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EP-4735587-A1 - N-TERMINAL TRUNCATED GLYCOGEN DEBRANCHING ENZYMES FOR THE TREATMENT OF GLYCOGEN STORAGE DISEASE III

EP4735587A1EP 4735587 A1EP4735587 A1EP 4735587A1EP-4735587-A1

Abstract

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

Inventors

  • GARDIN, Antoine
  • ROUILLON, Jérémy

Assignees

  • GENETHON
  • Institut National de la Santé et de la Recherche Médicale
  • Université d'Evry Val d' Essonne

Dates

Publication Date
20260506
Application Date
20240628

Claims (15)

  1. 1. A functional truncated GDE polypeptide, wherein said functional truncated GDE polypeptide comprises a deletion with respect to a reference functional full-length human GDE sequence, and wherein said deletion consists of the deletion of amino acids in the N-terminal part of the reference functional full-length human GDE sequence in such a way that the first six amino acids at the N-terminus of the functional truncated GDE polypeptide are: - MQYYFL (SEQ ID NO: 7) ; - MFLQGN (SEQ ID NO: 8) ; - MQGNEK (SEQ ID NO: 9) ; - MGNEKS (SEQ ID NO: 10); - MNEKSG (SEQ ID NO: 11); - MKSGGG (SEQ ID NO: 12); - MSGGGY (SEQ ID NO: 13).
  2. 2. The functional truncated GDE polypeptide of 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 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.
  3. 3. The functional truncated GDE polypeptide of claim 1, wherein the reference functional full-length human GDE has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:4, preferably SEQ ID NO: 1, or has an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 1 or SEQ ID NO:4, preferably to SEQ ID NO: 1.
  4. 4. The functional truncated GDE polypeptide of any one of the preceding claims, wherein the functional truncated GDE polypeptide further comprises a deletion or a combination of deletions with respect to the reference functional full-length human GDE sequence, such as a deletion or a combination of deletions in the C-terminal part of the GDE sequence, or a deletion or a combination of deletions in the central domain of the GDE sequence ; in particular wherein the functional truncated GDE polypeptide further comprises a deletion or a combination of deletions with respect to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, and wherein the deletion(s) is (are) selected from any deletion referred to as Al, A2, A3, A4, A5, A6, and A7 in table 2.
  5. 5. The functional truncated GDE polypeptide of any one of the preceding claims, wherein the functional truncated GDE polypeptide 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: 14-20.
  6. 6. The functional truncated GDE polypeptide of any one of the preceding claims, wherein the functional truncated GDE polypeptide 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: 14, and comprises the sequence of SEQ ID NO:21 ; - an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 15, and comprises the sequence of SEQ ID NO:22 ; - an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 16, and comprises the sequence of SEQ ID NO:23 ; - an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 17, and comprises the sequence of SEQ ID NO:24 ; - an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 18, and comprises the sequence of SEQ ID NO:25 ; - an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO: 19, and comprises the sequence of SEQ ID NO:26 ; or - an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or at least 99 percent sequence identity to SEQ ID NO:20, and comprises the sequence of SEQ ID NO:27.
  7. 7. The functional truncated GDE polypeptide of any one of the preceding claims, wherein the functional truncated GDE polypeptide has an amino acid sequence as shown in SEQ ID NO: 14-20.
  8. 8. A nucleic acid molecule encoding the functional truncated GDE polypeptide of any one of claims 1 to 7.
  9. 9. An expression cassette, comprising, preferably in this order : a promoter; optionally, an intron; the nucleic acid molecule of claim 8; and a polyadenylation signal.
  10. 10. A vector, in particular a viral vector, comprising the nucleic acid molecule of claim 8 or the expression cassette of claim 9.
  11. 11. The vector of claim 10, which is an AAV vector.
  12. 12. An isolated cell transformed with the nucleic acid molecule of claim 8, the expression cassette of claim 9 or the vector of claims 10-11, wherein the cell is in particular a liver cell, a muscle cell, a cardiac cell or CNS cell.
  13. 13. The functional truncated GDE polypeptide of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the expression cassette of claim 9, the vector of claims 10-11, or the cell of claim 12, for use as a medicament.
  14. 14. The functional truncated GDE polypeptide of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the expression cassette of claim 9, the vector of claims 10-11, or the cell of claim 12, for use in a method for treating a disease caused by a mutation in the AGL gene encoding GDE.
  15. 15. The functional truncated GDE polypeptide of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the expression cassette of claim 9, the vector of claims 10-11, or the cell of claim 12, for use in a method for treating GSDIII (Cori disease).

