N-terminal truncated GDE for the treatment of glycogenosis III

Abstract

The present invention relates to a functional N-terminal truncated GDE polypeptide for the treatment of glycogenosis III.

Description

Technical Field

[0001] The present invention relates to the treatment of glycogenosis III (GSDIII).

Background Art

[0002] Mutations in the AGL gene cause a deficiency of glycogen debranching enzyme (GDE), an enzyme involved in glycogen breakdown, or “amylo-alpha-1,6-glucosidase, 4-alpha-glucanotransferase”. GDE has two independent catalytic activities that occur at different sites on the protein: 4-alpha-glucotransferase activity and amylo-1,6-glucosidase activity. Genetic deficiency of GDE causes incomplete glycogen breakdown in glycogenosis III (GSDIII), resulting in the accumulation of abnormal glycogen with short outer chains in various organs, mainly the liver and muscle. This disease is characterized by hepatomegaly, hypoglycemia, short stature, variable myopathy, and cardiomyopathy. Most patients have GSDIII (type IIIa) involving both the liver and muscle, but some patients (about 15 percent) have liver lesions only (type IIIb). Liver symptoms usually occur in childhood. Cirrhosis and hepatocellular carcinoma have been reported in some cases (Chen et al., 2009, Scriver's Online Metabolic & Molecular Bases of Inherited Disease, New York: McGraw-Hill; Kishnani et al., 2010, Genet Med 12, 446 - 463). Muscle weakness may also be present in childhood. It becomes more apparent in adults, manifesting in the 20s or 30s. The morbidity from progressive muscle weakness is significant, and patients may become wheelchair-bound in later stages. Patients may also develop cardiomyopathy. There is a large clinical variability in the severity of the symptoms that these patients develop. Progressive myopathy and / or cardiomyopathy and / or peripheral neuropathy are the main causes of morbidity in adults (Kishnani et al., 2010, Genet Med 12, 446 - 463; Cornelio et al., 1984, Arch Neurol 41, 1027 - 1032; Coleman et al., 1992, Ann Intern Med 116, 896 - 900).Reports of neurological symptoms potentially associated with the disease have been obtained from clinicians caring for patients with GSDIII, and they have reported fluctuations in attention, executive function deficits, and emotional skill deficiencies (Michon et al., 2015, J Inherit Metab Dis, 38(3):573-580). Based on this, widespread glycogen accumulation throughout the nervous system has been described for the GDE- / - mouse model of the disease (Pagliarani et al., 2014, Biochim Biophys Acta, 1842(11):2318-2328; Liu et al., 2014, Mol Genet Metab, 111(4):467-476), but a careful characterization of the phenotypes associated with glycogen accumulation is not yet available. Current treatments are symptomatic, and there is no effective treatment for the disease. Hypoglycemia can be managed by frequent meals rich in carbohydrates containing cornstarch supplements or nocturnal intragastric tube feeding. Patients with myopathy are treated by nocturnal enteral infusion in addition to a high-protein diet during the day. For some patients, a temporary improvement in symptoms has been described, but there are no systematic studies or long-term data demonstrating that a high-protein diet prevents or treats progressive myopathy (Kishnani et al., 2010, Genet Med 12, 446-463). These approaches do little to change the long-term course and morbidity of these diseases.

[0003] Therefore, long-term treatment with GSDIII is still required. Gene therapy aimed at stably replacing the GDE protein in affected tissues seems to be a promising treatment approach. However, the large size of the GDE transgene is a major obstacle because it cannot fit within the size limits of most gene therapy vectors. In fact, the human AGL gene consists of 35 exons encoding a 7.4-kb mRNA that contains a 4,599-bp coding region for a 175-kDa GDE protein and a 2,371-bp 3' untranslated sequence (Bao Y et al., 1996, Genomics., 38(2):155-65). This is a practical problem because the minimum size of a GDE expression cassette (e.g., containing at least a promoter, GDE coding sequence, polyA signal, and two ITRs for an AAV vector) is larger than the 5-kb genomic size limit that can be packaged into an AAV gene therapy vector for in vivo gene delivery.

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

[0005] In patent applications WO2020 / 030661 and PCT / EP2021 / 073309, the inventors described another approach based on the use of a shortened GDE polypeptide that fits within a single viral vector. However, an alternative to the shortened GDE polypeptides previously described in WO2020 / 030661 and PCT / EP2021 / 073309 is desirable.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Patent Document 14

Patent Document 15

Patent Document 16

Non-Patent Document

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Non-Patent Document 21

Non-Patent Document 22

Non-Patent Document 23

Non-Patent Document 24

Non-Patent Document 25

Non-Patent Document 26

Non-Patent Document 27

Non-Patent Document 28

Non-Patent Document 29

Non-Patent Document 30

Non-Patent Document 31

Non-Patent Document 32

Non-Patent Document 33

Non-Patent Document 34

Non-Patent Document 35

Non-Patent Document 36

Non-Patent Document 37

Non-Patent Document 38

Non-Patent Document 39

Non-Patent Document 40

Non-Patent Document 41

Non-Patent Document 42

Non-Patent Document 43

Non-Patent Document 44

Summary of the Invention

Means for Solving the Problems

[0008] The present invention relates to a functional truncated GDE polypeptide containing a deletion with respect to a reference functional full-length human GDE sequence, wherein the deletion is such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are - MGSFQY (SEQ ID NO: 7); - MEKSGG (SEQ ID NO: 8); - MILRVG (SEQ ID NO: 9); - MGADNH (SEQ ID NO: 10); or - MLDCVT (SEQ ID NO: 11) and consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence in such a form.

[0009] In certain embodiments, the deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence in such a form that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8) or MILRVG (SEQ ID NO: 9), and preferably, the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8).

[0010] In certain embodiments, a reference functional full-length human GDE has an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In certain embodiments, a reference functional full-length human GDE has an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO: 1, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4, preferably SEQ ID NO: 1.

[0011] In certain embodiments, a functional truncated GDE polypeptide further comprises a deletion or combination of deletions relative to a reference functional full-length human GDE sequence, for example, a deletion or combination of deletions at the C-terminal portion of the GDE sequence, or a deletion or combination of deletions in the central domain of the GDE sequence. In certain embodiments, a functional truncated GDE polypeptide further comprises a deletion or combination of deletions relative to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and the deletion is selected from any of the deletions designated as Δ1, Δ2, Δ3, Δ4, Δ5, Δ6 and Δ7 in Table 2 below.

[0012] In certain embodiments, the functional truncated GDE polypeptide has an amino acid sequence as set forth in SEQ ID NOs: 12-16, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NOs: 12-16. In certain embodiments, the functional truncated GDE polypeptide has an amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14, preferably SEQ ID NO: 13, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14, preferably SEQ ID NO: 13.

[0013] The present invention also relates to a nucleic acid molecule encoding the functional truncated GDE polypeptide of the present invention.

[0014] The present invention - a promoter; - optionally, an intron; - the nucleic acid molecule according to the claims of the present invention; and - a polyadenylation signal also relates to an expression cassette preferably containing them in this order.

[0015] Another aspect of the present invention relates to a vector, particularly a viral vector, containing the nucleic acid molecule or expression cassette of the present invention. In certain embodiments, the vector is an AAV vector.

[0016] 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 particularly a liver cell, muscle cell, heart cell or CNS cell.

[0017] The present invention also relates to a functional truncated GDE polypeptide, nucleic acid molecule, expression cassette, vector or cell as defined above for use as a medicament. The present invention also relates to a functional truncated GDE polypeptide, nucleic acid molecule, expression cassette, vector or cell as defined above for use in a method for treating a disease caused by a mutation in the AGL gene encoding GDE. In a further specific embodiment, the present invention relates to a functional truncated GDE polypeptide, nucleic acid molecule, expression cassette, vector or cell as defined above for use in a method for treating GSDIII (Cori disease). BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0019] In patent applications WO2020 / 030661 and PCT / EP2021 / 073309, the inventors demonstrated that a truncated GDE polypeptide having a size compatible with encapsulation into the capsid in AAV vectors while retaining its enzymatic activity can be used.

[0020] Despite the lack of knowledge about the three-dimensional structure of the GDE protein, the inventors identified a new N-terminal truncated GDE polypeptide having high protein expression levels and very good efficacy in reducing glycogen in GSDIII mice.

[0021] Accordingly, 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 (Cori disease).

[0022] 1 - N-terminal truncated GDE polypeptide The shortened GDE polypeptide according to the present invention is a functional GDE polypeptide whose coding sequence is small enough to be efficiently packaged into a gene therapy vector, particularly a single AAV vector.

[0023] The "functional" GDE polypeptide means, at least in part, a polypeptide that retains at least one, preferably all, of the enzymatic activities of the GDE protein. As a result, the functional GDE polypeptide realized by the present invention can restore glycogen accumulation and muscle strength in vivo. As defined herein, the GDE enzymatic activity is the 4-alpha-glucotransferase activity and the amylo-1,6-glucosidase activity involved in glycogenolysis. The transferase activity of GDE transfers three glucose units of glycogen from one chain to another. This leaves one glucose unit at the branch point, which is then released as glucose by glucosidase activity. In certain embodiments, the functional GDE polypeptide of the present invention has the same functionality as the full-length GDE polypeptide, particularly the full-length human GDE polypeptide. For example, the functional GDE polypeptide of the present invention has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of, or at least 100% compared to 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, with respect to one or preferably both of the above enzymatic activities. The activity of the GDE protein of the present invention can exceed 100% of the activity of the full-length human GDE protein, particularly the full-length human GDE protein of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, for example, exceeding 110%, 120%, 130%, 140%, 150%, 200%, 500%, 700%, or even exceeding 1000%. In certain embodiments, the functional GDE polypeptide of the present invention has the same functionality as the full-length GDE polypeptide, particularly the full-length human GDE polypeptide in muscle tissues such as the heart or quadriceps muscle.For example, the functional GDE polypeptide of the present invention can have an activity of at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% with respect to one or preferably both of the above enzyme activities in muscle tissues such as the heart or quadriceps muscle, or at least 100% compared to the full-length human GDE protein, particularly the full-length human GDE protein of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. The activity of the GDE protein of the present invention in muscle tissues such as the heart or quadriceps muscle can exceed 100% of the activity of the full-length human GDE protein, particularly the full-length human GDE protein of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, for example, exceed 110%, 120%, 130%, 140%, 150%, 200%, 500%, 700%, or even exceed 1000%.

[0024] The "functional" GDE polypeptide means a non-pathological GDE polypeptide. In particular, the functional GDE polypeptide of the present invention is not the GDE polypeptide found in patients with GSDIII (Cori disease) such as GSDIIIa or GSDIIIb.

[0025] One of ordinary skill in the art can readily determine whether a polypeptide is a functional GDE polypeptide. Suitable methods will be apparent to one of ordinary skill in the art. For example, one suitable in vitro method involves inserting a nucleic acid encoding the polypeptide into a vector, such as a plasmid or viral vector, transfecting or transducing the vector into a host cell, such as 293T or HeLa cells, or other cells, such as Huh7, and assaying for GDE activity. Suitable methods are described in more detail in the Experimental section below. For example, GDE activity can be determined by measuring the glucose produced after incubating a homogenized tissue or cell extract pre-transfected with a vector expressing a functional GDE polypeptide with limit dextrin (glycogen digested by glycogen phosphorylase). Other methods include testing GDE efficacy by evaluating the muscle strength of treated GDE-KO animals by wire hang, for example, 1, 2, or 3 months after administration, by evaluating the recovery of glycogen accumulation in muscle and / or heart tissue, and / or by evaluating the normalization of blood glucose in treated GDE-KO animals after administration of the vector, for example, 1, 2, or 3 months after administration. Additionally, GDE expression can be evaluated by Western blot in the tissues of GDE KO animals, for example, 1, 2, or 3 months after administration of the vector.

[0026] With respect to the present invention, "reference full-length human GDE sequence" encompasses all native isoforms of human GDE. Bao and colleagues (Genomics, 1997, 38, 155-165) identified the existence of six transcript variants encoding three GDE protein isoforms. Transcript variants 1-4 encode the same protein, namely, GDE isoform 1. Transcript variants 5 and 6 encode GDE isoforms 5 and 6, respectively.

[0027] With respect to the present invention, the "reference full-length human GDE sequence" does not encode a pathogenic GDE polypeptide. In particular, the reference full-length human GDE sequence does not encode the pathogenic variants found in patients with GSDIII that contain mutations, deletions or insertions compared to the wild-type non-pathogenic full-length GDE sequence.

[0028] Accordingly, the term "reference full-length human GDE polypeptide" encompasses all native isoforms of human GDE including the precursor form, as well as GDE proteins or fragments thereof that have been modified or mutated by insertions, deletions and / or substitutions and are functional derivatives of GDE. In particular, the reference full-length human GDE sequence is selected from the group consisting of SEQ ID NO: 1 (corresponding to wild-type GDE isoform 1, UniProtKB accession number: P35573-1), SEQ ID NO: 4 (corresponding to a variant of GDE isoform 1), SEQ ID NO: 2 (corresponding to wild-type GDE isoform 5, UniProtKB accession number: P35573-2), SEQ ID NO: 5 (corresponding to a variant of GDE isoform 5), SEQ ID NO: 3 (corresponding to wild-type GDE isoform 6, UniProtKB accession number: P35573-3), and SEQ ID NO: 6 (corresponding to a variant of GDE isoform 6).

[0029] In certain embodiments, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity 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, particularly to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In certain embodiments, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to 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 units of the number of amino acids. In certain embodiments, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to 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 units of the number of amino acids. In certain embodiments, the reference full-length human GDE has at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to 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 units of the number of amino acids.

[0030] The term "identical" and its grammatical variations refer to sequence identity between two nucleic acid molecules or between two polypeptide molecules. When the same base or amino acid occupies the position in both of the two sequences being compared, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of matching positions shared by the two sequences × 100 divided by the number of positions compared. For example, if 6 out of 10 positions in two sequences match, the two sequences are 60% identical. Generally, the comparison is made by aligning the two sequences so as to obtain the maximum identity. Various bioinformatics tools known to those skilled in the art, such as BLAST or FASTA, can be used to align nucleic acid sequences.

