Treatment of glycogen storage disease (GSD)

Abstract

To provide treatment of glycogen storage disease (GSD).SOLUTION: The present invention relates to a kit of parts comprising (i) a pharmacological chaperone or a pharmaceutically acceptable salt thereof and (ii) a therapeutic acid alpha glucosidase (GAA) polypeptide or a nucleic acid molecule encoding a therapeutic GAA polypeptide, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof and Ambroxol (ABX) or a derivative thereof.SELECTED DRAWING: None

Description

[Background technology]

[0001] Glycogen storage diseases (GSDs) are metabolic disorders caused by deficiencies of enzymes affecting glycogen synthesis, glycogenolysis, or glycolysis, typically in muscle and / or liver cells. GSDs are classified into various types, ranging from GSD type 0 to GSD type XV (Table 1).

[0002] [Table 1]

[0003] Pompe disease, also known as GSD II or acid maltase deficiency, is an autosomal recessive metabolic myopathy caused by a deficiency of the lysosomal enzyme acid α-glucosidase (GAA). GAA is an exo-1,4 and 1,6-α-glucosidase that hydrolyzes glycogen to glucose within the lysosome. GAA deficiency leads to glycogen accumulation within the lysosomes, causing progressive damage to respiratory, cardiac, and skeletal muscles. The disease ranges from a rapidly progressive infantile course that is usually fatal by age 1–2 years to a slowly progressive, heterogeneous course that causes significant morbidity and early mortality in children and adults [1] and [2].

[0004] Central nervous system (CNS) disorders, particularly respiratory neuromuscular disorders, are prominent in GSDs, especially in patients with early- and late-onset Pompe disease. Studies have shown that glycogen accumulation in the central nervous system causes respiratory problems in patients with Pompe disease, leading to respiratory dysfunction by 4–6 months of age. [3] [4] Therefore, it is important to develop treatments that improve the correction of respiratory problems in Pompe disease, especially those that improve the correction of the pathogenic accumulation of glycogen in the nervous system tissues of GSD patients.

[0005] Current human therapy for treating Pompe disease involves the administration of recombinant human GAA, a so-called enzyme replacement therapy (ERT). ERT has shown efficacy in severe infantile GSD II. However, the benefits of ERT are limited by the need for frequent infusions and the development of inhibitory antibodies against recombinant hGAA [5]. Furthermore, ERT does not efficiently improve systemic function, likely due to a combination of poor biodistribution of the protein after peripheral intravenous delivery, lack of uptake from some tissues, and high immunogenicity. In particular, ERT has been shown to be less effective in treating CNS disorders, particularly in correcting glycogen accumulation in nervous system tissues of GSD patients, because recombinant GAA does not cross the blood-brain barrier [6] and [7].

[0006] Gene therapy approaches for treating Pompe disease are also being studied.For example, WO2018 / 046774 discloses the use of the nucleic acid molecule encoding truncated GAA polypeptide fused with signal peptide to improve tissue uptake of GAA.However, glycogen accumulation is only partially rescued in the central nervous system.

[0007] Another approach developed to treat Pompe disease is the administration of pharmacological chaperones to facilitate GAA folding and increase its stability. Several pharmacological chaperones have been tested, such as 1-deoxynojirimycin (DNJ) or its derivatives (WO 2006 / 125141), particularly the DNJ derivative called NB-DNJ (AT2221 or miglustat). Miglustat has also been tested in combination with recombinant GAA polypeptides to promote GAA activity and improve muscle function. [8] However, DNJ or its derivatives have not increased GAA uptake into nervous system tissues.

[0008] ABX or the combination of ABX and DNJ has also been tested in a Pompe cell model expressing a mutant form of GAA.[9] However, ABX or ABX and DNJ did not increase tissue uptake of GAA, particularly in nervous system tissues. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2018 / 046774 [Patent Document 2] WO2006 / 125141 [Non-patent literature]

[0010] [Non-Patent Document 1] Hirschhorn, R. and Reuser, AJ 2001 In The Metabolic and Molecular Basis for Inherited Disease (Scriver, CR, Beaudet, AL, Sly, WS & Valle, D. Eds.), pp. 3389-3419. McGraw-Hill, New York, pp. 3403-3405. [Non-patent document 2] Van der Ploeg and Reuser, 2008 Lancet 372:1342~1351 [Non-patent document 3] Van den Hout et al., 2003, Pediatrics;112:332~340[PubMed:12897283] [Non-patent document 4] DeRuisseau et al. 2009;Proc Natl Acad Sci USA, 106:9419~9424. [PubMed:19474295] [Non-Patent Document 5] Amalfitano, A. et al. 2001 Genet. In Med. 3:132~138 [Non-patent document 6] Kikuchi et al., 1998, Clinical and metabolic correction of Pompe disease by enzyme therapy in acid maltase-deficient quail. The Journal of Clinical Investigation;101:827~833 [PubMed:9466978] [Non-Patent Document 7] Raben et al., 2003, Molecular Genetics and Metabolism;80:159~169[PubMed:14567965] [Non-patent document 8] Xu et al., JCI Insight 2019;4(5):e125358 [Non-Patent Document 9] Lukas et al., The American Society of Gene and Cell Therapy 2015, Vol. 23, No. 3, pp. 456-464) Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, there remains a need to provide better therapies for treating GSDs such as Pompe disease, particularly for increasing GAA uptake into tissues of the nervous system, treating CNS disorders in GSDs, improving respiratory neuromuscular function, and / or reducing respiratory dysfunction in subjects with GSDs. [Means for solving the problem]

