functional variant of SEQ ID NO: 14 may consist of amino acids at positions 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 contain any mutations, insertions, or deletions within 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, or 1-200 of SEQ ID NO: 14. In a particular 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 contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0071] In a preferred embodiment, the functionally shortened GDE polypeptide comprises or consists of SEQ ID NO: 14.

[0072] "Δ1b10" shortened polypeptide In certain embodiments, the functionally truncated GDE polypeptide of the present invention comprises a deletion from a reference functional full-length human GDE sequence, the deletion consisting of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MFLQGN" (SEQ ID NO: 8).

[0073] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - The deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MFLQGN" (SEQ ID NO: 8).

[0074] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2-94 of the reference functional full-length GDE sequence. - The amino acids at position 1 and 95 of the reference functional full-length GDE sequence are not deleted.

[0075] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-94 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 95-110, 95-130, 95-150, 95-170, 95-190, 95-210, 95-230, 95-250, 95-270, or 95-280 of the reference functional full-length GDE sequence are not deleted.

[0076] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-94 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 95-280 of the reference functional full-length GDE sequence are not deleted.

[0077] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-94 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide contains the sequence "MFLQGN" (SEQ ID NO: 8).

[0078] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-94 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide comprises the sequence "MFLQGN" (SEQ ID NO: 8), - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0079] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-94 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence of sequence number 22.

[0080] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-94 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide comprises the sequence of sequence number 22, - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0081] In certain embodiments, the functionally truncated GDE polypeptide includes or comprises SEQ ID NO: 15, or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 15, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 15.

[0082] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 15, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 15 has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 15, in particular, the same enzymatic activity involved in glycogenolysis in muscle tissue such as the heart or quadriceps. In certain embodiments, a functional variant of SEQ ID NO: 15 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 15 to restore glycogen storage and muscle strength in vivo. In certain embodiments, a functional variant of SEQ ID NO: 15 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 15.

[0083] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 15, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 15, has the same N-terminus as SEQ ID NO: 15. In certain embodiments, the N-terminal amino acid of the functional variant of SEQ ID NO: 15 is "MFLQGN" (SEQ ID NO: 8). In another particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 15 corresponds to the sequence of SEQ ID NO: 22.

[0084] In a particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 15 may 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 contain any mutations, insertions, or deletions within 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, or 1-200 of SEQ ID NO: 15. In a particular 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 contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0085] In a preferred embodiment, the functionally shortened GDE polypeptide includes or comprises SEQ ID NO: 15.

[0086] "Δ1b11" shortened polypeptide In certain embodiments, the functionally truncated GDE polypeptide of the present invention comprises a deletion from a reference functional full-length human GDE sequence, the deletion consisting of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MQGNEK" (SEQ ID NO: 9).

[0087] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - The deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MQGNEK" (SEQ ID NO: 9).

[0088] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-96 of the reference functional full-length GDE sequence. - The amino acids at position 1 and 97 of the reference functional full-length GDE sequence are not deleted.

[0089] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-96 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 97-130, 97-150, 97-170, 97-190, 97-210, 97-230, 97-250, 97-270, or 97-280 of the reference functional full-length GDE sequence are not deleted.

[0090] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-96 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 97-280 of the reference functional full-length GDE sequence are not deleted.

[0091] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-96 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide contains the sequence "MQGNEK" (SEQ ID NO: 9).

[0092] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-96 of the reference functional full-length human GDE sequence. - The aforementioned functional truncated GDE polypeptide contains the sequence "MQGNEK" (SEQ ID NO: 9), - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0093] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-96 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence of Sequence ID No. 23.

[0094] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-96 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide comprises the sequence of sequence number 23, - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0095] In certain embodiments, the functionally truncated GDE polypeptide includes or comprises SEQ ID NO: 16, or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 16, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 16.

[0096] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 16, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 16, has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 16, in particular, the same enzymatic activity involved in glycogenolysis in muscle tissue such as the heart or quadriceps. In certain embodiments, a functional variant of SEQ ID NO: 16 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 16 to restore glycogen storage and muscle strength in vivo. In certain embodiments, a functional variant of SEQ ID NO: 16 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 16.

[0097] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 16, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 16, has the same N-terminus as SEQ ID NO: 16. In certain embodiments, the N-terminal amino acid of the functional variant of SEQ ID NO: 16 is "MQGNEK" (SEQ ID NO: 9). In another particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 16 corresponds to the sequence of SEQ ID NO: 23.

[0098] In a further embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 16 may 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 contain any mutations, insertions, or deletions within 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, or 1-200 of SEQ ID NO: 16. In a particular 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 contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0099] In a preferred embodiment, the functionally shortened GDE polypeptide includes or comprises SEQ ID NO: 16.

[0100] "Δ1b12" shortened polypeptide In certain embodiments, the functionally truncated GDE polypeptide of the present invention comprises a deletion from a reference functional full-length human GDE sequence, wherein the deletion consists of a deletion of amino acids in the N-terminal region of the reference functional full-length human GDE sequence such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MGNEKS" (SEQ ID NO: 10).

[0101] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - The deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally shortened GDE polypeptide become "MGNEKS" (SEQ ID NO: 10).

[0102] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2-97 of the reference functional full-length GDE sequence. - The amino acids at position 1 and 98 of the reference functional full-length GDE sequence are not deleted.

[0103] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-97 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 98-130, 98-150, 98-170, 98-190, 98-210, 98-230, 98-250, 98-270, or 98-280 of the reference functional full-length GDE sequence are not deleted.

[0104] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-97 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 98-280 of the reference functional full-length GDE sequence are not deleted.

[0105] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-97 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence "MGNEKS" (SEQ ID NO: 10).

[0106] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-97 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence "MGNEKS" (SEQ ID NO: 10), - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0107] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-97 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence of sequence number 24.

[0108] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-97 of the reference functional full-length human GDE sequence. - The functional truncated GDE polypeptide comprises the sequence of sequence number 24, - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0109] In certain embodiments, the functionally truncated GDE polypeptide includes or comprises SEQ ID NO: 17, or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 17, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 17.

[0110] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 17, for example at least 75% or at least 80%, for example 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 with SEQ ID NO: 17, has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 17, in particular the same enzymatic activity involved in glycogenolysis in muscle tissue such as the heart or quadriceps. In certain embodiments, a functional variant of SEQ ID NO: 17 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 17 to restore glycogen storage and muscle strength in vivo. In certain embodiments, a functional variant of SEQ ID NO: 17 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 17.

[0111] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 17, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 17, has the same N-terminus as SEQ ID NO: 17. In certain embodiments, the N-terminal amino acid of the functional variant of SEQ ID NO: 17 is "MGNEKS" (SEQ ID NO: 10). In another particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 17 corresponds to the sequence of SEQ ID NO: 24.

[0112] In a further embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 17 may 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: 17. In other words, according to this embodiment, the functional variant of SEQ ID NO: 17 does not contain any mutations, insertions, or deletions within 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, or 1-200 of SEQ ID NO: 17. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 17 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 17. In other words, according to this embodiment, the functional variant of SEQ ID NO: 17 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0113] In a preferred embodiment, the functionally shortened GDE polypeptide includes or comprises SEQ ID NO: 17.

[0114] "Δ1b13" shortened polypeptide In certain embodiments, the functionally truncated GDE polypeptide of the present invention comprises a deletion from a reference functional full-length human GDE sequence, wherein the deletion consists of a deletion of amino acids in the N-terminal region of the reference functional full-length human GDE sequence such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MNEKSG" (SEQ ID NO: 11).

[0115] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - The deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally shortened GDE polypeptide become "MNEKSG" (SEQ ID NO: 11).

[0116] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2-98 of the reference functional full-length GDE sequence. - The amino acids at position 1 and 99 of the reference functional full-length GDE sequence are not deleted.

[0117] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-98 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 99-130, 99-150, 99-170, 99-190, 99-210, 99-230, 99-250, 99-270, or 99-280 of the reference functional full-length GDE sequence are not deleted.

[0118] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-98 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 99-280 of the reference functional full-length GDE sequence are not deleted.

[0119] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-98 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence "MNEKSG" (SEQ ID NO: 11).

[0120] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-98 of the reference functional full-length human GDE sequence. - The aforementioned functionally shortened GDE polypeptide contains the sequence "MNEKSG" (SEQ ID NO: 11), - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0121] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-98 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence of SEQ ID NO: 25.

[0122] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of amino acid deletions at positions 2-98 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide comprises the sequence of sequence number 25, - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0123] In certain embodiments, the functionally truncated GDE polypeptide includes or comprises SEQ ID NO: 18, or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 18, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 18.

[0124] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 18, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 18, has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 18, in particular, the same enzymatic activity involved in glycogenolysis in muscle tissue such as the heart or quadriceps. In certain embodiments, a functional variant of SEQ ID NO: 18 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 18 to restore glycogen storage and muscle strength in vivo. In certain embodiments, a functional variant of SEQ ID NO: 18 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 18.

[0125] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 18, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 18, has the same N-terminus as SEQ ID NO: 18. In certain embodiments, the N-terminal amino acid of the functional variant of SEQ ID NO: 18 is "MNEKSG" (SEQ ID NO: 11). In another particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 18 corresponds to the sequence of SEQ ID NO: 25.

[0126] In a further embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 18 may 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: 18. In other words, according to this embodiment, the functional variant of SEQ ID NO: 18 does not contain any mutations, insertions, or deletions within 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, or 1-200 of SEQ ID NO: 18. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 18 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 18. In other words, according to this embodiment, the functional variant of SEQ ID NO: 18 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0127] In a preferred embodiment, the functionally shortened GDE polypeptide includes or comprises SEQ ID NO: 18.