[0031] In certain embodiments, the reference full-length human GDE sequence has an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 4, particularly SEQ ID NO: 1 corresponding to GDE isoform 1.

[0032] In certain embodiments, the truncated GDE polypeptides of the invention that are truncated relative to the reference full-length human GDE sequence may include one or more additional amino acid modifications relative to the reference full-length human GDE sequence. In particular, in addition to the deletions further described below, the functional truncated GDE polypeptides may include one or more amino acid modifications, such as amino acid insertions, deletions and / or substitutions, compared to the reference full-length human GDE sequence. For example, the functional truncated GDE polypeptides may include 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), particularly 1 to 5 (e.g., 1, 2, 3, 4 or 5) additional amino acid modifications, provided that the functionality of the truncated GDE polypeptide is maintained.

[0033] The functional truncated GDE polypeptides of the invention are N-terminal truncated GDE polypeptides. An "N-terminal truncated GDE polypeptide" means a GDE polypeptide that contains a deletion relative to the reference functional full-length human GDE sequence, wherein the deletion consists of a deletion of amino acids at the N-terminal portion of the reference functional full-length human GDE sequence. The "N-terminal portion" of the reference functional full-length human GDE sequence refers to a region consisting of the first 280 amino acid residues (i.e., the most N-terminal 280 amino acid residues of the reference full-length GDE sequence), the first 200 amino acid residues (i.e., the most N-terminal 200 amino acid residues of the reference full-length GDE sequence), the first 150 amino acid residues (i.e., the most N-terminal 150 amino acid residues of the reference full-length GDE sequence), preferably the first 125 amino acid residues (i.e., the most N-terminal 125 amino acid residues of the reference full-length GDE sequence), and most preferably the first 123 amino acid residues (i.e., the most N-terminal 123 amino acid residues of the reference full-length GDE sequence).

[0034] As detailed below, the N-terminal truncated GDE polypeptides of the invention retain methionine as the first residue at the N-terminal side terminus.

[0035] The functional truncated GDE polypeptide of the present invention contains a deletion with respect to the reference functional full-length human GDE sequence, and the deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are - MGSFQY (SEQ ID NO: 7); - MEKSGG (SEQ ID NO: 8); - MILRVG (SEQ ID NO: 9); - MGADNH (SEQ ID NO: 10); or - MLDCVT (SEQ ID NO: 11) in the form of

[0036] Preferably, the functional truncated GDE polypeptide of the present invention contains a deletion with respect to the reference functional full-length human GDE sequence, and the deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are MEKSGG (SEQ ID NO: 8).

[0037] 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 in which the first 6 amino acids at the N-terminal end are different from the 6 amino acids at the N-terminal end of the reference functional full-length human GDE sequence, and the first 6 amino acids at the N-terminal end of the truncated polypeptide are SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11, preferably SEQ ID NO: 8.

[0038] In certain embodiments, the functional truncated GDE polypeptide of the present invention contains a deletion with respect to the reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; - the deletion is such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are - MGSFQY (SEQ ID NO: 7); - MEKSGG (SEQ ID NO: 8); - MILRVG (SEQ ID NO: 9); - MGADNH (SEQ ID NO: 10); or - MLDCVT (SEQ ID NO: 11) It consists of a deletion of amino acids at the N-terminus of the reference functional full-length human GDE sequence in such a form that it becomes

[0039] In a preferred embodiment, the functional truncated GDE polypeptide of the present invention contains a deletion with respect to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; - The deletion consists of a deletion of amino acids at the N-terminus of the reference functional full-length human GDE sequence in such a form that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide become MEKSGG (SEQ ID NO: 8).

[0040] In a specific embodiment, the functional truncated GDE polypeptide of the present invention contains a deletion with respect to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; - The deletion is (i) a deletion of any amino acid between the first methionine and the sequence "GSFQY" (SEQ ID NO: 54); (ii) a deletion of any amino acid between the first methionine and the sequence "EKSGG" (SEQ ID NO: 55); (iii) a deletion of any amino acid between the first methionine and the sequence "ILRVG" (SEQ ID NO: 56); (iv) a deletion of any amino acid between the first methionine and the sequence "GADNH" (SEQ ID NO: 57); or (v) a deletion of any amino acid between the first methionine and the sequence "LDCVT" (SEQ ID NO: 58) and consists of.

[0041] According to this embodiment, a deletion consisting of a deletion of any amino acid between the first methionine and the sequence "GSFQY" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "GSFQY" are deleted, but the first methionine and the sequence "GSFQY" (SEQ ID NO: 54) are not deleted.

[0042] According to this embodiment, a deletion consisting of a deletion of any amino acid between the first methionine and the sequence "EKSGG" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "EKSGG" are deleted, but the first methionine and the sequence "EKSGG" (SEQ ID NO: 55) are not deleted.

[0043] According to this embodiment, a deletion consisting of a deletion of any amino acid between the first methionine and the sequence "ILRVG" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "ILRVG" are deleted, but the first methionine and the sequence "ILRVG" (SEQ ID NO: 56) are not deleted.

[0044] According to this embodiment, a deletion consisting of a deletion of any amino acid between the first methionine and the sequence "GADNH" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "GADNH" are deleted, but the first methionine and the sequence "GADNH" (SEQ ID NO: 57) are not deleted.

[0045] According to this embodiment, a deletion consisting of a deletion of any amino acid between the first methionine and the sequence "LDCVT" means that all consecutive amino acids between the first methionine at the N-terminus and the sequence "LDCVT" are deleted, but the first methionine and the sequence "LDCVT" (SEQ ID NO: 58) are not deleted.

[0046] In a preferred embodiment, the functional truncated GDE polypeptide 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 has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; - The deletion consists of a deletion of any amino acid between the first methionine and the sequence "EKSGG".

[0047] In a particular embodiment, the functional truncated GDE polypeptide 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 has the amino acids of SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length; - The deletion is (i) consists of a deletion of amino acids at positions 2 to 88, and the amino acids at positions 1 and 89 are not deleted; (ii) consists of a deletion of amino acids at positions 2 to 99, and the amino acids at positions 1 and 100 are not deleted; (iii) consists of a deletion of amino acids at positions 2 to 111, and the amino acids at positions 1 and 112 are not deleted; (iv) consists of a deletion of amino acids at positions 2 to 115, and the amino acids at positions 1 and 116 are not deleted; or (v) consists of a deletion of amino acids at positions 2 to 123, and the amino acids at positions 1 and 124 are not deleted.

[0048] In a preferred embodiment, the functional truncated GDE polypeptide 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 has the amino acids of SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length; - The deletion consists of a deletion of amino acids at positions 2 to 99, and the amino acids at positions 1 and 100 are not deleted.

[0049] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises only one deletion in the N-terminal portion of a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length; - 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, - the deletion in the N-terminal portion of the reference functional full-length human GDE sequence is (i) a deletion of amino acids at positions 2-88 of the reference functional full-length human GDE sequence; (ii) a deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence; (iii) a deletion of amino acids at positions 2-111 of the reference functional full-length human GDE sequence; (iv) a deletion of amino acids at positions 2-115 of the reference functional full-length human GDE sequence; (v) a deletion of amino acids at positions 2-123 of the reference functional full-length human GDE sequence and consists of.

[0050] In a preferred embodiment, the functional truncated GDE polypeptide of the present invention comprises only one deletion in the N-terminal portion of a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length; - 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, - the deletion in the N-terminal portion of the reference functional full-length human GDE sequence consists of a deletion of amino acids at positions 2-99 of the reference functional full-length human GDE sequence.

[0051] "One and only deletion" means that there is one deletion in the N-terminal region corresponding to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence (i.e., the 280 amino acid residues at the most N-terminal of the reference full-length GDE sequence). For clarity, a functional truncated GDE containing one and only one deletion of the amino acids at positions 2 to 88 in the N-terminal region of the reference functional full-length human GDE sequence is, - the amino acids at positions 2 to 88 of the reference functional full-length sequence are deleted, - the amino acids at positions 1 and 89 to 280 are not deleted, corresponding to the truncated polypeptide.

[0052] In certain embodiments, the functional truncated GDE polypeptide of the present invention contains 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 the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, - the deletion is designated as Δ1b4, Δ1b5, Δ1b6, Δ1b7, Δ1b8 in Table 1, preferably Δ1b5:

[0053]

Table 1

[0054] For clarity, Table 1 (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 "Δ1b4" truncated GDE polypeptide corresponds to a functional truncated GDE polypeptide derived from SEQ ID NO: 1 or SEQ ID NO: 4 in which all consecutive amino acids at positions 2 to 88 are deleted relative to SEQ ID NO: 1 or SEQ ID NO: 4.

[0055] "Δ1b4" truncated polypeptide In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, said deletion consisting of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are MGSFQY (SEQ ID NO: 7).

[0056] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE has an amino acid sequence as set forth 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 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - said deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MGSFQY" (SEQ ID NO: 7).

[0057] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - said deletion consists of a deletion of amino acids at positions 2 to 88 of the reference functional full-length GDE sequence, - the amino acid at position 1 and the amino acid at position 89 of the reference functional full-length GDE sequence are not deleted.

[0058] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 88 of the reference functional full-length human GDE sequence, - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, - The amino acids at positions 89 to 110, 89 to 130, 89 to 150, 89 to 170, 89 to 190, 89 to 210, 89 to 230, 89 to 250, 89 to 270, or 89 to 280 of the reference functional full-length GDE sequence are not deleted.

[0059] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 88 of the reference functional full-length human GDE sequence, - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, - The amino acids at positions 89 to 280 of the reference functional full-length GDE sequence are not deleted.

[0060] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 88 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence "MGSFQY" (SEQ ID NO: 7).

[0061] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 88 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence "MGSFQY" (SEQ ID NO: 7), - The functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0062] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion with respect to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 88 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 17.

[0063] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion with respect to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 88 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 17. - The functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0064] In certain embodiments, the functional truncated GDE polypeptide comprises, or consists of, SEQ ID NO: 12, or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 12, such as at least 75% or at least 80% sequence identity, 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 to SEQ ID NO: 12.

[0065] In certain embodiments, a functional variant having at least 70% sequence identity to SEQ ID NO: 12, such as at least 75% or at least 80% sequence identity, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 12 has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 12, particularly the same enzymatic activity involved in glycogen breakdown in muscle tissues such as heart or quadriceps muscle. In certain embodiments, the functional variant of SEQ ID NO: 12 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 12 to restore glycogen accumulation and muscle strength in vivo. In certain embodiments, the functional variant of SEQ ID NO: 12 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 12.

[0066] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 12, such as at least 75% or at least 80% sequence identity, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 12 has the same N-terminal portion as SEQ ID NO: 12. In certain embodiments, the most N-terminal amino acid of the functional variant of SEQ ID NO: 12 is "MGSFQY" (SEQ ID NO: 7). In another particular embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 12 corresponds to the sequence of SEQ ID NO: 17.

[0067] In certain embodiments, the most N-terminal amino acid of the functional variant of SEQ ID NO: 12 can consist of the 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, 1-200 of SEQ ID NO: 12. In other words, according to this embodiment, the functional variant of SEQ ID NO: 12 does not contain any mutations, insertions, or deletions within the regions corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 12. In certain embodiments, the N-terminal side terminus of the functional variant of SEQ ID NO: 12 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 12. In other words, according to this embodiment, the functional variant of SEQ ID NO: 12 does not contain any mutations, insertions, or deletions within the regions corresponding to amino acids 1-50, 1-100, or 1-150.

[0068] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 12.

[0069] "Δ1b5" truncated polypeptide In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, said deletion consisting of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MEKSGG" (SEQ ID NO: 8).

[0070] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE has an amino acid sequence as set forth 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 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - said deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MEKSGG" (SEQ ID NO: 8).

[0071] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - said deletion consists of a deletion of amino acids at positions 2 to 99 of the reference functional full-length GDE sequence, - the amino acids at positions 1 and 100 of the reference functional full-length GDE sequence are not deleted.

[0072] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 99 of the reference functional full-length human GDE sequence, - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, - The amino acids at positions 100 to 110, 100 to 130, 100 to 150, 100 to 170, 100 to 190, 100 to 210, 100 to 230, 100 to 250, 100 to 270, or positions 100 to 280 of the reference functional full-length GDE sequence are not deleted.

[0073] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 99 of the reference functional full-length human GDE sequence, - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, - The amino acids at positions 100 to 280 of the reference functional full-length GDE sequence are not deleted.

[0074] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 99 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence "MEKSGG" (SEQ ID NO: 8).

[0075] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 99 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence "MEKSGG" (SEQ ID NO: 8), - The functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 of SEQ ID NO: 1 or SEQ ID NO: 4.

[0076] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 99 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 18.

[0077] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 99 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 18, - The functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 of SEQ ID NO: 1 or SEQ ID NO: 4.

[0078] In certain embodiments, the functional truncated GDE polypeptide comprises, or consists of, SEQ ID NO: 13, or a functional variant thereof having at least 70% sequence identity, such as at least 75% or at least 80% sequence identity, for example, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 13.

[0079] In certain embodiments, a functional variant having at least 70% sequence identity, such as at least 75% or at least 80% sequence identity, for example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 13 has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 13, particularly the same enzymatic activity involved in glycogen breakdown in muscle tissues such as the heart or quadriceps muscle. In certain embodiments, the functional variant of SEQ ID NO: 13 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 13 to restore glycogen accumulation and muscle strength in vivo. In certain embodiments, the functional variant of SEQ ID NO: 13 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 13.

[0080] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 13, such as at least 75% or at least 80% sequence identity, for example 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 13, has the same N-terminal portion as SEQ ID NO: 13. In certain embodiments, the most N-terminal amino acid of the functional variant of SEQ ID NO: 13 is "MEKSGG" (SEQ ID NO: 8). In another particular embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 13 corresponds to the sequence of SEQ ID NO: 18.

[0081] In certain embodiments, the most N-terminal amino acid of the functional variant of SEQ ID NO: 13 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 13. In other words, according to this embodiment, the functional variant of SEQ ID NO: 13 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 13. In certain embodiments, the N-terminal side terminus of the functional variant of SEQ ID NO: 13 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 13. In other words, according to this embodiment, the functional variant of SEQ ID NO: 13 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0082] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 13.