[0012] In a first aspect, the present invention relates to a kit-of-parts comprising (i) a pharmacological chaperone or a pharmaceutically acceptable salt thereof, and (ii) a therapeutic acid alpha-glucosidase (GAA) polypeptide or a nucleic acid molecule encoding a therapeutic GAA polypeptide, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof and ambroxol (ABX) or a derivative thereof. - As a medicine, in the treatment of glycogen storage diseases (GSD), and / or - a method for treating central nervous system (CNS) disorders in GSD, may be used.

[0013] In a second aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: - in the treatment of glycogen storage disease (GSD) in a subject receiving therapeutic acid alpha-glucosidase (GAA) treatment for treating said GSD, - in a method of increasing GAA uptake into tissues of the nervous system in a subject receiving therapeutic GAA treatment for treating GSD; and / or - in a method for treating central nervous system (CNS) disorders of GSD in a subject receiving therapeutic GAA treatment for treating GSD, 1. A composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use, comprising: The present invention relates to a composition wherein the pharmacological chaperones are DNJ or a derivative thereof and ABX or a derivative thereof. [Brief explanation of the drawings]

[0014] [Figure 1]Figure 1 shows the experimental design for the study on WT mice. Six- to eight-week-old C57Bl / 6J male mice were intravenously injected with PBS or an AAV8 vector expressing secreted GAA (AAV-GAA) at 5 x 10 vg / kg. Two months after vector injection, mice were either left untreated or treated for four weeks with seven chaperone molecules or their combinations dissolved in drinking water. Each week of treatment consisted of three days of treatment and four days of rest (washout). Three months after gene therapy treatment, mice were bled and sacrificed for tissue collection. [Figure 2] Figure 1 shows that chaperone treatment improves circulating GAA levels in wild-type mice. Three months after vector injection, blood GAA levels were measured by Western blot in mice treated as shown in Figure 1. Data were expressed as the ratio of human GAA band intensity to the intensity of a nonspecific band used for normalization. Error bars represent the mean standard deviation. Statistical analysis was performed by ANOVA (* = p < 0.05 vs. AAV-GAA-injected mice given regular water during the third month, DNJ-ABX-treated group (n = 3), voglibose- and acarbose-treated group (n = 4), n = 5 per group; CTRL represents mice injected with PBS and not given AAV-GAA). [Figure 3-1] Figure 3 shows that chaperone treatment improves GAA uptake into tissues in wild-type mice. Three months after vector injection, lysosomal GAA levels were measured by Western blot in the hearts (Figure 3A) and triceps muscles (Figure 3B) of mice treated as shown in Figure 1. Data are expressed as the ratio of human GAA band intensity to GAPDH intensity, which was used for normalization. Error bars represent the mean standard deviation. Statistical analysis was performed by ANOVA (* = p < 0.05 vs. AAV-GAA-injected mice given regular water during month 3; n = 5 per group, except for the DNJ-ABX-treated group (n = 3), the voglibose- and acarbose-treated group (n = 4)). [Figure 3-2]Figure 3 shows that chaperone treatment results in improved GAA uptake into tissues in wild-type mice. Three months after vector injection, lysosomal GAA levels were measured by Western blot in the diaphragm (Fig. 3C) and quadriceps (Fig. 3D) of mice treated as shown in Fig. 1. [Figure 3-3] Figure 3 shows that chaperone treatment improves GAA uptake into tissues in wild-type mice. Three months after vector injection, lysosomal GAA levels were measured by Western blot in the brain (Figure 3E) and spinal cord (Figure 3F) of mice treated as shown in Figure 1. [Figure 4] Figure 1 shows the experimental design for the study on GAA-deficient (knockout) mice. Three- to four-week-old GAA-deficient male mice were intravenously injected with 1 × 10 vg / kg of an AAV8 vector expressing secreted GAA in combination with PBS or a pharmacological chaperone (PC) dissolved in drinking water. Two groups of untreated GAA wild-type mice and GAA-deficient mice injected with the AAV-GAA vector or PBS served as controls. The PC molecule was orally administered to the mice using a "3-day on / 4-day off" regimen consisting of three consecutive days of treatment followed by four consecutive days of drinking water alone. Two months after gene therapy treatment in combination with the pharmacological chaperone, the mice were bled and sacrificed, and tissues were collected. [Figure 5] Figure 4 shows that chaperone treatment improves circulating GAA levels in GAA KO mice. Two months after vector injection, blood GAA levels were measured by Western blot in mice treated as shown in Figure 4. Data were expressed as the ratio of human GAA band intensity to the intensity of a nonspecific band used for normalization. Error bars represent the mean standard deviation. Statistical analysis was performed by ANOVA (* = p < 0.05 vs. AAV-GAA-injected mice given regular water during the second month; n = 8 per group except for the ABX-treated group (n = 7); GAA+ / + mice (WT) and GAA- / - mice (KO) were injected with PBS and given regular water). [Figure 6]Figure 4 shows that chaperone treatment improves circulating GAA activity in GAA KO mice. Two months after vector injection, blood GAA activity was measured in the blood of mice treated as shown in Figure 4. Error bars represent the mean standard deviation. Statistical analysis was performed by ANOVA (* = p < 0.05 vs. AAV-GAA-injected mice given regular water during the second month; n = 8 per group except for the ABX-treated group (n = 7); GAA+ / + mice (WT) and GAA- / - mice (KO) were injected with PBS and given regular water). [Figure 7-1] Figure 7 shows that chaperone treatment improves GAA uptake into tissues in GAA KO mice. Two months after vector injection, treated mice were sacrificed and tissues were harvested as shown in Figure 4. Results of GAA activity detected in the heart (Figure 7A) and diaphragm (Figure 7B) are shown. Error bars represent the mean standard deviation. Statistical analysis was performed by ANOVA (*=p<0.05 vs. AAV-GAA-injected mice given regular water during month 3, n=8 per group except for the ABX-treated group (n=7)). [Figure 7-2] Figure 7 shows that chaperone treatment improves GAA uptake into tissues of GAA KO mice. Two months after vector injection, treated mice were sacrificed and tissues were collected as shown in Figure 4. Results of GAA activity detected in triceps (Figure 7C) and quadriceps (Figure 7D) muscles are shown. [Figure 8-1] Figure 8 shows that chaperone treatment results in reduced glycogen accumulation in tissues of GAA KO mice. Two months after vector injection, treated mice were sacrificed and tissues were harvested as shown in Figure 4. Glycogen content was measured in the heart (Figure 8A) and diaphragm (Figure 8B). Error bars represent the mean standard deviation. Statistical analysis was performed by ANOVA (*=p<0.05 vs. AAV-GAA-injected mice given regular water during month 3, n=8 per group except for the ABX-treated group (n=7)). [Figure 8-2]Figure 8 shows that chaperone treatment results in reduced glycogen accumulation in tissues of GAA KO mice. Two months after vector injection, treated mice were sacrificed and tissues were collected as shown in Figure 4. Glycogen content was measured in the triceps (Figure 8C) and quadriceps (Figure 8D) muscles. [Figure 9] Figure 1 shows the experimental design for the study on GAA-deficient (knockout) mice. Three- to four-month-old GAA-deficient male mice were intravenously treated with ERT (alglucosidase alfa, 20 mg / kg) in combination with a pharmacological chaperone (PC) dissolved in drinking water. Untreated GAA wild-type mice, untreated GAA-deficient mice, and GAA-deficient mice treated with ERT alone served as controls. Blood was collected 3 hours after ERT. [Figure 10] Figure 10 shows that chaperone treatment results in improved circulating GAA levels and activity in GAA KO mice. Three hours after ERT, blood GAA levels (Figure 10A) and activity (Figure 10B) were measured in mice treated as shown in Figure 9. GAA level results were expressed as the ratio of human GAA band intensity to the nonspecific band intensity used for normalization. Error bars represent the mean standard deviation. Statistical analysis was performed by ANOVA (*=p<0.05 vs. ERT mice receiving regular water during the protocol, n=8 per group; untreated GAA+ / + mice (WT) and GAA- / - mice (KO) received regular water). DETAILED DESCRIPTION OF THE INVENTION