[0128] "Δ1b14" shortened polypeptide In certain embodiments, the functionally truncated GDE polypeptide of the present invention comprises a deletion from a reference functional full-length human GDE sequence, wherein the deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MKSGGG" (SEQ ID NO: 12).

[0129] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - The deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally shortened GDE polypeptide become "MKSGGG" (SEQ ID NO: 12).

[0130] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 100 of the reference functional full-length GDE sequence. - The amino acids at position 1 and 101 of the reference functional full-length GDE sequence are not deleted.

[0131] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 100 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 101-130, 101-150, 101-170, 101-190, 101-210, 101-230, 101-250, 101-270, or 101-280 of the reference functional full-length GDE sequence are not deleted.

[0132] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 100 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 101-280 of the reference functional full-length GDE sequence are not deleted.

[0133] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 100 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence "MKSGGG" (SEQ ID NO: 12).

[0134] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 100 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence "MKSGGG" (SEQ ID NO: 12), - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0135] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 100 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence of SEQ ID NO: 26.

[0136] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 100 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide comprises the sequence of sequence number 26, - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0137] In certain embodiments, the functionally truncated GDE polypeptide includes or comprises SEQ ID NO: 19, or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 19, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 19.

[0138] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 19, for example at least 75% or at least 80%, for example 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 with SEQ ID NO: 19, has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 19, in particular the same enzymatic activity involved in glycogenolysis in muscle tissue such as the heart or quadriceps. In certain embodiments, a functional variant of SEQ ID NO: 19 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 19 to restore glycogen storage and muscle strength in vivo. In certain embodiments, a functional variant of SEQ ID NO: 19 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 19.

[0139] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 19, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 19, has the same N-terminus as SEQ ID NO: 19. In certain embodiments, the N-terminal amino acid of the functional variant of SEQ ID NO: 19 is "MKSGGG" (SEQ ID NO: 12). In another particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 19 corresponds to the sequence of SEQ ID NO: 26.

[0140] In a further embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 19 may 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: 19. In other words, according to this embodiment, the functional variant of SEQ ID NO: 19 does not contain any mutations, insertions, or deletions within 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, or 1-200 of SEQ ID NO: 19. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 19 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 19. In other words, according to this embodiment, the functional variant of SEQ ID NO: 19 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0141] In a preferred embodiment, the functionally shortened GDE polypeptide includes or comprises SEQ ID NO: 19.

[0142] "Δ1b15" shortened polypeptide In certain embodiments, the functionally truncated GDE polypeptide of the present invention comprises a deletion from a reference functional full-length human GDE sequence, the deletion consisting of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MSGGGY" (SEQ ID NO: 13).

[0143] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - The deletion consists of an amino acid deletion in the N-terminal region of the reference functional full-length human GDE sequence, such that the first six amino acids of the N-terminus of the functionally truncated GDE polypeptide become "MSGGGY" (SEQ ID NO: 13).

[0144] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 101 of the reference functional full-length GDE sequence. - The amino acids at position 1 and position 102 of the reference functional full-length GDE sequence are not deleted.

[0145] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 101 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 102-130, 102-150, 102-170, 102-190, 102-210, 102-230, 102-250, 102-270, or 102-280 of the reference functional full-length GDE sequence are not deleted.

[0146] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 101 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted. - The amino acids at positions 102-280 of the reference functional full-length GDE sequence are not deleted.

[0147] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 101 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence "MSGGGY" (SEQ ID NO: 13).

[0148] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 101 of the reference functional full-length human GDE sequence. - The aforementioned functional truncated GDE polypeptide contains the sequence "MSGGGY" (SEQ ID NO: 13), - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0149] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 101 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide contains the sequence of sequence number 27.

[0150] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is either 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. - The deletion consists of a deletion of amino acids at positions 2 to 101 of the reference functional full-length human GDE sequence. - The functionally shortened GDE polypeptide comprises the sequence of sequence number 27, - The functionally shortened GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0151] In certain embodiments, the functionally truncated GDE polypeptide includes or comprises SEQ ID NO: 20, or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 20, for example, at least 75% or at least 80% sequence identity, for example, 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 with SEQ ID NO: 20.

[0152] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 20, such as at least 75% or at least 80% sequence identity, for example 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 with SEQ ID NO: 20, has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 20, particularly the same enzymatic activity involved in glycogenolysis in muscle tissues such as the heart or quadriceps muscle. In certain embodiments, the functional variant of SEQ ID NO: 20 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 20 to restore glycogen accumulation and muscle strength in vivo. In certain embodiments, the functional variant of SEQ ID NO: 20 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 20.

[0153] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 20, such as at least 75% or at least 80% sequence identity, for example 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 with SEQ ID NO: 20, has the same N-terminal portion as SEQ ID NO: 20. In certain embodiments, the most N-terminal amino acid of the functional variant of SEQ ID NO: 20 is "MSGGGY" (SEQ ID NO: 13). In another particular embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 20 corresponds to the sequence of SEQ ID NO: 27.

[0154] In a further embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 20 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, 1-200 of SEQ ID NO: 20. In other words, according to this embodiment, the functional variant of SEQ ID NO: 20 does not contain any mutations, insertions or deletions within 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: 20. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 20 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 20. In other words, according to this embodiment, the functional variant of SEQ ID NO: 20 does not contain any mutations, insertions or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0155] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 20.

[0156] Combined with other deletions In a particular embodiment, the functional truncated GDE polypeptide of the present invention as described above, which contains a deletion in the N-terminal part of the reference functional full-length human GDE sequence, may further contain a deletion or a combination of deletions in other parts of the reference functional full-length human GDE sequence.

[0157] In a particular embodiment, the functional truncated GDE polypeptide - is a deletion of only one and only one of the consecutive amino acids 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, - another deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence and includes.

[0158] In certain embodiments, the functionally shortened GDE polypeptide is - Deletions of one or a single consecutive amino acid in the N-terminus of the reference functional full-length human GDE sequence, wherein the N-terminus corresponds to the first 280 amino acid residues of the reference functional full-length human GDE sequence, - Another deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence Includes, The reference functional full-length human GDE sequence was selected from the group consisting of SEQ ID NOs: 1 to 6. The deletions are referred to as Δ1b9, Δ1b10, Δ1b11, Δ1b12, Δ1b13, Δ1b14, or Δ1b15 in Table 1.

[0159] In certain embodiments, the functionally shortened GDE polypeptide of the present invention comprises at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 related to SEQ ID NO: 1 or SEQ ID NO: 4.

[0160] In certain embodiments, the functionally shortened GDE polypeptide of the present invention, which includes a deletion in the N-terminus of a reference functional full-length human GDE sequence, may further include a deletion or a combination of deletions in the C-terminus and / or in the central domain of the reference functional full-length GDE sequence.

[0161] In certain embodiments, the C-terminal region of the reference functional full-length GDE sequence corresponds to the region consisting of the last 112 amino acids, i.e., the 112 amino acids at the very C-terminus of the reference functional full-length human GDE sequence.

[0162] In certain embodiments, the central domain of the full-length reference GDE sequence of SEQ ID NO: 1 or SEQ ID NO: 4 corresponds to the region consisting of amino acids from positions 710 to 865 of SEQ ID NO: 1 or SEQ ID NO: 4.

[0163] In certain embodiments, the functionally shortened GDE polypeptide is - A single deletion and a single deletion in the N-terminus of the reference functional full-length human GDE sequence, wherein the N-terminus corresponds to a region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence, - Another deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence Includes, The reference functional full-length human GDE sequence was selected from the group consisting of SEQ ID NOs: 1 to 6. Deletions at the N-terminus are referred to as Δ1b9, Δ1b10, Δ1b11, Δ1b12, Δ1b13, Δ1b14, or Δ1b15 in Table 1; Additional deletions or combinations of deletions in other parts of the reference functional full-length human GDE sequence are selected from any of the deletions designated Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, and Δ7 in Table 2:

[0164] [Table 2]

[0165] 2 - Nucleic acid molecules encoding N-terminal truncated GDE polypeptides In another embodiment, the present invention relates to a nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above.

[0166] The term "nucleic acid molecule" (or nucleic acid sequence) refers to single-stranded or double-stranded DNA or RNA molecules, in particular DNA encoding a functionally truncated GDE polypeptide according to the present invention.

[0167] In preferred embodiments, 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 an appropriate regulatory sequence. The size of the nucleic acid molecule encoding the functional truncated GDE polypeptide is preferably less than about 4.5 kb, and more preferably less than about 4.4 kb.

[0168] The "gene therapy vector" means any vector suitable for gene therapy. In particular, the gene therapy vector can be a plasmid or a recombinant virus, for example, a viral vector derived from a retrovirus or a lentivirus. Preferably, the viral vector is an AAV vector, for example, an AAV vector suitable for transduction into liver tissue or muscle cells. Extensive experience in clinical trials and preclinical models of muscle diseases indicates that adeno-associated virus (AAV) is the optimal vector for in vivo gene therapy for GSDIII. These vectors efficiently transduce the liver and muscle, their production can be scaled up and down, and compared to other gene therapy vectors, they have a relatively low immunogenic profile. However, one of the biggest limitations in the use of AAV for gene replacement is their limited capsid packaging size limit (about 5 kb). In fact, during the production of recombinant AAV, genomes larger than 5 kb are often packaged into capsids with low efficiency, and the resulting AAV may contain fragmented genomes, thereby reducing the effectiveness of gene transfer.