[0083] "Δ1b6" truncated polypeptide In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, said deletion consisting of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MILRVG" (SEQ ID NO: 9).

[0084] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE has an amino acid sequence as set forth 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 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - said deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MILRVG" (SEQ ID NO: 9).

[0085] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - said deletion consists of a deletion of amino acids at positions 2 to 111 of the reference functional full-length GDE sequence, - the amino acid at position 1 and the amino acid at position 112 of the reference functional full-length GDE sequence are not deleted.

[0086] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 111 of the reference functional full-length human GDE sequence, - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, - The amino acids at positions 112 to 130, 112 to 150, 112 to 170, 112 to 190, 112 to 210, 112 to 230, 112 to 250, 112 to 270, or positions 112 to 280 of the reference functional full-length GDE sequence are not deleted.

[0087] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 111 of the reference functional full-length human GDE sequence, - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, - The amino acids at positions 112 to 280 of the reference functional full-length GDE sequence are not deleted.

[0088] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 111 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence "MILRVG" (SEQ ID NO: 9).

[0089] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 111 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide includes the sequence "MILRVG" (SEQ ID NO: 9), - The functional truncated GDE polypeptide of the present invention includes at least the amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0090] In certain embodiments, the functional truncated GDE polypeptide of the present invention includes a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 111 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide includes the sequence of SEQ ID NO: 19.

[0091] In certain embodiments, the functional truncated GDE polypeptide of the present invention includes a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 111 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide includes the sequence of SEQ ID NO: 19, - The functional truncated GDE polypeptide of the present invention includes at least the amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0092] In certain embodiments, the functional truncated GDE polypeptide comprises, or consists of, SEQ ID NO: 14, or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 14, such as at least 75% or at least 80% sequence identity, 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 to SEQ ID NO: 14.

[0093] In certain embodiments, a functional variant having at least 70% sequence identity to SEQ ID NO: 14, such as at least 75% or at least 80% sequence identity, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 14 has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 14, particularly the same enzymatic activity involved in glycogenolysis in muscle tissues such as heart or quadriceps muscle. In certain embodiments, the functional variant of SEQ ID NO: 14 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 14 to restore glycogen accumulation and muscle strength in vivo. In certain embodiments, the functional variant of SEQ ID NO: 14 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 14.

[0094] In certain embodiments, functional variants having at least 70% sequence identity with SEQ ID NO: 14, such as at least 75% or at least 80% sequence identity, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 14, have the same N-terminal portion as SEQ ID NO: 14. In certain embodiments, the most N-terminal amino acid of the functional variant of SEQ ID NO: 14 is "MILRVG" (SEQ ID NO: 9). In another certain embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 14 corresponds to the sequence of SEQ ID NO: 19.

[0095] In a further embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 14 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 14. In other words, according to this embodiment, the functional variant of SEQ ID NO: 14 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 14. In certain embodiments, the N-terminal side terminus of the functional variant of SEQ ID NO: 14 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 14. In other words, according to this embodiment, the functional variant of SEQ ID NO: 14 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0096] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 14.

[0097] "Δ1b7" truncated polypeptide In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, said deletion consisting of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MGADNH" (SEQ ID NO: 10).

[0098] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE has an amino acid sequence as set forth 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 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - said deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MGADNH" (SEQ ID NO: 10).

[0099] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - said deletion consists of a deletion of amino acids at positions 2 to 115 of the reference functional full-length GDE sequence, - the amino acid at position 1 and the amino acid at position 116 of the reference functional full-length GDE sequence are not deleted.

[0100] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or is a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 115 of the reference functional full-length human GDE sequence, - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, - The amino acids at positions 116 to 130, 116 to 150, 116 to 170, 116 to 190, 116 to 210, 116 to 230, 116 to 250, 116 to 270, or 116 to 280 of the reference functional full-length GDE sequence are not deleted.

[0101] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 115 of the reference functional full-length human GDE sequence, - The amino acid at position 1 of the reference functional full-length GDE sequence is not deleted, - The amino acids at positions 116 to 280 of the reference functional full-length GDE sequence are not deleted.

[0102] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 115 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence "MGADNH" (SEQ ID NO: 10).

[0103] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 115 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence "MGADNH" (SEQ ID NO: 10), - The functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0104] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 115 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 20.

[0105] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 115 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide comprises the sequence of SEQ ID NO: 20, - The functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0106] In certain embodiments, the functional truncated GDE polypeptide comprises, or consists of, SEQ ID NO: 15, or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 15, such as at least 75% or at least 80% sequence identity, 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 to SEQ ID NO: 15.

[0107] In certain embodiments, a functional variant having at least 70% sequence identity to SEQ ID NO: 15, such as at least 75% or at least 80% sequence identity, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 15 has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 15, particularly the same enzymatic activity involved in glycogenolysis in muscle tissues such as the heart or quadriceps muscle. In certain embodiments, the functional variant of SEQ ID NO: 15 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 15 to restore glycogen accumulation and muscle strength in vivo. In certain embodiments, the functional variant of SEQ ID NO: 15 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 15.

[0108] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 15, such as at least 75% or at least 80% sequence identity, for example 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 15, has the same N-terminal portion as SEQ ID NO: 15. In certain embodiments, the most N-terminal amino acid of the functional variant of SEQ ID NO: 15 is "MGADNH" (SEQ ID NO: 10). In another certain embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 15 corresponds to the sequence of SEQ ID NO: 20.

[0109] In a further embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 15 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 15. In other words, according to this embodiment, the functional variant of SEQ ID NO: 15 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 15. In certain embodiments, the N-terminal side terminus of the functional variant of SEQ ID NO: 15 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 15. In other words, according to this embodiment, the functional variant of SEQ ID NO: 15 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0110] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 15.

[0111] "Δ1b8" truncated polypeptide In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, said deletion consisting of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MLDCVT" (SEQ ID NO: 11).

[0112] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE has an amino acid sequence as set forth 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 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO: 4; - said deletion consists of a deletion of amino acids in the N-terminal portion of the reference functional full-length human GDE sequence such that the first 6 amino acids at the N-terminus of the functional truncated GDE polypeptide are "MLDCVT" (SEQ ID NO: 11).

[0113] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - said deletion consists of a deletion of amino acids at positions 2 to 123 of the reference functional full-length GDE sequence, - the amino acid at position 1 and the amino acid at position 124 of the reference functional full-length GDE sequence are not deleted.

[0114] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - the deletion consists of a deletion of amino acids 2 to 123 of a reference functional full-length human GDE sequence; - the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted; - the amino acids at positions 124-130, 124-150, 124-170, 124-190, 124-210, 124-230, 124-250, 124-270, or 124-280 of the reference functional full-length GDE sequence are not deleted.

[0115] In certain embodiments, the functional truncated GDE polypeptides of the invention comprise a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length; - the deletion consists of a deletion of amino acids 2 to 123 of a reference functional full-length human GDE sequence; - the amino acid at position 1 of the reference functional full-length GDE sequence is not deleted; - The amino acids 124 to 280 of the reference functional full-length GDE sequence are not deleted.

[0116] In certain embodiments, the functional truncated GDE polypeptides of the invention comprise a deletion relative to a reference functional full-length human GDE sequence, - the reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4 or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length; - the deletion consists of a deletion of amino acids 2 to 123 of a reference functional full-length human GDE sequence; - said functional truncated GDE polypeptide comprises the sequence "MLDCVT" (SEQ ID NO: 11).

[0117] In certain embodiments, the functional truncated GDE polypeptides of the invention comprise a deletion relative to a reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 123 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide includes the sequence "MLDCVT" (SEQ ID NO: 11), - The functional truncated GDE polypeptide of the present invention includes at least amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0118] In certain embodiments, the functional truncated GDE polypeptide of the present invention includes a deletion with respect to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 123 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide includes the sequence of SEQ ID NO: 21.

[0119] In certain embodiments, the functional truncated GDE polypeptide of the present invention includes a deletion with respect to the reference functional full-length human GDE sequence, - The reference functional full-length human GDE sequence is SEQ ID NO: 1 or SEQ ID NO: 4, or a functional variant of SEQ ID NO: 1 or SEQ ID NO: 4 having the same length, - The deletion consists of a deletion of amino acids at positions 2 to 123 of the reference functional full-length human GDE sequence, - The functional truncated GDE polypeptide includes the sequence of SEQ ID NO: 21, - The functional truncated GDE polypeptide of the present invention includes at least amino acid residues at positions 429 to 666, 866 to 892, 1088 to 1194, and 1235 to 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0120] In certain embodiments, the functional truncated GDE polypeptide comprises, or consists of, SEQ ID NO: 16, or a functional variant thereof having at least 70% sequence identity to SEQ ID NO: 16, such as at least 75% or at least 80% sequence identity, 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 to SEQ ID NO: 16.

[0121] In certain embodiments, a functional variant having at least 70% sequence identity to SEQ ID NO: 16, such as at least 75% or at least 80% sequence identity, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to SEQ ID NO: 16 has substantially the same functionality or enzymatic activity as the GDE polypeptide of SEQ ID NO: 16, particularly the same enzymatic activity involved in glycogenolysis in muscle tissues such as heart or quadriceps muscle. In certain embodiments, the functional variant of SEQ ID NO: 16 has substantially the same ability as the GDE polypeptide of SEQ ID NO: 16 to restore glycogen accumulation and muscle strength in vivo. In certain embodiments, the functional variant of SEQ ID NO: 16 has substantially the same expression level as the GDE polypeptide of SEQ ID NO: 16.

[0122] In certain embodiments, a functional variant having at least 70% sequence identity with SEQ ID NO: 16, such as at least 75% or at least 80% sequence identity, for example 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with SEQ ID NO: 16, has the same N-terminal portion as SEQ ID NO: 16. In certain embodiments, the most N-terminal amino acid of the functional variant of SEQ ID NO: 16 is "MLDCVT" (SEQ ID NO: 11). In another certain embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 16 corresponds to the sequence of SEQ ID NO: 21.

[0123] In a further embodiment, the most N-terminal amino acid of the functional variant of SEQ ID NO: 16 can consist of amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 16. In other words, according to this embodiment, the functional variant of SEQ ID NO: 16 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-175, 1-200 of SEQ ID NO: 16. In certain embodiments, the N-terminal side terminus of the functional variant of SEQ ID NO: 16 consists of amino acids 1-50, 1-100, or 1-150 of SEQ ID NO: 16. In other words, according to this embodiment, the functional variant of SEQ ID NO: 16 does not contain any mutations, insertions, or deletions within the region corresponding to amino acids 1-50, 1-100, or 1-150.

[0124] In a preferred embodiment, the functional truncated GDE polypeptide comprises or consists of SEQ ID NO: 16.

[0125] Combinations with other deletions In certain embodiments, the functional truncated GDE polypeptide of the present invention, which contains a deletion in the N-terminal portion of the reference functional full-length human GDE sequence, may further contain a deletion or a combination of deletions in other portions of the reference functional full-length human GDE sequence.

[0126] In certain embodiments, the functional truncated GDE polypeptide - is a deletion of only one of the consecutive amino acids 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 is included.

[0127] In certain embodiments, the functional truncated GDE polypeptide - is a deletion of only one of the consecutive amino acids 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 is included, the reference functional full-length human GDE sequence is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, and the deletions are designated as Δ1b4, Δ1b5, Δ1b6, Δ1b7, Δ1b8 in Table 1.

[0128] In certain embodiments, the functional truncated GDE polypeptide of the present invention comprises at least the amino acid residues at positions 429 - 666, 866 - 892, 1088 - 1194, and 1235 - 1420 with respect to SEQ ID NO: 1 or SEQ ID NO: 4.

[0129] In certain embodiments, the functional truncated GDE polypeptide of the invention, which contains a deletion in the N-terminal portion of the reference functional full-length human GDE sequence, may further contain a deletion or a combination of deletions in the C-terminal portion of the reference functional full-length GDE sequence and / or in the central domain of the reference full-length GDE sequence.

[0130] In certain embodiments, the C-terminal portion of the reference functional full-length GDE sequence corresponds to the region consisting of the last 112 amino acids, i.e., the most C-terminal 112 amino acids of the reference functional full-length human GDE sequence.

[0131] In certain embodiments, 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 the amino acids at positions 710 to 865 of SEQ ID NO: 1 or SEQ ID NO: 4.

[0132] In certain embodiments, the functional truncated GDE polypeptide - is a deletion at only one position in the N-terminal portion of the reference functional full-length human GDE sequence, wherein the N-terminal portion corresponds to the region consisting of the first 280 amino acid residues of the reference functional full-length human GDE sequence, and - another deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence and includes the reference functional full-length human GDE sequence is selected from the group consisting of SEQ ID NOs: 1 to 6, the deletion in the N-terminal portion is designated as Δ1b4, Δ1b5, Δ1b6, Δ1b7, Δ1b8 in Table 1 (Table 1); the additional deletion or combination of deletions in other parts of the reference functional full-length human GDE sequence is selected from any of the deletions designated as Δ1, Δ2, Δ3, Δ4, Δ5, Δ6, and Δ7 in Table 2 (Table 2):

[0133]

Table 2

[0134] A nucleic acid molecule encoding a 2-N-terminal truncated GDE polypeptide In another aspect, the invention relates to a nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above.

[0135] 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 functional truncated GDE polypeptide according to the invention.

[0136] In a preferred embodiment, the nucleic acid molecule encoding the functional truncated GDE polypeptide is small enough to be packaged into a gene therapy vector, such as an AAV vector, in combination with appropriate regulatory sequences. The size of the nucleic acid molecule encoding the functional truncated GDE polypeptide is preferably less than about 4.5 kb, preferably less than about 4.4 kb.

