WO2021165841A1

WO2021165841A1 - Compound for the treatment of a glycogenosis

Info

Publication number: WO2021165841A1
Application number: PCT/IB2021/051322
Authority: WO
Inventors: Rosella Franconi; Giovanni Giuliano; Carmela MARINO; Olivia Costantina DEMURTAS; Doriana TRIGGIANI
Original assignee: ENEA - Agenzia nazionale per le nuove tecnologie, l'energia e lo sviluppo economico sostenibile; Aig Associazione Italiana Glicogenosi
Priority date: 2020-02-19
Filing date: 2021-02-17
Publication date: 2021-08-26
Also published as: EP4106801A1; IT202000003371A1

Abstract

The present invention relates to a compound selected from a polynucleotide coding for a glycogen debranching enzyme (GDE), a vector and a host cell comprising the polynucleotide. The present invention further relates to the use of the compound for the treatment of a glycogenosis (GSD), preferably GSDIII.

Description

DESCRIPTION

TITLE

COMPOUND FOR THE TREATMENT OF A GLYCOGENOSIS FIELD OF THE INVENTION

The present invention relates to a compound selected from a polynucleotide coding for the glycogen debranching enzyme (GDE), a vector and a host cell genetically engineered so as to express the GDE enzyme. The present invention further relates to the use of the compound for the treatment of a glycogenosis (GSD), preferably GSDIII.

PRIOR ART

Glycogenoses (or glycogen storage diseases, GSD) are a group of at least fifteen rare metabolic diseases due to alterations of enzymes that act at different levels of the metabolic pathway of glycogen degradation. Glycogen represents a storage form of sugars that the body uses according to need to release glucose in order to maintain blood glucose at normal levels during fasting and/or for energy purposes for muscular contraction. An accumulation of glycogen in tissues causes severe alterations, mainly in the liver, in muscles and in other organs.

In particular, glycogenosis type III (GSDIII, or Cori or Forbes disease), is an ultra rare disease, with an estimated prevalence of about ± 1 out of 80,000-100,000 born, due to the absence of the glycogen debranching enzyme (GDE).

GDE is a cytoplasmic monomeric protein with two distinct catalytic activities: it contains a transferase domain and a glucosidase domain. The lack or an alteration of the activity of the GDE enzyme leads to abnormal glycogen with shorter chains (called p hosphorylase-limit dextrin, PLD) which accumulates in skeletal and/or heart muscle and/or in the liver.

The disease manifests itself a few months after birth or during childhood with an enlargement of the liver, hypoglycaemia and retarded growth. The muscles generally weaken starting from the second-third decade of life and, in addition to muscle hypotonia, some affected individuals can develop a hypertrophic cardiomyopathy over time. The symptoms present themselves with a highly variable severity. The disease is caused by mutations of the “AGL” ( amylo-1,6 - glucosidase, 4-alpha-glucanotransferase) gene, which codes for the GDE enzyme, and is transmitted in an autosomal recessive manner: it is necessary that both genes, of paternal and maternal origin, lead to a pathological change in the sequence in order for the disease to manifest itself. The parents are therefore healthy carriers.

There is presently no cure for GSDIII and only a treatment of a dietary type is available. Enzyme replacement therapy (ERT) has recently been developed for various genetic diseases (e.g. lysosomal). For example, for GSDII (Pompe disease or lysosomal glycogen storage disease), a drug based on the enzyme recombinant human lysosomal a-glucosidase (GAA) has existed for a few years. However, the problems associated with the development of ERT, when available, are the high costs of the treatments and the fact that the recombinant enzyme must be active and not immunogenic.