[0169] The sequences of the nucleic acid molecules of the present invention encoding functional truncated GDE polypeptides can be optimized for in vivo GDE polypeptide expression. Sequence optimization can involve numerous changes in the nucleic acid sequence, including codon optimization, increased GC content, decreased CpG island number, decreased alternative open reading frame (ARF) number, and / or decreased splice donor and splice acceptor site number. Due to the degeneracy of the genetic code, different nucleic acid molecules may encode the same protein. It is also well known that the genetic codes of different organisms are often biased towards the use of one of several codons encoding the same amino acid compared to the use of others. Codon optimization introduces changes into the nucleotide sequence that take advantage of the codon bias present in a given cellular situation, so that the resulting codon-optimized nucleotide sequence is more likely to be expressed at a relatively high level in such a given cellular situation compared to a non-codon-optimized sequence. In preferred embodiments of the present invention, such optimized nucleotide sequences encoding a functional truncated GDE polypeptide are codon-optimized to enhance their expression in human cells compared to uncodon-optimized nucleotide sequences encoding the same functional truncated GDE polypeptide, for example, by effectively utilizing human-specific codon usage frequency bias. The nucleic acid sequence encoding full-length human GDE isoform 1 is shown in SEQ ID NO: 51. An example of the corresponding codon-optimized sequence is shown in SEQ ID NO: 52.

[0170] In certain embodiments, the nucleic acid molecule of the present invention encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the nucleic acid molecule of the present invention comprises or consists of the sequence shown in SEQ ID NO: 28 or SEQ ID NO: 29, which encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 14. In certain embodiments, the nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 28 or SEQ ID NO: 29, preferably SEQ ID NO: 29. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 28 or SEQ ID NO: 29, preferably SEQ ID NO: 29, for example, at least 96, 97, 98, 99, or 100 percent identity.

[0171] In certain embodiments, the nucleic acid molecule of the present invention encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the nucleic acid molecule of the present invention comprises or consists of the sequence shown in SEQ ID NO: 30 or SEQ ID NO: 31, which encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 15. In certain embodiments, the nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31, preferably SEQ ID NO: 31. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31, preferably SEQ ID NO: 31, for example, at least 96, 97, 98, 99, or 100 percent identity.

[0172] In certain embodiments, the nucleic acid molecule of the present invention encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 16. In certain embodiments, the nucleic acid molecule of the present invention comprises or consists of the sequence shown in SEQ ID NO: 32 or SEQ ID NO: 33, which encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 16. In certain embodiments, the nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 32 or SEQ ID NO: 33, preferably SEQ ID NO: 33. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 32 or SEQ ID NO: 33, preferably SEQ ID NO: 33, for example, at least 96, 97, 98, 99, or 100 percent identity.

[0173] In certain embodiments, the nucleic acid molecule of the present invention encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the nucleic acid molecule of the present invention comprises or consists of the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 35, which encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 17. In certain embodiments, the nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 35, preferably SEQ ID NO: 35. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 35, preferably SEQ ID NO: 35, for example, at least 96, 97, 98, 99, or 100 percent identity.

[0174] In certain embodiments, the nucleic acid molecule of the present invention encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 18. In certain embodiments, the nucleic acid molecule of the present invention comprises or consists of the sequence shown in SEQ ID NO: 36 or SEQ ID NO: 37 encoding a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 18. In certain embodiments, the nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 36 or SEQ ID NO: 37, preferably SEQ ID NO: 37. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 36 or SEQ ID NO: 37, preferably SEQ ID NO: 37, for example, at least 96, 97, 98, 99, or 100 percent identity.

[0175] In certain embodiments, the nucleic acid molecule of the present invention encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 19. In certain embodiments, the nucleic acid molecule of the present invention comprises or consists of the sequence shown in SEQ ID NO: 38 or SEQ ID NO: 39 encoding a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 19. In certain embodiments, the nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 38 or SEQ ID NO: 39, preferably SEQ ID NO: 39. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 38 or SEQ ID NO: 39, preferably SEQ ID NO: 39, for example, at least 96, 97, 98, 99, or 100 percent identity.

[0176] In certain embodiments, the nucleic acid molecule of the present invention encodes a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 20. In certain embodiments, the nucleic acid molecule of the present invention comprises or consists of the sequence shown in SEQ ID NO: 40 or SEQ ID NO: 41 encoding a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 20. In certain embodiments, the nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 40 or SEQ ID NO: 41, preferably SEQ ID NO: 41. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity with respect to the nucleotide sequence of SEQ ID NO: 40 or SEQ ID NO: 41, preferably SEQ ID NO: 41, for example, at least 96, 97, 98, 99, or 100 percent identity.

[0177] Nucleic acid molecules encoding functional truncated GDE polypeptides as defined above may have at least 80, at least 85, at least 90, or at least 95 percent identity with any of the nucleotide sequences of SEQ ID NOs. 28-41. In certain embodiments, the nucleic acid molecules of the present invention have at least 95 percent identity with any of the nucleotide sequences of SEQ ID NOs. 28-41, for example, at least 96, 97, 98, 99, or 100 percent identity.

[0178] Preferably, a nucleic acid molecule encoding a functionally truncated GDE polypeptide as defined above may have at least 80, at least 85, at least 90, or at least 95 percent identity with any of the nucleotide sequences of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, or SEQ ID NO: 41. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity, for example, at least 96, 97, 98, 99, or 100 percent identity with any of the nucleotide sequences of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, or SEQ ID NO: 41.

[0179] 3 - Nucleic acid constructs The present invention also relates to nucleic acid constructs comprising the nucleic acid molecules of the present invention described above. A nucleic acid construct may correspond to an expression cassette comprising the nucleic acid sequence of the present invention, which is operably ligated to one or more expression regulatory sequences and / or other sequences that enhance expression. As used herein, the term “operably ligated” refers to the ligation of polynucleotide elements in a functional relationship. A nucleic acid is “operably ligated” if it is in a functional relationship with another nucleic acid sequence. For example, a promoter or another transcription regulatory sequence is operably ligated to a coding sequence if it affects the transcription of the coding sequence. Such expression regulatory sequences, such as promoters, enhancers (e.g., cis-regulatory modules (CRMs)), introns, poly(A) signals, etc., are known in the art.

[0180] In certain embodiments, the expression cassette may include a promoter. The promoter may be a universal promoter or a tissue-specific promoter, in particular a promoter that can promote expression in cells or tissues where GDE expression is desirable, for example, in cells or tissues where GDE expression is desirable in GDE-deficient patients. Preferably, the promoter is a universal promoter.

[0181] In the first specific embodiment, the promoter is a muscle-specific promoter. A non-limiting example of a muscle-specific promoter is the muscle creatine kinase (MCK) promoter. A non-limiting example of a preferred muscle creatine kinase promoter is the human muscle creatine kinase promoter and the truncated mouse muscle creatine kinase [(tMCK) promoter] (Wang B et al., Construction and analysis of compact muscle-selective promoters for AAV vectors. Gene Ther. November 2008; 15(22): pp. 1489-99) (Representative GenBank accession number AF188002). Human muscle creatine kinase has gene ID number 1158 (Representative GenBank accession number NC_000019.9, accessed December 26, 2012). Other examples of muscle-specific promoters include the synthetic promoter C5.12 (SPc5-12, hereafter referred to as "C5.12"), e.g., the SPc5-12 promoter (disclosed in Wang et al., Gene Therapy, Vol. 15, pp. 1489-1499 (2008)), the MHCK7 promoter (Salva et al., Mol Ther. February 2007; 15(2):320-9), the myosin light chain (MLC) promoter, e.g., MLC2 (gene ID number 4633; representative GenBank accession number NG_007554.1, accessed December 26, 2012); the myosin heavy chain (MHC) promoter, e.g., alpha-MHC (gene ID number 4624; representative GenBank accession number NG_023444.1, accessed December 26, 2012); and the desmin promoter (gene ID Gene ID 1674; Representative GenBank accession number NG_008043.1, accessed: December 26, 2012); Cardiac troponin C promoter (gene ID number 7134; Representative GenBank accession number NG_008963.1, accessed: December 26, 2012); Troponin I promoter (gene ID numbers 7135, 7136, and 7137; Representative GenBank accession numbers NG_016649.1, NG_011621.1, and NG_007866).2. Accessed: December 26, 2012); myoD gene family promoter (Weintraub et al., Science, 251, 761 (1991); gene ID number 4654; representative GenBank accession number NM_002478, accessed: December 26, 2012); alpha-actin promoter (gene ID numbers 58, 59, and 70; representative GenBank accession numbers NG_006672.1, NG_011541.1, and NG_007553.1, accessed: December 26, 2012); beta-actin promoter (gene ID number 60; representative GenBank accession number NG_007992.1, accessed: December 26, 2012); gamma-actin promoter (gene ID number 7 1 and 72; representative GenBank accession numbers NG_011433.1 and NM_001199893, accessed December 26, 2012); muscle-specific promoter located in intron 1 of ocular Pitx3 (gene ID number 5309) (Coulon et al.; this muscle-selective promoter corresponds to residues 11219-11527 of representative GenBank accession number NG_008147, accessed December 26, 2012); and promoter described in U.S. Patent Application Publication No. 2003 / 0157064, and CK6 promoter (Wang et al., 2008 doi: 10.1038 / gt.2008).104) is one example. 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 includes a combination of an MCK-derived enhancer and the SPc5-12 promoter. In a specific embodiment of the present invention, the muscle-specific promoter is selected from the group consisting of the SPc5-12 promoter, the MHCK7 promoter, the E-syn promoter, the muscle creatine kinase myosin light chain (MLC) promoter, the myosin heavy chain (MHC) promoter, the cardiac troponin C promoter, the troponin I promoter, the myoD gene family promoter, the alpha-actin promoter, the beta-actin promoter, the gamma-actin promoter, the muscle-specific promoter located in intron 1 of the eye-type Pitx3, the CK6 promoter, the CK8 promoter, and the Acta1 promoter. In a further embodiment, the muscle-specific promoter is selected from the group consisting of the SPc5-12, desmin, and MCK promoters. In further embodiments, the muscle-specific promoter is selected from the group consisting of SPc5-12 and MCK promoters. In preferred embodiments, the muscle-specific promoter is the SPc5-12 promoter.