[0137] "Gene therapy vector" means any vector suitable for gene therapy. In particular, a gene therapy vector can be a plasmid or a recombinant virus, such as a viral vector derived from a retrovirus or a lentivirus. Preferably, the viral vector is an AAV vector, such as an AAV vector suitable for transduction into liver tissue or muscle cells. Extensive experience in clinical trials and preclinical models of muscle diseases indicates that adeno-associated virus (AAV) is the optimal vector for in vivo gene therapy for GSDIII. These vectors efficiently transduce liver and muscle, their production can be scaled up and down, and compared to other gene therapy vectors, they have a relatively low immunogenic profile. However, one of the greatest limitations in the use of AAV for gene replacement is their limited capsid packaging size limit (about 5 kb). Indeed, during the production of recombinant AAV, genomes larger than 5 kb are encapsidated into capsids with low efficiency, and the resulting AAV may contain fragmented genomes, thereby reducing the effectiveness of gene transfer.

[0138] The sequence of the nucleic acid molecule of the present invention encoding the functional truncated GDE polypeptide can be optimized for the expression of the GDE polypeptide in vivo. Sequence optimization can include a number of changes in the nucleic acid sequence, including codon optimization, increasing the GC content, decreasing the number of CpG islands, decreasing the number of alternative open reading frames (ARFs), and / or decreasing 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. It is also well known that the genetic codes of different organisms are often biased in the use of one of several codons that encode the same amino acid compared to the use of others. By codon optimization, changes are introduced into the nucleotide sequence such that the resulting codon-optimized nucleotide sequence takes advantage of the codon bias present in a given cellular context and has a higher likelihood of being expressed at a relatively high level in such a given cellular context compared to the non-codon-optimized sequence. In a preferred embodiment of the present invention, such an optimized nucleotide sequence encoding the functional truncated GDE polypeptide is codon-optimized to improve its expression in human cells compared to the non-codon-optimized nucleotide sequence encoding the same functional truncated GDE polypeptide, for example, by taking advantage of the human-specific codon usage frequency bias. The nucleic acid sequence encoding full-length human GDE isoform 1 is as shown in SEQ ID NO: 32. An example of the corresponding codon-optimized sequence is as shown in SEQ ID NO: 33.

[0139] In certain embodiments, the nucleic acid molecule of the present invention encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the nucleic acid molecule of the present invention comprises, or consists of, the sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, which encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, a nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity to the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 23, preferably SEQ ID NO: 22. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity, e.g., at least 96, 97, 98, 99, or 100 percent identity, to the nucleotide sequence of SEQ ID NO: 22 or SEQ ID NO: 23, preferably SEQ ID NO: 22.

[0140] In certain embodiments, the nucleic acid molecule of the present invention encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the nucleic acid molecule of the present invention comprises, or consists of, the sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 25, which encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, a nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90, or at least 95 percent identity to the nucleotide sequence of SEQ ID NO: 24 or SEQ ID NO: 25, preferably SEQ ID NO: 24. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity, e.g., at least 96, 97, 98, 99, or 100 percent identity, to the nucleotide sequence of SEQ ID NO: 24 or SEQ ID NO: 25, preferably SEQ ID NO: 24.

[0141] In certain embodiments, the nucleic acid molecule of the present invention encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the nucleic acid molecule of the present invention comprises, or consists of, the sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 27, which encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, a nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90 or at least 95 percent identity to the nucleotide sequence of SEQ ID NO: 26 or SEQ ID NO: 27, preferably SEQ ID NO: 26. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity, e.g., at least 96, 97, 98, 99 or 100 percent identity, to the nucleotide sequence of SEQ ID NO: 26 or SEQ ID NO: 27, preferably SEQ ID NO: 26.

[0142] In certain embodiments, the nucleic acid molecule of the present invention encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the nucleic acid molecule of the present invention comprises, or consists of, the sequence set forth in SEQ ID NO: 28 or SEQ ID NO: 29, which encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, a nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90 or at least 95 percent identity to the nucleotide sequence of SEQ ID NO: 28 or SEQ ID NO: 29, preferably SEQ ID NO: 28. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity, e.g., at least 96, 97, 98, 99 or 100 percent identity, to the nucleotide sequence of SEQ ID NO: 28 or SEQ ID NO: 29, preferably SEQ ID NO: 28.

[0143] In certain embodiments, the nucleic acid molecule of the present invention encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the nucleic acid molecule of the present invention comprises, or consists of, the sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31 that encodes a functional truncated GDE polypeptide having the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, a nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above has at least 80, at least 85, at least 90 or at least 95 percent identity to the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31, preferably SEQ ID NO: 30. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity, e.g., at least 96, 97, 98, 99 or 100 percent identity, to the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31, preferably SEQ ID NO: 30.

[0144] A nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above may have at least 80, at least 85, at least 90 or at least 95 percent identity to any of the nucleotide sequences of SEQ ID NOs: 22 - 31. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity, e.g., at least 96, 97, 98, 99 or 100 percent identity, to any of the nucleotide sequences of SEQ ID NOs: 22 - 31.

[0145] Preferably, a nucleic acid molecule encoding a functional truncated GDE polypeptide as defined above may have at least 80, at least 85, at least 90 or at least 95 percent identity to any of the nucleotide sequences of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30. In certain embodiments, the nucleic acid molecule of the present invention has at least 95 percent identity, e.g., at least 96, 97, 98, 99 or 100 percent identity, to any of the nucleotide sequences of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30.

[0146] 3 - Nucleic acid construct The present invention also relates to a nucleic acid construct comprising the nucleic acid molecule of the present invention as described above. The nucleic acid construct may correspond to an expression cassette comprising the nucleic acid sequence of the present invention operably linked to one or more expression control sequences and / or other sequences that enhance expression. As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter, or another transcriptional regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Such expression control sequences, such as promoters, enhancers (e.g., cis-regulatory modules (CRM)), introns, polyA signals, etc., are known in the art.

[0147] In certain embodiments, the expression cassette may comprise a promoter. The promoter may be a ubiquitous promoter or a tissue-specific promoter, particularly a promoter that can promote expression in cells or tissues where expression of GDE is desired, for example, in cells or tissues where GDE expression is desired in GDE-deficient patients. Preferably, the promoter is a ubiquitous promoter.

[0148] In the first specific embodiment, the promoter is a muscle-specific promoter. Non-limiting examples of muscle-specific promoters include the muscle creatine kinase (MCK) promoter. Non-limiting examples of suitable 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. November 2008;15(22):1489-1499) (representative GenBank accession number AF188002). Human muscle creatine kinase has a gene ID number 1158 (representative GenBank accession number NC_000019.9, access date: December 26, 2012). Other examples of muscle-specific promoters include the synthetic promoter C5.12 (spC5.12, alternatively referred to herein as "C5.12"), for example, spC5.12 or the spC5.12 promoter (disclosed in Wang et al., Gene Therapy, Volume 15, pages 1489-1499 (2008)), the MHCK7 promoter (Salva et al., Mol Ther. February 2007;15(2):320-329), the myosin light chain (MLC) promoter, for example, MLC2 (gene ID number 4633; representative GenBank accession number NG_007554.1, access date: December 26, 2012); the myosin heavy chain (MHC) promoter, for example, alpha-MHC (gene ID number 4624; representative GenBank accession number NG_023444.1, access date: December 26, 2012); the desmin promoter (gene ID number 1674; representative GenBank accession number NG_008043.1, access date: December 26, 2012); the cardiac troponin C promoter (gene ID number 7134; representative GenBank accession number NG_008963.1, access date: December 26, 2012); the troponin I promoter (gene ID numbers 7135, 7136, and 7137; representative GenBank accession numbers NG_016649.1, NG_011621.1, and NG_007866.2. Access date: December 26, 2012); myoD gene family promoter (Weintraub et al., Science, 251, 761 (1991); Gene ID number 4654; Representative GenBank accession number NM_002478, Access date: December 26, 2012); alpha-actin promoter (Gene ID numbers 58, 59, and 70; Representative GenBank accession numbers NG_006672.1, NG_011541.1, and NG_007553.1, Access date: December 26, 2012); beta-actin promoter (Gene ID number 60; Representative GenBank accession number NG_007992.1, Access date: December 26, 2012); gamma-actin promoter (Gene ID numbers 71 and 72; Representative GenBank accession numbers NG_011433.1 and NM_001199893, Access date: December 26, 2012); a muscle-specific promoter present within intron 1 of ocular Pitx3 (Gene ID number 5309) (Coulon et al.; This muscle-selective promoter corresponds to residues 11219 - 11527 of Representative GenBank accession number NG_008147, Access date: December 26, 2012); and the promoter described in US Patent Application Publication No. 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, Volume 15, pages 1489 - 1499 (2008), which includes a combination of an enhancer from MCK and the spC5.12 promoter. In a specific embodiment of the present invention, the muscle-specific promoter is spC5.It is selected from the group consisting of 12 promoter, MHCK7 promoter, E-syn promoter, muscle creatine kinase myosin light chain (MLC) promoter, myosin heavy chain (MHC) promoter, cardiac troponin C promoter, troponin I promoter, myoD gene family promoter, alpha-actin promoter, beta-actin promoter, gamma-actin promoter, muscle-specific promoter present in intron 1 of ocular Pitx3, CK6 promoter, CK8 promoter and Acta1 promoter. In a further embodiment, the muscle-specific promoter is selected from the group consisting of spC5.12, desmin and MCK promoter. In a further embodiment, the muscle-specific promoter is selected from the group consisting of spC5.12 and MCK promoter. In a preferred embodiment, the muscle-specific promoter is spC5.12 promoter.

[0149] In another specific embodiment, the promoter is a liver-specific promoter. Non-limiting examples of liver-specific promoters include the HSE promoter (liver-specific promoter), alpha-1 antitrypsin promoter (hAAT), transthyretin promoter, albumin promoter, thyroxine-binding globulin (TBG) promoter, LSP promoter (comprising two copies of the thyroxine-binding globulin promoter sequence and the alpha1-microglobulin / bikunin enhancer sequence and a leader sequence - Ill, C. R. 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.), and the like. Other useful liver-specific promoters, for example, those listed in the Liver Specific Gene Promoter Database (http: / / rulai.cshl.edu / LSPD / ) collected by the Cold Spring Harbor Laboratory, are known in the art. A preferred liver-specific promoter for the present invention is a promoter comprising an H1 enhancer and a TTR promoter. An example of such a liver-specific promoter is the HSE promoter. The HSE promoter contains the mouse TTR promoter and the H1 enhancer described in SEQ ID NO: 51, and is derived from the initial research paper by Robert H. Costa et al., 1986 (Transcriptional control of the mouse prealbumin (transthyretin) gene: both promoter sequences and a distinct enhancer are cell specific. Mol Cell Biol. 1986;6(12):4697-4708).In a preferred embodiment, the liver-specific promoter is an HSE promoter having the sequence of SEQ ID NO: 53, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 53, for example, at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity to SEQ ID NO: 53.

[0150] In another specific embodiment, the promoter is a neuron-specific promoter. Non-limiting examples of neuron-specific promoters will be apparent to those skilled in the art and include, but are not limited to, the following among numerous ones: synapsin-1 (Syn) promoter, neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). In a specific embodiment, the neuron-specific promoter is the Syn promoter. Other neuron-specific promoters include, but are not limited to, the synapsin-2 promoter, tyrosine hydroxylase promoter, dopamine β-hydroxylase promoter, hypoxanthine phosphoribosyltransferase promoter, low affinity NGF receptor promoter, and choline acetyltransferase promoter (Bejanin et al., 1992; Carroll et al., 1995; Chin and Greengard, 1994; Foss-Petter et al., 1990; Harrington et al., 1987; Mercer et al., 1991; Patei et al., 1986). Representative promoters specific for motor neurons include, but are not limited to, the promoter of calcitonin gene-related peptide (CGRP), a known factor derived from motor neurons. Other promoters that function in motor neurons include the promoters of choline acetyltransferase (ChAT), neuron-specific enolase (NSE), synapsin, and Hb9.Other neuron-specific promoters useful in the present invention include, but are not limited to, GFAP (for astrocytes), calbindin 2 (for interneurons), Mnx1 (motor neurons), nestin (neurons), parvalbumin, somatostatin, and Plp1 (oligodendrocytes and Schwann cells).

[0151] In a preferred embodiment, the promoter is a ubiquitous promoter. Representative ubiquitous promoters include the cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, the cytomegalovirus enhancer / promoter (CMV) (with the CMV enhancer as needed) [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 retrovirus Rous sarcoma virus (RSV) LTR promoter (with the RSV enhancer as needed), the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, the EF1 alpha (EF1a) promoter or a short version of the EF1a promoter, and the Ins84 promoter (as described in WO2020 / 219949). In a preferred embodiment, the promoter is a shortened version of the CMV promoter.

[0152] In addition, the promoter may be an endogenous promoter, such as the albumin promoter or the GDE promoter.

[0153] Short promoters, such as short versions of known promoters, are of particular interest in the present invention. In a preferred embodiment, the promoter is less than about 500 pb, preferably less than about 450 pb, preferably less than about 400 pb.

[0154] In a preferred embodiment, the promoter is a shorter version of any of the promoters described herein, for example, a shorter version of the CMV promoter, or a shorter version of the EF1a promoter, or a promoter comprising the H1 enhancer and the TTR promoter.

[0155] In a preferred embodiment, the promoter is a short version of the CMV promoter. More preferably, the promoter is the CMV promoter having the sequence of SEQ ID NO: 43, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 43, such as at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity to SEQ ID NO: 43, in a short version.

[0156] In another specific embodiment, the promoter is a short version of the EF1a promoter. Preferably, the promoter is the EF1a promoter having the sequence as shown in SEQ ID NO: 52, or a functional variant thereof having at least 80% sequence identity to SEQ ID NO: 52, such as at least 85%, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity to SEQ ID NO: 52, in a short version.

[0157] In another specific embodiment, the promoter is the Ins84 promoter as described in WO2020 / 219949.

[0158] The expression cassette containing the nucleic acid molecule of the present invention can be adapted depending on the target population of GSDIII patients, depending on the clinical symptoms of GSDIII disease, and / or depending on the target tissue. For example, in the case of patients having clinical symptoms of GSDIII disease mainly 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 molecule of the present invention in muscle, such as the above-mentioned mini-CMV promoter, in particular, the mini-CMV promoter of SEQ ID NO: 43. In the case of patients having clinical symptoms 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 molecule of the present invention in the liver, such as the above-mentioned HSE promoter, in particular, the HSE promoter of SEQ ID NO: 53. In the case of patients having clinical symptoms of GSDIII disease in both muscle and liver, a promoter capable of inducing the expression of the nucleic acid molecule of the present invention in both tissues is preferably used.