An alternative is represented by gene therapy, in particular non-viral gene therapy, which, among its advantages, shows little immunogenicity. At present there exist two main DNA transfer systems: one based on viral vectors and one on non-viral vectors. At the basis of the various recently approved gene therapies there are vectors based on adeno-associated viruses (AAV), generally considered the safest. In fact, with these vectors, phase I clinical studies have also been conducted on gene therapy for glycogenosis types I and II (GSDI and GSDII), whereas in the case of GSDIII only one preclinical study on a mouse model has been conducted. Said study demonstrated that the acquisition of a functional GDE enzyme effectively restores the “healthy” phenotype. However, since the insertion limit imposed by the AAV vector is about 5 kb, the cDNA of the AGL gene was divided into two parts (with an overlapping region) and it was necessary to introduce a step of a homologous recombination process, which partly nullifies the advantage of the high transfection efficiency of the viral vector.

Although the most recent successes in the treatment of rare diseases due to hereditary genetic effects have been reached using approaches based on gene addition mediated by recombinant viral vectors, the problems associated with these systems (poor safety, poor ability to accommodate exogenous DNA and high costs) are driving towards the development of alternative systems.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a compound selected from: a) a polynucleotide coding for a glycogen debranching enzyme (GDE), a recombinant or synthetic derivative of GDE, a fragment or an allele variant of said GDE; b) a vector comprising said polynucleotide; c) a genetically engineered host cell that expresses said polynucleotide.

A second aspect of the present invention relates to a pharmaceutical composition comprising the compound described above.

A third aspect of the present invention relates to the compound as described above for use as a medicament.

A fourth aspect of the present invention relates to the compound described above or the recombinant GDE enzyme for use in the treatment of a glycogenosis (GSD). Preferably, the GSD is GSDIII (Cori or Forbes disease).

A fifth aspect of the present invention relates to a pharmaceutical composition comprising the compound described above and/or the GDE enzyme, for use in the treatment of a glycogenosis (GSD). The composition is preferably administered in association or in combination with electropermeabilisation of the plasma membrane.

A further aspect of the present invention relates to a method for the treatment of a GSD, preferably GSDIII (Cori or Forbes disease). Said method comprises at least a step of administering an effective amount of the compound and/or of the GDE enzyme according to the present invention or of a composition that comprises the compound and/or GDE enzyme as described above, to a subject (individual) affected or suspected to be affected by a GSD, preferably GSDIII, possibly in combination with electropermeabilisation.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is described below in detail and illustrated with reference to the appended figures, in which:

Figure 1 shows the expression of the GDE protein obtained from the expression of the sAGL (sGDE) gene in different E. coli strains transformed with suitable expression vectors comprising the codon-optimised AGL gene ( sAGL·, SEQ ID NO. 1 ): A. Immunoblotting on total soluble proteins (TSP 10 pg) extracted from bacterial cells BL21 and BL21/Gro7, containing the constructs pQE-30-His6-s/tGZ. or pQE50 -sAGL after induction of expression at 37 °C or 28 °C for 16 hours. B. Immunoblotting on the soluble (S) and insoluble (I) fraction of proteins extracted after induction at 28 °C of BL21/Gro7 cells containing the pQE-30-Flis6- sAGL plasmid. M: Marker of molecular weight;

Figure 2 shows a comparison between the recombinant sGDE protein purified from bacteria (Flis6-sGDE) and the endogenous GDE protein present in red cells and white cells of human blood;

Figure 3 shows the result of the enzymatic test for assessing glycogen debranching activity. C3PV: cell line of human fibroblasts; WBC: human white blood cells; GM02523: fibroblasts from GSDIII patients.

Figure 4 shows the stability of the recombinant sGDE enzyme purified from bacteria. (+): rat liver homogenate; (-): GM02523, fibroblasts derived from GSDIII patients; 1 : sGDE fresh preparation; 2: sGDE stored at 4 °C for 12 months; 3: lyophilised sGDE resuspended in a reaction buffer.

Figure 5 shows the overexpression of the recombinant GDE enzyme in human cells (HEK 293) in culture after transfection with the plasmid containing the codon-optimised s AGL gene, pVAX-s AGL (H10, H11 ) and with the one containing the AGL gene with the human sequence ( hAGL ), pVA X-hAGL (B4) compared to non-transfected cells (control), evaluated by immunofluorescence microscopy).

