N-terminally truncated glycogen debranching enzymes for treatment of glycogen storage disease III

Abstract

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

Description

Technical Field

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

[0002] Mutations in the AGL gene result in a genetic defect in glycogen debranching enzyme (GDE), or amylase-α-1,6-glucanase, 4-α-glucantransferase, enzymes involved in glycogen degradation. GDE exhibits two independent catalytic activities occurring at different sites on the protein: 4-α-glucantransferase activity and amylase-1,6-glucantransferase activity. In glycogen storage disease III (GSDIII), a genetic defect in GDE causes incomplete glycogenolysis, leading to the accumulation of abnormal glycogen with short outer chains in various organs, primarily the liver and muscles. The disease is characterized by hepatomegaly, hypoglycemia, short stature, variable myopathy, and cardiomyopathy. Most patients have GSDIII (type IIIa) involving both the liver and muscles, while some (~15%) involve only the liver (type IIIb). Hepatic symptoms typically appear in childhood. Cirrhosis and hepatocellular carcinoma have been reported in some cases (Chen et al., 2009, *Scriver's Online Metabolic & Molecular Bases of Inherited Disease*, New York: McGraw-Hill; Kishnani et al., 2010, *Genet Med* 12, 446-463). Myasthenia gravis may occur in childhood. It becomes more common in adults, with onset in the twenties or thirties. There is a significant incidence of progressive myasthenia gravis, and patients in advanced stages may become wheelchair-bound. Cardiomyopathy may also occur in patients. The severity of symptoms in these patients exhibits significant clinical variability. Progressive myopathy and / or cardiomyopathy are the leading causes of disease in adults (Kishnani et al., 2010, Genet Med 12, 446-463; Cornelio et al., 1984, Arch Neurol 41, 1027-1032; Coleman et al., 1992, Ann Intern Med 116, 896-900). Reports of possible neurological manifestations associated with the disease have emerged from clinicians working with patients with GSDIII, who have reported fluctuations in attention, executive deficits, and impaired emotional skills (Michon et al., 2015, J Inherit Metab Dis, 38(3): 573-580). Therefore, in the disease Agl - / -In mouse models, significant accumulation of glycogen in the liver, skeletal muscle, and heart, and lower levels in the central nervous system have been recorded (Pagliarani et al., 2014, Biochim Biophys Acta, 1842(11):2318-2328; Liu et al., 2014, Mol Genet Metab, 111(4): 467-476), although detailed characterization of neurocognitive phenotypes associated with glycogen accumulation remains lacking. Current treatments are symptomatic and there is no effective therapy for the disease. Hypoglycemia can be controlled by frequent high-carbohydrate meals supplemented with corn starch or nighttime gastric tube feeding, and by a high-protein diet during the day followed by overnight enteral infusion. Transient improvements in muscle symptoms have been recorded in some patients following the use of a high-protein diet, but no systematic studies or long-term data have confirmed its ability to prevent or treat progressive myopathy (Kishnani et al., 2010, Genet Med 12, 446-463). These methods have little effect on altering the long-term course and incidence of these diseases.

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

[0004] The inventors have previously proposed using dual AAV vectors to overcome this size limitation (WO2018 / 162748). This approach uses two vectors (each containing half the expression cassette) to transduce the same cell. While the use of dual AAV vectors is promising, for economic and practical reasons, a gene therapy strategy that implements only one viral vector is more preferable.

[0005] In patent applications WO2020 / 030661 and WO2022 / 043280, the inventors described another method based on using a truncated GDE peptide suitable for a single viral vector. However, alternatives to the truncated GDE peptides previously described in WO2020 / 030661 and WO2022 / 043280 are desirable. Summary of the Invention

[0006] This invention relates to a functionally truncated GDE polypeptide, wherein the functionally truncated GDE polypeptide contains a deletion relative to a reference functional full-length human GDE sequence, and wherein the deletion consists of an amino acid deletion in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functionally truncated GDE polypeptide are: - MQYYFL (SEQ ID NO: 7); - MFLQGN (SEQ ID NO: 8); - MQGNEK (SEQ ID NO: 9); - MGNEKS (SEQ ID NO: 10); - MNEKSG (SEQ ID NO: 11); - MKSGGG (SEQ ID NO: 12); or - MSGGGY (SEQ ID NO: 13).

[0007] In a preferred embodiment, the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MNEKSG (SEQ ID NO: 11).

[0008] In one specific embodiment, the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In another specific embodiment, the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO: 1, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO: 1.

[0009] In one particular embodiment, the functionally truncated GDE peptide further comprises, relative to the reference functional full-length human GDE sequence, deletions or combinations of deletions, such as deletions or combinations of deletions in the C-terminal portion of the GDE sequence or deletions or combinations of deletions in the central domain of the GDE sequence. In one particular embodiment, the functionally truncated GDE peptide further comprises, 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, wherein the deletion is selected from any deletion referred to as Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, and Δ7 in Table 2 below.

[0010] In one specific embodiment, the functional truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NO: 14-20, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 14-20. In one specific embodiment, the functional truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NO: 14-20. Preferably, the functional truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NO: 18, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 18.

[0011] In one particular embodiment, the functionally truncated GDE peptide according to any one of the preceding claims has: - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 14, and containing the sequence of SEQ ID NO: 21; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 15, and containing the sequence of SEQ ID NO: 22; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 16, and containing the sequence of SEQ ID NO: 23; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 17, and containing the sequence of SEQ ID NO: 24; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 18, and containing the sequence of SEQ ID NO: 25; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 19, and containing the sequence of SEQ ID NO: 26; or - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 20, and containing the sequence of SEQ ID NO: 27.

[0012] This invention also relates to a nucleic acid molecule that encodes the functionally truncated GDE polypeptide of this invention.

[0013] The present invention also relates to an expression box, which preferably comprises in the following order: - Promoter; - Optional introns; - The nucleic acid molecule as claimed in the claims of this invention; and - Polyadenylation signaling.

[0014] Another aspect of the invention relates to a vector, particularly a viral vector, comprising the nucleic acid molecule or expression cassette of the invention. In one particular embodiment, the vector is an AAV vector.

[0015] The present invention also relates to an isolated cell transformed with the nucleic acid molecule, expression cassette or vector of the present invention, wherein the cell is in particular hepatocyte, myocyte, cardiomyocyte or CNS cell.

[0016] This invention also relates to functionally truncated GDE peptides, nucleic acid molecules, expression cassettes, vectors, or cells as defined above, used as pharmaceuticals. This invention also relates to methods for treating diseases caused by mutations in the AGL gene encoding GDE, using functionally truncated GDE peptides, nucleic acid molecules, expression cassettes, vectors, or cells as defined above. In another specific embodiment, this invention relates to methods for treating GSDIII (Corrie's disease), using functionally truncated GDE peptides, nucleic acid molecules, expression cassettes, vectors, or cells as defined above. Attached Figure Description

[0017] Figure 1 . The N-terminal region of the GDE enzyme in Candida glabrata (top) and humans (bottom). The truncation sites of the “Δ1b2” and “Δ1b3” proteins, as well as the truncation sites of the truncated proteins (“Δ1b9” to “Δ1b15”), are shown.

[0018] Figure 2 . (A) Experimental design involving intramuscular injection of an rAAV vector containing truncated forms of GDE (“Δ1b9” to “Δ1b15”) into GSDIII mice. Truncated “Δ1b3” protein and full-length GDE (FS) were used as controls. Histological analysis was performed 1 month after intramuscular injection. KO was injected with saline (PBS). Agl - / - ) and WT Agl + / + (A) Mice, as negative controls. (B) Western blot analysis of GDE and plaque protein expression from lysates of the left tibialis anterior (TA) muscle one month after intramuscular injection of an rAAV vector containing a truncated GDE peptide. (C) Based on Figure 2 (B) Western blot for quantification of GDE protein expression.

[0019] Figure 3 . (A) Experimental design involving intravenous injection of rAAV vectors containing truncated forms of GDE (“Δ1b3” and “Δ1b13”) into GSDIII mice. Tissue analysis was performed 2 months post-intravenous injection. KO (Agl- / -) and WT (Agl+ / +) mice were injected with saline (PBS) as negative controls. (B) Glycogen content in quadriceps femoris muscle lysate 2 months post-intravenous injection of rAAV vectors containing truncated GDE peptides. Detailed Implementation

[0020] In patent applications WO2020 / 030661 and WO2022 / 043280, the inventors have demonstrated the use of truncated GDE peptides whose size is compatible with encapsulation in an AAV vector while maintaining their enzyme activity.

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

[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 (Corrie's disease).

[0023] 1-N-terminal truncated GDE peptide

[0024] The truncated GDE polypeptide according to the present invention is a functional GDE polypeptide whose coding sequence is small enough to be efficiently packaged in gene therapy vectors, particularly in a single AAV vector.

[0025] "Functional" GDE polypeptides are defined as polypeptides that at least partially retain at least one enzymatic activity of the GDE protein, preferably all enzymatic activities of the GDE protein. Therefore, the functional GDE polypeptides implemented in this invention can salvage glycogen accumulation and muscle strength in vivo. As defined herein, GDE enzymatic activity refers to 4-α-glucan transferase activity and amylase-1,6-glucanase activity involved in glycogen degradation. The transferase activity of GDEs repositions three glucose units of glycogen from one chain to another. This leaves a glucose unit at the branch point, which is subsequently released as glucose via glucosidase activity. In one particular embodiment, the functional GDE polypeptides of this invention have the same function as full-length GDE polypeptides, particularly full-length human GDE polypeptides. For example, compared with full-length human GDE proteins, particularly those 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 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 activity of one, preferably two, of the aforementioned enzymes, or at least 100% of the activity. The activity of the GDE protein of the present invention may even be more than 100% of the activity of full-length human GDE proteins, particularly those 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, more than 110%, 120%, 130%, 140%, 150%, 200%, 500%, 700%, or even more than 1000%. In one particular embodiment, the functional GDE polypeptide of the present invention has the same function as the full-length GDE polypeptide, particularly the full-length human GDE polypeptide in muscle tissue such as cardiac muscle or quadriceps femoris. For example, compared with 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 functional GDE polypeptide of the present invention in muscle tissue such as cardiac muscle or quadriceps femoris relative to one, preferably both, of the above enzymes can be at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99%, or at least 100%.The activity of the GDE protein of the present invention in muscle tissue, such as cardiac muscle or quadriceps femoris, can be more than 100% of the activity of the full-length human GDE protein, especially the full-length human GDE protein of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, for example, more than 110%, 120%, 130%, 140%, 150%, 200%, 500%, 700% or even more than 1000%.

[0026] "Functional" GDE peptides also refer to non-pathological GDE peptides. Specifically, the functional GDE peptides of the present invention are not GDE peptides found in patients with GSDIII (Corrie's disease), such as GSDIIIa or GSDIIIb.

[0027] Those skilled in the art can readily determine whether a polypeptide is a functional GDE polypeptide. Suitable methods will be apparent to those skilled in the art. For example, a suitable in vitro method involves inserting the nucleic acid encoding the polypeptide into a vector, such as a plasmid or viral vector, transfecting or transducing host cells, such as 293T or HeLa cells, or other cells, such as Huh7, with said vector, and measuring GDE activity. Alternatively, GDE activity can be determined by measuring the glucose produced after incubating homogenized tissue or cell extracts previously transfected with a vector expressing a functional GDE polypeptide with limited dextrin (glycogen digested by glycogen phosphorylase). Other methods include testing the efficacy of GDE by assessing muscle strength in treated GDE-KO animals by wire suspension after administration of said vector, for example, one, two, or three months after administration, and by assessing the rescue of glycogen accumulation in muscle and / or myocardial tissue and / or assessing the normalization of blood glucose in treated GDE-KO animals after administration of said vector, for example, one, two, or three months after administration. Furthermore, GDE expression in tissues of GDE KO animals can be assessed by Western blotting after administration of the vector, for example, one, two, or three months later. Some suitable methods are described in more detail in the experimental section below.

[0028] In the context of this invention, "reference full-length human GDE sequence" encompasses all natural isotypes of human GDE. Bao et al. (Genomics, 1997, 38, 155-165) identified the existence of six transcriptomorphs encoding three GDE protein isotypes. Transcriptomorphs 1-4 encode the same protein, namely GDE isotype 1. Transcriptomorphs 5 and 6 encode GDE isotypes 5 and 6, respectively.

[0029] In the context of this invention, the “reference full-length human GDE sequence” does not encode pathological GDE peptides. Specifically, the reference full-length human GDE sequence does not encode pathological variants found in GSDIII patients that contain mutations, deletions, or insertions compared to wild-type non-pathological full-length human GDE sequences.

[0030] Therefore, the term "reference full-length human GDE polypeptide" encompasses all natural isotypes of human GDE, including precursor forms, as well as GDE proteins or fragments thereof modified or mutated through insertion, deletion, and / or substitution as functional derivatives of GDE. Specifically, the reference full-length human GDE sequence is selected from SEQ ID NO: 1 (corresponding to wild-type GDE isotype 1, UniProtKB identifier: P35573-1), SEQ ID NO: 4 (corresponding to a variant of GDE isotype 1), SEQ ID NO: 2 (corresponding to wild-type GDE isotype 5, UniProtKB identifier: P35573-2), SEQ ID NO: 5 (corresponding to a variant of GDE isotype 5), SEQ ID NO: 3 (corresponding to wild-type GDE isotype 6, UniProtKB identifier: P35573-3), and SEQ ID NO: 6 (corresponding to a variant of GDE isotype 6).