[0182] In a second specific embodiment, the promoter is a liver-specific promoter. Non-limiting examples of liver-specific promoters include the HSE promoter (liver-specific promoter), alpha-1 antitrypsin promoter (hAAT), transthyretin promoter, albumin promoter, thyroxine-binding globulin (TBG) promoter, and LSP promoter (containing a thyroid hormone-binding globulin promoter sequence, two copies of an alpha-1-microglobulin / bicinine enhancer sequence, and a 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.). Other useful liver-specific promoters, such as those listed in the Liver Specific Gene Promoter Database (http: / / rulai.cshl.edu / LSPD / ) collected by the Cold Spring Harbor Laboratory, are known in the art. A preferred liver-specific promoter for 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 a mouse TTR promoter and the H1 enhancer described in Sequence ID No. 63, and is derived from an early research paper by Robert H. Costa et al., 1986 (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 an HSE promoter having the sequence of SEQ ID NO: 65, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 65, for example, at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity to SEQ ID NO: 65.

[0183] In another specific embodiment, the promoter is a neuron-specific promoter. Non-limiting examples of neuron-specific promoters, among many that would be apparent to those skilled in the art, include, but are not limited to, the synapsin-1 (Syn) promoter, the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). In a particular embodiment, the neuron-specific promoter is the Syn promoter. Other neuron-specific promoters include, but are not limited to, the synapsin-2 promoter, tyrosine hydroxylase promoter, dopamine β-hydrolase promoter, hypoxanthine phosphoribosyltransferase promoter, low-affinity NGF receptor promoter, and 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). A representative promoter specific to motor neurons is, but is not limited to, the promoter of calcitonin gene-related peptide (CGRP), a known motor neuron-derived factor. Other promoters that function in motor neurons include the promoters of 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 (for motor neurons), nestin (for neurons), parvalbumin, somatostatin (Somatostation), and Plp1 (for oligodendrocytes and Schwann cells).

[0184] In preferred embodiments, the promoter is a universal promoter. Representative universal promoters include the cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, the cytomegalovirus enhancer / promoter (CMV) (with CMV enhancer as needed) [see, e.g., Boshart et al., Cell, 41: pp. 521-530 (1985)] or a short version of the CMV promoter, the PGK promoter, the SV40 initial promoter, the retrovirus Roussarcoma virus (RSV) LTR promoter (with RSV enhancer as needed), the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the EF1 alpha (EF1a) promoter or a short version of the EF1a promoter, and the Ins84 promoter (as described in WO2020 / 219949). In a further preferred embodiment, the promoter is a shortened version of the CMV promoter.

[0185] In addition, the promoter may be an endogenous promoter, such as an albumin promoter or a GDE promoter.

[0186] Short promoters, such as short versions of known promoters, are of particular interest in this invention. In preferred embodiments, the promoter is less than about 500 pb, preferably less than about 450 pb, and preferably less than about 400 pb.

[0187] In a preferred embodiment, the promoter is a shorter version of any of the promoters described herein, for example, a shorter version of the CMV promoter, or a shorter version of the EF1a promoter, or a promoter comprising an H1 enhancer and a TTR promoter.

[0188] 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 of SEQ ID NO: 62, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 62, for example, at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity to SEQ ID NO: 62.

[0189] 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 as shown in SEQ ID NO: 64, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 64, for example, at least 85%, in particular 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: 64.

[0190] In another specific embodiment, the promoter is an Ins84 promoter, as described in WO2020 / 219949.

[0191] Expression cassettes containing the nucleic acid molecule of the present invention can be adapted depending on the target population of GSDIII patients, the clinical symptoms of GSDIII disease, and / or the target tissue. For example, in patients with clinical symptoms of GSDIII disease primarily in muscle (e.g., adolescent or adult GSDIII patients), the expression cassette preferably contains a muscle-specific promoter or any promoter capable of inducing potent expression of the nucleic acid molecule of the present invention in muscle, for example, the miniCMV promoter described above, in particular the miniCMV promoter of SEQ ID NO: 62. In patients with clinical symptoms of GSDIII disease in the liver, the expression cassette preferably contains a liver-specific promoter or any promoter capable of inducing potent expression of the nucleic acid molecule of the present invention in the liver, for example, the HSE promoter described above, in particular the HSE promoter of SEQ ID NO: 65. In patients with clinical symptoms of GSDIII disease in both muscle and liver, a promoter capable of inducing expression of the nucleic acid molecule of the present invention in both tissues is preferably used.

[0192] In certain embodiments, the promoter associates with an enhancer sequence, such as a cis-regulatory module (CRM) or an artificial enhancer sequence. Examples of CRMs useful in carrying out the present invention include those described in Rincon et al., Mol Ther. January 2015;23(1):43-52, Chuah et al., Mol Ther. September 2014;22(9):1605-13, or Nair et al., Blood. May 15, 2014;123(20):3195-9. Other regulatory elements that can enhance the muscle-specific expression of genes, particularly in cardiac and / or skeletal muscle, are disclosed in WO2015110449. Specific examples of nucleic acid regulatory elements, including artificial sequences, include those obtained by rearranging transcription factor-binding sites (TFBSs) present in the sequences disclosed in WO2015110449. The rearrangement may include changing the order of the TFBSs and / or changing the position of one or more TFBSs relative to other TFBSs and / or changing the copy number of one or more TFBSs. For example, nucleic acid regulatory elements for enhancing muscle-specific gene expression, particularly cardiac and skeletal muscle-specific gene expression, may include binding sites for E2A, HNH 1, NF1, C / EBP, LRF, MyoD, and SREBP; or E2A, NF1, p53, C / EBP, LRF, and SREBP; or E2A, HNH 1, HNF3a, HNF3b, NF1, C / EBP, LRF, MyoD, and SREBP; or E2A, HNF3a, NF1, C / EBP, LRF, MyoD, and SREBP; or E2A, HNF3a, NF1, CEBP, LRF, MyoD, and SREBP; or HNF4, NF1, RSRFC4, C / EBP, LRF, and MyoD; or 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 include binding sites for E2A, NF1, SRFC, p53, C / EBP, LRF, and MyoD; or E2A, NF1, C / EBP, LRF, MyoD, and SREBP; or E2A, HNF3a, C / EBP, LRF, MyoD, SEREBP, and Tal1_b; or E2A, SRF, p53, C / EBP, LRF, MyoD, and SREBP; or HNF4, NF1, RSRFC4, C / EBP, LRF, and SREBP; or E2A, HNF3a, HNF3b, NF1, SRF, C / EBP, LRF, MyoD, and SREBP; or E2A, CEBP, and MyoD. In further examples, these nucleic acid regulatory elements include at least two, e.g., two, three, four or more copies of one or more of the previously listed TFBS. Other regulatory elements that can enhance the liver-specific expression of genes in particular are disclosed in WO2009130208.

[0193] In certain embodiments, the enhancer is a short-size enhancer. In particular, enhancers for use in the present invention may consist of 10 to 175 nucleotides, for example, 40 to 100 nucleotides, and especially 50 to 80 nucleotides. In certain embodiments, the enhancer is a 72-nucleotide HS-CRM8 enhancer comprising SEQ ID NO: 63, or a functional variant of SEQ ID NO: 63 having enhancer activity. In other embodiments, the enhancer is a functional variant of a 72-nucleotide HS-CRM8 enhancer that is at least 80% identical to SEQ ID NO: 63, for example, at least 85% identical to SEQ ID NO: 63, especially at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% identical.

[0194] In another specific embodiment, the nucleic acid construct includes introns, particularly introns positioned between the promoter and the GDE coding sequence. Introns may be introduced to increase mRNA stability and protein production. In further embodiments, the introns include human beta-globin b2 (or HBB2) introns, coagulation factor IX (FIX) introns, SV40 introns, hCMV intron A (hCMVI), TPL introns (TPLI), CHEF1 gene intron 1 (CHEFI), MVM introns (Wu et al., 2008), 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), and β-globin / immunoglobulin heavy chain hybrid introns (5'-donor site from human β-globin introns and 3'-acceptor site from immunoglobulin heavy chain variable region introns, Wu et al., 2008, Mol Ther, 16(2):280-289; Kurachi et al., 1995, J Biol Chem., 270(10): pp. 5276-5281; hybrid introns consisting of adenovirus splice donors and immunoglobulin G splices (Wong et al., 1985, Chromosoma, 92(2): pp. 124-135; Yew et al., 1997, Hum Gene Ther, 8(5): pp. 575-584; Choi T. et al., 1991, Mol Cell Biol, 11(6): pp. 3070-3074; Huang et al., 1990, Mol Cell Biol., 10(4): pp. 1805-1810); hybrid 19S / 16S SV40 introns (5'-donor site from 19S intron and 3'-acceptor site from 16S intron, Yew et al., 1997, Hum Gene Ther, 8(5): pp. 575-584), or chicken beta-globin introns. In another further embodiment, the intron is a modified intron (in particular a modified HBB2 or FIX intron) designed to reduce the number of alternative open reading frames (ARFs) found in the intron, or even to eliminate such ARFs completely.Preferably, ARFs that are longer than 50 bp and have a start codon and a stop codon in frame are removed. ARFs can be removed by modifying the intron sequence. For example, the modification can be carried out by nucleotide substitution, insertion, or deletion, preferably by nucleotide substitution. As an example, one or more nucleotides, in particular one nucleotide, in the ATG or GTG start codon present in the sequence of the intron of interest can be replaced to obtain a non-start codon. For example, ATG or GTG can be replaced with CTG, which is not a start codon, within the sequence of the intron of interest.