[0159] In certain embodiments, the promoter is associated with an enhancer sequence, such as a cis-regulatory module (CRM) or an artificial enhancer sequence. Examples of CRMs useful in the practice of the present invention include those described in Rincon et al., Mol Ther. January 2015;23(1):43-52, Chuah et al., Mol Ther. September 2014;22(9):1605-13 or Nair et al., Blood. May 15, 2014;123(20):3195-9. In particular, other regulatory elements capable of enhancing the muscle-specific expression of a gene, particularly expression in cardiac and / or skeletal muscle, are those disclosed 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. Said rearrangement may include changing the order of the TFBSs, and / or changing the position of one or more TFBSs relative to other TFBSs, and / or changing the copy number of one or more of the TFBSs. For example, a nucleic acid regulatory element for enhancing muscle-specific gene expression, particularly cardiac and skeletal muscle-specific gene expression, may comprise binding sites for E2A, HNH 1, NF1, C / EBP, LRF, MyoD, and SREBP; or E2A, NF1, p53, C / EBP, LRF, and SREBP; or E2A, HNH 1, HNF3a, HNF3b, NF1, C / EBP, LRF, MyoD, and SREBP; or E2A, HNF3a, NF1, C / EBP, LRF, MyoD, and SREBP; or E2A, HNF3a, NF1, CEBP, LRF, MyoD, and SREBP; or HNF4, NF1, RSRFC4, C / EBP, LRF, and MyoD; or NF1, PPAR, p53, C / EBP, LRF, and MyoD.For example, nucleic acid regulatory elements for enhancing muscle-specific gene expression, particularly skeletal muscle-specific gene expression, may include binding sites for E2A, NF1, SRFC, p53, C / EBP, LRF, and MyoD; or E2A, NF1, C / EBP, LRF, MyoD, and SREBP; or E2A, HNF3a, C / EBP, LRF, MyoD, SEREBP, and Tal1_b; or E2A, SRF, p53, C / EBP, LRF, MyoD, and SREBP; or HNF4, NF1, RSRFC4, C / EBP, LRF, and SREBP; or E2A, HNF3a, HNF3b, NF1, SRF, C / EBP, LRF, MyoD, and SREBP; or E2A, CEBP, and MyoD. In a further example, these nucleic acid regulatory elements may include at least two, such as two, three, four or more copies of one or more of the TFBSs listed above. Other regulatory elements capable of enhancing the liver-specific expression of a gene in particular are those disclosed in WO2009130208.

[0160] In certain embodiments, the enhancer is a short-sized enhancer. In particular, the enhancer for use in the present invention may consist of 10 to 175 nucleotides, such as 40 to 100 nucleotides, particularly 50 to 80 nucleotides. In certain embodiments, the enhancer is the 72-nucleotide HS-CRM8 enhancer consisting of SEQ ID NO: 51 or a functional variant of SEQ ID NO: 51 having enhancer activity. In another embodiment, the enhancer is a functional variant of the 72-nucleotide HS-CRM8 enhancer that is at least 80% identical to SEQ ID NO: 51, such as at least 85% identical to SEQ ID NO: 51, particularly at least 90% identical, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% identical.

[0161] In another specific embodiment, the nucleic acid construct comprises an intron, particularly an intron placed between a promoter and a GDE coding sequence. The intron can be introduced to increase mRNA stability and protein production. In a further embodiment, the intron is a human beta-globin b2 (or HBB2) intron, a coagulation 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 FIX shortened intron 1 (Wu et al., 2008, Mol Ther, 16(2):280-289; Kurachi et al., 1995, J Biol Chem., 270(10):5276-5281), a beta-globin / immunoglobulin heavy chain hybrid intron (5'-donor site from the human beta-globin intron and 3'-acceptor site from the immunoglobulin heavy chain variable region intron, Wu et al., 2008, Mol Ther, 16(2):280-289; Kurachi et al., 1995, J Biol Chem., 270(10):5276-5281), a hybrid intron consisting of an adenovirus splice donor and an immunoglobulin G splice (Wong et al., 1985, Chromosoma, 92(2):124-135; Yew et al., 1997, Hum Gene Ther, 8(5):575-584; Choi T. et al., 1991, Mol Cell Biol, 11(6):3070-3074; Huang et al., 1990, Mol Cell Biol., 10(4):1805-1810), a hybrid 19S / 16S SV40 intron (5'-donor site from the 19S intron and 3'-acceptor site from the 16S intron, Yew et al., 1997, Hum Gene Ther, 8(5):575-584), or a chicken beta-globin intron. In yet a further embodiment, the intron is a modified intron (particularly a modified HBB2 or FIX intron) designed to reduce the number of alternative open reading frames (ARFs) found in the intron or even to completely remove the ARF.Preferably, an ARF having a length exceeding 50 bp and having a stop codon in-frame with the start codon is removed. The ARF can be removed by modifying the intron sequence. For example, the modification can be performed by nucleotide substitution, insertion or deletion, preferably by nucleotide substitution. As an example, one or more nucleotides, particularly one nucleotide, in the ATG or GTG start codon present in the sequence of the intron of interest are replaced, and as a result, a non-start codon can be obtained. For example, ATG or GTG can be replaced with CTG, which is not a start codon, within the sequence of the intron of interest.

[0162] The classical HBB2 intron is represented by SEQ ID NO: 34. For example, this HBB2 intron can be modified by deleting the start codons (ATG and GTG codons) within the intron. In a particular embodiment, the modified HBB2 intron has the sequence represented by SEQ ID NO: 35. The classical FIX intron is derived from the first intron of human FIX and is represented by SEQ ID NO: 36. The FIX intron can be modified by deleting the start codons (ATG and GTG codons) within the intron. In a particular embodiment, the modified FIX intron has the sequence represented by SEQ ID NO: 37. The classical chicken - beta - globin intron used in nucleic acid constructs is represented by SEQ ID NO: 38. The chicken - beta - globin intron can be modified by deleting the start codons (ATG and GTG codons) within the intron. In a particular embodiment, the modified chicken - beta - globin intron has the sequence represented by SEQ ID NO: 39.

[0163] The inventors have previously shown in WO2015 / 162302 that such modified introns, particularly modified HBB2 or FIX introns, have advantageous properties and can significantly improve the expression of transgenes.

[0164] In certain embodiments, the nucleic acid construct of the present invention is an expression cassette that, in the 5' to 3' direction, comprises a promoter optionally preceded by an enhancer, the coding sequence of the present invention (i.e., a nucleic acid molecule encoding a functional truncated GDE polypeptide), and a polyadenylation signal, such as the pA58 polyadenylation signal (pA58 polyA), the bovine growth hormone polyadenylation signal (bGH polyA), the SV40 polyadenylation signal, or another naturally occurring or artificial polyadenylation signal, in that order. Preferably, the polyadenylation signal is bGH polyA or pA58 polyA, more preferably pA58 polyA. In certain embodiments, the polyadenylation signal is bGH polyA as set forth in SEQ ID NO: 41. In certain embodiments, a very short polyA signal is used. For example, a very short polyA signal containing less than 20 nucleotides is used. In certain embodiments, the polyadenylation signal is the human soluble neuropilin-1 (sNRP) polyadenylation signal (sNRP polyA; SEQ ID NO: 40). In a preferred embodiment, the polyadenylation signal is the pA58 polyadenylation signal as set forth in SEQ ID NO: 42.

[0165] In certain embodiments, the nucleic acid construct of the present invention is an expression cassette that, in the 5' to 3' direction, comprises a promoter optionally preceded by an enhancer, an intron, the coding sequence of the present invention, and a polyadenylation signal. In another embodiment, the nucleic acid construct of the present invention is an expression cassette that, in the 5' to 3' direction, comprises a promoter, the coding sequence of the present invention, and a polyadenylation signal. In another embodiment, the nucleic acid construct of the present invention is an expression cassette that, in the 5' to 3' direction, comprises an enhancer, a promoter, the coding sequence of the present invention, and a polyadenylation signal. In a further particular embodiment, the nucleic acid construct of the present invention is an expression cassette that, in the 5' to 3' direction, comprises an enhancer, a promoter, an intron, the coding sequence of the present invention, and a polyadenylation signal. In a further particular embodiment of the present invention, an expression cassette that, in the 5' to 3' direction, comprises a promoter, an optional intron, the coding sequence of the present invention, and a polyA signal.

[0166] In another embodiment, the nucleic acid construct of the present invention is an expression cassette comprising, in the 5' to 3' direction, a 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 the 5' to 3' direction, an enhancer, a 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).

[0167] In a further specific embodiment, the expression cassette comprises, in the 5' to 3' direction, a SpC5-12 promoter; a sequence encoding the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16; and bGH polyA or pA58 polyA, particularly pA58 polyA. In another embodiment, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter; a sequence encoding the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16; and bGH polyA or pA58 polyA, particularly pA58 polyA.

[0168] In a preferred embodiment, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide as defined above, such as a sequence encoding the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16; and bGH polyA or pA58 polyA, particularly pA58 polyA.

[0169] In certain embodiments, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide as defined above, such as a sequence selected from the group consisting of SEQ ID NOs: 22 to 31; and bGH polyA or pA58 polyA, particularly pA58 polyA.

[0170] In certain embodiments, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding the functional truncated GDE polypeptide of SEQ ID NO: 12; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 22 or SEQ ID NO: 23 encoding the functional truncated GDE polypeptide of SEQ ID NO: 12; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette comprises, in the 5' to 3' direction, the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 22 or SEQ ID NO: 23 encoding the functional truncated GDE polypeptide of SEQ ID NO: 12; and pA58 polyA of SEQ ID NO: 42.

[0171] In a preferred embodiment, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 13; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 24 or SEQ ID NO: 25 encoding a functional truncated GDE polypeptide of SEQ ID NO: 13; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette comprises, in the 5' to 3' direction, the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 24 or SEQ ID NO: 25 encoding a functional truncated GDE polypeptide of SEQ ID NO: 13; and pA58 polyA of SEQ ID NO: 42.

[0172] In a further embodiment, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 14; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette comprises, in the 5' to 3' direction, a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 26 or SEQ ID NO: 27 encoding a functional truncated GDE polypeptide of SEQ ID NO: 14; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette comprises, in the 5' to 3' direction, the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 26 or SEQ ID NO: 27 encoding a functional truncated GDE polypeptide of SEQ ID NO: 14; and pA58 polyA of SEQ ID NO: 42.

[0173] In a further embodiment, the expression cassette, in the 5' to 3' direction, comprises a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 15; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette, in the 5' to 3' direction, comprises a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 28 or SEQ ID NO: 29 encoding a functional truncated GDE polypeptide of SEQ ID NO: 15; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette, in the 5' to 3' direction, comprises the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 28 or SEQ ID NO: 29 encoding a functional truncated GDE polypeptide of SEQ ID NO: 15; and pA58 polyA of SEQ ID NO: 42.

[0174] In a further embodiment, the expression cassette, in the 5' to 3' direction, comprises a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence encoding a functional truncated GDE polypeptide of SEQ ID NO: 16; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette, in the 5' to 3' direction, comprises a CMV promoter, such as a mini-CMV promoter, particularly the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 30 or SEQ ID NO: 31 encoding a functional truncated GDE polypeptide of SEQ ID NO: 16; and bGH polyA or pA58 polyA, particularly pA58 polyA. In certain embodiments, the expression cassette, in the 5' to 3' direction, comprises the mini-CMV promoter of SEQ ID NO: 43; a sequence of SEQ ID NO: 30 or SEQ ID NO: 31 encoding a functional truncated GDE polypeptide of SEQ ID NO: 16; and pA58 polyA of SEQ ID NO: 42.

[0175] In particular embodiments, the expression cassette comprises or consists of a sequence as set forth in SEQ ID NO: 44 to SEQ ID NO: 48. In further embodiments, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity to SEQ ID NO: 44 to SEQ ID NO: 48.

[0176] In a particular embodiment, the expression cassette comprises or consists of a sequence as set forth in SEQ ID NO: 44. In a further embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity to SEQ ID NO: 44.

[0177] In a preferred embodiment, the expression cassette comprises or consists of a sequence as set forth in SEQ ID NO: 45. In a further embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity to SEQ ID NO: 45, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity.

[0178] In a further embodiment, the expression cassette comprises or consists of a sequence as set forth in SEQ ID NO: 46. In a further embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity, such as at least 85% sequence identity, particularly at least 90%, more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even at least 99% sequence identity to SEQ ID NO: 46.

[0179] In a further embodiment, the expression cassette comprises or consists of a sequence as shown in SEQ ID NO: 47. In a further embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity to 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.

[0180] In a further embodiment, the expression cassette comprises or consists of a sequence as shown in SEQ ID NO: 48. In a further embodiment, the expression cassette comprises or consists of a sequence having at least 80% sequence identity 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.

[0181] When designing the nucleic acid constructs of the present invention, those skilled in the art will take care to consider the size limitations of the vectors used to deliver said constructs to cells or organs. In particular, it is known to those skilled in the art that a major limitation of AAV vectors is their cargo capacity, which can vary depending on the AAV serotype, but is thought to be limited to around the size of the parental viral genome. For example, 5 kb is the maximum size that is normally considered to be packaged in the AAV8 capsid (Wu Z. et al., Mol Ther., 2010, 18(1): 80-86; Lai Y. et al., Mol Ther., 2010, 18(1): 75-79; Wang Y. et al., Hum Gene Ther Methods, 2012, 23(4): 225-33). In addition, during the production of recombinant AAV, genomes larger than 5 kb are encapsidated into capsids with low efficiency, and the resulting AAV may contain fragmented genomes, thereby reducing the efficiency of gene transfer. Thus, those skilled in the art will take care to select the components of the nucleic acid constructs of the present invention such that the resulting nucleic acid sequence, including the sequences encoding the AAV 5'- and 3'-ITRs, does not exceed, preferably, 110% of the cargo capacity of the AAV vector to be implemented, and in particular, does not exceed 5 kb. AAV vectors with larger cargo capacities can also be used in connection with the present invention. For example, AAV particles lacking the Vp2 subunit have been shown to successfully package larger genomes (i.e., 6 kb) while maintaining the integrity of the genome encapsulated in the capsid (Grieger et al., 2005, J Virol., 79(15):9933-9944).