[0031] In one specific embodiment, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, particularly with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In one specific embodiment, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 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 one specific embodiment, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 5, and has the same length as SEQ ID NO: 2 or SEQ ID NO: 5 in terms of the number of amino acids. In one specific embodiment, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 3 or SEQ ID NO: 6, and has the same length as SEQ ID NO: 3 or SEQ ID NO: 6 in terms of the number of amino acids.

[0032] The term "identical" and its variations refer to sequence identity between two nucleic acid molecules or two polypeptide molecules. Two compared sequences are considered identical at that position when a position is occupied by the same base or the same amino acid. 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, multiplied by 100. For example, if six out of ten positions in two sequences match, the two sequences have 60% identity. Typically, comparisons are performed when the two sequences are aligned to provide maximum identity. Various bioinformatics tools known to those skilled in the art can be used to align nucleic acid sequences, such as BLAST or FASTA.

[0033] In one particular embodiment, the reference full-length human GDE sequence has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, particularly SEQ ID NO: 1, corresponding to GDE subtype 1.

[0034] In one particular embodiment, the truncated GDE polypeptide of the present invention, which is truncated relative to a reference full-length human GDE sequence, may contain one or more additional amino acid modifications relative to the reference full-length human GDE sequence. Specifically, in addition to deletions further described below, the functionally truncated GDE polypeptide may contain one or more amino acid modifications, such as amino acid insertions, deletions, and / or substitutions, compared to the reference full-length human GDE sequence. For example, the functionally truncated GDE polypeptide may contain 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acid modifications, particularly 1 to 5 (e.g., 1, 2, 3, 4, or 5) additional amino acid modifications, provided that the function of the truncated GDE polypeptide is preserved.

[0035] The functional truncated GDE polypeptide of the present invention is an N-terminal truncated GDE polypeptide. "N-terminal truncated GDE polypeptide" means a GDE polypeptide containing a deletion relative to a reference full-length functional human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference full-length functional human GDE sequence. The "N-terminal portion" of the reference full-length functional GDE sequence refers to the region consisting of the first 280 amino acid residues (i.e., the N-terminal 280 amino acid residues of the reference full-length GDE sequence), the first 200 amino acid residues (i.e., the N-terminal 200 amino acid residues of the reference full-length GDE sequence), the first 150 amino acid residues (i.e., the N-terminal 150 amino acid residues of the reference full-length GDE sequence), preferably the first 125 amino acid residues (i.e., the N-terminal 125 amino acid residues of the reference full-length GDE sequence), and most preferably the first 123 amino acid residues (i.e., the N-terminal 123 amino acid residues of the reference full-length GDE sequence).

[0036] As detailed below, the N-terminal truncated GDE polypeptide of the present invention retains methionine as the first residue at the N-terminal end.

[0037] The functional truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of an amino acid deletion in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE peptide are: - MQYYFL (SEQ ID NO: 7); - MFLQGN (SEQ ID NO: 8); - MQGNEK (SEQ ID NO: 9); - MGNEKS (SEQ ID NO: 10); - MNEKSG (SEQ ID NO: 11); - MKSGGG (SEQ ID NO: 12); or - MSGGGY (SEQ ID NO: 13).

[0038] Preferably, the functional truncated GDE polypeptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MNEKSG (SEQ ID NO: 11).

[0039] In other words, the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE results in a truncated GDE polypeptide, wherein the first six amino acids at the N-terminus are different from the first six amino acids at the N-terminus of the reference functional full-length human GDE sequence, and wherein the first six amino acids at the N-terminus of the truncated polypeptide are SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

[0040] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence wherein the reference functional full-length human GDE sequence has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; - And wherein the deletion consists of an amino acid deletion in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are: - MQYYFL (SEQ ID NO: 7); - MFLQGN (SEQ ID NO: 8); - MQGNEK (SEQ ID NO: 9); - MGNEKS (SEQ ID NO: 10); - MNEKSG (SEQ ID NO: 11); - MKSGGG (SEQ ID NO: 12); or - MSGGGY (SEQ ID NO: 13).

[0041] In a preferred embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence wherein the reference functional full-length human GDE sequence has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; - And the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MNEKSG (SEQ ID NO: 11).

[0042] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence wherein the reference functional full-length human GDE sequence has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; - And the aforementioned deficiency consists of the following deficiencies: (i) The deletion of any amino acid between the first methionine and the sequence “QYYFL” (SEQ ID NO: 66); (ii) The deletion of any amino acid between the first methionine and the sequence “FLQGN” (SEQ ID NO: 67); (iii) The deletion of any amino acid between the first methionine and the sequence “QGNEK” (SEQ ID NO: 68); (iv) The deletion of any amino acid between the first methionine and the sequence “GNEKS” (SEQ ID NO: 69); (v) The deletion of any amino acid between the first methionine and the sequence “NEKSG” (SEQ ID NO: 70); (vi) The deletion of any amino acid between the first methionine and the sequence “KSGGG” (SEQ ID NO: 71); (vii) The deletion of any amino acid between the first methionine and the sequence “SGGGY” (SEQ ID NO: 72).

[0043] According to this implementation method Depend on The deletion of any amino acid between the first methionine and the sequence "QYYFL". compositionThe deletion means that all consecutive amino acids between the first methionine at the N-terminus and the sequence “QYYFL” are deleted, while the first methionine and the sequence “QYYFL” (SEQ ID NO: 66) are not deleted.

[0044] According to this implementation method Depend on The deletion of any amino acid between the first methionine and the sequence "FLQGN". composition The deletion means that all consecutive amino acids between the first methionine at the N-terminus and the sequence “FLQGN” are missing, while the first methionine and the sequence “FLQGN” (SEQ ID NO: 67) are not missing.

[0045] According to this implementation method Depend on The deletion of any amino acid between the first methionine and the sequence "QGNEK". composition The deletion means that all consecutive amino acids between the first methionine at the N-terminus and the sequence “QGNEK” are deleted, while the first methionine and the sequence “QGNEK” (SEQ ID NO: 68) are not deleted.

[0046] According to this implementation method Depend on The deletion of any amino acid between the first methionine and the sequence "GNEKS". composition The deletion means that all consecutive amino acids between the first methionine at the N-terminus and the sequence “GNEKS” are deleted, while the first methionine and the sequence “GNEKS” (SEQ ID NO: 69) are not deleted.

[0047] According to this implementation method Depend on The deletion of any amino acid between the first methionine and the sequence “NEKSG”. composition The deletion means that all consecutive amino acids between the first methionine at the N-terminus and the sequence “NEKSG” are missing, while the first methionine and the sequence “NEKSG” (SEQ ID NO: 70) are not missing.

[0048] According to this implementation method Depend on The deletion of any amino acid between the first methionine and the sequence "KSGGG". composition The deletion means that all consecutive amino acids between the first methionine at the N-terminus and the sequence “KSGGG” are deleted, while the first methionine and the sequence “KSGGG” (SEQ ID NO: 71) are not deleted.

[0049] According to this implementation method Depend on The deletion of any amino acid between the first methionine and the sequence "SGGGY". composition The deletion means that all consecutive amino acids between the first methionine at the N-terminus and the sequence “SGGGY” are missing, while the first methionine and the sequence “SGGGY” (SEQ ID NO: 72) are not missing.

[0050] In a preferred embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence wherein the reference functional full-length human GDE sequence has an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; - And the deletion described therein consists of the deletion of any amino acid between the first methionine and the sequence “NEKSG” (SEQ ID NO: 70).

[0051] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence wherein the amino acid sequence of the functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length is described. - The aforementioned missing information consists of the following missing information: (i) The deletion of amino acids at positions 2-91, except that amino acids at positions 1 and 92 are not deleted; (ii) The deletion of amino acids at positions 2-94, except that amino acids at positions 1 and 95 are not deleted; (iii) The amino acid at positions 2-96 is deleted, but the amino acids at positions 1 and 97 are not deleted; (iv) The amino acids at positions 2-97 are deleted, except for the amino acids at positions 1 and 98; (v) The deletion of amino acids at positions 2-98, with amino acids at positions 1 and 99 not being deleted; (vi) The deletion of amino acids at positions 2-100, except that amino acids at positions 1 and 101 are not deleted; or (vii) The amino acids at positions 2-101 are deleted, while the amino acids at positions 1 and 102 are not deleted.

[0052] In a preferred embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence wherein the amino acid sequence of the functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length is described. - The deletion consists of the deletion of amino acids at positions 2-98, and the amino acids at positions 1 and 99 are not deleted.

[0053] In one particular embodiment, the functionally truncated GDE peptide of the present invention comprises one and only one deletion of the N-terminal portion of the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The N-terminal portion corresponds to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence. - And the deletion in the N-terminal portion of the reference functional full-length human GDE sequence consists of the following deletions: (i) The deletion of amino acids at positions 2-91 of the reference functional full-length human GDE sequence; (ii) The deletion of amino acids at positions 2-94 of the reference functional full-length human GDE sequence; (iii) The deletion of amino acids at positions 2-96 of the reference functional full-length human GDE sequence; (iv) The deletion of amino acids at positions 2-97 of the reference functional full-length human GDE sequence; (v) The deletion of amino acids at positions 2-98 of the reference functional full-length human GDE sequence; (vi) The deletion of amino acids at positions 2-100 of the reference functional full-length human GDE sequence; or (vii) The deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence.

[0054] In a preferred embodiment, the functionally truncated GDE peptide of the present invention comprises one and only one deletion of the N-terminal portion of the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The N-terminal portion corresponds to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence. - And the deletion in the N-terminal portion of the reference functional full-length human GDE sequence consists of the deletion of amino acids at positions 2-98 of the reference functional full-length human GDE sequence.

[0055] "One and only one deletion" means that there is a deletion in the N-terminal portion of the region corresponding to the first 280 amino acid residues of the reference full-length functional human GDE sequence (i.e., the N-terminal 280 amino acid residues of the reference full-length GDE sequence). For clarity, a functional truncated GDE containing one and only one deletion of amino acids at positions 2-91 of the N-terminal portion of the reference full-length functional human GDE sequence corresponds to the following truncated polypeptide: - The amino acids at positions 2-91 of the reference functional full-length sequence are deleted, and - The amino acids at positions 1 and 92-280 were not deleted.

[0056] In one particular embodiment, the functionally truncated GDE peptide of the present invention includes one and only one deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 6. - And the aforementioned missing values ​​are referred to as Δ1b9, Δ1b10, Δ1b11, Δ1b12, Δ1b13, Δ1b14, or Δ1b15 in Table 1: Table 1:

[0057] For clarity, Table 1 should be understood as follows. When the reference full-length GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, the functional “Δ1b9” truncated GDE polypeptide corresponds to a functional truncated GDE polypeptide derived from SEQ ID NO: 1 or SEQ ID NO: 4, which, relative to SEQ ID NO: 1 or SEQ ID NO: 4, has all consecutive amino acids from positions 2-91 deleted.

[0058] "Δ1b9" truncated polypeptide

[0059] In one particular embodiment, the functional truncated GDE polypeptide of the present invention includes a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are MQYYFL (SEQ ID NO: 7).

[0060] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and - The deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MQYYFL” (SEQ ID NO: 7).

[0061] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-91 of the reference functional full-length GDE sequence, and - The amino acids at positions 1 and 92 of the reference functional full-length GDE sequence were not deleted.

[0062] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids 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 was not deleted, and - Amino acids from positions 92 to 110, 92 to 130, 92 to 150, 92 to 170, 92 to 190, 92 to 210, 92 to 230, 92 to 250, 92 to 270, or 92 to 280 of the reference functional full-length GDE sequence are not deleted.

[0063] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids 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 was not deleted, and - The amino acids from positions 92 to 280 of the reference functional full-length GDE sequence were not deleted.

[0064] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-91 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence “MQYYFL” (SEQ ID NO: 7).

[0065] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-91 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described therein contains the sequence “MQYYFL” (SEQ ID NO: 7). - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0066] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-91 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence of SEQ ID NO: 21.

[0067] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-91 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described herein contains the sequence of SEQ ID NO: 21. - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0068] In one particular embodiment, the functionally truncated GDE polypeptide 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 with SEQ ID NO: 14, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 14, or is composed of said sequence or functional variant.

[0069] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 14, such as at least 75% or at least 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has substantially the same function or enzymatic activity as the GDE peptide of SEQ ID NO: 14, particularly the same enzymatic activity involved in glycogen degradation in muscle tissue such as cardiac muscle or quadriceps femoris. In one specific embodiment, the functional variant of SEQ ID NO: 14 has substantially the same ability to salvage glycogen accumulation and muscle strength in vivo as the GDE peptide of SEQ ID NO: 14. In one specific embodiment, the functional variant of SEQ ID NO: 14 has substantially the same expression level as the GDE peptide of SEQ ID NO: 14.

[0070] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 14, such as at least 75% or at least 80% sequence identity with SEQ ID NO: 14 (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has the same N-terminal portion as SEQ ID NO: 14. In one specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 14 is “MQYYFL” (SEQ ID NO: 7). In another specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 14 corresponds to the sequence of SEQ ID NO: 21.