[0015] definition The term "pharmacological chaperone" refers to a small molecule that stabilizes a prefolded protein by binding to it and stabilizing it against thermal denaturation and proteolysis. According to the present invention, the pharmacological chaperone may be in the form of a salt, e.g., a pharmaceutically acceptable salt.

[0016] The term "pharmacological chaperone" according to the present invention refers to both (i) 1-deoxynojirimycin (DNJ) or a derivative thereof and (ii) ambroxol (ABX) or a derivative thereof.

[0017] The term "pharmaceutically acceptable salt" refers to a salt of an acid or base known for its use in preparing active ingredients for use in therapy. Examples of pharmaceutically acceptable acids suitable as the source of the anion include those disclosed in Handbook of Pharmaceutical Salts: Properties, Selection and Use (P.H. Stahl and C.G. Wermuth, Weinheim / Zurich: Wiley-VCH / VHCA, 200). Salts approved by a regulatory agency of the federal or state government or listed in the United States or European Pharmacopoeia or other pharmacopeias generally recognized for use in animals and humans. Examples include acetate, adipate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, and lactate. , malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, and xinafoate. Suitable base salts are formed from bases that form non-toxic salts. Examples include aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, and zinc salts. Hemisalts of acids and bases, such as hemisulfate and hemicalcium salts, can also be formed.