[0182] 4 - Vector The present invention also relates to vectors comprising a nucleic acid molecule or construct as disclosed herein. In certain embodiments, the vector comprises a nucleic acid molecule or construct encoding a functional truncated GDE polypeptide as defined above.

[0183] In particular, the vector of the present invention is a vector suitable for protein expression, preferably for use in gene therapy. In one embodiment, the vector is a plasmid vector. In another embodiment, the vector is a nanoparticle containing the nucleic acid molecule of the present invention, particularly messenger RNA encoding the functional truncated GDE polypeptide of the present invention. In another embodiment, the vector is a transposon-based system, such as the highly active Sleeping Beauty (SB100X) transposon system (Mates et al., 2009), which enables the integration of the nucleic acid molecule or construct of the present invention into the genome of the target cell. In another embodiment, the vector is a viral vector suitable for gene therapy that targets any cell of interest, such as liver tissue or cells, muscle cells, CNS cells (e.g., brain cells), or hematopoietic stem cells, such as cells of the erythroid lineage (e.g., erythrocytes). In this case, the nucleic acid construct of the present invention also contains sequences suitable for the efficient production of viral vectors, as is well known in the art.

[0184] Viral vectors, such as retroviral vectors, such as lentiviral vectors, or non-pathogenic parvoviruses, more preferably AAV vectors, are preferred for the delivery of the nucleic acid molecule or construct of the present invention. Adeno-associated virus (AAV) of the human parvovirus is a naturally replication-defective dependovirus that can integrate into the genome of infected cells to establish latent infection. This last-mentioned property appears to be unique among mammalian viruses because integration occurs at a specific site (19q13.3-qter) in the human genome called AAVS1, which is located on chromosome 19.

[0185] Therefore, AAV vectors have attracted great interest as potential vectors for human gene therapy. The beneficial 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 ability to infect a wide range of cell lines derived from different tissues.

[0186] Among the serotypes of AAV isolated from humans or non-human primates (NHPs) and well-characterized, human serotype 2 was the first AAV developed as a gene transfer vector. Other currently used AAV serotypes include AAV-1, AAV-2 variants (e.g., quadruple mutant capsid-optimized AAV-2 containing an engineered capsid with the Y44+500+730F+T491V changes disclosed in Ling et al., Hum Gene Ther Methods, July 18, 2016), -3 and AAV-3 variants (e.g., AAV3-ST variant containing an engineered AAV3 with two amino acid changes, S663V+T492V, disclosed in Vercauteren et al., 2016, Mol. Ther. 24(6), 1042), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (e.g., AAV6 variant containing the triple mutant AAV6 capsid Y731F / Y705F / T492V form disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, 16026), -7, -8, -9, -2G9, -10, e.g., cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV serotypes, e.g., AAVpo4 and AAVpo6, and tyrosine, lysine and serine capsid variants of AAV serotypes, etc. In addition, other non-native engineered variants and chimeric AAVs may also be useful. The AAV virus can be engineered using conventional molecular biology techniques that allow these particles to be optimized for cell-specific delivery of nucleic acid sequences, to minimize immunogenicity, to adjust stability and particle lifetime, for efficient degradation, and for accurate delivery to the nucleus.

[0187] AAV fragments desirable for assembly into vectors include the cap protein, which includes vp1, vp2, vp3 and the hypervariable region; the rep protein, which includes rep 78, rep 68, rep 52 and rep 40; and the sequences encoding these proteins. These fragments can be readily utilized in various vector systems and host cells.

[0188] AAV-based recombinant vectors lacking the Rep protein are integrated into the host genome with low efficiency and mainly exist as stable circular episomes, which can persist for years within target cells. Instead of using AAV natural serotypes, artificial AAV serotypes, including but not limited to those having non-naturally occurring capsid proteins, can be used in connection with the present invention. Such artificial capsids can be generated by any suitable technique using a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) in combination with a heterologous sequence obtainable from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, a non-AAV viral source, or a non-viral source. Artificial AAV serotypes can be, but are not limited to, chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids.

[0189] In the context of the present invention, the AAV vector comprises an AAV capsid capable of transducing a target cell of interest, i.e., a cell of an immunotolerogenic tissue (e.g., hepatocytes), and a cell of a tissue of therapeutic interest, such as a muscle cell, a CNS cell or a heart cell. In certain embodiments, the AAV vector comprises an AAV capsid capable of transducing a muscle cell or a heart cell. According to certain embodiments, the AAV vector is of AAV-1, -2, AAV-2 variant (e.g., the quadruple mutant capsid-optimized AAV-2 comprising an engineered capsid with the Y44+500+730F+T491V changes, disclosed in Ling et al., July 18, 2016, Hum Gene Ther Methods. [Epub ahead of print]), -3 and AAV-3 variant (e.g., the AAV3-ST variant comprising an engineered AAV3 capsid with the two amino acid changes S663V+T492V, disclosed in Vercauteren et al., 2016, Mol. Ther. 24(6), 1042), -3B and AAV-3B variant, -4, -5, -6 and AAV-6 variant (e.g., the AAV6 variant comprising the triple mutant AAV6 capsid Y731F / Y705F / T492V form, disclosed in Rosario et al., 2016, Mol Ther Methods Clin Dev. 3, 16026), -7, -8, -9, -9P1, -2G9, -10, e.g., cy10 and -rh10, -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP, AAV2i8, porcine AAV, e.g., AAVpo1 (as described in WO2021 / 219762), AAVpo4 and AAVpo6, and of tyrosine, lysine and serine capsid variants of AAV serotypes. In certain embodiments, the AAV vector is of the AAV6, AAV8, AAV9, AAV9P1, AAVrh74 or AAV2i8 serotype (i.e., the AAV vector has a capsid of the AAV6, AAV8, AAV9, AAV9P1, AAVrh74 or AAV2i8 serotype). In a further particular embodiment, the AAV vector is a pseudotyped vector, i.e., its genome and capsid are derived from different serotypes of AAV.For example, a pseudotyped AAV vector can be a vector in which the genome is derived from one of the above-described AAV serotypes and the capsid is derived from a different serotype. For example, the genome of a pseudotyped vector may have a capsid derived from the AAV6, AAV8, AAV9, AAV9P1, AAVrh74, or AAV2i8 serotype, and its genome may be derived from a different serotype. In certain embodiments, the AAV vector has a capsid of the AAV6, AAV8, AAV9, or AAVrh74 serotype, particularly of the AAV6, AAV8, AAV9, or AAV9P1 serotype, more particularly of the AAV6, AAV9, or AAV9P1 serotype.

[0190] In certain embodiments where the vector is for use in delivering a therapeutic transgene to muscle cells, the AAV vector can be selected, among others, from the group consisting of AAV8, AAV9, and AAVrh74.

[0191] In another certain embodiment where the vector is for use in delivering a transgene to liver cells, the AAV vector can be selected, among others, from the group consisting of AAV1, AAV5, AAV8, AAV9, AAVrh10, AAVrh39, AAVrh43, AAVrh74, AAV-LK03, AAV2G9, AAV.PHP, AAV-Anc80, and AAV3B.

[0192] In a further certain embodiment where the vector is for use in delivering a transgene to the CNS, the AAV vector can be selected, among others, from the group consisting of AAV9, AAV9P1, AAV10, and AAV2G9.

[0193] In another embodiment, the capsid is a modified capsid. For the purposes of the present invention, a "modified capsid" can 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.

[0194] In certain embodiments, the AAV vector is a chimeric vector, i.e., its capsid contains VP capsid proteins derived from at least two different AAV serotypes, or contains at least one chimeric VP protein having VP protein regions or domains derived from at least two AAV serotypes. Examples of such chimeric AAV vectors useful for transducing liver cells are described in Shen et al., Molecular Therapy, 2007 and Tenney et al., Virology, 2014. For example, the chimeric AAV vector can be derived from a combination of an AAV8 capsid sequence and a sequence of an AAV serotype different from the AAV8 serotype, such as any of those specifically described above. In another embodiment, the capsid of the AAV vector contains one or more variant VP capsid proteins that exhibit high liver affinity, such as those described in WO2015013313, in particular, the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6 capsid variants.

[0195] In another embodiment, the modified capsid can be obtained from capsid modifications inserted by error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al., 2011). In certain embodiments, the capsid comprises a P1 insertion, such as that described in WO2019 / 193119 or WO2020 / 200499 or WO2022 / 053630. Additionally, the capsid variant can include a single amino acid change, such as a tyrosine variant (e.g., as described in Zhong et al., 2008). In certain embodiments, the vector is AAV9rh74 comprising a P1 insertion (e.g., as described in Sellier, P et al., "Muscle-specific, liver-detargeted adeno-associated virus gene therapy rescues Pompe phenotype in adult and neonate Gaa- / - mice.", Journal of inherited metabolic disease, 10.1002 / jimd.12625., May 19, 2023). In another specific embodiment, the vector is a P1-displaying AAV9 variant called "AAVMYO" as described in Weinmann et al. (Weinmann, Jonas et al., "Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants.", Nature communications 11, 1 5432., October 28, 2020).

[0196] In certain embodiments, the AAV vector is an AAV vector such as that described in WO2019 / 193119 or an AAV vector such as that described in WO2020 / 200499.

[0197] In a further embodiment, the AAV vector is an AAV vector as described in WO2020 / 216861 or an AAV vector as described in WO2022 / 003211. In particular, the AAV vector may have a hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, for example, a hybrid capsid of AAV8 and AAV2 / 13.

[0198] In addition, the genome of the AAV vector can be either a single-stranded genome or a self-complementary double-stranded genome (McCarty et al., Gene Therapy, 2003). Self-complementary double-stranded AAV vectors are generated by deleting the terminal resolution site from one of the AAV terminal repeats. These modified vectors, whose replicating genomes are half the length of the wild-type AAV genome, tend to package DNA dimers. In a preferred embodiment, the AAV vector incorporated into the practice of the present invention has a single-stranded genome, and more preferably, is AAV8, AAV9, AAVrh74, AAVrh74-P1, AAV9rh74-P1, AAV2i8 capsid, or a hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, in particular, AAV8, AAV9, AAV9rh74-P1 or a hybrid capsid as described in WO2020 / 216861 or WO2022 / 003211, and more particularly, the AAV9 capsid.

[0199] The AAV vector used to package the GDE sequence of the present invention can also be modified to increase its cargo 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 genome encapsulated in the capsid (Grieger et al., 2005).

[0200] Additional suitable sequences, as known in the art, can be introduced into the nucleic acid construct of the present invention to obtain a functional viral vector. Suitable sequences include AAV ITRs.

[0201] In certain embodiments, the AAV vector comprises the muscle-specific promoter described above, particularly a muscle-specific promoter that results in some leakage of expression into liver cells.

[0202] In another particular embodiment of the invention, the AAV vector comprises the liver-specific promoter described above. The immune tolerance-inducing and metabolic properties of the liver are advantageously incorporated to develop an optimized vector for expressing GDE in hepatocytes and for inducing immune tolerance to the protein, due to this embodiment.

[0203] In certain embodiments, a dual recombinant AAV vector system comprising two AAV vectors as described in WO 2018 / 162748 is used to deliver a nucleic acid molecule or construct encoding a functional truncated GDE polypeptide as defined above. In particular, the dual AAV vector system comprises - a first AAV vector comprising, between the 5' AAV ITR and the 3' AAV ITR, a first nucleic acid sequence encoding the N-terminal portion of the truncated GDE polypeptide as defined above, and - a second AAV vector comprising, between the 5' AAV ITR and the 3' AAV ITR, a second nucleic acid sequence encoding a portion of the truncated GDE polypeptide as defined above and the first and second nucleic acid sequences encoding the GDE comprise a polynucleotide region that allows for the production of the full-length truncated GDE polypeptide as defined above.

[0204] In another particular embodiment, the AAV vector is a single AAV vector comprising a nucleic acid molecule or construct encoding a functional truncated GDE polypeptide as defined above.

[0205] 5 - Cells The present invention also relates to cells transformed or transfected by the nucleic acid molecules, constructs or vectors of the present invention, particularly isolated cells, such as liver cells, heart cells, CNS cells or muscle cells. In certain embodiments, the cells are isolated human cells. In further particular embodiments, the cells are not human embryonic stem cells. The cells of the present invention express the functional truncated GDE polypeptide described above. The cells of the present invention can be delivered to a subject in need thereof, such as a patient lacking GDE, by any suitable route of administration, such as by injection into the liver, CNS, heart, muscle or bloodstream of the subject. In certain embodiments, the present invention comprises transfecting liver or muscle cells, particularly the liver or muscle cells of the subject to be treated, and administering the transfected liver and / or muscle cells into which the nucleic acid has been introduced to the subject. In certain embodiments, the liver cells are liver cells from the patient to be treated or liver stem cells that have been further transformed and differentiated into liver cells in vitro for subsequent administration to the patient. In another embodiment, the cells are muscle cells from the patient to be treated or muscle stem cells that have been further transformed and differentiated into muscle cells in vitro as needed for subsequent administration to the patient.

[0206] 6 - Pharmaceutical Composition The present invention also provides a pharmaceutical composition comprising a nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, or cell of the present invention. Such a composition may comprise a therapeutically effective amount of a therapeutic agent (the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, or cell of the present invention) and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency for use in animals and humans or listed in the U.S. or European Pharmacopeia or other generally recognized pharmacopeias. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic agent is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, as well as aqueous dextrose and glycerol solutions, can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like.