[0071] In one particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 14 may be composed 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 in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, or 1-200 of SEQ ID NO: 14. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 14 is composed 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 in the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0072] In a preferred embodiment, the functionally truncated GDE polypeptide comprises or is composed of SEQ ID NO: 14.

[0073] "Δ1b10" truncated polypeptide

[0074] In one particular embodiment, the functional truncated GDE polypeptide of the present invention includes a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MFLQGN” (SEQ ID NO: 8).

[0075] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and - The deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MFLQGN” (SEQ ID NO: 8).

[0076] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-94 of the reference functional full-length GDE sequence, and - The amino acids at position 1 and position 95 of the reference functional full-length GDE sequence were not deleted.

[0077] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids 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 was not deleted, and - Amino acids at positions 95-110, 95 to 130, 95 to 150, 95 to 170, 95 to 190, 95 to 210, 95 to 230, 95 to 250, 95 to 270 or 95 to 280 from the reference functional full-length GDE sequence are not deleted.

[0078] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids 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 was not deleted, and - The amino acids from positions 95 to 280 of the reference functional full-length GDE sequence were not deleted.

[0079] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-94 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence “MFLQGN” (SEQ ID NO: 8).

[0080] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-94 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described therein contains the sequence “MFLQGN” (SEQ ID NO: 8). - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0081] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-94 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence of SEQ ID NO: 22.

[0082] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-94 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described herein comprises the sequence of SEQ ID NO: 22. - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0083] In one particular embodiment, the functionally truncated GDE polypeptide comprises SEQ ID NO: 15 or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 15, such as having at least 75% or at least 80% sequence identity with SEQ ID NO: 15, 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, or is composed of said sequence or functional variant.

[0084] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 15, such as at least 75% or at least 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has substantially the same function or enzymatic activity as the GDE peptide of SEQ ID NO: 15, particularly the same enzymatic activity involved in glycogen degradation in muscle tissue such as cardiac muscle or quadriceps femoris. In one specific embodiment, the functional variant of SEQ ID NO: 15 has substantially the same ability to salvage glycogen accumulation and muscle strength in vivo as the GDE peptide of SEQ ID NO: 15. In one specific embodiment, the functional variant of SEQ ID NO: 15 has substantially the same expression level as the GDE peptide of SEQ ID NO: 15.

[0085] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 15, such as at least 75% or at least 80% sequence identity with SEQ ID NO: 15 (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has the same N-terminal portion as SEQ ID NO: 15. In one specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 15 is “MFLQGN” (SEQ ID NO: 8). In another specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 15 corresponds to the sequence of SEQ ID NO: 22.

[0086] In one particular embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 15 may be composed 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: 15. In other words, according to this embodiment, the functional variant of SEQ ID NO: 15 does not contain any mutations, insertions, or deletions in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, or 1-200 of SEQ ID NO: 15. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 15 is composed 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 in the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0087] In a preferred embodiment, the functionally truncated GDE polypeptide comprises or is composed of SEQ ID NO: 15.

[0088] "Δ1b11" truncated polypeptide

[0089] In one particular embodiment, the functional truncated GDE polypeptide of the present invention includes a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MQGNEK” (SEQ ID NO: 9).

[0090] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and - The deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MQGNEK” (SEQ ID NO: 9).

[0091] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-96 of the reference functional full-length GDE sequence, and - The amino acids at positions 1 and 97 of the reference functional full-length GDE sequence were not deleted.

[0092] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion is composed of the deletion of amino acids at positions 2-96 of the reference functional full-length GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence was not deleted, and - Amino acids at positions 97 to 130, 97 to 150, 97 to 170, 97 to 190, 97 to 210, 97 to 230, 97 to 250, 97 to 270 or 97 to 280 from the reference functional full-length GDE sequence are not deleted.

[0093] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-96 of the reference functional full-length GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence was not deleted, and - The amino acids from positions 97 to 280 of the reference functional full-length GDE sequence were not deleted.

[0094] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-96 of the reference functional full-length GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence “MQGNEK” (SEQ ID NO: 9).

[0095] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-96 of the reference functional full-length GDE sequence. - The functionally truncated GDE polypeptide described therein contains the sequence “MQGNEK” (SEQ ID NO: 9). - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0096] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-96 of the reference functional full-length GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence of SEQ ID NO: 23.

[0097] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-96 of the reference functional full-length GDE sequence. - The functionally truncated GDE polypeptide described herein contains the sequence of SEQ ID NO: 23. - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0098] In one particular embodiment, the functionally truncated GDE polypeptide comprises SEQ ID NO: 16 or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 16, such as having at least 75% or at least 80% sequence identity with SEQ ID NO: 16, 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, or is composed of said sequence or functional variant.

[0099] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 16, such as at least 75% or at least 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has substantially the same function or enzymatic activity as the GDE peptide of SEQ ID NO: 16, particularly the same enzymatic activity involved in glycogen degradation in muscle tissue such as cardiac muscle or quadriceps femoris. In one specific embodiment, the functional variant of SEQ ID NO: 16 has substantially the same ability to salvage glycogen accumulation and muscle strength in vivo as the GDE peptide of SEQ ID NO: 16. In one specific embodiment, the functional variant of SEQ ID NO: 16 has substantially the same expression level as the GDE peptide of SEQ ID NO: 16.

[0100] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 16, such as at least 75% or at least 80% sequence identity with SEQ ID NO: 16 (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has the same N-terminal portion as SEQ ID NO: 16. In one specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 16 is “MQGNEK” (SEQ ID NO: 9). In another specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 16 corresponds to the sequence of SEQ ID NO: 23.

[0101] In another embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 16 may be composed 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: 16. In other words, according to this embodiment, the functional variant of SEQ ID NO: 16 does not contain any mutations, insertions, or deletions in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, or 1-200 of SEQ ID NO: 16. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 16 is composed 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 in the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0102] In a preferred embodiment, the functionally truncated GDE polypeptide comprises or is composed of SEQ ID NO: 16.

[0103] "Δ1b12" truncated polypeptide

[0104] In one particular embodiment, the functional truncated GDE polypeptide of the present invention includes a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MGNEKS” (SEQ ID NO: 10).

[0105] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and - The deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MGNEKS” (SEQ ID NO: 10).

[0106] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-97 of the reference functional full-length human GDE sequence, and - The amino acids at positions 1 and 98 of the reference functional full-length GDE sequence were not deleted.

[0107] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids 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 was not deleted, and - Amino acids at positions 98 to 130, 98 to 150, 98 to 170, 98 to 190, 98 to 210, 98 to 230, 98 to 250, 98 to 270 or 98 to 280 from the reference functional full-length GDE sequence are not deleted.

[0108] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids 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 was not deleted, and - The amino acids from positions 98 to 280 of the reference functional full-length GDE sequence were not deleted.

[0109] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-97 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence “MGNEKS” (SEQ ID NO: 10).

[0110] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-97 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described therein contains the sequence “MGNEKS” (SEQ ID NO: 10). - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0111] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-97 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence of SEQ ID NO: 24.

[0112] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-97 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described herein comprises the sequence of SEQ ID NO: 24. - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0113] In one particular embodiment, the functionally truncated GDE polypeptide comprises SEQ ID NO: 17 or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 17, such as having at least 75% or at least 80% sequence identity with SEQ ID NO: 17, 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, or is composed of said sequence or functional variant.

[0114] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 17, such as at least 75% or at least 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has substantially the same function or enzymatic activity as the GDE peptide of SEQ ID NO: 17, particularly the same enzymatic activity involved in glycogen degradation in muscle tissue such as cardiac muscle or quadriceps femoris. In one specific embodiment, the functional variant of SEQ ID NO: 17 has substantially the same ability to salvage glycogen accumulation and muscle strength in vivo as the GDE peptide of SEQ ID NO: 17. In one specific embodiment, the functional variant of SEQ ID NO: 17 has substantially the same expression level as the GDE peptide of SEQ ID NO: 17.

[0115] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 17, such as at least 75% or at least 80% sequence identity with SEQ ID NO: 17 (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has the same N-terminal portion as SEQ ID NO: 17. In one specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 17 is “MGNEKS” (SEQ ID NO: 10). In another specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 17 corresponds to the sequence of SEQ ID NO: 24.

[0116] In another embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 17 may be composed 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: 17. In other words, according to this embodiment, the functional variant of SEQ ID NO: 17 does not contain any mutations, insertions, or deletions in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, or 1-200 of SEQ ID NO: 17. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 17 is composed 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 in the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0117] In a preferred embodiment, the functionally truncated GDE polypeptide comprises or is composed of SEQ ID NO: 17.

[0118] "Δ1b13" truncated polypeptide

[0119] In one particular embodiment, the functional truncated GDE polypeptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MNEKSG” (SEQ ID NO: 11).

[0120] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and - The deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MNEKSG” (SEQ ID NO: 11).

[0121] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-98 of the reference functional full-length human GDE sequence, and - The amino acids at positions 1 and 99 of the reference functional full-length GDE sequence were not deleted.

[0122] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids 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 was not deleted, and - Amino acids at positions 99 to 130, 99 to 150, 99 to 170, 99 to 190, 99 to 210, 99 to 230, 99 to 250, 99 to 270 or 99 to 280 from the reference functional full-length GDE sequence are not deleted.

[0123] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids 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 was not deleted, and - The amino acids from positions 99 to 280 of the reference functional full-length GDE sequence were not deleted.

[0124] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-98 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence “MNEKSG” (SEQ ID NO: 11).

[0125] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-98 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described herein contains the sequence “MNEKSG” (SEQ ID NO: 11). - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0126] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-98 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence of SEQ ID NO: 25.

[0127] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-98 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described herein comprises the sequence of SEQ ID NO: 25. - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0128] In one particular embodiment, the functionally truncated GDE polypeptide comprises SEQ ID NO: 18 or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 18, such as having at least 75% or at least 80% sequence identity with SEQ ID NO: 18, 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, or is composed of said sequence or functional variant.

[0129] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 18, such as at least 75% or at least 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has substantially the same function or enzymatic activity as the GDE peptide of SEQ ID NO: 18, particularly the same enzymatic activity involved in glycogen degradation in muscle tissue such as cardiac muscle or quadriceps femoris. In one specific embodiment, the functional variant of SEQ ID NO: 18 has substantially the same ability to salvage glycogen accumulation and muscle strength in vivo as the GDE peptide of SEQ ID NO: 18. In one specific embodiment, the functional variant of SEQ ID NO: 18 has substantially the same expression level as the GDE peptide of SEQ ID NO: 18.

[0130] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 18, such as at least 75% or at least 80% sequence identity with SEQ ID NO: 18 (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has the same N-terminal portion as SEQ ID NO: 18. In one specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 18 is “MNEKSG” (SEQ ID NO: 11). In another specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 18 corresponds to the sequence of SEQ ID NO: 25.

[0131] In another embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 18 may be composed 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: 18. In other words, according to this embodiment, the functional variant of SEQ ID NO: 18 does not contain any mutations, insertions, or deletions in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, or 1-200 of SEQ ID NO: 18. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 18 is composed 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 in the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0132] In a preferred embodiment, the functionally truncated GDE polypeptide comprises or is composed of SEQ ID NO: 18.

[0133] "Δ1b14" truncated polypeptide

[0134] In one particular embodiment, the functional truncated GDE polypeptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MKSGGG” (SEQ ID NO: 12).

[0135] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and - The deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MKSGGG” (SEQ ID NO: 12).

[0136] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-100 of the reference functional full-length human GDE sequence, and - The amino acids at position 1 and position 101 of the reference functional full-length GDE sequence were not deleted.

[0137] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids from position 2 to position 100 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence was not deleted, and - Amino acids at positions 101 to 130, 101 to 150, 101 to 170, 101 to 190, 101 to 210, 101 to 230, 101 to 250, 101 to 270 or 101 to 280 from the reference functional full-length GDE sequence are not deleted.

[0138] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids from position 2 to position 100 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence was not deleted, and - The amino acids from positions 101 to 280 of the reference functional full-length GDE sequence were not deleted.

[0139] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids from position 2 to position 100 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide contained in the sequence “MKSGGG” (SEQ ID NO: 12).

[0140] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids from position 2 to position 100 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described therein contains the sequence “MKSGGG” (SEQ ID NO: 12). - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0141] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids from position 2 to position 100 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence of SEQ ID NO: 26.

[0142] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids from position 2 to position 100 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described herein comprises the sequence of SEQ ID NO: 26. - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0143] In one particular embodiment, the functionally truncated GDE polypeptide comprises SEQ ID NO: 19 or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 19, such as having at least 75% or at least 80% sequence identity with SEQ ID NO: 19, 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, or is composed of said sequence or functional variant.

[0144] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 19, such as at least 75% or at least 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has substantially the same function or enzymatic activity as the GDE peptide of SEQ ID NO: 19, particularly the same enzymatic activity involved in glycogen degradation in muscle tissue such as cardiac muscle or quadriceps femoris. In one specific embodiment, the functional variant of SEQ ID NO: 19 has substantially the same ability to salvage glycogen accumulation and muscle strength in vivo as the GDE peptide of SEQ ID NO: 19. In one specific embodiment, the functional variant of SEQ ID NO: 19 has substantially the same expression level as the GDE peptide of SEQ ID NO: 19.