[0018] The term "DNJ" according to the present invention means 1-deoxynojirimycin (CAS number 19130-96-2, INN duvoglustat). The term "DNJ derivative" according to the present invention means a compound derived from DNJ. DNJ derivatives include N-methyl-DNJ, N-butyl-DNJ, N-cyclopropylmethyl-DNJ, N-(2-(N,N-dimethylamido)ethyloxy-DNJ, N-4-t-butyloxycarbonyl-piperidinylmethyl-DNJ, N-2-R-tetrahydrofuranylmethyl-DNJ, N-2-R-tetrahydrofuranylmethyl-DNJ, N-(2-(2,2,2-trifluoroethoxy)ethyl-DNJ, N-2-methoxyethyl-DNJ, N-2-ethoxyethyl-DNJ, N-4-trifluoromethylbenzyl-DNJ, N-alpha-cyano-4-trifluoromethylbenzyl-DNJ, N-4-trifluoromethoxybenzyl-DNJ, N-4-n-pentoxybenzyl-DNJ, and N-4-n-pentoxybenzyl-DNJ. The DNJ may be selected from N-butoxybenzyl-DNJ, Cl-nonyl-DNJ, or N-butyl-DNJ (CAS No. 72599-27-0, INN miglustat). DNJ or a derivative thereof can be prepared by methods known in the art, for example, as described in WO2006 / 125141. DNJ or a derivative thereof may be in the form of a salt, such as DNJ hydrochloride (CAS No. 73285-50-4) or N-butyl-DNJ hydrochloride (CAS No. 210110-90-0). In some embodiments, the DNJ is duvoglustat (CAS No. 19130-96-2), duvoglustat hydrochloride (CAS No. 73285-50-4), miglustat (CAS No. 72599-27-0), or miglustat hydrochloride (CAS No. 210110-90-0).

[0019] The term "ABX" according to the present invention refers to ambroxol (CAS number 18683-91-5). The term "ABX derivative" refers to a compound derived from ABX. The ABX derivative may be bromhexine (CAS number 3572-43-8). ABX or a derivative thereof may be in the form of a salt, such as ABX hydrochloride (CAS number 23828-92-4). ABX or a derivative thereof may be prepared by methods known in the art, for example, as described in US2004 / 0242700. In one embodiment, the pharmacological chaperone is duvoglustat (CAS No. 19130-96-2) or duvoglustat hydrochloride (CAS No. 73285-50-4); and ambroxol hydrochloride (CAS No. 23828-92-4). In another embodiment, the pharmacological chaperone is miglustat (CAS No. 72599-27-0) or miglustat hydrochloride (CAS No. 210110-90-0); and ambroxol hydrochloride (CAS No. 23828-92-4).

[0020] The term "NAC" according to the present invention refers to N-acetylcysteine ​​(CAS number 616-91-1(L)). The term "NAC derivative" refers to a compound derived from NAC. NAC derivatives may be obtained by coupling NAC with an amino acid by forming an ester, amide, and / or hybrid bond between the amino acid and NAC. Any amino acid or amino acid analogue can be used.

[0021] The term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the United States or European Pharmacopoeia or other pharmacopeia generally recognized for animal and human use. A "pharmaceutical composition" refers to a composition containing a pharmaceutically acceptable carrier. For example, a carrier can be a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can 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, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and 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, nonfat dry milk, glycerol, propylene glycol, water, ethanol, and the like. Where the pharmaceutical composition is adapted for oral administration, tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form, for example, of solutions, syrups, or suspensions, or can be presented as a dry product for constitution with water or another suitable vehicle before use.Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl- or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents, as needed. The compositions of the present invention are preferably pharmaceutical compositions.

[0022] The term "polypeptide" refers to an amino acid sequence, i.e., a chain of amino acids linked by peptide bonds. The amino acid sequence of a therapeutic GAA polypeptide or its coding sequence can be derived from any source, including avian and mammalian species. As used herein, the term "avian" includes, but is not limited to, chicken, duck, goose, quail, turkey, and pheasant. As used herein, the term "mammal" or "mammals" includes, but is not limited to, humans, monkeys and other non-human primates, cows, sheep, goats, horses, cats, dogs, lagomorphs, etc. In embodiments of the invention, the therapeutic GAA polypeptide is a human, murine, or quail, particularly a human therapeutic GAA polypeptide.

[0023] The term "acid α-glucosidase" or "GAA" refers to exo-1,4-α-D-glucosidase, which hydrolyzes both α-1,4 and α-1,6 linkages in oligosaccharides to liberate glucose. GAA deficiency results in glycogen storage disease type II (GSDII), also known as Pompe disease (although this term formally refers to the infantile-onset form). GAA catalyzes the complete breakdown of glycogen, slowing it down at branch points. The 28-kb human acid α-glucosidase gene on chromosome 17 encodes a 3.6-kb mRNA and produces a 951-amino acid polypeptide.

[10]

[11] This enzyme undergoes cotranslational N-linked glycosylation in the endoplasmic reticulum. It is synthesized as a 110 kDa precursor form and matures to the final lysosomal 76 and 67 kDa forms by extensive glycosylation modification, phosphorylation, and proteolytic processing via an approximately 90 kDa endosomal intermediate

[10] ,

[12] ;

[13] and

[14] .

[0024] The term "glycogen storage disease" or "GSD" refers to a metabolic disease caused by a deficiency in an enzyme affecting glycogen synthesis, glycogenolysis, or glycolysis. In particular, the glycogen storage disease can be one or more of the types of GSD disclosed in Table 1. According to the present invention, the GSD is preferably GSDI (von Gierke disease), GSDII (Pompe disease), GSDIII (Cawley disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, or a fatal congenital glycogen storage disease of the heart. More particularly, the glycogen storage disease is selected from the group consisting of GSDI, GSDII, and GSDIII, and even more particularly, from the group consisting of GSDII and GSDIII. In a more particular embodiment, the glycogen storage disease is GSDII.