[0195] The classic HBB2 intron is shown in SEQ ID NO: 53. For example, this HBB2 intron can be modified by deleting the start codons (ATG and GTG codons) within the intron. In certain embodiments, the modified HBB2 intron has the sequence shown in SEQ ID NO: 54. The classic FIX intron is derived from the first intron of human FIX and is shown in SEQ ID NO: 55. The FIX intron can be modified by deleting the start codons (ATG and GTG codons) within the intron. In certain embodiments, the modified FIX intron has the sequence shown in SEQ ID NO: 56. The classic chicken-betaglobin intron used in nucleic acid constructs is shown in SEQ ID NO: 57. The chicken-betaglobin intron can be modified by deleting the start codons (ATG and GTG codons) within the intron. In certain embodiments, the modified chicken-betaglobin intron has the sequence shown in SEQ ID NO: 58.

[0196] The inventors previously demonstrated in WO2015 / 162302 that such modified introns, in particular modified HBB2 or FIX introns, possess advantageous properties and can significantly improve the expression of transgenes.

[0197] In certain embodiments, the nucleic acid construct of the present invention is an expression cassette comprising a promoter with an enhancer optionally preceding it in the 5' to 3' direction, the coding sequence of the present invention (i.e., a nucleic acid molecule encoding a functional truncated GDE polypeptide), and a polyadenylation signal, e.g., pA58 polyadenylation signal (pA58 polyA), bovine growth hormone polyadenylation signal (bGH polyA), 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 certain embodiments, the polyadenylation signal is bGH polyA as shown in SEQ ID NO: 60. In certain embodiments, a very short polyA signal is used. For example, a very short polyA signal containing fewer than 20 nucleotides is used. In certain embodiments, the polyadenylation signal is the human soluble neuropilin-1 (sNRP) polyadenylation signal (sNRP PolyA; SEQ ID NO: 59). In preferred embodiments, the polyadenylation signal is the pA58 polyadenylation signal as shown in SEQ ID NO: 61.

[0198] In certain embodiments, the nucleic acid construct of the present invention is an expression cassette comprising a promoter, an intron, the coding sequence of the present invention, and a polyadenylation signal, with an enhancer optionally preceding it, in the 5' to 3' direction. In another embodiment, the nucleic acid construct of the present invention is an expression cassette comprising a promoter, the coding sequence of the present invention, and a polyadenylation signal, in the 5' to 3' direction. In yet another embodiment, the nucleic acid construct of the present invention is an expression cassette comprising an enhancer, a promoter, the coding sequence of the present invention, and a polyadenylation signal, in the 5' to 3' direction. In a further specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising an enhancer, a promoter, an intron, the coding sequence of the present invention, and a polyadenylation signal, in the 5' to 3' direction. In a further specific embodiment of the present invention, an expression cassette comprising a promoter, an optional intron, the coding sequence of the present invention, and a poly(A) signal, in the 5' to 3' direction.

[0199] In another embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in the 5' to 3' direction, an SPc5-12 promoter or CMV promoter, e.g., a mini-CMV promoter, the coding sequence of the present invention, and a polyadenylation signal (e.g., bGH polyA or pA58 polyA, particularly pA58 polyA). In another embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in the 5' to 3' direction, an enhancer, an SPc5-12 promoter or CMV promoter, e.g., a mini-CMV promoter, the coding sequence of the present invention, and a polyadenylation signal (e.g., bGH polyA or pA58 polyA, particularly pA58 polyA).

[0200] In a further specific embodiment, the expression cassette comprises, in the 5' to 3' direction, an SPc5-12 promoter; a sequence encoding the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; and bGH polyA or pA58 polyA, in particular pA58 polyA. In another embodiment, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter; a sequence encoding the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; and bGH polyA or pA58 polyA, in particular pA58 polyA.

[0201] In a preferred embodiment, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functional truncated GDE polypeptide as defined above, e.g., a sequence encoding the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; and bGH polyA or pA58 polyA, in particular pA58 polyA.

[0202] In certain embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functional truncated GDE polypeptide as defined above, e.g., a sequence selected from the group consisting of SEQ ID NOs: 28 to 41; and bGH polyA or pA58 polyA, in particular pA58 polyA.

[0203] In certain embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 14; and bGH polyA or pA58 polyA, in particular pA58 polyA. In certain embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 14, sequence SEQ ID NO: 28 or SEQ ID NO: 29; and bGH polyA or pA58 polyA, in particular pA58 polyA. In certain embodiments, the expression cassette includes, in the 5' to 3' direction, a mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 14, sequence SEQ ID NO: 28 or SEQ ID NO: 29; and pA58 polyA of SEQ ID NO: 61.

[0204] In a preferred embodiment, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 15; and bGH polyA or pA58 polyA, particularly pA58 polyA. In a particular embodiment, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 15, SEQ ID NO: 30 or SEQ ID NO: 31; and bGH polyA or pA58 polyA, particularly pA58 polyA. In a particular embodiment, the expression cassette includes, in the 5' to 3' direction, a mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 15, SEQ ID NO: 30 or SEQ ID NO: 31; and pA58 polyA of SEQ ID NO: 61.

[0205] In further embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 16; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 16, of SEQ ID NO: 32 or SEQ ID NO: 33; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 16, of SEQ ID NO: 32 or SEQ ID NO: 33; and pA58 polyA of SEQ ID NO: 61.

[0206] In further embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 17; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 17, sequence SEQ ID NO: 34 or SEQ ID NO: 35; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 17, sequence SEQ ID NO: 34 or SEQ ID NO: 35; and pA58 polyA of SEQ ID NO: 61.

[0207] In further embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 18; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 18, sequence SEQ ID NO: 36 or SEQ ID NO: 37; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 18, sequence SEQ ID NO: 36 or SEQ ID NO: 37; and pA58 polyA of SEQ ID NO: 61.

[0208] In further embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 19; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 19, sequence SEQ ID NO: 38 or SEQ ID NO: 39; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 19, sequence SEQ ID NO: 38 or SEQ ID NO: 39; and pA58 polyA of SEQ ID NO: 61.

[0209] In further embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 20; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a CMV promoter, e.g., a mini-CMV promoter, in particular the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 20, of SEQ ID NO: 40 or SEQ ID NO: 41; and bGH polyA or pA58 polyA, in particular pA58 polyA. In specific embodiments, the expression cassette includes, in the 5' to 3' direction, a mini-CMV promoter of SEQ ID NO: 62; a sequence encoding the functionally truncated GDE polypeptide of SEQ ID NO: 20, of SEQ ID NO: 40 or SEQ ID NO: 41; and pA58 polyA of SEQ ID NO: 61.

[0210] In certain embodiments, the expression cassette contains or comprises the sequences as shown in SEQ ID NOs. 42 to 48. In further embodiments, the expression cassette contains or comprises sequences having at least 80% sequence identity to SEQ ID NOs. 42 to 48, for example, at least 85% sequence identity, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity.

[0211] In certain embodiments, the expression cassette contains or comprises the sequence as shown in SEQ ID NO: 42. In further embodiments, the expression cassette contains or comprises a sequence having at least 80% sequence identity to SEQ ID NO: 42, for example, at least 85% sequence identity, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity.

[0212] In a preferred embodiment, the expression cassette contains or comprises the sequence as shown in SEQ ID NO: 43. In a further embodiment, the expression cassette contains or comprises a sequence having at least 80% sequence identity to SEQ ID NO: 43, for example, at least 85% sequence identity, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity.

[0213] In a further embodiment, the expression cassette contains or comprises the sequence as shown in SEQ ID NO: 44. In a further embodiment, the expression cassette contains or comprises a sequence having at least 80% sequence identity to SEQ ID NO: 44, for example, at least 85% sequence identity, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity.

[0214] In a further embodiment, the expression cassette contains or comprises the sequence as shown in SEQ ID NO: 45. In a further embodiment, the expression cassette contains or comprises a sequence having at least 80% sequence identity to SEQ ID NO: 45, for example, at least 85% sequence identity, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity.

[0215] In a further embodiment, the expression cassette contains or comprises the sequence as shown in SEQ ID NO: 46. In a further embodiment, the expression cassette contains or comprises a sequence having at least 80% sequence identity to SEQ ID NO: 46, for example, at least 85% sequence identity, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity.

[0216] In a further embodiment, the expression cassette contains or comprises the sequence as shown in SEQ ID NO: 47. In a further embodiment, the expression cassette contains or comprises a sequence having at least 80% sequence identity to SEQ ID NO: 47, for example, at least 85% sequence identity, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity.

[0217] In a further embodiment, the expression cassette contains or comprises the sequence as shown in SEQ ID NO: 48. In a further embodiment, the expression cassette contains or comprises a sequence having at least 80% sequence identity to SEQ ID NO: 48, for example, at least 85% sequence identity, in particular at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even more than 99% sequence identity.

[0218] When designing nucleic acid constructs of the present invention, those skilled in the art will take into consideration the size limitations of the vector used to deliver the constructs to cells or organs. In particular, it is known to those skilled in the art that the main limitation of AAV vectors is their cargo capacity, which may vary depending on the AAV serotype but is generally limited to the size of the parental viral genome. For example, 5 kb is the maximum size that is typically considered when packaged in an AAV8 capsid (Wu Z. et al., Mol Ther., 2010, 18(1): pp. 80-86; Lai Y. et al., Mol Ther., 2010, 18(1): pp. 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4): 225-33). In addition, during recombinant AAV production, genomes larger than 5kb are encapsulated in the capsid with low efficiency, and the resulting AAV may contain fragmented genomes, thereby reducing the efficiency of gene transfer. Therefore, those skilled in the art will take care when implementing the present invention to select components of the nucleic acid construct of the present invention such that the resulting nucleic acid sequence, including sequences encoding AAV 5'- and 3'-ITRs, preferably does not exceed 110% of the cargo capacity of the AAV vector being executed, and particularly preferably does not exceed 5kb. AAV vectors with larger cargo capacity can also be used in connection with the present invention. For example, AAV particles lacking the Vp2 subunit have been shown to successfully package larger genomes (i.e., 6kb) while maintaining the integrity of the genome encapsulated in the capsid (Grieger et al., 2005, J Virol., 79(15):9933-9944).