[0145] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 19, such as at least 75% or at least 80% sequence identity with SEQ ID NO: 19 (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has the same N-terminal portion as SEQ ID NO: 19. In one specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 19 is “MKSGGG” (SEQ ID NO: 12). In another specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 19 corresponds to the sequence of SEQ ID NO: 26.

[0146] In another embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 19 may be composed 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: 19. In other words, according to this embodiment, the functional variant of SEQ ID NO: 19 does not contain any mutations, insertions, or deletions in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, or 1-200 of SEQ ID NO: 19. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 19 is composed 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 in the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0147] In a preferred embodiment, the functionally truncated GDE polypeptide comprises or is composed of SEQ ID NO: 19.

[0148] "Δ1b15" truncated polypeptide

[0149] In one particular embodiment, the functional truncated GDE polypeptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MSGGGY” (SEQ ID NO: 13).

[0150] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4; and - The deletion consists of the deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are “MSGGGY” (SEQ ID NO: 13).

[0151] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence, and - The amino acids at position 1 and position 102 of the reference functional full-length GDE sequence were not deleted.

[0152] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence was not deleted, and - Amino acids at positions 102 to 130, 102 to 150, 102 to 170, 102 to 190, 102 to 210, 102 to 230, 102 to 250, 102 to 270 or 102 to 280 from the reference functional full-length GDE sequence are not deleted.

[0153] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to the reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence. - The amino acid at position 1 of the reference functional full-length GDE sequence was not deleted, and - Among them, amino acids from positions 102 to 280 of the reference functional full-length GDE sequence were not deleted.

[0154] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide contained in the sequence “MSGGGY” (SEQ ID NO: 13).

[0155] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described therein contains the sequence “MSGGGY” (SEQ ID NO: 13). - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0156] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence. - And the functionally truncated GDE polypeptide described therein contains the sequence of SEQ ID NO: 27.

[0157] In one particular embodiment, the functionally truncated GDE peptide of the present invention contains a deletion relative to a reference functional full-length human GDE sequence. - The reference functional full-length human GDE sequence is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 or SEQ ID NO: 1 or SEQ ID NO: 4 of the same length. - The deletion consists of the deletion of amino acids at positions 2-101 of the reference functional full-length human GDE sequence. - The functionally truncated GDE polypeptide described herein contains the sequence of SEQ ID NO: 27. - And wherein the functionally truncated GDE polypeptide of the present invention contains at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0158] In one particular embodiment, the functionally truncated GDE polypeptide comprises SEQ ID NO: 20 or a functional variant thereof having at least 70% sequence identity with SEQ ID NO: 20, such as having at least 75% or at least 80% sequence identity with SEQ ID NO: 20, 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, or is composed of said sequence or functional variant.

[0159] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 20, such as at least 75% or at least 80% sequence identity (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has substantially the same function or enzymatic activity as the GDE peptide of SEQ ID NO: 20, particularly the same enzymatic activity involved in glycogen degradation in muscle tissue such as cardiac muscle or quadriceps femoris. In one specific embodiment, the functional variant of SEQ ID NO: 20 has substantially the same ability to salvage glycogen accumulation and muscle strength in vivo as the GDE peptide of SEQ ID NO: 20. In one specific embodiment, the functional variant of SEQ ID NO: 20 has substantially the same expression level as the GDE peptide of SEQ ID NO: 20.

[0160] In one specific embodiment, the functional variant having at least 70% sequence identity with SEQ ID NO: 20, such as at least 75% or at least 80% sequence identity with SEQ ID NO: 20 (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity), has the same N-terminal portion as SEQ ID NO: 20. In one specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 20 is “MSGGGY” (SEQ ID NO: 13). In another specific embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 20 corresponds to the sequence of SEQ ID NO: 27.

[0161] In another embodiment, the N-terminal amino acid of the functional variant of SEQ ID NO: 20 may be composed 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: 20. In other words, according to this embodiment, the functional variant of SEQ ID NO: 20 does not contain any mutations, insertions, or deletions in the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, or 1-200 of SEQ ID NO: 20. In a particular embodiment, the N-terminus of the functional variant of SEQ ID NO: 20 is composed 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 in the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0162] In a preferred embodiment, the functionally truncated GDE polypeptide comprises or is composed of SEQ ID NO: 20.

[0163] Combinations with other missing elements

[0164] In one particular embodiment, the functionally truncated GDE polypeptide of the present invention, which includes a deletion in the N-terminal portion of the reference functional full-length human GDE sequence as described above, may further include deletions or combinations of deletions in other portions of the reference functional full-length human GDE sequence.

[0165] In one particular implementation, the functionally truncated GDE peptide comprises: - The deletion of one and only one consecutive amino acid in the N-terminal portion of the reference functional full-length human GDE sequence, wherein the N-terminal portion corresponds to the first 280 amino acid residues of the reference functional full-length human GDE sequence. - Another deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence.

[0166] In one particular implementation, the functionally truncated GDE peptide comprises: - The deletion of one and only one consecutive amino acid in the N-terminal portion of the reference functional full-length human GDE sequence, wherein the N-terminal portion corresponds to the first 280 amino acid residues of the reference functional full-length human GDE sequence, and - Another deletion or combination of deletions in other portions of the reference functional full-length human GDE sequence. The reference functional full-length human GDE sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 6. And the aforementioned missing values ​​are referred to as Δ1b9, Δ1b10, Δ1b11, Δ1b12, Δ1b13, Δ1b14 or Δ1b15 in Table 1.

[0167] In one particular embodiment, the functionally truncated GDE polypeptide of the present invention comprises at least amino acid residues at positions 429-666, 866-892, 1088-1194, and 1235-1420 relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0168] In one particular embodiment, the functionally truncated GDE polypeptide of the present invention, which includes a deletion in the N-terminal portion of a reference full-length human GDE sequence, may further include a deletion or a combination of deletions in the C-terminal portion of the reference full-length GDE sequence and / or in the central domain of the reference full-length GDE sequence.

[0169] In one particular embodiment, the C-terminal portion of the reference full-length GDE sequence corresponds to the last 112 amino acids of the reference full-length human GDE sequence, i.e., the region consisting of the 112 most C-terminal amino acids.

[0170] In one particular embodiment, the central domain of the reference full-length GDE sequence of SEQ ID NO: 1 or SEQ ID NO: 4 corresponds to the region consisting of amino acids at positions 710 to 865 of SEQ ID NO: 1 or SEQ ID NO: 4.

[0171] In one particular implementation, the functionally truncated GDE peptide comprises: - One and only one deletion of the N-terminal portion of the reference functional full-length human GDE sequence, wherein the N-terminal portion corresponds to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence, and - Another deletion or combination of deletions in other portions of the reference functional full-length human GDE sequence. The reference functional full-length human GDE sequence is selected from SEQ ID NO: 1 to SEQ ID NO: 6. And the missing parts in the N-terminal portion are referred to as Δ1b9, Δ1b10, Δ1b11, Δ1b12, Δ1b13, Δ1b14 or Δ1b15 in Table 1; Furthermore, the additional deletions or combinations of deletions in other portions of the reference functional full-length human GDE sequence are selected from any deletions referred to as Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, and Δ7 in Table 2: Table 2:

[0172] 2- Nucleic acid molecules encoding the N-terminally truncated GDE polypeptide

[0173] On the other hand, the present invention relates to nucleic acid molecules encoding functionally truncated GDE polypeptides as defined above.

[0174] The term "nucleic acid molecule" (or nucleic acid sequence) refers to a DNA or RNA molecule in single-stranded or double-stranded form, particularly DNA encoding a functionally truncated GDE polypeptide according to the present invention.

[0175] In a preferred embodiment, the nucleic acid molecule encoding the functionally truncated GDE polypeptide is small enough to be packaged in a gene therapy vector, such as an AAV vector, in combination with a suitable regulatory sequence. The size of the nucleic acid molecule encoding the functionally truncated GDE polypeptide is preferably less than about 4.5 kb, and more preferably less than about 4.4 kb.

[0176] "Gene therapy vector" refers to any vector suitable for gene therapy. Specifically, the gene therapy vector can be a plasmid or a recombinant virus, such as a viral vector derived from a retrovirus or lentivirus. Preferably, the viral vector is an AAV vector, such as an AAV vector suitable for transducing liver tissue or muscle cells. Extensive experience in clinical trials and preclinical models of muscle diseases has demonstrated that adeno-associated virus (AAV) is the preferred vector for in vivo gene therapy of GSDIII. These vectors efficiently transduce liver and muscle, their production is scalable, and they exhibit relatively low immunogenicity compared to other gene therapy vectors. However, one of the biggest limitations of using AAV for gene replacement is their limited capsid size (approximately 5 kb). In fact, during recombinant AAV production, genomes larger than 5 kb typically exhibit low capsiding efficiency, and the resulting AAV may contain fragmented genomes, reducing gene transfer efficiency.

[0177] The sequence of the nucleic acid molecule encoding a functionally truncated GDE polypeptide of the present invention can be optimized for in vivo expression of the GDE polypeptide. Sequence optimization may include numerous changes in the nucleic acid sequence, including codon optimization, increasing GC content, reducing the number of CpG islands, reducing the number of optional open reading frames (ARFs), and / or reducing the number of splice donor and splice acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules may encode the same protein. Furthermore, it is well known that the genetic code of different organisms often biases the use of one of several codons encoding the same amino acid over the others. By introducing changes in the nucleotide sequence through codon optimization, which utilizes the codon bias present in a given cellular background, the resulting codon-optimized nucleotide sequence is more likely to be expressed at a relatively higher level than the uncodon-optimized sequence in such a given cellular background. In a preferred embodiment of the invention, such optimized nucleotide sequences encoding functionally truncated GDE polypeptides are codon-optimized to improve their expression in human cells compared to uncodon-optimized nucleotide sequences encoding the same functionally truncated GDE polypeptide, for example by utilizing human-specific codon usage bias. The nucleic acid sequence encoding the full-length human GDE subtype 1 is shown in SEQ ID NO: 51. An example of the corresponding codon-optimized sequence is shown in SEQ ID NO: 52.

[0178] In one specific embodiment, 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 one specific embodiment, 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, encoding a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 14. In one specific embodiment, the nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above has at least 80%, at least 85%, at least 90%, or at least 95% identity with the nucleotide sequence of SEQ ID NO: 28 or SEQ ID NO: 29, preferably SEQ ID NO: 29. In one specific embodiment, the nucleic acid molecule of the present invention has at least 95% identity with 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% identity.

[0179] In one specific embodiment, 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 one specific embodiment, 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, encoding a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 15. In one specific embodiment, the nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95% identity with the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31, preferably SEQ ID NO: 31. In one specific embodiment, the nucleic acid molecule of the present invention has at least 95% identity with 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% identity.

[0180] In one specific embodiment, 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 one specific embodiment, 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, encoding a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 16. In one specific embodiment, the nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above has at least 80%, at least 85%, at least 90%, or at least 95% identity with the nucleotide sequence of SEQ ID NO: 32 or SEQ ID NO: 33, preferably SEQ ID NO: 33. In one specific embodiment, the nucleic acid molecule of the present invention has at least 95% identity with 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% identity.

[0181] In one specific embodiment, 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 one specific embodiment, 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, encoding a functionally truncated GDE polypeptide having the amino acid sequence shown in SEQ ID NO: 17. In one specific embodiment, the nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above has at least 80%, at least 85%, at least 90%, or at least 95% identity with the nucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 35, preferably SEQ ID NO: 35. In one specific embodiment, the nucleic acid molecule of the present invention has at least 95% identity with 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% identity.

[0182] In one specific embodiment, 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 one specific embodiment, 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 one specific embodiment, the nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95% identity with the nucleotide sequence of SEQ ID NO: 36 or SEQ ID NO: 37, preferably SEQ ID NO: 37. In one specific embodiment, the nucleic acid molecule of the present invention has at least 95% identity with 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% identity.

[0183] In one specific embodiment, 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 one specific embodiment, 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 one specific embodiment, the nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95% identity with the nucleotide sequence of SEQ ID NO: 38 or SEQ ID NO: 39, preferably SEQ ID NO: 39. In one specific embodiment, the nucleic acid molecule of the present invention has at least 95% identity with 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% identity.

[0184] In one specific embodiment, 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 one specific embodiment, 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 one specific embodiment, the nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95% identity with the nucleotide sequence of SEQ ID NO: 40 or SEQ ID NO: 41, preferably SEQ ID NO: 41. In one specific embodiment, the nucleic acid molecule of the present invention has at least 95% identity with 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% identity.

[0185] The nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above may have at least 80%, at least 85%, at least 90%, or at least 95% identity with any of the nucleotide sequences in SEQ ID NO: 28 to 41. In one particular embodiment, the nucleic acid molecule of the present invention has at least 95% identity with any of the nucleotide sequences in SEQ ID NO: 28 to 41, for example, at least 96%, 97%, 98%, 99%, or 100% identity.

[0186] Preferably, the nucleic acid molecule encoding the functionally truncated GDE polypeptide as defined above may have at least 80, at least 85, at least 90, or at least 95% identity with any of the nucleotide sequences in SEQ ID 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 one particular embodiment, the nucleic acid molecule of the present invention has at least 95% identity with any of the nucleotide sequences in 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, for example, at least 96, 97, 98, 99, or 100% identity.