[0025] The term "GAA polypeptide" refers, without further strictness, to GAA with a signal peptide (i.e., a GAA precursor) and GAA without a signal peptide, and may be a wild-type or mutant GAA polypeptide.

[0026] The term "wild-type GAA polypeptide" refers to a naturally occurring form of GAA, e.g., SEQ ID NO: 2 (corresponding to GenBank accession number NP_000143.2), or SEQ ID NO: 30, is a wild-type human GAA polypeptide (also found in the Uniprot entry for GAA accession number P10253; corresponding to GenBank CAA68763.1; SEQ ID NO: 30). Within SEQ ID NO: 2 and SEQ ID NO: 30, amino acid residues 1-27 correspond to the signal peptide of the wild-type GAA polypeptide. The term "wild-type GAA polypeptide," without further clarification, refers ambiguously to GAA with a signal peptide (i.e., a GAA precursor) and GAA without a signal peptide.

[0027] The term "mutant GAA polypeptide" refers to a GAA polypeptide having at least one amino acid modification, such as a substitution, deletion, or addition, compared to a wild-type GAA polypeptide. Exemplary variant GAA polypeptides include SEQ ID NO: 29 (GenBank AAA52506.1), SEQ ID NO: 31 (GenBank: EAW89583.1), and SEQ ID NO: 32 (GenBank ABI53718.1).

[0028] Other useful mutant GAA polypeptides include those described by Hoefsloot et al.

[10] ; Van Hove et al.

[15] and GenBank Accession No. NM_008064 (mouse). Other mutant GAA polypeptides include those described in WO2012 / 145644, WO00 / 34451, and US 6,858,425. Other useful mutant GAA polypeptides include those described in the literature, such as GAA II described by Kunita et al.

[16] , and GAA polymorphisms and SNPs described by Hirschhorn, R., and Reuser [1].

[0029] The term "therapeutic GAA polypeptide" refers to a GAA polypeptide that can be used in the treatment of GSD. In particular, a therapeutic GAA polypeptide of the present invention has the functionality of a wild-type GAA polypeptide. The functionality of wild-type GAA is to hydrolyze both α-1,4 and α-1,6 linkages of oligosaccharides and polysaccharides, more particularly glycogen, to liberate glucose. A therapeutic GAA polypeptide may have at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 100% of the hydrolysis activity against glycogen compared to the wild-type GAA polypeptide of SEQ ID NO: 2, 30, 29, 31, or SEQ ID NO: 32. The activity of a therapeutic GAA polypeptide may even be greater than 100% of the activity of the wild-type GAA protein of SEQ ID NO: 2, 29, 30, 31, or 32, e.g., greater than 110%, 120%, 130%, 140%, or even 150%. Therapeutic GAA polypeptides may be purified from recombinant cell expression systems (e.g., mammalian or insect cells (see U.S. Patent Nos. 5,580,757, 6,395,884, 6,458,574, 6,461,609, 210,666, 6,083,725, 6,451,600, 5,236,838, and 5,879,680)), human placenta, or animal milk (see U.S. Patent No. 6,188,045). A therapeutic GAA polypeptide currently approved for the treatment of Pompe disease is a recombinant GAA polypeptide called alglucosidase alfa (sold under the trade names Lumizyme® or Myozyme® by Genzyme, Inc.).

[0030] A therapeutic GAA polypeptide of the present invention comprises a GAA polypeptide portion and, ultimately, a signal peptide portion fused to the N-terminus of the GAA polypeptide portion. Thus, a therapeutic GAA polypeptide may comprise (i) a GAA polypeptide portion and a signal peptide portion fused to the N-terminus of the GAA polypeptide portion, or (ii) a GAA polypeptide portion without a signal peptide portion fused to the N-terminus of the GAA polypeptide portion. In some embodiments, a therapeutic GAA polypeptide may also comprise additional sequences to improve biodistribution, stability, and / or tissue uptake of the therapeutic GAA polypeptide.

[0031] The term "GAA polypeptide portion" refers to a GAA polypeptide that lacks a signal peptide. It may be a wild-type GAA polypeptide portion or a mutant GAA polypeptide portion.

[0032] The term "wild-type GAA polypeptide portion" refers to naturally occurring GAA lacking a signal peptide, for example, SEQ ID NO: 1 and SEQ ID NO: 33 are both wild-type human GAA polypeptide portions.

[0033] The term "mutated GAA polypeptide portion" refers to a GAA polypeptide portion that has at least one amino acid modification, such as a substitution, deletion, or addition, compared to a wild-type GAA polypeptide portion. Any mutant GAA polypeptide can be used as the basis for defining a mutant GAA polypeptide portion. In some embodiments, the mutant GAA polypeptide portion is a truncated GAA polypeptide. In the context of the present invention, a "truncated GAA polypeptide" refers to a GAA polypeptide comprising one or more consecutive amino acids truncated from the N-terminus of a parent GAA polypeptide lacking a signal peptide. In one embodiment, the parent GAA polypeptide lacking a signal peptide is a wild-type GAA polypeptide lacking a signal peptide, such as a wild-type human GAA polypeptide lacking a signal peptide represented by SEQ ID NO: 1 or SEQ ID NO: 33. In another embodiment, the parent GAA polypeptide is a mutant GAA polypeptide lacking a signal peptide. Any mutant GAA polypeptide known in the art can be used as a basis for defining the parent GAA polypeptide lacking a signal peptide. Exemplary mutant GAA polypeptides include SEQ ID NO: 29 (GenBank AAA52506.1), SEQ ID NO: 31 (GenBank: EAW89583.1), and SEQ ID NO: 32 (GenBank ABI53718.1). Other useful mutants include those described by Hoefsloot et al.