[0207] The composition may also contain small amounts of wetting or emulsifying agents, or pH buffers, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic agent, preferably in a purified form, together with a suitable amount of carrier for the proper administration to the subject. In certain embodiments, the nucleic acid, vector or cell of the invention is formulated into a composition comprising phosphate buffered saline supplemented with 0.25% human serum albumin. In another specific embodiment, the nucleic acid, vector or cell of the invention is formulated into a composition comprising lactated Ringer's solution and a nonionic surfactant, such as Pluronic® F68, at a final concentration of 0.01 - 0.0001% by weight of the total composition, for example, at a concentration of 0.001% by weight. The formulation may further contain serum albumin, particularly human serum albumin, for example, 0.25% human serum albumin. Other suitable formulations for either storage or administration are known in the art, particularly from WO 2005 / 118792 or Allay et al., 2011.

[0208] In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, a composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also contain solubilizing agents and local anesthetics such as lidocaine to relieve the pain at the injection site.

[0209] In certain embodiments, the nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides 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, functional truncated GDE polypeptides or cells of the present invention can be delivered in a controlled release system.

[0210] In certain embodiments, the nucleic acid molecule is delivered as an mRNA corresponding to a transcript encoding the functional truncated GDE polypeptide of the present invention. Specifically, the mRNA of the present invention can be delivered using liposomes, such as lipid nanoparticles (LNPs).

[0211] Methods of administering the nucleic acid molecules, nucleic acid constructs, vectors, functional truncated polypeptides or cells of the present invention include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural and oral routes. In certain embodiments, administration is by intravenous or intramuscular route. The nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides or cells of the present invention, whether vectorized or not, can be administered by any convenient route, such as by injection or bolus injection, by absorption from epithelial or mucosal skin layers (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local administration.

[0212] In certain embodiments, it may be desirable to locally administer the pharmaceutical composition of the present invention to an area in need of treatment, such as the liver or muscle. This can be achieved, for example, by an implant system, said implant system being of a porous, non-porous or gelatinous material containing a membrane, such as a sialastic membrane, or fibers.

[0213] In certain embodiments, the functional truncated GDE polypeptides of the invention are used in enzyme replacement therapy (ERT), particularly for treating GSDIII. The term "enzyme replacement therapy" or "ERT" generally refers to the introduction of a purified enzyme into an individual lacking such enzyme. The polypeptides administered in the present invention can be obtained from natural sources, by recombinant expression, produced in vitro, or purified from isolated tissues or fluids. In particular, when used in ERT, the polypeptides of the invention can be administered parenterally, for example, by intraperitoneal, intramuscular, intravascular (i.e., intravenous or intraarterial) administration. In particular, the polypeptide is administered by intravenous injection. Said administration can be repeated frequently, for example, daily, weekly, biweekly or monthly, particularly weekly or biweekly.

[0214] The amount of the therapeutic agent of the invention (i.e., the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide or cell of the invention) that would be effective in the treatment of GSDIII can be administered by standard clinical techniques. In addition, in vivo and / or in vitro assays can be utilized as needed to aid in predicting the optimal dosage range. The exact dosage to be utilized in the formulation will also depend on the route of administration and the severity of the disease and should be determined according to the judgment of the practitioner and the circumstances of each patient. The dosage of the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide or cell of the invention administered to a subject in need thereof will vary depending on several factors including, but not limited to, the route of administration, the particular disease being treated, the age of the subject, or the expression levels required to achieve a therapeutic effect. One of ordinary skill in the art can readily determine the required dosage range based on such factors and their knowledge in the art. In the case of a treatment involving administration of a viral vector such as an AAV vector to a subject, a typical dosage of the vector is at least 1×10 8 vector genomes (vg / kg), for example, at least 1×10 9 vg / kg, at least 1×1010 vg / kg, at least 1×10 11 vg / kg, at least 1×10 12 vg / kg, at least 1×10 13 vg / kg, or at least 1×10 14 vg / kg.

[0215] 7 - Treatment method The present invention also relates to a method for treating GSDIII, which comprises the step of delivering a therapeutically effective amount of the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, pharmaceutical composition or cell of the present invention to a subject in need thereof.

[0216] Cirrhosis and hepatocellular carcinoma may also develop in patients with GSDIII. Therefore, the present invention also relates to a method for treating cirrhosis and hepatocellular carcinoma in GSDIII patients, which comprises the step of delivering a therapeutically effective amount of the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, pharmaceutical composition or cell of the present invention to a subject in need thereof.

[0217] The present invention relates to a method for treating GSDIII, which method does not induce an immune response against the transgene (i.e., against the functional truncated GDE polypeptide encoded by the nucleic acid molecule) or induces a reduced immune response against the transgene, and which method comprises delivering a therapeutically effective amount of the nucleic acid, vector, functional truncated GDE polypeptide, pharmaceutical composition or cell of the present invention to a subject in need thereof. The present invention also relates to a method for treating GSDIII, which method comprises repeated administration of a therapeutically effective amount of the nucleic acid, vector, functional truncated GDE polypeptide, pharmaceutical composition or cell of the present invention to a subject in need thereof. In this aspect, the nucleic acid molecule, nucleic acid construct or vector of the present invention comprises a promoter that functions in liver cells, thereby enabling immune tolerance to the expressed functional truncated GDE polypeptide produced from those cells. Also in this aspect, the pharmaceutical composition used in this aspect comprises a nucleic acid molecule, nucleic acid construct or vector comprising a promoter that functions in liver cells. In the case of delivery of cells, particularly liver cells, heart cells, CNS cells or muscle cells, said cells are cells previously collected from a subject in need of treatment and are engineered by introducing the nucleic acid molecule, nucleic acid construct or vector of the present invention therein, whereby they can be made capable of producing a functional truncated GDE polypeptide. According to certain embodiments, in the aspect comprising repeated administration, said administration may be repeated at least once or a plurality of times and may further be carried out according to a regular schedule such as once a week, once a month or once a year. A regular schedule may also include administration once every 2, 3, 4, 5, 6, 7, 8, 9 or 10 years or for a number of years exceeding 10 years. In another specific embodiment, for each administration of the viral vector of the present invention, the administration is carried out using a different virus for each successive administration due to the immune response that may occur against the previously administered viral vector, thereby avoiding a decrease in effectiveness. For example, a first administration of an AAV vector comprising an AAV8 capsid may be followed by an administration of a vector comprising an AAV9 capsid.

[0218] According to the present invention, the treatment may include a curative, palliative or prophylactic effect. Thus, therapeutic and prophylactic treatments include alleviating the symptoms of GSDIII, or preventing or otherwise reducing the risk of onset of a particular glycogenosis. The term "prophylactic" can be considered to reduce the severity or manifestation of a particular condition. "Prophylactic" also includes preventing recurrence in a patient previously diagnosed with that condition. "Therapeutic" can also reduce the severity of an existing condition. The term "treatment" is used herein to refer to any regimen that can provide a benefit to an animal, particularly a mammal, more particularly a human subject.

[0219] The present invention also relates to an ex vivo gene therapy method for the treatment of GSDIII, comprising the steps of introducing the nucleic acid molecule, nucleic acid construct or vector of the present invention into isolated cells of a patient in need thereof, such as isolated hematopoietic stem cells, and introducing said cells into said patient in need thereof.

[0220] The present invention also relates to the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, cell or pharmaceutical composition of the present invention for use as a medicament.

[0221] The present invention also relates to the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, cell or pharmaceutical composition of the present invention for use in a method for treating a disease caused by a mutation in the AGL gene encoding GDE, particularly for treating GSDIII (Cori disease), such as GSDIIIa and GSDIIIb, particularly GSDIIIa.

[0222] The present invention further relates to the use of the nucleic acid molecule, nucleic acid construct, vector, functional truncated GDE polypeptide, cell or pharmaceutical composition of the present invention in the manufacture of a medicament useful for the treatment of GSDIII (Cori disease).

[0223] The present invention also relates to the nucleic acid molecules, nucleic acid constructs, vectors, functional truncated GDE polypeptides, cells or pharmaceutical compositions of the present invention for use in a method for delivering a GDE protein to diseased tissues, particularly muscle and liver tissues, particularly muscle.

Examples

[0224] The present invention will be described in more detail by further referring to the following experimental examples and the accompanying drawings. These examples are provided for illustrative purposes only and are not intended to be limiting.

[0225] In patent applications WO2020 / 030661 and PCT / EP2021 / 073309, it was shown that it is possible to generate GDE proteins in a shortened form while retaining enzymatic activity. In particular, it was shown that the truncated proteins "Δ1b2" and "Δ1b3" efficiently reduce the amount of glycogen in different muscle tissues. "Δ1b2" corresponds to the GDE polypeptide of SEQ ID NO: 1 with amino acids 2-81 deleted. "Δ1b3" corresponds to the GDE polypeptide of SEQ ID NO: 1 with amino acids 2-103 deleted. However, both proteins were shown to be expressed less or have lower stability in vivo than full-size GDE. Therefore, the aim was to modify the N-terminal truncation site in order to find a truncated protein that is more stable and better expressed, and ultimately to increase the ability of the protein to reduce glycogen in vivo.

[0226] Five N-terminal truncated GDE proteins were designed as described in the following table:

[0227]

Table 3

[0228] To evaluate the activity of the truncated GDE, AAV vectors expressing the truncated GDE (Δ1b5 or Δ1b3) or the full-size protein (AAV-GDE-full-size) were produced. The AAV9rh74-P1 vector (i.e., an AAV vector having a hybrid capsid of AAV9 and AAVrh74 modified with the peptide P1) was used throughout the experiment. The transgene was cloned into a transgene expression cassette optimized for muscle expression and composed of a miniCMV promoter and a pA58 polyadenylation signal. The cassettes of the Δ1b5, Δ1b3, and GDE-full-size constructs are as shown by SEQ ID NO: 45, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.

[0229] (Example 1) The vector was injected into the left anterior tibialis muscle of 4- to 6-month-old GSDIII mice at a dose of 3×10 11 vg / mouse (Figure 1). Wild-type (WT, PBS) or knockout mice (KO, PBS) injected with PBS were used as controls. The mice were sacrificed 1 month after injection to measure the glycogen content in the left anterior tibialis muscle.

[0230] Measurement of glycogen content The glycogen content was measured indirectly as glucose released after complete digestion by Aspergillus Niger amyloglucosidase (Sigma-Aldrich, ref A1602) in tissue homogenates. Samples were incubated at 95 °C for 10 min and then cooled to 4 °C. After addition of amyloglucosidase (final concentration 4 U / mL) and potassium acetate pH 5.5 (final concentration 25 mM) at 37 °C for 90 min, the reaction was stopped by incubating the samples at 95 °C for 10 min. A control reaction without amyloglucosidase was prepared for each sample and incubated under the same conditions. The released glucose was determined using a glucose assay kit (Sigma-Aldrich) and the resulting absorbance was obtained at a wavelength of 540 nm using an EnSpire alpha plate reader (PerkinElmer). The glucose released after amyloglucosidase was then normalized by the total protein concentration.

[0231] Measurement of GDE expression Mouse tissues were homogenized in phosphate-buffered saline (PBS, ThermoFisher Scientific) containing cOmplete™ protease inhibitor cocktail (Roche, ref 4693132001). Protein concentration was determined using Pierce™ BCA Protein Assay (Thermo Fisher Scientific) according to the manufacturer's instructions. A 50 μg fraction of total protein for both Agl− / − mice injected with PBS and Agl− / − mice injected with rAAV, and 10 μg of total protein for Agl+ / + mice injected with PBS were loaded into each well. SDS-PAGE electrophoresis was performed on a 4–12% Bis-Tris gradient polyacrylamide gel (NuPAGE™, Invitrogen). 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-vinculin mouse monoclonal antibody (V9131, Sigma). The membrane was washed and incubated with the appropriate secondary antibody (LI-COR Biosciences) and visualized by an Odyssey imaging system (LI-COR Biosciences).

[0232] Quantification of vector genome copy number. DNA was extracted from tissue homogenates using the Nucleomag Pathogen extraction kit (#744210.4) from Macherey-Nagel according to the manufacturer's instructions. The vector genomic copy number was determined using a qPCR assay. The PCR primers used in the reaction were located in the glucosyltransferase domain of the full-length and truncated codon-optimized GDE (SEQ-33; forward: 5'-CTG AAG CTG TGG GAG TTC TT-3' (SEQ ID NO: 59) and reverse: 5'-CTC TTG GTC ACT CTT CTG TTC TC-3' (SEQ ID NO: 60)). As an internal control, primers located within the mouse titin gene (forward: 5'-AAA ACG AGC AGT GAC GTG AGC-3' (SEQ ID NO: 61) and reverse: 5'-TTC AGT CAT GCT GCT AGC GC-3' (SEQ ID NO: 62)) were used.

[0233] Results The results show a significant decrease in glycogen content in mice injected with the truncated GDE construct compared to the control group injected with PBS. The GDE proteins "Δ1b3", "Δ1b5" and the full-length form of GDE (GDE full size) showed significant efficacy in terms of glycogen depletion in the injected muscle (Figure 2).

[0234] The Δ1b5 GDE construct shows much better protein expression compared to the Δ1b3 GDE construct even though the truncation sites are very close (Figure 1) (Figures 3 and 4). This experiment shows that it is impossible to predict the expression level of a new truncated protein based solely on the position of the truncation site.

[0235] Furthermore, quantification of the vector genomic copy number (VGCN) in the injected muscle showed less VGCN in the tibialis anterior muscle injected with the Δ1b5 GDE construct compared to the Δ1b3 GDE construct (Figure 5). This emphasizes that the higher expression of the Δ1b5 GDE construct observed in Figures 3 - 4 is not due to a larger amount of the injected rAAV vector.

[0236] (Example 2) The efficacy of rAAV vectors encoding Δ1b3-GDE (Figure 6) or Δ1b5-GDE (Figure 7) was evaluated in a mouse model of GSDIII. Four-month-old male Agl - / - mice were injected via the tail vein with an rAAV vector encoding either Δ1b3-GDE (Figure 6) or Δ1b5-GDE (Figure 7) at a dose of 1×10 14 vg / kg. Agl + / + and Agl - / - mice injected with PBS were used as controls. As described above, glycogen content in the heart and triceps muscle was measured at the time of euthanasia. A wire hang test, expressed as the number of drops per minute, was performed 3 months after vector injection. Using a wire with a thickness of 4 mm, the number of drops was recorded over a period of 3 minutes. The average number of drops per minute was reported for each animal.