[0187] 3- Nucleic acid constructs

[0188] This invention also relates to a nucleic acid construct comprising the nucleic acid molecule of the invention as described above. The nucleic acid construct may correspond to an expression cassette comprising a nucleic acid sequence of the invention operatively linked to one or more expression control sequences and / or other sequences that enhance expression. As used herein, the term "operatively linked" refers to a polynucleotide element linked in a functional relationship. A nucleic acid is "operatively linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or another transcriptional regulatory sequence is operatively linked to the coding sequence if it affects the transcription of the coding gene. Such expression control sequences are known in the art, such as promoters, enhancers (e.g., cis-regulatory modules (CRMs)), introns, polyA signals, etc.

[0189] In one particular embodiment, the expression cassette may include a promoter. The promoter may be a ubiquitous or tissue-specific promoter, particularly one capable of promoting expression in cells or tissues requiring GDE expression, such as in cells or tissues requiring GDE expression in GDE-deficient patients.

[0190] In a first specific embodiment, the promoter is a muscle-specific promoter. Non-limiting examples of muscle-specific promoters include the muscle creatine kinase (MCK) promoter. Suitable non-limiting examples of muscle creatine kinase promoters are 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. 2008 Nov;15(22):1489-99) (representative GenBank accession number AF188002). Human muscle creatine kinase has gene ID number 1158 (representative GenBank accession number NC_000019.9, registered on December 26, 2012). Other examples of muscle-specific promoters include the synthetic promoter C5.12 (SPc5-12, also referred to as “C5.12” in this paper), such as the SPc5-12 promoter (published in Wang et al., Gene Therapy, Vol. 15, pp. 1489–1499 (2008)), the MHCK7 promoter (Salva et al., Mol Ther. 2007 Feb;15(2):320-9), myosin light chain (MLC) promoters such as MLC2 (gene ID 4633; representative GenBank accession NG_007554.1, registered on December 26, 2012), myosin heavy chain (MHC) promoters such as α-MHC (gene ID 4624; representative GenBank accession NG_023444.1, registered on December 26, 2012), and the desmin promoter (gene ID 167). 4; Representative GenBank accession number NG_008043.1, registered on December 26, 2012), cardiac troponin C promoter (gene ID 7134; representative GenBank accession number NG_008963.1, registered on December 26, 2012), troponin I promoter (gene IDs 7135, 7136 and 7137; representative GenBank accession numbers NG_016649.1, NG_011621.1 and NG_007866).2. Registered on December 26, 2012), myoD gene family promoters (Weintraub et al., Science, 251, 761 (1991); gene ID 4654; representative GenBank accession number NM_002478, registered on December 26, 2012), α-actin promoters (gene IDs 58, 59 and 70; representative GenBank accession numbers NG_006672.1, NG_011541.1 and NG_007553.1, registered on December 26, 2012), β-actin promoters (gene ID 60; representative GenBank accession number NG_007992.1, registered on December 26, 2012). The following are listed: the γ-actin promoter (gene IDs 71 and 72; representative GenBank accessions NG_011433.1 and NM_001199893, registered on December 26, 2012); the muscle-specific promoter located in intron 1 of the eye-shaped Pitx3 (gene ID 5309) (Coulon et al., the muscle-selective promoter corresponds to residues 11219-11527 of the representative GenBank accession NG_008147, registered on December 26, 2012); and the promoters described in US Patent Publication US 2003 / 0157064 and the CK6 promoter (Wang et al., 2008 doi: 10.1038 / gt.2008.104). In another specific embodiment, the muscle-specific promoter is the E-Syn promoter described in Wang et al., Gene Therapy, Vol. 15, pp. 1489–1499 (2008), which comprises a combination of an MCK-derived enhancer and an SPc5-12 promoter. In a specific embodiment of the invention, the muscle-specific promoter is selected from the SPc5-12 promoter, MHCK7 promoter, E-syn promoter, muscle creatine kinase promoter, myosin light chain (MLC) promoter, myosin heavy chain (MHC) promoter, cardiac troponin C promoter, troponin I promoter, myoD gene family promoter, α-actin promoter, β-actin promoter, γ-actin promoter, muscle-specific promoter located in intron 1 of the eye-shaped Pitx3, CK6 promoter, CK8 promoter, and Acta1 promoter. In another embodiment, the muscle-specific promoter is selected from SPc5-12, desmin, and MCK promoters. In another embodiment, the muscle-specific promoter is selected from SPc5-12 and MCK promoters. In a preferred embodiment, the muscle-specific promoter is the SPc5-12 promoter.

[0191] 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), the α-1 antitrypsin promoter (hAAT), the thyroxine transporter promoter, the albumin promoter, the thyroxine-binding globulin (TBG) promoter, the LSP promoter (containing the thyroxine-binding globulin promoter sequence, two copies of the α1-microglobulin / bikunin enhancer sequence and the leader sequence—Ill, CR et al., (1997), Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A, Blood Coag. Fibrinol. 8: S23–S30.), etc. Other useful liver-specific promoters are known in the art, such as those listed in the liver-specific gene promoter database compiled by Cold Spring Harbor Laboratory (http: / / rulai.cshl.edu / LSPD / ). In the context of this invention, a preferred liver-specific promoter is one that includes an H1 enhancer and a TTR promoter. An example of such a liver-specific promoter is the HSE promoter. The HSE promoter contains the mouse TTR promoter and H1 enhancer described in SEQ NO: 63 and is derived from the original paper by Robert H. Costa et al. in 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 SEQ ID NO: 65, or a functional variant thereof having at least 80% sequence identity with SEQ ID NO: 65, for example, having at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity with SEQ ID NO: 65.

[0192] In another specific embodiment, the promoter is a neuron-specific promoter. Non-limiting examples of neuron-specific promoters include, but are not limited to, the following promoters: synaptic protein-1 (Syn) promoter, neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), as well as other neuron-specific promoters that will be obvious to those skilled in the art. In one specific embodiment, the neuron-specific promoter is the Syn promoter. Other neuron-specific promoters include, but are not limited to, the synaptocin-2 promoter, the tyrosine hydroxylase promoter, the dopamine β-hydroxylase promoter, the hypoxanthine phosphoribosyltransferase promoter, the low-affinity NGF receptor promoter, and the choline acetyltransferase promoter (Bejanin et al., 1992; Carroll et al., 1995; Chin and Greengard, 1994; Foss-Petter et al., 1990; Harrington et al., 1987; Mercer et al., 1991; Patei et al., 1986). Representative motor neuron-specific promoters include, but are not limited to, the promoter of calcitonin gene-related peptide (CGRP), a known motor neuron-derived factor. Other promoters functioning in motor neurons include choline acetyltransferase (ChAT), neuron-specific enolase (NSE), synaptocin, and Hb9 promoters. Other neuron-specific promoters useful in this invention include, but are not limited to: GFAP (for astrocytes), calcium-binding protein 2 (for interneurons), Mnx1 (motor neurons), nestin (neurons), parvoprotein, somatostatin, and Plp1 (oligodendrocytes and Schwann cells).

[0193] In a preferred embodiment, the promoter is a ubiquitous promoter. Representative ubiquitous promoters include the cytomegalovirus enhancer / chicken β-actin (CAG) promoter, the cytomegalovirus enhancer / promoter (CMV) (optionally having a CMV enhancer) [see, for example, Boshart et al., Cell, 41:521-530 (1985)] or a short version of the CMV promoter, the PGK promoter, the SV40 early promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter, the EF1α (EF1a) promoter or a short version of the EF1a promoter, and the Ins84 promoter (as described in WO2020 / 219949). In another preferred embodiment, the promoter is a truncated version of the CMV promoter.

[0194] In addition, the promoter can also be an endogenous promoter, such as the albumin promoter or the GDE promoter.

[0195] Short promoters are of particular interest in this invention, such as shorter versions of known promoters. In a preferred embodiment, the promoter is less than about 500 pb, preferably less than about 450 pb, and more preferably less than about 400 pb.

[0196] In a preferred embodiment, the promoter is a shorter version of any promoter described herein, such as a shorter version of the CMV promoter or the EF1a promoter or a shorter version of a promoter that includes the H1 enhancer and the TTR promoter.

[0197] In a preferred embodiment, the promoter is a short version of the CMV promoter. More preferably, the promoter is a short version of the CMV promoter having the sequence SEQ ID NO: 62, or a functional variant thereof having at least 80% sequence identity with SEQ ID NO: 62, for example, having at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity with SEQ ID NO: 62.

[0198] In another specific embodiment, the promoter is a short version of the EF1a promoter. Preferably, the promoter is a short version of the EF1a promoter having the sequence shown in SEQ ID NO: 64, or a functional variant thereof having at least 80% sequence identity with SEQ ID NO: 64, for example, having at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity with SEQ ID NO: 64.

[0199] In another particular implementation, the promoter is the Ins84 promoter as described in WO2020 / 219949.

[0200] The expression cassette containing the nucleic acid molecules of the present invention can be tailored to the target population of GSDIII patients, the clinical presentation of GSDIII disease, and / or the target tissue. For example, for patients with clinical presentations 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 strong expression of the nucleic acid molecules of the present invention in muscle, such as the miniCMV promoter described above, particularly the miniCMV promoter of SEQ ID NO: 62. For patients with clinical presentations of GSDIII disease in the liver, the expression cassette preferably contains a liver-specific promoter or any promoter capable of inducing strong expression of the nucleic acid molecules of the present invention in the liver, such as the HSE promoter described above, particularly the HSE promoter of SEQ ID NO: 65. For patients with clinical presentations of GSDIII disease in both muscle and liver, a promoter capable of inducing expression of the nucleic acid molecules of the present invention in both tissues is preferably used.

[0201] In one particular embodiment, the promoter is associated with an enhancer sequence such as a cis-regulatory module (CRM) or an artificial enhancer sequence. CRMs useful in the practice of this invention include those described in Rincon et al., Mol Ther. 2015 Jan;23(1):43-52, Chuah et al., Mol Ther. 2014 Sep;22(9):1605-13, or Nair et al., Blood. 2014 May 15;123(20):3195-9. Other regulatory elements capable of particularly enhancing muscle-specific expression of genes, especially in cardiac and / or skeletal muscle, are those described in WO2015110449. Specific examples of nucleic acid regulatory elements comprising artificial sequences include regulatory elements obtained by rearranging transcription factor binding sites (TFBSs) present in the sequences disclosed in WO2015110449. The rearrangement may encompass altering 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, HNH1, NF1, C / EBP, LRF, MyoD, and SREBP, or binding sites for E2A, NF1, p53, C / EBP, LRF, and SREBP, or binding sites for E2A, HNH1, p53, C / EBP, LRF, and SREBP, or binding sites for E2A, HNH1, p53, C / EBP, LRF, and SREBP. 1. Binding sites for HNF3a, HNF3b, NF1, C / EBP, LRF, MyoD, and SREBP, or binding sites for E2A, HNF3a, NF1, C / EBP, LRF, MyoD, and SREBP, or binding sites for E2A, HNF3a, NF1, CEBP, LRF, MyoD, and SREBP, or binding sites for HNF4, NF1, RSRFC4, C / EBP, LRF, and MyoD, or binding sites for NF1, PPAR, p53, C / EBP, LRF, and MyoD.For example, nucleic acid regulatory elements for enhancing the expression of muscle-specific genes, particularly skeletal muscle-specific genes, may also include binding sites for E2A, NF1, SRFC, p53, C / EBP, LRF, and MyoD; or binding sites for E2A, NF1, C / EBP, LRF, MyoD, and SREBP; or binding sites for E2A, HNF3a, C / EBP, LRF, MyoD, SEREBP, and Tal1_b; or binding sites for E2A, SRF, p53, C / EBP, LRF, MyoD, and SREBP; or binding sites for HNF4, NF1, RSRFC4, C / EBP, LRF, and SREBP; or binding sites for E2A, HNF3a, HNF3b, NF1, SRF, C / EBP, LRF, MyoD, and SREBP; or binding sites for E2A, CEBP, and MyoD. In other instances, these nucleic acid regulatory elements comprise at least two, for example two, three, four, or more copies of one or more of the TFBS described above. Other regulatory elements capable of particularly enhancing liver-specific expression of genes are those disclosed in WO2009130208.

[0202] In one particular embodiment, the enhancer is a short-size enhancer. Specifically, the enhancer used in this invention can consist of 10 to 175 nucleotides, for example 40 to 100 nucleotides, particularly 50 to 80 nucleotides. In one particular embodiment, the enhancer is a 72-nucleotide HS-CRM8 enhancer consisting of SEQ ID NO: 63, or a functional variant of SEQ ID NO: 63 with enhancer activity. In another embodiment, the enhancer is a functional variant of the 72-nucleotide HS-CRM8 enhancer that has at least 80% identity with SEQ ID NO: 63, for example at least 85% identity, particularly at least 90% identity, and more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% identity.