Figure 6 shows the overexpression of the GDE recombinant enzyme in HEK cells transfected with the pVA X-sAGL (H10, H11 ) plasmid compared to non-transfected cells (control) or those transfected with the pVA X-hAGL (B4) plasmid evaluated by immunoblotting (WB, “Western blot) and Ponceau red staining (control);

Figure 7 shows the increase in glycogen debranching activity in HEK cells transfected with the pVA X-sAGL (H10 and H11) plasmid and with the pVA X-hAGL (B4) plasmid compared to non- transfected cells (C) of the same type, verified by means of a specific enzymatic test. The positive control is represented by the recombinant GDE protein purified from bacteria (His6- sGDE).

Figure 8 shows the expression of the recombinant GDE enzyme in GDE-deficient cells (fibroblasts derived from GSDIII patients), GM00111 (a.) and GM02523 (b.) after transfection with the pVA X-sAGL plasmid using the transfecting agent Mirus 1 (column 1 ) or Mirus 2 (column 2). C-: non-transfected cells (negative control). The recombinant His6-sGDE protein purified from bacteria (sGDE) and the HEK cells (C+) represent the positive control.

Figure 9 shows the increase in/restoration of glycogen debranching activity in GM00111 (a.) and GM02523 (b.) cells after transfection with the pVA X-sAGL plasmid mediated by transfecting agents (Mirus 1 or Mirus 2), and verified by means of a specific enzymatic test. C-: non-transfected cells, negative control; C+: HEK cells, positive control.

DEFINITIONS

In the context of the present invention, “orthologs” means similar genes or proteins that can be found in mutually correlated organisms.

In the context of the present invention, “recombinant” means a protein obtained following the transcription and translation of a fragment of recombinant DNA.

In the context of the present invention, “recombinant DNA” means a DNA sequence obtained artificially by chemical synthesis or by combining genetic material of different origins, as may occur in the case of a plasmid containing a gene of interest.

In the context of the present invention, “electroporation” or “electropermeabilisation” (reversible) means the application of a sudden electric discharge to cells and/or tissues and/or organs with the aim of opening the plasma membrane of the cells simultaneously in numerous points, thereby enabling DNA molecules to penetrate. In the context of the present invention, “polynucleotide coding for a glycogen debranching enzyme” or “sAGL” means the codon-optimised AGL.

In the context of the present invention, “hAGL” means the non-codon-optimised human AGL gene.

In the context of the present invention, “sGDE” means the GDE protein obtained from the expression of the sAGL gene.

In the context of the present invention, “His6-sGDE’ means: the sGDE protein fused to a 6 histidine tag at the N-terminus.

In the context of the present invention, “hGDE’ means the GDE protein obtained from the expression of the hAGL gene.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to a compound selected from: a) a polynucleotide coding for a glycogen debranching enzyme (GDE), or a recombinant or synthetic derivative of GDE, a fragment or an allele variant of said GDE; b) a vector comprising said polynucleotide; c) a genetically engineered host cell that expresses said polynucleotide

In one embodiment, the polynucleotide coding for said GDE is selected from DNA and RNA; it is preferably DNA.

In one embodiment, the polynucleotide comprises a nucleotide sequence substantially homologous or identical to SEQ ID N0.1 (Table 1). For example, the polynucleotide comprises a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to SEQ ID NO. 1.

The polynucleotide preferably comprises a nucleotide sequence that is at least 80%, more preferably at least 90% homologous or identical to SEQ ID NO. 1 .

In one embodiment, the polynucleotide consists in a nucleotide sequence substantially homologous or identical to SEQ ID N0.1. For example, the polynucleotide consists in a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to SEQ ID NO. 1.

The polynucleotide preferably consists in a nucleotide sequence that is at least 80%, more preferably at least 90% homologous or identical to SEQ ID NO. 1 .