[0219] 4 - Vector The present invention also relates to vectors comprising nucleic acid molecules or constructs as disclosed herein. In certain embodiments, the vector comprises nucleic acid molecules or constructs encoding functional truncated GDE polypeptides as defined above.

[0220] In particular, the vectors of the present invention are suitable for protein expression, preferably for use in gene therapy. In one embodiment, the vector is a plasmid vector. In another embodiment, the vector is a nanoparticle containing the nucleic acid molecule of the present invention, in particular messenger RNA encoding the functional truncated GDE polypeptide of the present invention. In another embodiment, the vector is a transposon-based system, e.g., the highly active Sleeping Beauty (SB100X) transposon system (Mates et al., 2009), which enables the incorporation of the nucleic acid molecule or construct of the present invention into the genome of a target cell. In another embodiment, the vector is a viral vector suitable for gene therapy targeting any cell of interest, e.g., liver tissue or cells, muscle cells, CNS cells (e.g., brain cells), or hematopoietic stem cells, e.g., erythrocyte-derived cells (e.g., erythrocytes). In this case, the nucleic acid construct of the present invention also contains sequences suitable for efficient production of viral vectors, as is well known in the art.

[0221] Viral vectors, such as retroviral vectors, such as lentiviral vectors, or nonpathogenic parvoviruses, more preferably AAV vectors, are preferred for the delivery of nucleic acid molecules or constructs of the present invention. Adeno-associated virus (AAV) of human parvovirus is a naturally replication-defective dependent virus that can integrate into the genome of infected cells to establish latent infection. This last characteristic appears to be unique among mammalian viruses because the integration occurs at a specific site in the human genome (19q13.3-qter) located on chromosome 19, called AAVS1.

[0222] Therefore, AAV vectors are attracting considerable interest as potential vectors for human gene therapy. The virus's beneficial properties include its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and its ability to infect a wide range of cell lines originating from different tissues.

[0223] Among the AAV serotypes isolated from humans or non-human 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., quadruple mutant capsid-optimized AAV-2 containing an engineered capsid with the Y44+500+730F+T491V change, disclosed by Ling et al., July 18, 2016, Hum Gene Ther Methods.), -3 and AAV-3 variants (e.g., AAV3-ST variant containing an engineered AAV3 with two amino acid changes, S663V+T492V, disclosed by Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042), -3B and AAV-3B variants, and -4, -5, -6 and AAV-6 variants (e.g., Rosario et al., 2016, Mol Ther Methods Clin Dev.). Examples include AAV6 variants (including the triple mutant AAV6 capsid Y731F / Y705F / T492V, as disclosed on page 3, 16026), -7, -8, -9, -2G9, -10, e.g., cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV serotypes, e.g., AAVpo4 and AAVpo6, and tyrosine, lysine, and serine capsid variants of AAV serotypes. In addition, other non-naturally engineered variants and chimeric AAVs may also be useful. AAV viruses can be engineered using conventional molecular biology techniques that allow for the optimization of these particles for cell-specific delivery of nucleic acid sequences, to minimize immunogenicity, to adjust stability and particle lifetime, for efficient degradation, and for precise delivery to the nucleus.

[0224] Desired AAV fragments for vector assembly include cap proteins containing vp1, vp2, vp3 and a hypervariable region; rep proteins containing rep 78, rep 68, rep 52, and rep 40; and sequences encoding these proteins. These fragments can be readily utilized in various vector systems and host cells.

[0225] Recombinant vectors based on AAV lacking the Rep protein are incorporated into the host genome with low efficacy and exist primarily as stable circular episomes, which can persist in target cells for many years. Instead of using natural AAV serotypes, artificial AAV serotypes containing, but not limited to, AAVs with capsid proteins that do not exist naturally can be used in connection with the present invention. Such artificial capsids can be generated by any preferred technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with heterologous sequences that can be obtained from different selected AAV serotypes, non-contiguous portions of the same AAV serotype, from non-AAV viral sources, or from non-viral sources. Artificial AAV serotypes may, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids.

[0226] In relation to the present invention, the AAV vector comprises an AAV capsid that can be transduced into target cells of interest, namely cells of immunotolerative tissues (e.g., hepatocytes), and cells of tissues of therapeutic interest, such as muscle cells, CNS cells, or cardiac cells. In certain embodiments, the AAV vector comprises an AAV capsid that can be transduced into muscle cells or cardiac cells. According to a particular embodiment, the AAV vector may be AAV-1, -2, AAV-2 variant (e.g., quadruple mutant capsid optimized AAV-2 containing an engineered capsid with the Y44+500+730F+T491V change, disclosed in Ling et al., July 18, 2016, Hum Gene Ther Methods. [Pre-print Epub]), -3 and AAV-3 variant (e.g., AAV3-ST variant containing 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 variant, -4, -5, -6 and AAV-6 variant (e.g., Rosario et al., 2016, Mol Ther Methods Clin Dev. 3. AAV6 variants including the triple mutant AAV6 capsid Y731F / Y705F / T492V, disclosed on page 16026, -7, -8, -9, -9P1, -2G9, -10, e.g., cy10 and -rh10, -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP.B, AAV.PHPeB, AAV2i8, porcine AAV, e.g., AAVpo1 (as described in WO2021 / 219762), AAVpo4 and AAVpo6, and tyrosine, lysine and serine capsid variants of AAV serotypes. In certain embodiments, 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 even more specific embodiments, the AAV vector is a pseudotyped vector, i.e., its genome and capsid are derived from different serotypes of AAV.For example, a pseudotyped AAV vector may be a vector in which the genome originates from one of the above-mentioned AAV serotypes and the capsid originates from another serotype. For example, the genome of a pseudotyped vector may have a capsid originating from the AAV6, AAV8, AAV9, AAV9P1, AAVrh74, or AAV2i8 serotype, and the genome may originate from a different serotype. In certain embodiments, the AAV vector has a capsid originating from the AAV6, AAV8, AAV9, or AAVrh74 serotype, more particularly from the AAV6, AAV8, AAV9, or AAV9P1 serotype, and more particularly from the AAV6, AAV9, or AAV9P1 serotype.

[0227] In certain embodiments where the vector is intended for use in delivering therapeutic transgenes to muscle cells, the AAV vector may be selected from among a number of options, particularly from the group consisting of AAV8, AAV9, and AAVrh74.

[0228] In another specific embodiment, where the vector is intended for use in delivering a transgene to liver cells, the AAV vector may be selected from among a number of options, particularly AAV1, AAV5, AAV8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAVrh74, AAV-LK03, AAV2G9, AAV.PHP, AAV-Anc80, and AAV3B.

[0229] In a further specific embodiment, where the vector is intended for use in delivering the transgene to the CNS, the AAV vector may be selected from among several groups, particularly AAV9, AAV9P1, AAV10, AAV2G9, AAV.PHP.B, and AAV.PHPeB.

[0230] In another embodiment, the capsid is a modified capsid. With respect to the present invention, “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.

[0231] In certain embodiments, the AAV vector is a chimeric vector, i.e., its capsid contains VP capsid proteins derived from at least two different AAV serotypes, or contains at least one chimeric VP protein having VP protein regions or domains derived from at least two AAV serotypes. Examples of such chimeric AAV vectors useful for transduction into liver cells 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 an AAV8 capsid sequence and a sequence of an AAV serotype different from the AAV8 serotype, e.g., any of those specifically described above. In another embodiment, the AAV vector capsid comprises one or more variant VP capsid proteins that exhibit high liver affinity, e.g., those described in WO2015013313, in particular the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid variants.

[0232] In another embodiment, the modified capsid may be obtained from a capsid modification inserted by error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). In certain embodiments, the capsid includes a P1 insertion, as described in WO2019 / 193119, WO2020 / 200499, or WO2022 / 053630. In addition, the capsid variant may include a single amino acid change, e.g., a tyrosine variant (e.g., as described in Zhong et al., 2008). In certain embodiments, the vector is AAV9rh74 containing 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, May 19, 2023). In another specific embodiment, the vector is a P1-presenting AAV9 variant called "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, October 28, 2020). In another specific embodiment, the vector is an RGD peptide-containing AAV variant known as "MyoAAV," as described by Tabebordbar et al. (Tabebordbar, Mohammadsharif, "Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species," Cell 184, pp. 4919-4938, September 16, 2021).

[0233] In certain embodiments, the AAV vector is an AAV vector as described in WO2019 / 193119 or an AAV vector as described in WO2020 / 200499.

[0234] In further embodiments, the AAV vector is an AAV vector as described in WO2020 / 216861 or an AAV vector as described in WO2022 / 003211. In particular, the AAV vector may have a hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, for example, a hybrid capsid of AAV8 and AAV2 / 13.

[0235] In addition, the genome of an AAV vector can be either a single-stranded genome or a self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting a terminal separation site from one of the AAV terminal repeats. These modified vectors, whose replicated genome is half the length of the wild-type AAV genome, tend to package DNA dimers. In preferred embodiments, the AAV vector incorporated into the implementation of the present invention has a single-stranded genome and more 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 an AAV8, AAV9, AAV9rh74-P1, or hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, and more particularly an AAV9 capsid.

[0236] The AAV vector used to package the GDE sequence of the present invention can also be modified to increase its cargo capacity. For example, an AAV vector lacking the Vp2 subunit has been shown to successfully package a larger genome (i.e., 6kb) while maintaining the integrity of the genome encapsulated in the capsid (Grieger et al., 2005).