[0203] In another specific embodiment, the nucleic acid construct includes introns, particularly introns positioned between the promoter and the GDE coding sequence. Introns can be introduced to improve mRNA stability and protein production. In another embodiment, the intron is a human β-globin b2 (or HBB2) intron, a condensation factor IX (FIX) intron, an SV40 intron, an hCMV intron A (hCMVI), a TPL intron (TPLI), a CHEF1 gene intron 1 (CHEFI), an MVM intron (Wu et al., 2008), a truncated FIX intron 1 (Wu et al., 2008, Mol Ther, 16(2):280–289; Kurachi et al., 1995, J Biol Chem., 270(10):5276–5281), or a β-globin / immunoglobulin heavy chain hybrid intron (5´ - donor site from human β-globin intron and 3´ - acceptor site from immunoglobulin heavy chain variable region intron, Wu et al., 2008, Mol Ther, 16(2):280–289; Kurachi et al., 1995, J Biol Chem., 270(10):5276–5281), a heterozygous intron composed of adenovirus splicing donor and immunoglobulin G splicing (Wong et al., 1985, Chromosoma, 92(2):124–135; Yew et al., 1997, Hum Gene Ther, 8(5):575–584; Choi T. et al., 1991, Mol Cell Biol, 11(6):3070–3074; Huang et al., 1990, Mol Cell Biol., 10(4):1805–1810), a heterozygous 19S / 16S SV40 intron (5´-donor site from 19S intron and 3´-recipient site from 16S intron, Yew et al., 1997, Hum Gene Ther, 8(5):575–584) or chicken β-globin introns. In another embodiment, the intron is a modified intron (particularly a modified HBB2 or FIX intron) designed to reduce the number of optional open reading frames (ARFs) present in the intron or even remove them entirely. Preferably, the ARF with a length span exceeding 50 bp and having a stop codon in the same frame as the start codon is removed. ARFs can be removed by modifying the sequence of the intron. For example, modification can be performed by nucleotide substitution, insertion, or deletion, preferably by nucleotide substitution. As an example, one or more nucleotides, particularly one nucleotide, in the ATG or GTG start codon present in the sequence of the intron of interest can be replaced to produce a non-start codon.For example, the ATG or GTG within the sequence of the intron of interest can be replaced with the CTG, which is not a start codon.

[0204] The classic HBB2 intron is shown in SEQ ID NO: 53. For example, this HBB2 intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In one particular embodiment, 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 eliminating the start codons (ATG and GTG codons) within the intron. In one particular embodiment, the modified FIX intron has the sequence shown in SEQ ID NO: 56. The classic chicken β-globin intron used in nucleic acid constructs is shown in SEQ ID NO: 57. The chicken β-globin intron can be modified by eliminating the start codons (ATG and GTG codons) within the intron. In one particular embodiment, the modified chicken β-globin intron has the sequence shown in SEQ ID NO: 58.

[0205] The inventors previously demonstrated in WO2015 / 162302 that such modified introns, particularly modified HBB2 or FIX introns, have advantageous properties and can significantly improve the expression of the introduced gene.

[0206] In one specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in the 5' to 3' direction, a promoter optionally preceded by an enhancer, the coding sequence of the present invention (i.e., a nucleic acid molecule encoding a functionally truncated GDE polypeptide), and a polyadenylation signal, such as 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 one specific embodiment, the polyadenylation signal is bGH polyA as shown in SEQ ID NO: 60. In one specific embodiment, a very short polyA signal is used. For example, a very short polyA signal containing fewer than 20 nucleotides is used. In one particular embodiment, the polyadenylation signal is the human soluble neurocilia protein-1 (sNRP) polyadenylation signal (sNRP polyA; SEQ ID NO: 59). In a preferred embodiment, the polyadenylation signal is the pA58 polyadenylation signal as shown in SEQ ID NO: 61.

[0207] In one specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in a 5' to 3' orientation, a promoter optionally preceded by an enhancer, an intron, the coding sequence of the present invention, and a polyadenylation signal. In another specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in a 5' to 3' orientation, a promoter, the coding sequence of the present invention, and a polyadenylation signal. In yet another specific embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in a 5' to 3' orientation, an enhancer, a promoter, the coding sequence of the present invention, and a polyadenylation signal. In yet another specific embodiment, the expression cassette comprises, in a 5' to 3' orientation, a promoter, an intron, the coding sequence of the present invention, and a polyA signal.

[0208] In another embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in a 5' to 3' orientation, an SPc5-12 promoter or a CMV promoter such as 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 a 5' to 3' orientation, an enhancer, an SPc5-12 promoter or a CMV promoter such as 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).

[0209] In another specific embodiment, the expression cassette comprises, in a 5' to 3' orientation, 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, particularly pA58 polyA. In another embodiment, the expression cassette comprises, in a 5' to 3' orientation, 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, particularly pA58 polyA.

[0210] In a preferred embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide as defined above, such as a sequence encoding an 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, particularly pA58 polyA.

[0211] In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide as defined above, such as a sequence selected from SEQ ID NO: 28 to SEQ ID NO: 41; and bGH polyA or pA58 polyA, particularly pA58 polyA.

[0212] In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation,: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 14; and bGH polyA or pA58 polyA, particularly pA58 polyA. In another particular embodiment, the expression cassette comprises, in the 5' to 3' orientation, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 28 or SEQ ID NO: 29; and bGH polyA or pA58 polyA, particularly pA58 polyA. In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a mini-CMV promoter of SEQ ID NO: 62; a sequence of SEQ ID NO: 28 or SEQ ID NO: 29 encoding a functionally truncated GDE polypeptide of SEQ ID NO: 14; and pA58 polyA of SEQ ID NO: 61.

[0213] In a preferred embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a 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 comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 30 or SEQ ID NO: 31; and bGH polyA or pA58 polyA, particularly pA58 polyA. In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a mini-CMV promoter of SEQ ID NO: 62; a sequence of SEQ ID NO: 30 or SEQ ID NO: 31 encoding a functionally truncated GDE polypeptide of SEQ ID NO: 15; and pA58 polyA of SEQ ID NO: 61.

[0214] In another embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 16; and bGH polyA or pA58 polyA, particularly pA58 polyA. In a particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 32 or SEQ ID NO: 33; and bGH polyA or pA58 polyA, particularly pA58 polyA. In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a mini-CMV promoter of SEQ ID NO: 62; a sequence of SEQ ID NO: 32 or SEQ ID NO: 33 encoding a functionally truncated GDE polypeptide of SEQ ID NO: 16; and pA58 polyA of SEQ ID NO: 61.

[0215] In another embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 17; and bGH polyA or pA58 polyA, particularly pA58 polyA. In a particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 34 or SEQ ID NO: 35; and bGH polyA or pA58 polyA, particularly pA58 polyA. In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a mini-CMV promoter of SEQ ID NO: 62; a sequence of SEQ ID NO: 34 or SEQ ID NO: 35 encoding a functionally truncated GDE polypeptide of SEQ ID NO: 17; and pA58 polyA of SEQ ID NO: 61.

[0216] In another embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 18; and bGH polyA or pA58 polyA, particularly pA58 polyA. In a particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 36 or SEQ ID NO: 37; and bGH polyA or pA58 polyA, particularly pA58 polyA. In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a mini-CMV promoter of SEQ ID NO: 62; a sequence of SEQ ID NO: 36 or SEQ ID NO: 37 encoding a functionally truncated GDE polypeptide of SEQ ID NO: 18; and pA58 polyA of SEQ ID NO: 61.

[0217] In another embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 19; and bGH polyA or pA58 polyA, particularly pA58 polyA. In a particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 38 or SEQ ID NO: 39; and bGH polyA or pA58 polyA, particularly pA58 polyA. In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a mini-CMV promoter of SEQ ID NO: 62; a sequence of SEQ ID NO: 38 or SEQ ID NO: 39 encoding a functionally truncated GDE polypeptide of SEQ ID NO: 19; and pA58 polyA of SEQ ID NO: 61.

[0218] In another embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 20; and bGH polyA or pA58 polyA, particularly pA58 polyA. In a particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 62; a sequence encoding a functionally truncated GDE polypeptide of SEQ ID NO: 40 or SEQ ID NO: 41; and bGH polyA or pA58 polyA, particularly pA58 polyA. In one particular embodiment, the expression cassette comprises, in the 5' to 3' orientation: a mini-CMV promoter of SEQ ID NO: 62; a sequence of SEQ ID NO: 40 or SEQ ID NO: 41 encoding a functionally truncated GDE polypeptide of SEQ ID NO: 20; and pA58 polyA of SEQ ID NO: 61.

[0219] In one particular embodiment, the expression cassette comprises or consists of sequences as shown in SEQ ID NO: 42 to SEQ ID NO: 48. In another embodiment, the expression cassette comprises or consists of sequences having at least 80% sequence identity with SEQ ID NO: 42 to SEQ ID NO: 48, for example at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity.

[0220] In one particular embodiment, the expression cassette comprises or consists of the sequence shown in SEQ ID NO: 42. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity with SEQ ID NO: 42, for example at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity.

[0221] In one particular embodiment, the expression cassette comprises or consists of the sequence shown in SEQ ID NO: 43. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity with SEQ ID NO: 43, for example at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity.

[0222] In one particular embodiment, the expression cassette comprises or consists of the sequence shown in SEQ ID NO: 44. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity with SEQ ID NO: 44, for example at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity.

[0223] In a preferred embodiment, the expression cassette comprises or consists of the sequence shown in SEQ ID NO: 45. In another embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity with SEQ ID NO: 45, for example at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity with SEQ ID NO: 45.

[0224] In another embodiment, the expression cassette comprises or consists of the sequence shown in SEQ ID NO: 46. In yet another embodiment, the expression cassette comprises, or consists of, a sequence having at least 80% sequence identity with SEQ ID NO: 46, for example at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity.

[0225] In another embodiment, the expression cassette comprises or consists of the sequence shown in SEQ ID NO: 47. In yet another embodiment, the expression cassette comprises, or consists of, a sequence having at least 80% sequence identity with SEQ ID NO: 47, for example at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity.

[0226] In another embodiment, the expression cassette comprises or consists of the sequence shown in SEQ ID NO: 48. In yet another embodiment, the expression cassette comprises, or consists of, a sequence having at least 80% sequence identity with SEQ ID NO: 48, for example at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity.

[0227] In designing the nucleic acid constructs of this invention, those skilled in the art will consider the size limitations of the vectors used to deliver the constructs to cells or organs. Specifically, those skilled in the art will understand that the main limitation of AAV vectors is their carrying capacity, which can vary from one AAV serotype to another, but is considered to be limited to the size of the parent viral genome. For example, 5 kb is generally considered to be the largest size packaged in an AAV8 capsid (Wu Z. et al., MolTher., 2010, 18(1): 80-86; Lai Y. et al., MolTher., 2010, 18(1): 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4): 225-33). Furthermore, in the production of recombinant AAV, the efficiency of capsidation of genomes larger than 5 kb is low, and the resulting AAV may contain fragmented genomes, reducing the efficiency of gene transfer. Therefore, those skilled in the art will carefully select the components of the nucleic acid constructs of the present invention in practice, such that the resulting nucleic acid sequences (including sequences encoding AAV 5'- and 3'-ITRs) preferably do not exceed 110% of the carrying capacity of the AAV vector used, specifically preferably not exceeding 5 kb. In the context of the present invention, AAV vectors with larger carrying capacities can also be used. For example, AAV particles lacking the Vp2 subunit have been shown to successfully package larger genomes (i.e., 6 kb) while maintaining the integrity of the capsidated genome (Grieger et al., 2005, J Virol., 79(15):9933–9944).

[0228] 4-Carrier

[0229] The present invention also relates to vectors comprising the nucleic acid molecules or constructs disclosed herein. In one particular embodiment, the vector comprises a nucleic acid molecule or construct encoding a functionally truncated GDE polypeptide as defined herein.

[0230] Specifically, the vectors of the present invention are vectors suitable for protein expression, preferably suitable for 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, particularly a messenger RNA encoding the functionally truncated GDE polypeptide of the present invention. In yet another embodiment, the vector is a transposon-based system that allows the integration of the nucleic acid molecule or construct of the present invention into the genome of a target cell, such as the highly active Sleeping Beauty (SB100X) transposon system (Mates et al., 2009). In yet another embodiment, the vector is a viral vector suitable for gene therapy, targeting any cells of interest, such as liver tissue or cells, muscle cells, CNS cells (e.g., brain cells), or hematopoietic stem cells such as erythroid cells (e.g., red blood cells). In this case, the nucleic acid construct of the present invention also contains sequences suitable for generating highly efficient viral vectors, as is known in the art.

[0231] Viral vectors are preferably used to deliver the nucleic acid molecules or constructs of the present invention, such as retroviral vectors, lentiviral vectors, or nonpathogenic parvoviruses, more preferably AAV vectors. Human parvovirus adeno-associated virus (AAV) is a naturally occurring replication-defective 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 integration occurs at a specific site in the human genome called AAVS1, located on chromosome 19 (19q13.3-qter).

[0232] Therefore, there has been great interest in AAV vectors as potential vectors for human gene therapy. The advantageous properties of the virus include its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and its capacity to infect a wide range of cell lines derived from different tissues.

[0233] Among the serotypes of AAVs isolated from and fully characterized from humans or non-human primates (NHP), 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., AAV-2 with a quadruple mutation in an engineered capsid containing the Y44+500+730F+T491V variation, disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods.), -3 and AAV-3 variants (e.g., AAV3-ST variant with an engineered AAV3 capsid containing the two amino acid variations S663V+T492V, disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p. 1042), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (e.g., AAV6 variant with an AAV6 capsid containing the Y731F / Y705F / T492V triple mutation, disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev.). 3, p. 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 mutants of AAV serotypes. Furthermore, other non-natural engineered variants and chimeric AAVs may also be useful.