[10] , Van Hove et al.

[15] , and GenBank Accession No. NM_008064 (mouse). Other mutant GAA polypeptides include those described in WO2012 / 145644, WO00 / 34451 and US 6,858,425. In particular embodiments, the parent GAA polypeptide lacking a signal peptide is derived from the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 30. More particularly, the parent GAA polypeptide lacking a signal peptide is SEQ ID NO: 1 or SEQ ID NO: 33, preferably SEQ ID NO: 1.

[0034] In particular, truncated GAA polypeptides of the present invention have 1 to 75 consecutive amino acids truncated at their N-terminus compared to the parent GAA polypeptide lacking its signal peptide. Specifically, truncated GAA polypeptides have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 1 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 consecutive amino acids may be truncated. In preferred embodiments, a truncated GAA polypeptide has 6, 7, 8, 9, 10, 40, 41, 42, 43, 44, 45, 46, or 47 consecutive amino acids truncated at its N-terminus compared to a parent GAA polypeptide lacking its signal peptide, and more particularly has 8, 42, or 43 consecutive amino acids truncated at its N-terminus compared to a parent GAA polypeptide lacking a signal peptide, particularly SEQ ID NO: 1 or SEQ ID NO: 33. In particular embodiments, truncated GAA polypeptides of the invention have the sequences set forth in SEQ ID NOs: 27, 28, 34, 35, and 36. SEQ ID NO: 27 corresponds to a truncated GAA polypeptide having 8 consecutive amino acids truncated from its N-terminus compared to SEQ ID NO: 1. SEQ ID NO: 28 corresponds to a truncated GAA polypeptide having 42 consecutive amino acids truncated from its N-terminus compared to SEQ ID NO: 1. SEQ ID NO: 34 corresponds to a truncated GAA polypeptide having 29 consecutive amino acids truncated from its N-terminus compared to SEQ ID NO: 1. SEQ ID NO: 35 corresponds to a truncated GAA polypeptide having 43 consecutive amino acids truncated from its N-terminus compared to SEQ ID NO: 1. SEQ ID NO: 36 corresponds to a truncated GAA polypeptide having 47 consecutive amino acids truncated from its N-terminus compared to SEQ ID NO: 1.

[0035] The term "signal peptide portion" according to the present invention refers to the endogenous (or native) signal peptide of a wild-type GAA polypeptide, e.g., the signal peptide encoded by the nucleic acid sequence of SEQ ID NO:4 (designated sp1), or an exogenous signal peptide of another protein. Specific exogenous signal peptides that are operable with the present invention include amino acids 1-20 from chymotrypsinogen B2 (SEQ ID NO:3), also designated sp7, the signal peptide of human alpha-1-antitrypsin (SEQ ID NO:5, also designated sp2), amino acids 1-25 from iduronate-2-sulfatase (SEQ ID NO:6, also designated sp6), and amino acids 1-23 from protease C1 inhibitor (SEQ ID NO:7, also designated sp8). The signal peptides of SEQ ID NO:3 and SEQ ID NO:5-SEQ ID NO:7 allow for higher secretion of chimeric GAA polypeptides both in vitro and in vivo when compared to GAA polypeptides containing their native signal peptides. In certain embodiments, the signal peptide has the sequence shown in SEQ ID NO: 3 to 7 or is a functional derivative thereof, i.e. a sequence which comprises deletions, insertions or substitutions of 1 to 5, particularly 1 to 4, particularly 1 to 3, particularly 1 to 2, particularly 1 amino acid compared to the sequence shown in SEQ ID NO: 3 to 7, insofar as the resulting sequence corresponds to a functional signal peptide, i.e. a signal peptide which allows the secretion of the GAA protein. In certain embodiments, the signal peptide portion is selected from the group consisting of SEQ ID NO: 3 to 7, preferably SEQ ID NO: 3.

[0036] In certain embodiments, the signal peptide portion fused to the N-terminus of the GAA polypeptide portion is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and the GAA polypeptide portion is selected from SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.

[0037] According to the present invention, the term "nucleic acid molecule" refers to a DNA or RNA molecule, particularly DNA, in single- or double-stranded form. According to the present invention, a nucleic acid molecule encodes a therapeutic GAA polypeptide as described above. For example, a nucleic acid molecule encoding a wild-type GAA polypeptide corresponds to SEQ ID NO: 8. A nucleic acid molecule encoding a therapeutic GAA polypeptide lacking the signal peptide may be nucleotide sequence 82-2859 of SEQ ID NO: 8, i.e., SEQ ID NO: 9.