[0237] Additional suitable sequences, such as those known in the art, can be introduced into the nucleic acid construct of the present invention to obtain a functional viral vector. Suitable sequences include AAV ITR.

[0238] In certain embodiments, the AAV vector includes the muscle-specific promoter described above, in particular a muscle-specific promoter that results in some leakage of expression to hepatocytes.

[0239] In another specific embodiment of the present invention, the AAV vector comprises the liver-specific promoter described above. The immune tolerance-inducing and metabolic properties of the liver are advantageously incorporated, thanks to this embodiment, for developing a highly efficient optimized vector for expressing GDE in hepatocytes and for inducing immune tolerance to the protein.

[0240] In certain embodiments, a dual recombinant AAV vector system comprising two AAV vectors, such as those described in WO 2018 / 162748, is used to deliver nucleic acid molecules or constructs encoding functional truncated GDE polypeptides as defined above. In particular, the dual AAV vector system is, - A first AAV vector containing between the 5' AAV ITR and the 3' AAV ITR a first nucleic acid sequence encoding the N-terminal portion of a truncated GDE polypeptide as defined above, and - A second AAV vector containing a second nucleic acid sequence encoding a portion of the truncated GDE polypeptide as defined above, between the 5' AAV ITR and the 3' AAV ITR. The first and second nucleic acid sequences encoding the GDE include a polynucleotide region that enables the production of a full-length truncated GDE polypeptide as defined above.

[0241] In another specific embodiment, the AAV vector is a single AAV vector comprising a nucleic acid molecule or construct encoding a functional truncated GDE polypeptide as defined above.

[0242] 5 - Cell The present invention also relates to cells transformed or transduced by nucleic acid molecules, constructs, or vectors of the present invention, particularly isolated cells, such as liver cells, cardiac cells, CNS cells, or muscle cells. In certain embodiments, the cells are isolated human cells. In further specific embodiments, the cells are not human embryonic stem cells. The cells of the present invention express the functional truncated GDE polypeptide described above. The cells of the present invention can be delivered to a subject in need, for example, a GDE-deficient patient, by any suitable route of administration, for example, by injection into the liver, CNS, heart, muscle, or bloodstream of the subject. In certain embodiments, the present invention includes transducing liver or muscle cells, particularly liver or muscle cells of a subject to be treated, and administering the transduced liver and / or muscle cells into which the nucleic acid has been introduced to the subject. In certain embodiments, the liver cells are liver cells from a patient to be treated, or liver stem cells that have been further transformed and differentiated into liver cells in vitro for subsequent administration to the patient. In another embodiment, the cells are muscle cells from the patient to be treated, or muscle stem cells that have been further transformed and, if necessary, differentiated into muscle cells in vitro for subsequent administration to the patient.

[0243] 6 - Pharmaceutical Compositions The present invention also provides pharmaceutical compositions comprising nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE polypeptides, or cells. Such compositions may comprise a therapeutically effective amount of the therapeutic agent (the nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE polypeptides, or cells) and a pharmaceutically acceptable carrier. In certain embodiments, the term “pharmaceutically acceptable” means approved by a federal or state government supervisory authority for use in animals and humans, or listed in the United States or European Pharmacopoeia or other generally recognized pharmacopoeias. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle through which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile solutions, such as water and oil, which may include oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous solutions of dextrose and glycerol, can also be used as liquid carriers, particularly in injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol.

[0244] The compositions may also contain small amounts of wetting or emulsifying agents or pH buffers, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic agent, preferably in a purified form, along with a suitable amount of carrier to form an appropriate dose for the target. In certain embodiments, the nucleic acids, vectors, or cells of the present invention are formulated in a composition comprising phosphate-buffered saline, supplemented with 0.25% human serum albumin. In another specific embodiment, the nucleic acids, vectors, or cells of the present invention are formulated into a composition comprising Ringer's lactate solution and a nonionic surfactant, such as Pluronic® F68, at a final concentration of 0.01–0.0001% by mass of the total composition, for example, at a concentration of 0.001% by mass. The formulation may further comprise serum albumin, in particular human serum albumin, for example, 0.25% human serum albumin. Other suitable formulations for either storage or administration are known in the art, particularly from WO 2005 / 118792 or Allay et al., 2011.

[0245] In preferred embodiments, the composition is formulated according to the usual procedures for pharmaceutical compositions suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.

[0246] In one embodiment, the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, or cell of the present invention can be delivered by vesicles, particularly liposomes. In yet another embodiment, the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, or cell of the present invention can be delivered by a controlled-release system.

[0247] In certain embodiments, the nucleic acid molecule is delivered as mRNA corresponding to a transcript encoding the functional truncated GDE polypeptide of the present invention. Specifically, the mRNA of the present invention can be delivered using liposomes, for example, lipid nanoparticles (LNPs).

[0248] The methods for administering 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, nasal, epidural, and oral routes. In certain embodiments, administration is by intravenous or intramuscular routes. The nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides, or cells of the present invention, whether vectorized or not, can be administered by any convenient route, for example, by injection or bolus injection, by absorption through the epithelium or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Administration may be systemic or topical.

[0249] In certain embodiments, it may be desirable to administer the pharmaceutical composition of the present invention topically to an area requiring treatment, such as the liver or muscle. This can be achieved, for example, by an indwelling system, which is made of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or fibers.

[0250] In certain embodiments, the functionally shortened GDE polypeptide of the present invention is used in enzyme replacement therapy (ERT), particularly to treat GSDIII. The term “enzyme replacement therapy” or “ERT” generally refers to the introduction of a purified enzyme into an individual deficient in such an enzyme. The polypeptide to be administered according to the present invention can be obtained by recombinant expression, produced in vitro or in transgenic animals, or purified from isolated tissue or fluid. In particular, when used in ERT, the polypeptide of the present invention can be administered parenterally, for example, by intraperitoneal, intramuscular, or intravascular (i.e., intravenous or intra-arterial) administration. In particular, the polypeptide is administered by intravenous injection. Such administration can be repeated frequently, for example, daily, weekly, every two weeks, or monthly, particularly weekly or every two weeks.

[0251] 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 would be effective in treating GSDIII can be administered by standard clinical techniques. In addition, in vivo and / or in vitro assays may be used as needed to help predict the optimal dosage range. The exact dose to be used in the formulation will depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and the condition 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 target in need will vary depending on several factors, including but not limited to the route of administration, the specific disease being treated, the age of the target, or the expression level required to achieve the therapeutic effect. Those skilled in the art can easily determine the required dosage range based on these factors, etc., based on their knowledge in the art. In the case of treatment involving administration of a viral vector such as an AAV vector, a typical dose of the vector is at least 1 × 10¹⁶ per kilogram of body weight. 8 Vector genome (vg / kg), e.g., at least 1 × 10⁻¹⁶ 9vg / kg, at least 1 × 10⁻⁶ 10 vg / kg, at least 1 × 10⁻⁶ 11 vg / kg, at least 1 × 10⁻⁶ 12 vg / kg, at least 1 × 10⁻⁶ 13 vg / kg, or at least 1 × 10⁻⁶ 14 It is vg / kg.

[0252] 7 - Treatment Method 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, functional truncated GDE polypeptide, pharmaceutical composition, or cells of the present invention to a target requiring such treatment.

[0253] Cirrhosis and hepatocellular carcinoma can also develop in patients with GSDIII. Therefore, the present invention also relates to a method for treating cirrhosis and hepatocellular carcinoma in patients with 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 cells of the present invention to a target in need.

[0254] The present invention relates to a method for treating GSDIII that does not induce an immune response to a transgene (i.e., to a functionally truncated GDE polypeptide encoded by a nucleic acid molecule) or induces a reduced immune response to a transgene, and also relates to a method comprising the step of delivering a therapeutically effective amount of the nucleic acid, vector, functionally truncated GDE polypeptide, pharmaceutical composition or cells of the present invention to a subject requiring such treatment. The present invention also relates to a method for treating GSDIII that comprises repeated administration of a therapeutically effective amount of the nucleic acid, vector, functionally truncated GDE polypeptide, pharmaceutical composition or cells of the present invention to a subject requiring such treatment. In this embodiment, the nucleic acid molecule, nucleic acid construct or vector of the present invention comprises a promoter that functions in liver cells, thereby enabling immune tolerance to the expressed functionally truncated GDE polypeptide produced from those cells. Also in this embodiment, the pharmaceutical composition used in this embodiment comprises a nucleic acid molecule, nucleic acid construct or vector that comprises a promoter that functions in liver cells. In the case of delivery of cells, particularly liver cells, cardiac cells, CNS cells, or muscle cells, the cells may be cells collected in advance from a subject requiring treatment and manipulated by introducing the nucleic acid molecules, nucleic acid constructs, or vectors of the present invention into them so as to be able to produce functional truncated GDE polypeptides. In some embodiments, in embodiments involving repeated administration, such administration may be repeated at least once or more times, and may even be considered to be carried out according to a regular schedule, such as once a week, once a month, or once a year. A regular schedule may also include administration once every two, three, four, five, six, seven, eight, nine, or ten years, or once every ten years or more. In another specific embodiment, for each administration of the viral vector of the present invention, the administration is carried out using a different virus for each successive administration to avoid a decrease in effectiveness due to any immune response that may occur against a previously administered viral vector. For example, a first administration of an AAV vector containing an AAV8 capsid may be carried out, followed by an administration of a vector containing an AAV9 capsid.

[0255] According to the present invention, treatments may include curative, mitigating, or prophylactic effects. Therefore, therapeutic and prophylactic treatments include alleviating the symptoms of GSDIII, or preventing or otherwise reducing the risk of developing certain glycogen storage diseases. The term “prophylactic” can be understood as reducing the severity or manifestation of a particular condition. “Prophylactic” also includes preventing recurrence of a particular condition in a patient previously diagnosed with that condition. “Therapeutic” can also mean reducing the severity of an existing condition. The term “treatment” is used herein to refer to any regimen that may benefit an animal, in particular a mammal, and more particularly a human subject.