[0234] AAV viruses can be engineered using conventional molecular biology techniques to optimize these particles for cell-specific delivery of nucleic acid sequences, minimize immunogenicity, regulate stability and particle lifetime, achieve efficient degradation, and ensure accurate delivery to the nucleus.

[0235] For assembly into a vector, an ideal AAV fragment includes cap proteins (including vp1, vp2, vp3, and hypervariable regions), rep proteins (including rep78, rep68, rep52, and rep40), and sequences encoding these proteins. These fragments can be readily used in a variety of vector systems and host cells.

[0236] AAV-based recombinant vectors lacking the Rep protein integrate into the host genome with low efficiency and exist primarily as stable circular free bodies that can persist in target cells for years.

[0237] As an alternative to using natural AAV serotypes, artificial AAV serotypes can be used in the context of this invention, including but not limited to AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be generated using any suitable technique by combining a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) with a heterologous sequence, which can be obtained from different selected AAV serotypes, non-contiguous portions of the same AAV serotype, or non-AAV viral sources or non-viral sources. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or “humanized” AAV capsids.

[0238] In the context of this invention, the AAV vector comprises an AAV capsid capable of transducing target cells of interest, i.e., cells of tolerance-prone tissues (e.g., hepatocytes) and cells of therapeutic target tissues, such as myocytes, CNS cells, or cardiomyocytes. In one particular embodiment, the AAV vector comprises an AAV capsid capable of transducing myocytes or cardiomyocytes.

[0239] According to one particular embodiment, the AAV vector is an AAV-1, -2, AAV-2 variant (e.g., a capsid-optimized AAV-2 comprising a quadruple mutation of an engineered capsid with the Y44+500+730F+T491V variation, disclosed in Ling et al., 2016 Jul 18, Hum Gene Ther Methods. [preprinted electronic version]), -3, and AAV-3 variants (e.g., an AAV3-ST variant comprising an engineered AAV3 capsid with the two amino acid variations S663V+T492V, disclosed in Vercauteren et al., 2016, Mol. Ther. Vol. 24(6), p.). The following are disclosed in WO2021 / 219762: -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (e.g., AAV6 variants containing a triple mutation in the form of Y731F / Y705F / T492V in the AAV6 capsid, published in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, p.16026), -7, -8, -9, -9P1, -2G9, -10 such as -cy10 and -rh10, -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP.B, AAV.PHPeB, AAV2i8, porcine AAV such as AAVpo1 (as described in WO2021 / 219762), AAVpo4 and AAVpo6, and tyrosine, lysine and serine capsid mutants of AAV serotypes. In one specific embodiment, the AAV vector is a serotype of AAV6, AAV8, AAV9, AAV9P1, AAVrh74, or AAV2i8 (i.e., the AAV vector has a capsid of a serotype of AAV6, AAV8, AAV9, AAV9P1, AAVrh74, or AAV2i8). In another specific embodiment, the AAV vector is a pseudotype vector, i.e., its genome and capsid are derived from different serotypes of AAV. For example, the pseudotype AAV vector may be a vector whose genome is derived from one of the AAV serotypes mentioned above and whose capsid is derived from another serotype. For example, the pseudotype vector may have a capsid derived from a serotype of AAV6, AAV8, AAV9, AAV9P1, AAVrh74, or AAV2i8, and its genome may be derived from different serotypes. In one particular embodiment, the AAV vector has a capsid of AAV6, AAV8, AAV9 or AAVrh74 serotype, particularly AAV6, AAV8, AAV9 or AAV9P1 serotype, and more particularly AAV6, AAV9 or AAV9P1 serotype.

[0240] In one particular embodiment in which a vector is used to deliver a therapeutically introduced gene into muscle cells, the AAV vector may be selected, in particular, from AAV8, AAV9, and AAVrh74.

[0241] In another specific embodiment in which the vector is used to deliver the transferred gene into hepatocytes, the AAV vector may be particularly selected from AAV1, AAV5, AAV8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAVrh74, AAV-LK03, AAV2G9, AAV.PHP, AAV-Anc80, and AAV3B.

[0242] In another specific implementation in which the vector is used to deliver the transferred gene to the CNS, the AAV vector may be particularly selected from AAV9, AAV9P1, AAV10 and AAV2G9, AAV.PHP.B, AAV.PHPeB.

[0243] In another embodiment, the capsid is a modified capsid. In the context of this invention, a "modified capsid" may be a chimeric capsid or a capsid comprising one or more variant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0244] In one particular embodiment, 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 that combines VP protein regions or domains derived from at least two AAV serotypes. Examples of such chimeric AAV vectors useful for transducing hepatocytes are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, a chimeric AAV vector may be derived from a combination of an AAV8 capsid sequence and a sequence from an AAV serotype other than AAV8, such as any of the serotypes specifically mentioned above. In another embodiment, the capsid of the AAV vector contains one or more variant VP capsid proteins, such as those described in WO2015013313, particularly the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid variants exhibiting high hepatophilia.

[0245] In another embodiment, the modified capsid may also be derived from capsid modifications via error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). In a particular embodiment, the capsid includes a P1 insertion, as described in WO2019 / 193119, WO2020 / 200499, or WO2022 / 053630. Furthermore, capsid variants may include single amino acid changes, such as tyrosine mutants (e.g., as described in Zhong et al., 2008). In one particular embodiment, the vector is AAV9rh74 containing a P1 insert (e.g., described in Sellier, P et al., “Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompephenotype in adult and neonate Gaa- / - mice”, Journal of inherited metabolic disease, 10.1002 / jimd.12625. 19 May. 2023). In another particular embodiment, the vector is an AAV9 mutant displaying P1, referred to as “AAVMYO”, as described by Weinmann et al. (Weinmann, Jonas et al., “Identification of amyotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants”, Nature Communications vol. 11, 15432. 28 Oct. 2020).In another particular embodiment, the vector is one of the AAV mutants containing the RGD peptide described in Tabebordbar et al. (Tabebordbar, Mohammadsharif et al., “Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species”, Cell vol. 184, 4919–4938, 16 Sept 2021), referred to as “MyoAAV”.

[0246] In one particular embodiment, the AAV carrier is the AAV carrier described in WO2019 / 193119 or the AAV carrier described in WO2020 / 200499.

[0247] In another embodiment, the AAV vector is the AAV vector described in WO2020 / 216861 or WO2022 / 003211. Specifically, the AAV vector may have a hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, such as a hybrid capsid between AAV8 and AAV2 / 13.

[0248] Furthermore, the genome of the AAV vector can be a single-stranded or self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting the terminal unwinding site from one of the terminal repeat sequences of the AAV. These replicated genomes are modified vectors that are half the length of the wild-type AAV genome and are prone to packaging DNA dimers. In a preferred embodiment, the AAV vector used in the practice of the present invention has a single-stranded genome and preferably includes a capsid of AAV8, AAV9, AAVrh74, AAVrh74-P1, AAV9rh74-P1, AAV2i8, or a heterozygous capsid as described in WO2020 / 216861 or WO2022 / 003211, particularly AAV8, AAV9, AAV9rh74-P1, or a heterozygous capsid as described in WO2020 / 216861 or WO2022 / 003211, and even more particularly the AAV9 capsid.

[0249] The AAV vectors used to package the GDE sequences of this invention can also be modified to increase their carrying capacity. For example, AAV vectors lacking the Vp2 subunit have been shown to successfully package larger genomes (i.e., 6 kb) while maintaining the integrity of the capsidated genome (Grieger et al., 2005).

[0250] As is known in the art, other suitable sequences can be introduced into the nucleic acid constructs of the present invention to obtain functional viral vectors. Suitable sequences include AAV ITR.

[0251] In one particular embodiment, the AAV vector contains a muscle-specific promoter as described above, particularly a muscle-specific promoter that exhibits some expression leakage in hepatocytes.

[0252] In another specific embodiment of the invention, the AAV vector comprises the liver-specific promoter as described above. Because this embodiment develops an efficient and optimized vector for expressing GDE in hepatocytes and inducing immune tolerance to the protein, it advantageously achieves hepatic tolerogenic and metabolic properties.

[0253] In one particular implementation, a dual recombinant AAV vector system comprising two AAV vectors, as described in WO 2018 / 162748, is used to deliver nucleic acid molecules or constructs encoding functionally truncated GDE peptides as defined above. Specifically, the dual AAV vector system comprises: - A first AAV vector, which contains a first nucleic acid sequence between the 5' and 3' AAV ITR encoding the N-terminal portion of a truncated GDE polypeptide as defined above, and - A second AAV vector, which contains a second nucleic acid sequence between the 5' and 3' AAV ITR encoding a portion of the truncated GDE polypeptide as defined above. Furthermore, the first and second nucleic acid sequences encoding the GDE contain multinucleotide regions that allow the generation of the full-length truncated GDE polypeptide as defined above.

[0254] In another specific embodiment, the AAV vector is a single AAV vector containing a nucleic acid molecule or construct encoding a functionally truncated GDE polypeptide as defined above.

[0255] 5-cell

[0256] This invention also relates to cells transformed or transduced using the nucleic acid molecules, constructs, or vectors of this invention, particularly isolated cells such as hepatocytes, cardiomyocytes, CNS cells, or myocytes. In one particular embodiment, the cells are isolated human cells. In another particular embodiment, the cells are not human embryonic stem cells. The cells of this invention express functionally truncated GDE polypeptides as described above. The cells of this invention can be delivered to a subject via any suitable route of administration, for example, by injection into the liver, CNS, heart, muscle, or bloodstream of a subject in need, such as a patient with GDE deficiency. In one particular embodiment, this invention comprises transducing liver or myocytes, particularly liver or myocytes of a subject to be treated, and administering the transduced liver and / or myocytes, in which the nucleic acid has been introduced, to the subject. In one particular embodiment, the hepatocytes are hepatocytes derived from a patient to be treated, or liver stem cells that have been further transformed and differentiated into hepatocytes in vitro for subsequent administration to the patient. In another embodiment, the cells are myocytes derived from a patient to be treated, or muscle stem cells that have been further transformed and optionally differentiated into myocytes in vitro for subsequent administration to the patient.

[0257] 6- Pharmaceutical Compositions

[0258] This invention also provides pharmaceutical compositions comprising the nucleic acid molecules, nucleic acid constructs, carriers, functionally truncated GDE peptides, or cells of this invention. Such compositions may comprise a therapeutically effective amount of the therapeutic agent (the nucleic acid molecules, nucleic acid constructs, carriers, functionally truncated GDE peptides, or cells of this invention) and a pharmaceutically acceptable carrier. In one particular embodiment, the term "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or European Pharmacopoeia or other recognized pharmacopoeia for use in animals and humans. The term "carrier" refers to a diluent, adjuvant, excipient, or solvent administered with the therapeutic agent. Such pharmaceutical carriers may be sterile liquids such as water and oils, including petroleum, animal, plant, or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol may also be used as liquid carriers, particularly for solutions for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, etc.

[0259] If desired, the composition may also contain small amounts of wetting agents, emulsifiers, or pH buffers. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EW Martin's *Remington's Pharmaceutical Sciences*. Such compositions contain a therapeutically effective amount, preferably in purified form, of a therapeutic agent and a suitable amount of a carrier to provide a form suitable for administration to a subject. In one particular embodiment, the nucleic acid, carrier, or cell of the invention is formulated in a composition comprising phosphate-buffered saline supplemented with 0.25% human serum albumin. In another specific embodiment, the nucleic acid, vector, or cell of the present invention is formulated in a composition comprising a lactated Ringer's solution and a nonionic surfactant such as pluronic F68, said surfactant having a final concentration of 0.01-0.0001% by weight of the total composition, for example, 0.001%. The formulation may also contain serum albumin, particularly human serum albumin, for example, 0.25% human serum albumin. Other suitable formulations for storage or administration are known in the art, particularly from WO 2005 / 118792 or Ally et al., 2011.

[0260] In a preferred embodiment, the composition is formulated according to conventional procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. Where necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.

[0261] In one embodiment, the nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE peptides, or cells of the present invention can be delivered in vesicles, particularly liposomes. In yet another embodiment, the nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE peptides, or cells of the present invention can be delivered in a controlled release system.

[0262] In one particular embodiment, the nucleic acid molecule is delivered as mRNA, corresponding to a transcript encoding the functionally truncated GDE polypeptide of the present invention. Specifically, the mRNA of the present invention can be delivered using liposomes, such as lipid nanoparticles (LNPs).

[0263] The methods of administration of the nucleic acid molecules, nucleic acid constructs, carriers, functional truncated peptides, or cells of the present invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. In one particular embodiment, the administration is via an intravenous or intramuscular route. The nucleic acid molecules, nucleic acid constructs, carriers, functional truncated peptides, or cells of the present invention, whether or not carrier-based, can be administered via any convenient route, such as by infusion or rapid concentration, via transepithelial or mucosal skin lining (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0264] In one particular embodiment, it may be desirable to apply the pharmaceutical composition of the present invention topically to areas requiring treatment, such as the liver or muscles. This can be achieved, for example, using implants that are porous, non-porous, or gel-like materials, including membranes such as silicone rubber membranes or fibers.