In one embodiment, the polynucleotide comprises at least one constitutive or tissue-specific promoter capable of controlling the expression of the cDNA coding for the AGL gene. In one embodiment, the vector comprising the polynucleotide described above is selected from: viral vector, plasmid, viral particles and phage.

Preferably, the vector is a plasmid vector.

In one embodiment of the invention, the genetically engineered host cell that expresses the polynucleotide described above is selected from: bacterial cell, fungal cell, animal cell, insect cell and plant cell; the host cell is preferably an animal cell.

A second aspect of the present invention relates to a pharmaceutical composition comprising the compound as defined above. Preferably, the composition of the present invention can further comprise at least one pharmacologically acceptable excipient, i.e. a compound, acceptable for pharmaceutical use, that is useful in the preparation of the composition and is generally biologically safe and nontoxic.

A third aspect of the present invention relates to the compound as described above in detail for use as a medicament.

A fourth aspect of the present invention relates to the compound as described above in detail or the glycogen debranching enzyme (GDE), an ortholog or synthetic GDE enzyme, for use in the treatment of a glycogenosis (GSD).

The GSD is preferably characterised by an accumulation of glycogen in muscle, the liver, heart and/or central nervous system.

In one embodiment, the GSD is selected from: GSDIa (Von Gierke disease), GSDI non-a, GSDII (Pompe disease), GSDIIb (Danon disease), GSDIII (Cori or Forbes disease), GSDIV (Andersen disease), GSDV (McArdle disease), GSDVI (Hers disease), GSDVII (Tarui disease), GSDIX, GSDXI (Fanconi-Bickel syndrome), GSDXI (aldolase deficiency), GSDXIII and GSDO.

In a preferred embodiment of the invention, the GSD is GSDIII (Cori or Forbes disease).

In one embodiment, the GDE enzyme comprises an amino acid sequence that is substantially identical to SEQ ID N0.2. For example, the GDE enzyme comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO. 2.

In a preferred embodiment, the GDE enzyme comprises an amino acid sequence that is at least 80%, more preferably at least 90% identical to SEQ ID NO. 2.

In one embodiment, the GDE enzyme consists in an amino acid sequence that is substantially identical to SEQ ID NO. 2. For example, the GDE enzyme consists in an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO. 2. In a preferred embodiment, the GDE enzyme consists in an amino acid sequence that is at least 80%, more preferably at least 90% identical to SEQ ID NO. 2.

In one embodiment of the invention, the GDE enzyme comprises modifications to the N- terminal and/or C-terminal region, for example apt to increase the activity of the GDE enzyme. Said modifications are preferably selected from deletions, additions, alterations of amino acids and combinations thereof. Alternatively, said GDE can be modified, preferably in its primary structure, by acetylation, carboxylation, phosphorylation and combinations thereof.

In a further embodiment, said GDE enzyme is conjugated/bound to a molecule, a metal, a marker, for example proteins, for the preparation of fusion proteins. In a further embodiment of the invention, said GDE enzyme is modified by means of molecular biology techniques in order to improve its resistance to proteolytic degradation and/or to optimise its solubility or to improve its pharmacokinetic characteristics.

According to one embodiment of the invention, said GDE enzyme can be modified with the aim of facilitating or improving delivery, preferably by means of PEGylation, or using container/shuttle/carrier systems, preferably such as liposomes, micelles, capsules, emulsions, matrices, gels and the like.

In a further embodiment, said GDE enzyme is coated with a structure capable of improving its stability and/or half-life and/or water solubility and/or immunological characteristics. Said structure is for example a pH-sensitive microsphere, a microsphere, a micro-tablet, a liposome, or else nano-delivery systems with conductive particles mediated by pulsed electric fields (PEFs) or fusion with cell-penetrating peptides (CPPs). In one embodiment, the enzyme is obtained by means of recombinant DNA techniques known to the person skilled in the art, preferably by cloning the polynucleotide (cDNA) coding for GDE in a plasmid vector for the expression of the recombinant enzyme in bacteria.