[0038] Nucleic acid molecules of the present invention encoding therapeutic GAA polypeptides can be optimized for in vivo expression of the therapeutic GAA polypeptide. Sequence optimization can include many modifications of the nucleic acid sequence, including codon optimization, increasing GC content, reducing the number of CpG islands, reducing the number of alternative open reading frames (ARFs), and reducing the number of splice donor and splice acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules can encode the same protein. It is also well known that the genetic codes of different organisms are often biased toward using one of several codons that encode the same amino acid over others. Codon optimization introduces changes into a nucleotide sequence that take advantage of the codon bias present in a given cellular context, thereby making the resulting codon-optimized nucleotide sequence more likely to be expressed at relatively high levels in such a given cellular context compared to a non-codon-optimized sequence. In a preferred embodiment of the present invention, such a sequence-optimized nucleotide sequence encodes a truncated GAA polypeptide and is codon-optimized to improve its expression in human cells compared to a non-codon-optimized nucleotide sequence encoding the same truncated GAA polypeptide, for example, by taking advantage of human-specific codon usage bias. In certain embodiments, a nucleic acid molecule encoding a therapeutic GAA polypeptide is codon-optimized and / or has increased GC content and / or has a reduced number of alternative open reading frames and / or has a reduced number of splice donor and / or splice acceptor sites compared to nucleotides 82-2859 of a wild-type human GAA polypeptide coding sequence, e.g., nucleotides 82-2859 of SEQ ID NO: 8. SEQ ID NO: 8 is a non-optimized nucleotide sequence encoding a wild-type human GAA polypeptide with a signal peptide. SEQ ID NO: 9 is a non-optimized nucleotide sequence encoding a wild-type human GAA polypeptide lacking a signal peptide. The optimized nucleotide sequence encoding a wild-type human GAA polypeptide may be SEQ ID NO: 10 (hGAA co1) or SEQ ID NO: 11 (hGAA co2). In particular embodiments, a nucleic acid molecule of the invention comprises the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 27; the sequence set forth in SEQ ID NO: 48 or SEQ ID NO: 49, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 28; the sequence set forth in SEQ ID NO: 50 or SEQ ID NO: 51, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 35; or the sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 53, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 36. In a preferred embodiment, a nucleic acid molecule of the invention comprises the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13, which encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 27.

[0039] In a preferred embodiment, a nucleic acid molecule encoding a therapeutic GAA polypeptide comprising a GAA polypeptide portion and a signal peptide portion fused to the N-terminus of the GAA polypeptide portion according to the present invention is as follows: - SEQ ID NO: 22, comprising (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 8 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1, and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 5; - SEQ ID NO: 23, comprising (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 8 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 12), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 5; - SEQ ID NO: 24, comprising (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 8 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 5; - SEQ ID NO: 25, which comprises SEQ ID NO: 12 (a nucleotide sequence encoding a truncated GAA polypeptide truncated by 8 consecutive amino acids at its N-terminus compared to the parent GAA polypeptide portion of SEQ ID NO: 1) and SEQ ID NO: 54 (a nucleotide sequence encoding sp7); - SEQ ID NO: 26, which comprises SEQ ID NO: 48 (a nucleotide sequence encoding a truncated GAA polypeptide truncated by 42 consecutive amino acids at its N-terminus compared to the parent GAA polypeptide portion of SEQ ID NO: 1) and SEQ ID NO: 54 (a nucleotide sequence encoding sp7); - SEQ ID NO: 37, comprising (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 29 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the non-optimized nucleotide sequence of SEQ ID NO: 9), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; - (i) SEQ ID NO: 38, comprising an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 29 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 12), and a signal peptide of SEQ ID NO: 3; - SEQ ID NO: 39, comprising (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 29 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 40, comprising (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 42 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the non-optimized nucleotide sequence of SEQ ID NO: 9), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 41, comprising (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 42 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 42, comprising (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 43 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the non-optimized nucleotide sequence of SEQ ID NO: 9), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 43, comprising (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 43 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 12), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; and - SEQ ID NO: 44, comprising (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 43 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 45, comprising (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 47 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the non-optimized nucleotide sequence of SEQ ID NO: 9), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; - SEQ ID NO: 46, comprising (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 47 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 12), and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3; and - SEQ ID NO: 47, comprising (i) an optimized nucleotide sequence (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 13) encoding a truncated GAA polypeptide truncated by 47 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1, and (ii) a nucleotide sequence encoding the signal peptide of SEQ ID NO: 3.

[0040] In another preferred embodiment, a nucleic acid molecule encoding a therapeutic GAA polypeptide comprising a GAA polypeptide portion of the present invention and a signal peptide portion fused to the N-terminus of the GAA polypeptide portion is as follows: - (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 8 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33, and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 29 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33, and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 42 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33, and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 43 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33, and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) a non-optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 47 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33, and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 8 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 8 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 29 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 29 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 42 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized sequence of SEQ ID NO: 12), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 42 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7. - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 43 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 12), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 43 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7. - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 47 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 12), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7; - (i) an optimized nucleotide sequence encoding a truncated GAA polypeptide truncated by 47 consecutive amino acids from its N-terminus compared to the parent hGAA of SEQ ID NO: 1 or compared to the parent hGAA of SEQ ID NO: 33 (a nucleotide sequence derived from the optimized nucleotide sequence of SEQ ID NO: 13), and (ii) a nucleotide sequence encoding a signal peptide of SEQ ID NO: 4, 5, 6 or 7.