[0256] The present invention also relates to an ex vivo gene therapy method for the treatment of GSDIII, comprising the steps of introducing a nucleic acid molecule, nucleic acid construct, or vector of the present invention into isolated cells of a patient in need thereof, for example, isolated hematopoietic stem cells, and introducing said cells into said patient in need thereof.

[0257] The present invention also relates to nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE polypeptides, cells, or pharmaceutical compositions for use as pharmaceuticals.

[0258] The present invention relates to nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides, cells, or pharmaceutical compositions for use in methods for treating diseases caused by mutations in the AGL gene encoding GDE, and more particularly in methods for treating GSDIII (GSDIIIa and GSDIIIb), especially GSDIIIa.

[0259] The present invention further relates to the use of nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE polypeptides, cells, or pharmaceutical compositions of the present invention in the manufacture of pharmaceuticals useful for the treatment of GSDIII (German disease of spongiform neuropathy).

[0260] The present invention also relates to nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides, cells, or pharmaceutical compositions for use in methods for delivering GDE proteins to diseased tissues, particularly muscle and liver tissue, and especially to muscle. [Examples]

[0261] The present invention will be further described in detail with reference to the following experimental examples and accompanying drawings. These examples are provided for illustrative purposes only and are not intended to limit the invention.

[0262] In patent applications WO2020 / 030661 and WO2022 / 043280, we demonstrated the ability to generate shortened forms of GDE protein while maintaining enzyme activity. In particular, we showed that the shortened proteins "Δ1b2" and "Δ1b3" efficiently reduce glycogen levels in different muscle tissues. "Δ1b2" corresponds to the GDE polypeptide of SEQ ID NO: 1, with amino acids 2-81 deleted. "Δ1b3" corresponds to the GDE polypeptide of SEQ ID NO: 1, with amino acids 2-103 deleted. However, both proteins were shown to be less expressed or less stable in vivo than full-size GDE. Therefore, the objective was to modify the N-terminal shortening site to find a shortened protein that is more stable and better expressed, and ultimately to increase the ability of the protein to reduce glycogen in vivo.

[0263] Seven N-terminal truncated GDE proteins were designed as described in the following table:

[0264] [Table 3]

[0265] Multiple truncated GDEs of the N-terminal domain were generated (Δ1b9-Δ1b15, Figure 1) and compared with the "Δ1b3" GDE truncated protein. To evaluate the activity of the truncated GDEs, AAV vectors expressing the truncated GDEs (Δ1b9-Δ1b15, or Δ1b3 used as a control) or the full-size protein (GDE-full-size) were produced. The AAV9rh74-P1 vector (i.e., an AAV vector with a hybrid capsid of AAV9 and AAVrh74 modified with peptide P1) was used throughout the experiment. The transgene was cloned into a transgene expression cassette optimized for muscle expression, which consisted of a miniCMV promoter and a pA58 polyadenylation signal. The cassettes of Δ1b9-Δ1b15, Δ1b13, and GDE-full-size constructs are as shown by SEQ ID NO: 42-48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.

[0266] (Example 1) The vectors were injected into the left anterior tibialis muscle of 4-month-old GSDIII mice at a dose of 3×10 11 vg / mouse (Figure 2A). Wild-type (Agl + / + , PBS) or knockout mice (Agl - / - , PBS) injected with PBS were used as controls. The mice were euthanized 1 month after injection for analysis of GDE expression in the left anterior tibialis muscle.

[0267] Measurement of GDE expression Mouse tissues were homogenized in phosphate-buffered saline (PBS, ThermoFisher Scientific) containing cOmplete™ protease inhibitor cocktail (Roche, ref 4693132001). Protein concentration was determined using Pierce™ BCA Protein Assay (Thermo Fisher Scientific) according to the manufacturer's instructions. For both Agl - / - mice injected with PBS and Agl - / - mice injected with AAV, a 50 μg fraction of the total protein was taken, and for Agl+ / + For mice (used as positive controls), 10 μg of total protein was loaded into each well. SDS-PAGE electrophoresis was performed on a 4–12% Bis-Tris gradient polyacrylamide gel (NuPAGE®, Invitrogen). After transfer, the membranes were blocked with Intercept blocking buffer (LI-COR Biosciences) and incubated with anti-GDE rabbit polyclonal antibody (16582-1-AP, Proteintech) and anti-vinculin mouse monoclonal antibody (V9131, Sigma). The membranes were washed and incubated with appropriate secondary antibodies (LI-COR Biosciences), and visualized using the Odyssey imaging system (LI-COR Biosciences).

[0268] result The results showed protein expression for all truncated GDE proteins (Figures 2B-C). All truncated GDE proteins showed protein expression levels at least equivalent to or even higher than those of the Δ1b3 GDE construct (Figures 2B-C).

[0269] (Example 2) Next, the inventors evaluated glycogen accumulation in the quadriceps muscles of GDE-KO animals injected with an AAV encoding the Δ1b13 GDE construct, compared to an AAV vector encoding the Δ1b3 GDE construct.

[0270] The vector was applied to 3-5 month old GSDIII mice in a 2.5 × 10⁶ dose. 13 The drug was administered intravenously at a dose of vg / kg (Figure 3A). KO(Agl - / - ) and WT(Agl + / + Mice were injected with saline solution (PBS) as a negative control. Two months after injection, the mice were euthanized for analysis of glycogen content in the quadriceps muscles.

[0271] Glycogen content was indirectly measured as glucose released after complete digestion of tissue homogenates using Aspergillus niger amyloglucosidase (Sigma-Aldrich, ref A1602). Samples were incubated at 95°C for 10 minutes and then cooled to 4°C. The reaction was stopped by adding amyloglucosidase (final concentration 4 U / mL) and potassium acetate pH 5.5 (final concentration 25 mM) at 37°C for 90 minutes, followed by incubation at 95°C for 10 minutes. A control reaction without amyloglucosidase was prepared for each sample and incubated under the same conditions. The released glucose was determined using a glucose assay kit (Sigma-Aldrich), and the resulting absorbance was obtained at a wavelength of 540 nm using an EnSpire alpha plate reader (PerkinElmer). The glucose released after amyloglucosidase was then normalized by the total protein concentration.

[0272] result The graph in Figure 3 shows glycogen levels measured in AAV-treated animals (either Δ1b13 GDE or Δ1b3 GDE) as well as in untreated wild-type (WT) and KO animals. The results show that the Δ1b13 construct, which showed better expression than Δ1b3 (see Figure 2), exhibits an increased ability to reduce glycogen accumulation in vivo in GSDIII mice compared to Δ1b3 (see Figure 3B).

Claims

1. A functional truncated GDE polypeptide comprising a deletion relative to a reference functional full-length human GDE sequence, wherein the deletion is the first six amino acids of the N-terminus of the functional truncated GDE polypeptide. - MQYYFL (array 7); - MFLQGN (sequence number 8); - MQGNEK (Sequence ID 9); - MGNEKS (Sequence ID 10); - MNEKSG (sequence number 11); - MKSGGG (Sequence ID 12); - MSGGGY (Sequence ID 13) A functionally shortened GDE polypeptide, comprising the deletion of an amino acid at the N-terminus of the aforementioned reference functional full-length human GDE sequence, in such a manner.

2. The functionally shortened 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, 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 percent 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.

3. The functionally shortened GDE polypeptide according to claim 1, wherein the reference functional full-length human GDE has the amino acid sequence 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 percent sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO:

1.

4. The above further includes deletions or combinations of deletions relative to the reference functional full-length human GDE sequence, for example, a deletion or combination of deletions in the C-terminal region of the GDE sequence, or a deletion or combination of deletions in the central domain of the GDE sequence; In particular, the sequence further includes deletions or combinations of deletions for sequence numbers 1, 2, 3, 4, 5, or 6, wherein the deletions are selected from any of the deletions referred to as Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, and Δ7 in Table 2. A functionally shortened GDE polypeptide according to any one of claims 1 to 3.

5. A functional truncated GDE polypeptide according to any one of claims 1 to 4, having an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity with SEQ ID NOs. 14 to 20.

6. - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 14, and containing the sequence of SEQ ID NO: 21; - Having an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 15, and containing the sequence of SEQ ID NO: 22; - Having an amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 16, and containing the sequence of SEQ ID NO: 23; - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 17, and containing the sequence of SEQ ID NO: 24; - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 18, and containing the sequence of SEQ ID NO: 25; - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 19, and containing the sequence of SEQ ID NO: 26; or - Having an amino acid sequence that has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity with SEQ ID NO: 20, and containing the sequence of SEQ ID NO: 27, A functionally shortened GDE polypeptide according to any one of claims 1 to 5.

7. A functional truncated GDE polypeptide according to any one of claims 1 to 6, having the amino acid sequence shown in sequence numbers 14 to 20.

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

9. - Promoter; - Introns as needed; - The nucleic acid molecule according to claim 8; and - Polyadenylation signal An expression cassette comprising, preferably, in this order.

10. A vector comprising the nucleic acid molecule described in claim 8 or the expression cassette described in claim 9, particularly a viral vector.

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

12. Isolated cells transformed with the nucleic acid molecule described in claim 8, the expression cassette described in claim 9, or the vector described in claims 10 to 11, wherein the cells are, in particular, liver cells, muscle cells, cardiac cells, or CNS cells.

13. A functional 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 to 11, or a cell according to claim 12, for use as a pharmaceutical.

14. A functional 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 to 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 functional 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 to 11, or a cell according to claim 12, for use in a method for treating GSDIII (German disease coliformis).

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