In a preferred embodiment of the invention, the GDE enzyme is obtained by cloning the non- codon-optimised human polynucleotide ( hAGL ) or the polynucleotide coding for GDE comprising SEQ ID NO. 1 ( sAGL ), as described above in detail.

In a further embodiment of the invention, said GDE enzyme is synthesised by means of conventional techniques for protein synthesis which are known to the skilled person. For example, the protein can be synthesised by chemical synthesis using solid-phase peptide synthesis.

In a further embodiment of the invention, the GDE enzyme is isolated or purified with methods known to the person skilled in the art. For example, the GDE enzyme can be purified by means of biochemical methods, such as filtration, affinity, immunoaffinity or by high-efficiency liquid chromatography (HPLC, FIP-HPLC, ion-exchange HPLC, size-exclusion HPLC). Table 1

In one embodiment, the compound or the GDE enzyme is delivered to a target tissue, preferably a tissue that contains accumulations of anomalous glycogen (PLD). The target tissue is preferably selected from: muscle, liver, heart and/or central nervous system; more preferably, the muscle is selected from: skeletal muscle, heart muscle and/or diaphragm.

In a preferred embodiment, the polynucleotide or the vector, as described above, is delivered to a target tissue that contains accumulations of glycogen.

The polynucleotide or the vector, as described above, is preferably delivered to a target tissue that contains accumulations of glycogen in association/combination with electropermeabilisation, i.e. with the application of electric fields, preferably pulsed electric fields (PEFs).

In a preferred embodiment, the pulsed electric fields are applied with a combination of high voltage electric pulses and low voltage pulses.

A fifth aspect of the present invention relates to a pharmaceutical composition comprising the compound or the GDE enzyme as defined above for use in the treatment of a glycogenosis. Preferably, the composition of the present invention can further comprise at least one pharmacologically acceptable excipient, i.e. a compound, acceptable for pharmaceutical use, that is useful in the preparation of the composition and is generally biologically safe and nontoxic. In one embodiment, the composition comprises at least a further therapeutic agent for the treatment of GSDs, in particular GSDIII (Cori or Forbes disease).

According to a preferred embodiment of the invention, the composition is formulated in liquid form, preferably as a solution, emulsion or sterile suspension.

In one embodiment, the composition is in lyophilised form, to be reconstituted to obtain a liquid formulation.

In one embodiment, the composition is administered by a parenteral route selected from the intravenous, subcutaneous and intramuscular routes.

In a preferred embodiment of the invention, the composition, preferably comprising the polynucleotide or the vector, as described above, is administered locally, preferably by local injection, directly into the tissue/organ to be treated.

The composition is preferably administered as a bolus or by continuous infusion.

In one embodiment, the tissue/organ to be treated is a tissue/organ that contains accumulations of glycogen. The tissue/organ to be treated is preferably selected from: muscle, liver, heart and/or central nervous system; more preferably, the muscle is selected from: skeletal muscle, heart muscle and/or diaphragm.

In a preferred embodiment of the invention, the composition is administered in association or in combination with electropermeabilisation.

In a preferred embodiment of the invention, the composition preferably comprising the polynucleotide or the vector, preferably the vector, is administered into the target tissue/organ in association or in combination with electropermeabilisation, i.e. with the application of electric fields, preferably pulsed electric fields (PEFs).

The electropermeabilisation is preferably applied locally, i.e. to the tissue/organ to be treated. In one embodiment, the electropermeabilisation is applied directly to the tissue/organ that contains accumulations of glycogen.

In a preferred embodiment, the pulsed electric fields are applied with a combination of high voltage pulses and low voltage pulses.