Description

N-TERMINAL TRUNCATED GLYCOGEN DEBRANCHING ENZYMES FOR THE TREATMENT OF GLYCOGEN STORAGE DISEASE III The present invention relates to the treatment of glycogen storage disease III (GSDIII). BACKGROUND OF THE INVENTION Mutations in the AGL gene cause genetic deficiency of glycogen debranching enzyme (GDE), or “amylo-alpha-l,6-glucosidase, 4-alpha-glucanotransferase”, an enzyme involved in glycogen degradation. GDE has two independent catalytic activities which occur at different sites on the protein: a 4-alpha-glucotransferase activity and an amylo-l,6-glucosidase activity. Genetic deficiency of GDE causes an incomplete glycogenolysis in glycogen storage disease III (GSDIII), resulting in accumulation of abnormal glycogen with short outer chain in various organs, mostly liver and muscle. The disease is characterized by hepatomegaly, hypoglycemia, short stature, variable myopathy and cardiomyopathy. Most patients have GSDIII involving both liver and muscle (type Illa), while some patients (~15 percent) have only liver involvement (type Illb). Liver symptoms normally occur in childhood. Liver 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 could be present during childhood. It becomes more prevalent in adults with onset in the third or fourth decade. There is significant morbidity from progressive muscle weakness and patients in later stages can become wheel chair bound. Patients can also develop cardiomyopathy. There is significant clinical variability in the severity of the symptoms that these patients develop. The progressive myopathy and/or cardiomyopathy are major causes of morbidity in adults (Kishnani et al., 2010, Genet Med 12, 446-463; Comelio et al., 1984, Arch Neurol 41, 1027-1032; Coleman et al., 1992, Ann Intern Med 116, 896-900). Reports of possible neurological manifestations associated with the disease derive from clinicians working with GSDIII patients, who reported attention fluctuations, deficiencies in executive functions and impaired emotional skills (Michon et al., 2015, J Inherit Metab Dis, 38(3): 573-580). Accordingly, in the Ag/ /_ mouse model of the disease, an extensive accumulation of glycogen was documented in the liver, skeletal muscles and heart, and to a lesser extent in the central nervous system (Pagliarani et al., 2014, Biochim Biophys Acta, 1842(11): 2318-2328; Liu et al., 2014, Mol Genet Metab, 111(4): 467-476) although a careful characterization of the neuro-cognitive phenotype associated with the accumulation of glycogen is still missing. Current treatment is symptomatic, and there is no effective therapy for the disease. Hypoglycemia can be controlled by frequent meals high in carbohydrates with cornstarch supplements or nocturnal gastric drip feedings, as well as a diet high in protein during the daytime plus overnight enteral infusion. In some patients, transient muscle improvement in symptoms has been documented after the use of a high protein diet, but there are no systemic studies or long-term data demonstrating that it is able to prevent or treat the progressive myopathy (Kishnani et al., 2010, Genet Med 12, 446- 463). These approaches do little to alter the long-term course and morbidity of these diseases. Therefore, there is still a need for a long-term treatment of GSDIII. Gene therapy aiming to stably replace the GDE protein in the affected tissues appears as a potential therapeutic approach. However, the large size of the GDE transgene constitutes a major impediment since it cannot fit in the size limit of most gene therapy vectors. Indeed, the human AGL gene is 85 kb in length and composed of 35 exons, encoding a 7.4-kb mRNA that includes a 4599-bp coding region and a 2371-bp 3' untranslated sequence to express a 175 kDa GDE protein (Bao Y et al., 1996, Genomics., 38(2): 155-65). This constitutes a real issue since the minimum size of a GDE expression cassette (including for example at least a promoter, the GDE coding sequence, a polyA signal and the two ITRs for an AAV vector) would be larger than 5 kb, the genome size limit that can be packaged into an AAV vector used for in vivo gene delivery. The inventors have previously proposed the use of dual AAV vectors to overcome this size limitation (WO2018/162748). Following this approach, two vectors, each containing half of the expression cassette, are used to transduce the same cell. Although the use of dual AAV vectors is promising, it would be preferable to provide a gene therapy strategy implementing only one viral vector for both economic and practical reasons. In patent applications W02020/030661 and W02022/043280, the present inventors described another approach based on the use of a truncated GDE polypeptide that fits in a single viral vector. Yet, alternatives to the truncated GDE polypeptides previously des