[0265] In one particular embodiment, the functionally truncated GDE peptides of the present invention are used for enzyme replacement therapy (ERT), particularly for the treatment of GSDIII. The terms "enzyme replacement therapy" or "ERT" generally refer to the introduction of a purified enzyme into an individual lacking that enzyme. The administerable peptides of the present invention can be obtained through recombinant expression, produced in vitro or in transgenic animals, or purified from isolated tissues or fluids. Specifically, when used for ERT, the peptides of the present invention can be administered parenterally, for example via intraperitoneal, intramuscular, or intravascular (i.e., intravenous or intraarterial) administration. Specifically, the peptides are administered via intravenous injection. The administration can be repeated frequently, for example daily, weekly, bi-weekly, or monthly, particularly weekly or bi-weekly.

[0266] The amount of the therapeutic agents of the present invention (i.e., the nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE peptides, or cells of the present invention) effective in the treatment of GSDIII can be determined by standard clinical techniques. Additionally, in vivo and / or in vitro assays may optionally be used to help predict the optimal dose range. The precise dose used in the formulation also depends on the route of administration and the severity of the disease, and should be determined based on the judgment of a practicing physician and the individual patient's condition. The dose of the nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE peptides, or cells of the present invention administered to the recipient subject will vary with several factors, including but not limited to the route of administration, the specific disease being treated, the age of the subject, or the expression level necessary to achieve a therapeutic effect. A person skilled in the art can readily determine the required dose range based on these and other factors, according to their knowledge in the field. In cases where treatment involves administration of a viral vector, such as an AAV vector, to the subject, the typical dose of said vector is at least 1 x 10⁻⁶. 8One vector genome per kilogram of body weight (vg / kg), for example, at least 1 x 102 9 vg / kg, at least 1x10 10 vg / kg, at least 1x10 11 vg / kg, at least 1x10 12 vg / kg, at least 1x10 13 vg / kg or at least 1x10 14 vg / kg.

[0267] 7- Treatment methods

[0268] The present invention also relates to a method for treating GSDIII, the method comprising the steps of delivering a therapeutically effective amount of the nucleic acid molecule, nucleic acid construct, vector, functionally truncated GDE peptide, pharmaceutical composition or cell of the present invention to a subject in need.

[0269] Cirrhosis and hepatocellular carcinoma can also occur in GSDIII patients. Therefore, the present invention also relates to a method for treating cirrhosis and hepatocellular carcinoma in GSDIII patients, the method comprising the steps 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 subject in need.

[0270] The present invention also relates to a method for treating GSDIII, the method not inducing an immune response against the introduced gene (i.e., against a functionally truncated GDE polypeptide encoded by the nucleic acid molecule), or inducing a reduced immune response against the introduced gene, the 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 in need. The present invention also relates to a method for treating GSDIII, the method comprising repeatedly administering a therapeutically effective amount of the nucleic acid, vector, functionally truncated GDE polypeptide, pharmaceutical composition, or cells of the present invention to a subject in need. In this regard, the nucleic acid molecule, nucleic acid construct, or vector of the present invention contains a functional promoter in hepatocytes, thereby allowing immune tolerance to the expressed functionally truncated GDE polypeptide generated therefrom. Similarly, in this regard, the pharmaceutical composition used in this aspect comprises a nucleic acid molecule, nucleic acid construct, or vector containing a functional promoter in hepatocytes. In the case of delivering cells, particularly liver, myocardial, CNS, or muscle cells, the cells may be cells previously collected from the subject requiring treatment and engineered to produce the functionally truncated GDE peptides by introducing the nucleic acid molecules, nucleic acid constructs, or vectors of the present invention therein. According to embodiments, in cases involving repeated administration, the administration may be repeated at least once or more, and may even be considered to be performed according to a regular schedule, such as weekly, monthly, or annually. The regular schedule may also include administration every 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or more. In another particular embodiment, each administration of the viral vector of the present invention uses a different virus for each subsequent dose, thereby avoiding efficacy reduction due to possible immune responses to the previously administered viral vector. For example, the first administration may use an AAV vector containing an AAV8 capsid, and subsequent administrations may use a vector containing an AAV9 capsid.

[0271] According to the present invention, treatment may include curative, alleviating, or preventative effects. Therefore, therapeutic and preventative treatments include the improvement or prevention of symptoms of GSDIII or otherwise reducing the risk of developing a specific glycogen storage disease. The term "preventative" can be considered as reducing the severity of a specific condition or decreasing its onset. "Preventative" also includes preventing the recurrence of a specific condition in patients previously diagnosed with it. "Therapeutic" can also reduce the severity of an existing condition. The term "treatment" is used herein to refer to any protocol that may be beneficial to animals, particularly mammals, and more particularly human subjects.

[0272] The present invention also relates to an ex vivo gene therapy method for treating GSDIII, the method comprising introducing the nucleic acid molecule, nucleic acid construct or vector of the present invention into isolated cells of a patient in need, such as isolated hematopoietic stem cells, and introducing the cells into the patient in need.

[0273] This invention also relates to nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE peptides, cell or pharmaceutical compositions of the present invention, which are used as pharmaceuticals.

[0274] This invention also relates to nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE polypeptides, cell or pharmaceutical compositions of the present invention, methods for treating diseases caused by mutations in the AGL gene encoding GDE, particularly methods for treating GSDIII (Corrie's disease) such as GSDIIIa and GSDIIIb, especially GSDIIIa.

[0275] The present invention also relates to the use of the nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE peptides, cells or pharmaceutical compositions of the present invention for the manufacture of a medicament for the treatment of GSDIII (Corrie's disease).

[0276] The present invention also relates to nucleic acid molecules, nucleic acid constructs, vectors, functionally truncated GDE peptides, cell or pharmaceutical compositions of the present invention, and methods for delivering said GDE protein to affected tissues, particularly muscle and liver tissue, particularly muscle.

[0277] Example

[0278] The invention is described in further detail below with reference to the experimental embodiments and accompanying drawings. These embodiments are provided for illustrative purposes only and are not intended to be limiting.

[0279] Patent applications WO2020 / 030661 and WO2022 / 043280 demonstrate the potential to generate truncated forms of GDE proteins while retaining enzymatic activity. Specifically, truncated proteins “Δ1b2” and “Δ1b3” are shown to efficiently reduce glycogen levels in various muscle tissues. “Δ1b2” corresponds to the GDE polypeptide of SEQ ID NO: 1, in which amino acids 2-81 are missing. “Δ1b3” corresponds to the GDE polypeptide of SEQ ID NO: 1, in which amino acids 2-103 are missing. However, both proteins have been shown to exhibit lower expression or stability in vivo compared to the full-size GDE. Therefore, the aim is to modify the N-terminal truncation site to discover more stable, better-expressed truncated proteins and ultimately improve the ability of said proteins to reduce glycogen in vivo.

[0280] As shown in the table below, seven N-terminal truncated GDE proteins were designed:

[0281] Several GDEs (Δ1b9 to Δ1b15) truncated in the N-terminal structural domain were generated. Figure 1 The GDE was compared with the truncated “Δ1b3” GDE protein. To assess the activity of the truncated GDE, AAV vectors expressing either the truncated GDE (Δ1b9 to Δ1b15 or Δ1b3 as a control) or the full-size protein (GDE-full-size) were generated. The AAV9rh74-P1 vector (i.e., an AAV vector with a hybrid capsid between AAV9 and AAVrh74 modified with peptide P1) was used throughout the experiment. The transfected gene was cloned into a transfected gene expression cassette optimized for muscle expression and consisting of a mini CMV promoter and a pA58 polyadenylation signal. The expression cassettes for Δ1b9 to Δ1b15, Δ1b3, and the GDE-full-size construct are shown in SEQ ID NO: 42-48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.

[0282] Example 1: The carrier is 3x10 11 A dose of vg / mouse was injected into the left tibialis anterior muscle of 4-month-old GSDIII mice. Figure 2 A). Wild-type PBS injection ( Agl + / + PBS) or knockout mice ( Agl - / - PBS was used as a control. Mice were euthanized one month after injection to analyze GDE expression in the left tibialis anterior muscle.

[0283] Measurement of GDE expression

[0284] Mouse tissue was placed in a solution containing cOmplete TM The protease inhibitor mixture (Roche, ref 4693132001) was homogenized in phosphate-buffered saline (PBS, ThermoFisher Scientific). Pierce was used according to the manufacturer's instructions. TM The BCA protein assay (Thermo Fisher Scientific) measures protein concentration. This is applicable to PBS and AAV injections. Agl - / - For both mice, loading 50 µg of total protein fraction into each well for PBS injection... Agl + / + For mice (used as a positive control), each well was loaded with a fraction of 10 µg of total protein. The fractions were stored on 4–12% Bis-Tris gradient polyacrylamide gels (NuPAGE).TM SDS-PAGE electrophoresis was performed on the membrane using an Invitrogen membrane. After transfer, the membrane was blocked with Intercept blocking buffer (LI-COR Biosciences) and incubated with anti-GDE rabbit polyclonal antibody (16582-1-AP, Proteintech) and anti-fouling mouse monoclonal antibody (V9131, Sigma). The membrane was washed, incubated with a suitable secondary antibody (LI-CORBiosciences), and visualized using an Odyssey imaging system (LI-COR Biosciences).

[0285] result

[0286] The results showed the protein expression of all truncated GDE proteins. Figure 2 BC). Compared to the protein expression of the Δ1b3 GDE construct, all truncated GDE proteins showed at least comparable or even higher protein expression levels. Figure 2 BC).

[0287] Example 2: We then evaluated glycogen accumulation in the quadriceps femoris of GDE-KO animals injected with AAV encoding the Δ1b13 GDE construct and compared it with the AAV vector encoding the Δ1b3 GDE construct.

[0288] The carrier is 2.5x10 13 The dose was administered intravenously to 3-5 month old GSDIII mice at a dose of vg / kg. Figure 3 A). Inject KO with saline (PBS). Agl - / - ) and WT Agl + / + Mice were used as a negative control. Two months after injection, the mice were euthanized to analyze the glycogen content in the quadriceps femoris muscle.

[0289] Glycogen content in tissue homogenates was indirectly measured as glucose released after complete digestion with Aspergillus niger amylase (Sigma-Aldrich, ref A1602). Samples were incubated at 95°C for 10 min and then cooled at 4°C. The reaction was terminated by incubating the samples at 95°C for 10 min after adding amylase (final concentration 4 U / mL) and potassium acetate (final concentration 25 mM) at pH 5.5 for 90 min at 37°C. A control reaction without amylase was prepared for each sample and incubated under the same conditions. Glucose release was measured using a glucose assay kit (Sigma-Aldrich), and the absorbance was acquired at 540 nm on an EnSpire alpha plate reader (PerkinElmer). Glucose released after amylase digestion was then normalized to total protein concentration.

[0290] result

[0291] Figure 3 The figure shows glycogen levels measured in AAV-treated animals (using Δ1b13 or Δ1b3 GDE) and untreated wild-type (WT) and KO animals. The results indicate that the Δ1b13 construct, which has been shown to express better than Δ1b3 (see [link to figure]), is superior. Figure 2 Compared to Δ1b3, it showed a stronger ability to reduce glycogen accumulation in GSDIII mice (see [link]). Figure 3 B).

Claims

1. A functionally truncated GDE polypeptide, wherein the functionally truncated GDE polypeptide contains a deletion relative to a reference functional full-length human GDE sequence, and wherein the deletion consists of an amino acid deletion in the N-terminal portion of the reference functional full-length human GDE sequence, such that the first six amino acids at the N-terminus of the functionally truncated GDE polypeptide are: - MQYYFL (SEQ ID NO: 7); - MFLQGN (SEQ ID NO: 8); - MQGNEK (SEQ ID NO: 9); - MGNEKS (SEQ ID NO: 10); - MNEKSG (SEQ ID NO: 11); - MKSGGG (SEQ ID NO: 12); - MSGGGY (SEQ ID NO: 13).

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

6.

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

1.

4. The functionally truncated GDE peptide according to any one of the preceding claims, The functionally truncated GDE peptide further comprises deletions or combinations thereof relative to the reference functional full-length human GDE sequence, such as deletions or combinations thereof in the C-terminal portion of the GDE sequence or deletions or combinations thereof in the central domain of the GDE sequence. Specifically, the functionally truncated GDE peptide further comprises a deletion or a combination of deletions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and the deletion is selected from any deletion referred to as Δ1, Δ2, Δ3, Δ4, Δ5, Δ6 and Δ7 in Table 2.

5. The functionally truncated GDE polypeptide according to any one of the preceding claims, wherein the functionally truncated GDE polypeptide has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 14-20.

6. The functionally truncated GDE polypeptide according to any one of the preceding claims, wherein the functionally truncated GDE polypeptide has: - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 14, and containing the sequence of SEQ ID NO: 21; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 15, and containing the sequence of SEQ ID NO: 22; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 16, and containing the sequence of SEQ ID NO: 23; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 17, and containing the sequence of SEQ ID NO: 24; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 18, and containing the sequence of SEQ ID NO: 25; - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 19, and containing the sequence of SEQ ID NO: 26; or - An amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99% sequence identity with SEQ ID NO: 20, and containing the sequence of SEQ ID NO:

27.

7. The functionally truncated GDE polypeptide according to any one of the preceding claims, wherein the functionally truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NO: 14-20.

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

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

10. A vector, particularly a viral vector, comprising a nucleic acid molecule according to claim 8 or an expression cassette according to claim 9.

11. The carrier according to claim 10, wherein it is an AAV carrier.

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

13. The functionally truncated GDE polypeptide according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, the expression cassette according to claim 9, the vector according to claims 10-11, or the cell according to claim 12, used as a drug.

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

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

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