Electropermeabilisation is capable of increasing, in a transient manner, the permeability of cell membranes, making possible the entry of the polynucleotide or vector into cells and into the treated tissues/organs. A non-viral approach, for example using a plasmid vector in association or in combination with electroporation, proves to be particularly advantageous, as it is safer than viral vectors and enables better control of the diffusion of the vector in tissues/organs. Furthermore, a local treatment with non-viral methods, such as plasmids, in association or in combination with electroporation, increases the effectiveness of the therapy and reduces the health costs and the adverse effects due to systemic administration. In other words, the Applicants have demonstrated that the compound of the present invention, delivered by means of (non-viral) physical or chemical methods, is capable of significantly increasing the expression of the GDE enzyme in the treated cells.

Alternatively, the composition is formulated for enteral administration, preferably for oral administration. In particular, the composition is formulated in solid form, preferably in the form of pills, capsules, tablets, granular powder, hard-shelled capsules, orally dissolving granules, sachets or lozenges.

A further aspect of the present invention relates to a method for the treatment of a GSD, preferably GSDIII (Cori or Forbes disease). Said method comprises at least a step of administering an effective amount of the compound or of the GDE enzyme according to the present invention or of a composition that comprises it, as described above in detail, to a subject (individual) affected or suspected to be affected by a GSD, preferably by GSDIII.

In one embodiment, the composition is administered intravenously as a bolus or by continuous infusion. The composition is preferably administered by local injection directly into the tissue affected by an accumulation of anomalous glycogen.

In a preferred embodiment of the invention, the composition is administered by injection or infusion in association or in combination with electropermeabilisation.

In a preferred embodiment of the invention, the composition comprising the polynucleotide or the vector, preferably the vector, more preferably the plasmid, is administered in association or in combination with electroporation (also known as “electropermeabilisation”), i.e. with the application of electric fields, preferably pulsed electric fields (PEFs).

Preferably, the electropermeabilisation is applied locally, i.e. on the tissue/organ to be treated. In one embodiment, the electropermeabilisation is applied directly to the tissue/organ that contains accumulations of glycogen.

Example

The synthetic sAGL gene (codon-optimised for expression in cells of the insect Spodoptera frugiperda) encoding the sGDE enzyme was synthesised by the American Genscript Co. The gene was then cloned under the control of inducible promoters in suitable plasmid vectors for the expression of the recombinant enzyme in bacteria, fused or not fused to a six histidine tag (His6) at the N-terminus, to enable purification by affinity chromatography. These vectors were used for the transformation of different strains of E. coli. Following induction of protein expression at different culture temperatures, the expression of a 176 KDa protein was obtained only when the His6 “tag” was present (Fig. 1 ). In the absence of the “tag”, the resulting GDE protein is smaller in size (about 120 KDa) (Fig. 1).

The Flis6-sGDE protein expressed in bacteria was purified by affinity chromatography and showed approximately the same molecular weight as the human GDE protein present in the blood cells of healthy persons (Fig. 2). The maintenance of catalytic activity (Fig. 3) was assessed using a protocol adapted from the one used for diagnosis at the Department of Clinical Biochemistry of the University of Manchester, NFIS Foundation Trust, Manchester, the United Kingdom and made available by Fiona Ivison. As previously described, the glycogen debranching enzyme shows two distinct catalytic activities, transferasic and glucosidasic. In particular, the test used measures the glucosidasic activity of the enzyme, which, by acting at the level of the branching points of the specific substrate thereof (PLD, phosphorylase-limit dextrin), releases molecules of glucose. These molecules, following a colorimetric reaction with the enzymes glucose oxidase and peroxidase and ABTS (2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) are measured by means of spectrophotometric readings at a wavelength of 740 nm: the absorbance values measured are in fact directly correlated with the concentration of glucose molecules released by the enzyme. A human cell line with a GDE deficiency (GM02523, fibroblasts derived from GSDIII patients) was used as a negative control for the test.

Furthermore, Flis6-sGDE is endowed with considerable stability, as demonstrated by the maintenance of catalytic activity even after storage at 4 °C for 12 months or after lyophilisation (Fig. 4).