[0237] Results The results show a significant decrease in glycogen content in the heart and triceps muscle in mice injected with the truncated Δ1b3-GDE construct (Figure 6B) and the Δ1b5-GDE construct (Figure 7B) compared to the control group injected with PBS. The Δ1b5-GDE construct showed better efficacy in terms of glycogen depletion in the heart and triceps muscle when compared to the Δ1b3-GDE construct.

[0238] In addition, treatment with rAAV-Δ1b3-GDE and rAAV-Δ1b5-GDE improved muscle strength as evaluated by wire hang. Three months after injection, the mice showed a lower drop frequency compared to the group injected with PBS (as shown in Figures 6C and 7C).

[0239] Importantly, treatment with rAAV-Δ1b5-GDE is shown to be far more efficient than rAAV-Δ1b3-GDE in restoring muscle strength. Three months after injection, mice injected with rAAV-Δ1b5-GDE showed a lower drop frequency (9.8 drops / min ± 1.8 vs. 31.4 drops / min ± 1.4 in the PBS-injected group, corresponding to a 69% decrease) compared to the PBS-injected group and thus almost reached wild-type levels (Figure 7C). In contrast, mice injected with rAAV-Δ1b3-GDE showed approximately 17 drops / min ± 1.9 three months after injection (vs. 23.8 ± 3.9 in the PBS-injected group, corresponding to only a 29% decrease).

[0240] These data clearly demonstrate that treatment with rAAV-Δ1b5-GDE drives muscle damage towards recovery at both the biochemical and functional levels in mice showing symptoms of adult GSDIII.

[0241] (Example 3) To confirm the efficacy of the rAAV-Δ1b5-GDE construct in a larger animal model, the vector was administered to a rat model of GSDIII. Six-week-old Agl rats treated with 1×10 14 vg / kg of rAAV-Δ1b5-GDE by tail vein injection were analyzed three months after injection (Figure 8A). Agl rats injected with PBS and Agl rats were used as controls. As described above, glycogen content in the heart and triceps muscle was measured at euthanasia. - / - rats injected with PBS and Agl - / - and Agl + / + rats were used as controls. As described above, glycogen content in the heart and triceps muscle was measured at euthanasia.

[0242] Histological analysis of the heart and triceps muscle was performed using HPS (hematoxylin-phloxin-saffron staining) and PAS (periodic acid Schiff staining). For muscle histological examination, the heart and triceps muscle were snap-frozen in pre-cooled isopentane in liquid nitrogen. Serial 8-μm sections were cut with a Leica CM3050 S cryostat (Leica Biosystems). To minimize sampling error, two sections of each specimen were obtained and stained with HPS or PAS according to standard procedures. Images were digitized using an Axioscan Z1 slide scanner (Zeiss, Germany) under a Zeiss Plan-Apochromat 10X / 0.45 M27 dry objective lens (ZEISS, Germany). The tiled scan images were reconstructed by ZEN software (ZEISS, Germany).

[0243] Results A significant 50% reduction in glycogen accumulation was observed 3 months after injection in the heart of rats injected with rAAV-Δ1b5-GDE (Figure 8B), which was also prominent in PAS staining (Figure 8C). Almost complete glycogen elimination was also observed in skeletal muscle, such as the triceps muscle (Figure 8D), along with normalization of muscle structure and reduction of glycogen accumulation in histological analysis (Figure 8E).

[0244] As a conclusion, Agl by rAAV vector encoding Δ1b5-GDE - / - The data obtained from the treatment of rats confirm those obtained in the mouse model of the disease and further support the clinical interpretation of rAAV-Δ1b5-GDE for treating muscle diseases in GSDIII patients.

[0245] (Example 4) The activity of the truncated Δ1b5-GDE was also evaluated regarding the human pathology using an in vitro human skeletal muscle model of GSDIII. Skeletal myoblasts and myotubes were derived from human induced pluripotent stem cells (hiPSCs) edited to knockdown the AGL gene by CRISPR / Cas9 technology (GSDIII CRISPR )

[0246] Transduction of hiPSC-derived skeletal muscle cells. GSDIII CRISPR hiPSCs were previously generated using CRISPR-mediated knockdown of the AGL gene (Rossiaud et al., Pathological modeling of Glycogen Storage Disease type III with CRISPR / Cas9 edited human pluripotent stem cells, Front. Cell Dev. Biol., May 11, 2023). Control hiPSCs were isogenic cell lines (CTRL1). GSDIII CRISPR Both GSDIII and CTRL1 hiPSCs were differentiated into skeletal myoblasts as previously described (Rossiaud et al.). After expansion in 96-well plates, hiPSC-derived skeletal myoblasts were transduced for 72 hours at either 75,000 or 15,000 MOI with an rAAV vector encoding either GFP or Δ1b5-GDE under the control of the miniCMV promoter. Subsequently, hiPSC-derived skeletal myoblasts were differentiated into myotubes as previously described (Rossiaud et al.).

[0247] Measurement of glycogen content in skeletal myotubes. After 4 days of differentiation into myotubes, hiPSC-derived skeletal myotubes were starved for 3 days in glucose-free DMEM medium containing 10% fetal bovine serum (ref 11966025, ThermoFisher Scientific) to induce glycogenolysis in CTRL1 myotubes as previously described (Rossiaud et al.). Cells were lysed using 0.3 M HCl and 450 mM Tris pH 8.0. Glycogen was then quantified using the Glycogen-Glo™ assay kit (ref CS1823B01, Promega) and normalized using the CellTiter-Glo™ Luminescent Cell Viability Assay (ref G7570, Promega).

[0248] Immunostaining assay. Skeletal muscle tubes derived from hiPSCs were fixed with 4% paraformaldehyde (Euromedex) for 10 minutes at room temperature. After two washes with PBS, the cells were permeabilized with 0.5% Triton X-100 for 5 minutes and blocked in PBS solution supplemented with 1% bovine serum albumin (BSA, Sigma) for 1 hour at room temperature. The skeletal muscle tubes were stained for specific skeletal myogenic markers overnight at 4°C using primary antibodies (desmin, ref AF3844, R&D, 1:200; MF20, ref 3ea, DSHB, 1:50; titin, ref T5650, US Biological, 1:50). After three washes with PBS, the staining was developed with appropriate Alexa Fluor secondary antibodies 1:1000 (donkey anti-goat AF488, ref A11055, Invitrogen, 1:1000; donkey anti-mouse AF488, ref A21202, Invitrogen, 1:1000) for 1 hour at room temperature in the dark, and the nuclei were visualized with Hoechst solution 1:3000 (Invitrogen). Cell imaging was performed using an LSM-800 confocal microscope (Zeiss) equipped with Zen Black software and a 20X objective lens.

[0249] Periodic acid Schiff (PAS) staining for skeletal muscle tubes. PAS staining of hiPSC-derived skeletal muscle tubes was performed using a PAS Staining Kit (Sigma-Aldrich) according to the manufacturer's instructions. Briefly, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. After two washes with PBS, cells were treated with periodic acid for 5 minutes at room temperature. After three washes with distilled water, cells were treated with Schiff reagent for 15 minutes at room temperature. Finally, after four washes with tap water, staining was visualized using an EVOS XL Core microscope (Invitrogen). Images were processed and analyzed using a custom FIJI script. First, the colors were separated, and only the green channel was retained as it had the strongest contrast. Then, the image was manually binarized to a binary image where the PAS signal was black and the background was white. A threshold was set and calculated to maximize the difference between genotypes, and then that threshold was applied to all images for quantification. Quantification of PAS staining was achieved using the following formula: PAS staining area / total area of the image × 100, resulting in the percentage of PAS staining within the image.

[0250] Results GSDIII CRISPR GSDIII and CTRL1 hiPSC-derived skeletal myoblasts were transduced with rAAV vectors expressing Δ1b5-GDE or GFP according to a previously reported protocol (Rossiaud et al.) (Figure 9A). After transduction with rAAV, skeletal myoblasts were differentiated into skeletal muscle tubes. Transduction with rAAV expressing GFP or Δ1b5-GDE did not alter the differentiation of myoblasts into myotubes, which showed similar expression of skeletal myogenic markers by immunostaining analysis (Figure 9B). GSDIII skeletal muscle tubes transduced with rAAV expressing Δ1b5-GDE CRISPR showed a significant reduction in glycogen content compared to GSDIII CRISPR myotubes transduced with the control vector (Figure 9C). PAS staining performed on the transduced skeletal muscle tubes confirmed these results (Figures 9D - 9E).

[0251] These data demonstrate that the truncated Δ1b5-GDE eliminates glycogen not only in mouse and rat muscle but also in the human skeletal muscle model of GSDIII, without any associated specific toxicity.

[0252] (Example 5) Large-scale rAAV vector production has a major impact on the yield and quality of the final product, which is a crucial parameter for transferring rAAV-based gene therapy to the clinic. To evaluate the effect of the use of truncated Δ1b5-GDE on vector production yield and quality, a large expression cassette expressing human full-length GDE (5.3 kb) was compared with a 5 kb expression cassette encoding truncated Δ1b5-GDE (Figure 10). Small-scale rAAV production (50 mL culture) was performed in triplicate for each vector, and viral titers were measured both before purification (prototype) and after purification (final product) for each triplicate. rAAV vector DNA was extracted and loaded onto a 1% agarose gel to evaluate genomic integrity. Analytical ultracentrifugation (AUC) was performed to analyze the ratio of full particles to empty particles.

[0253] Viral genome analysis on agarose gel. DNA was extracted using the High Pure Viral Nucleic Acid Kit (Roche, ref 11858874001). The purified viral DNA was then loaded onto a 1% agarose gel (Eurobio Scientific) and stained with SybrSafe® Gel Stain (Invitrogen) to visualize the viral DNA. The expected genomic size ranges from 5.0 to 5.3 kb.

[0254] Analytical ultracentrifugation (AUC). The AUC measures the sedimentation coefficient of macromolecules by tracking the optical density of a sample subjected to ultracentrifugation over time. The difference in sedimentation coefficient measured by Raleigh interference or absorbance at 260 nm is due to the content of the viral genome within the capsid. AUC analysis was performed using a Proteome Lab XL-I (Beckman Coulter, Indianapolis, Indiana). Aliquots of 400 μL of the rAAV vector and 400 μL of formulation buffer were loaded into a 2-sector velocity cell. Sedimentation velocity centrifugation was performed at 20,000 rpm and 20 °C. Using absorbance (260 nm) and Raleigh interference optics, the radial concentration was simultaneously recorded as a function of time until the lightest sedimenting component cleared the optical window (approximately 1.5 hours). Absorbance data required the use of extinction coefficients to calculate the molar concentrations and percentage values of empty capsids and genome-containing capsids. The molar concentrations of both genome-containing capsids and empty capsids were calculated using Beer's law, and the percentages of full-genome-containing capsids and empty capsids were calculated.

[0255] Results The use of Δ1b5-GDE cDNA enabled an approximately 10-fold increase in rAAV production yield (Figure 10B), and moreover, enabled efficient encapsulation of the non-truncated genome into capsids (Figure 10C). The improvement in yield and genome quality resulted in a dramatic improvement in the ratio of full particles to empty particles as measured by analytical ultracentrifugation (AUC), with full particles measured in AAV-Δ1b5-GDE being up to 37% compared to only 15% of full particles measured in AAV full-length GDE (Figure 10D).

[0256] Collectively, these data indicate that Δ1b5-GDE has in vivo efficacy similar to that of the full-size enzyme, while enabling high-yield and high-quality production of rAAV vectors, and thus represents a promising gene therapy candidate for the treatment of GSDIII.

Claims

1. A functional truncated GDE polypeptide comprising a deletion relative to a reference functional full-length human GDE sequence, wherein the deletion is the first six amino acids of the N-terminus of the functional truncated GDE polypeptide. - MGSFQY (Sequence ID 7); - MEKSGG (sequence number 8); - MILRVG (sequence number 9); - MGADNH (sequence number 10); or - MLDCVT (Sequence ID 11) A functionally shortened GDE polypeptide, comprising the deletion of an amino acid at the N-terminus of the aforementioned reference functional full-length human GDE sequence, in such a manner.

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

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

6.

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

1.

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

6. A functional truncated GDE polypeptide according to any one of claims 1 to 4, having an amino acid sequence as shown in SEQ ID NOs. 12 to 16, or having an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity with respect to SEQ ID NOs. 12 to 16.

7. A functional truncated GDE polypeptide according to any one of claims 1 to 4, having an amino acid sequence as shown in SEQ ID NO: 13 or SEQ ID NO: 14, preferably SEQ ID NO: 13, or having an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or at least 99 percent sequence identity with SEQ ID NO: 13 or SEQ ID NO: 14, preferably SEQ ID NO:

13.

8. A nucleic acid molecule encoding a functionally truncated GDE polypeptide as described in claim 1.

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

10. A vector comprising a nucleic acid molecule as described in claim 8, particularly a viral vector.

11. A vector comprising the expression cassette described in claim 9, more particularly a viral vector.

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

13. Isolated cells transformed with the nucleic acid molecule described in claim 8, wherein the cells are, in particular, liver cells, muscle cells, cardiac cells, or CNS cells.

14. A pharmaceutical composition comprising a functionally truncated GDE polypeptide according to any one of claims 1 to 4, a nucleic acid molecule according to claim 8, an expression cassette according to claim 9, a vector according to any one of claims 10 to 12, or a cell according to claim 13.

15. A pharmaceutical composition for use in a method for treating a disease caused by a mutation in the AGL gene encoding GDE, comprising a functional truncated GDE polypeptide according to any one of claims 1 to 4, a nucleic acid molecule according to claim 8, an expression cassette according to claim 9, a vector according to any one of claims 10 to 12, or a cell according to claim 13.

16. A pharmaceutical composition for use in a method for treating GSDIII (German disease of the cochlea), comprising a functionally truncated GDE polypeptide according to any one of claims 1 to 4, a nucleic acid molecule according to claim 8, an expression cassette according to claim 9, a vector according to any one of claims 10 to 12, or cells according to claim 13.

Images