The cDNA with the sequence optimised for expression in insect cells and the cDNA with the original sequence of the human AGL gene were cloned in a vector for expression in mammal cells, obtaining the constructs pVA X-sAGL and pVA X-hAGL, respectively. Intracellular delivery of these constructs was achieved with chemical methods (cationic liposome formulations, Lipofectamine) on a human cell line (human embryonic kidney cells 293, FIEK). Following transfection, only the synthetic sequence, not the original one of the human gene, showed to be expressed at high levels (Fig. 5, 6). The transfected human cells also showed a greater catalytic activity compared to the ‘background’ (in fact, the FIEK cells express GDE at low levels) (Fig. 7).

In addition, two GDE-deficient human cell lines (GM00111 and GM02523), characterised by us at a biochemical and ultrastructural level ( data not shown), were used for experiments on delivery by means of Lipofectamine. In this case the transfection did not have a positive result ( data not shown) and, for this reason, two new transfecting agents (MIRUS 1 : TranslT-X2® System; MIRUS 2: -TranslT®-2020 Reagent) were experimented with on these lines. Following transfection, the GDE-deficient human cells showed the expression of the enzyme (Fig. 8) and an increased catalytic activity compared to the non-transfected cells (Fig. 9).

At present delivery by electroporation is also undergoing experimentation.

Claims

1 . A compound selected from: a) a polynucleotide coding for a glycogen debranching enzyme (GDE), a recombinant or synthetic derivative of GDE, a fragment or an allele variant of said GDE, comprising a nucleotide sequence that is at least 80% identical to SEQ ID NO: 1 ; b) a vector comprising said polynucleotide; c) a genetically engineered host cell that expresses said polynucleotide.

2. The compound according to claim 1 , wherein the polynucleotide comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 1 .

3. The compound according to claim 1 or 2, wherein the polynucleotide comprises a nucleotide sequence that consists in SEQ ID NO: 1 .

4. The compound according to any one of claims 1-3 for use as a medicament.

5. The compound according to any one of claims 1-3 or the GDE enzyme, an ortholog or synthetic GDE enzyme, for use in the treatment of a glycogenosis (GSD).

6. The compound or the GDE enzyme for use according to claim 5, wherein the GDE enzyme comprises an amino acid sequence that is 80%, preferably at least 90% identical to SEQ ID NO. 2.

7. The compound or the GDE enzyme for use according to claim 5 or 6, wherein the GSD is GSDIII (Cori’s or Forbes’ disease).

8. The compound or the GDE enzyme for use according to any one of claims 5-7, in combination or association with electropermeabilisation.

9. The compound or the GDE enzyme for use according to claim 8, wherein the electropermeabilisation comprises a combination of high-voltage electric pulses and low- voltage electric pulses.

10. The compound or the GDE enzyme for use according to any one of claims 5-9, wherein a tissue/organ to be treated contains accumulations of glycogen.

11. The compound or the GDE enzyme for use according to claim 10, wherein the tissue/organ to be treated is selected from: muscle, liver, heart and/or central nervous system, the muscle is preferably selected from: skeletal and cardiac muscle and diaphragm.

12. The compound or GDE enzyme for use according to any one of claims 8-11 , wherein the electropermeabilisation is applied locally to the tissue/organ to be treated, preferably directly to the tissue/organ that contains accumulations of glycogen.

13. A pharmaceutical composition comprising the compound for use according to any one of claims 4-12.

14. A pharmaceutical composition comprising the GDE enzyme for use according to any one of claims 5-12.

15. The composition for use according to claim 13 or 14, formulated in liquid form, preferably in the form of a sterile solution, emulsion or suspension, or in lyophilised form in order to be reconstituted to obtain a liquid formulation.

16. The composition for use according to any one of claims 13-15, administered via a parenteral route selected from intravenous, subcutaneous, intramuscular, preferably administered locally by injection directly into the tissue/organ to be treated.