[0041] In a preferred embodiment, the therapeutic GAA polypeptide encoded by the nucleic acid molecule comprises a GAA polypeptide portion and a signal peptide portion fused to the N-terminus of the GAA polypeptide portion, wherein the signal peptide portion fused to the N-terminus of the GAA polypeptide portion is selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, preferably SEQ ID NO: 3, and the GAA polypeptide portion is selected from SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36, preferably SEQ ID NO: 27.

[0042] Most preferably, the nucleic acid molecule is selected from SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47, preferably SEQ ID NO:25.

[0043] Preferably, a nucleic acid molecule encoding a therapeutic GAA polypeptide according to the present invention comprising a GAA polypeptide portion and a signal peptide portion fused to the N-terminus of the GAA polypeptide portion is as follows: - SEQ ID NO: 25, which comprises SEQ ID NO: 12 (a nucleotide sequence encoding a truncated GAA polypeptide truncated by 8 consecutive amino acids at its N-terminus compared to the parent GAA polypeptide portion of SEQ ID NO: 1) and SEQ ID NO: 54 (a nucleotide sequence encoding sp7), or - SEQ ID NO: 26, which comprises SEQ ID NO: 48 (a nucleotide sequence encoding a truncated GAA polypeptide truncated by 42 consecutive amino acids at its N-terminus compared to the parent GAA polypeptide portion of SEQ ID NO: 1) and SEQ ID NO: 54 (a nucleotide sequence encoding sp7).

[0044] A nucleic acid molecule encoding a therapeutic GAA polypeptide of the present invention can be inserted into a nucleic acid construct for expressing the nucleic acid molecule (i.e., a transgene). The nucleic acid construct may include a promoter operably linked to one or more expression control sequences and / or other sequences that improve expression of the nucleic acid molecule and / or sequences that enhance secretion and / or tissue uptake of the therapeutic GAA polypeptide. 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 into a functional relationship with another nucleic acid sequence. For example, a promoter or other transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Such expression control sequences are known in the art and include, for example, promoters, enhancers (e.g., cis-regulatory modules (CRMs)), introns, polyA signals, etc.

[0045] In particular, the nucleic acid construct comprises a promoter and, optionally, an intron operably linked to the nucleic acid molecule. The promoter may be a ubiquitous or tissue-specific promoter, particularly a promoter capable of promoting expression in cells or tissues in which GAA expression is desired, such as cells or tissues in which GAA expression is desired in GAA-deficient patients. In particular embodiments, the promoter is a liver-specific promoter, such as the alpha-1 antitrypsin promoter (hAAT) (SEQ ID NO: 14), the transthyretin promoter, the albumin promoter, the thyroxine-binding globulin (TBG) promoter, or the LSP promoter (comprising a thyroid hormone-binding globulin promoter sequence, two copies of the alpha-1-microglobulin / bikunin enhancer sequence, and a leader sequence

[17] ). Other useful liver-specific promoters are known in the art, including, for example, those listed in the Liver-Specific Gene Promoter Database compiled by Cold Spring Harbor Laboratory (http: / / rulai.cshl.edu / LSPD / ). A preferred promoter in the context of the present invention is the hAAT promoter. In another embodiment, the promoter is is a promoter that directs expression in one tissue or cell of interest (e.g., muscle cells) and liver cells. For example, muscle cell-specific promoters, such as the desmin, Spc5-12, and MCK promoters, may exhibit some leakage of expression to liver cells, which may be advantageous in inducing immune tolerance in a subject to the GAA polypeptide expressed from the nucleic acid of the present invention. Other tissue-specific or non-tissue-specific promoters may be useful in practicing the present invention. For example, the nucleic acid construct may include a tissue-specific promoter that is a promoter different from a liver-specific promoter. For example, the promoter may be muscle-specific, such as the desmin promoter (and desmin promoter variants, such as the desmin promoter, containing natural or artificial enhancers), the Spc5-12 promoter, or the MCK promoter.In another embodiment, the promoter is a promoter specific to another cell lineage, such as the erythropoietin promoter for expression of a GAA polypeptide from erythroid cells. In another embodiment, the promoter is a ubiquitous promoter. Representative ubiquitous promoters include the cytomegalovirus enhancer / chicken beta actin (CAG) promoter, the cytomegalovirus enhancer / promoter (CMV), the PGK promoter, the SV40 early promoter, and the like. Furthermore, the promoter may be an endogenous promoter, such as the albumin promoter or the GAA promoter. In certain embodiments, the promoter is associated with an enhancer sequence, such as a cis-regulatory module (CRM) or an artificial enhancer sequence. For example, the promoter may be associated with an enhancer sequence, such as the human ApoE regulatory region (or human apolipoprotein E / CI locus, hepatic regulatory region HCR-1—Genbank accession number U32510, set forth in SEQ ID NO: 15). In certain embodiments, the enhancer sequence, such as the ApoE sequence, is associated with a liver-specific promoter, such as those listed above, particularly the hAAT promoter. Other CRMs useful in the practice of the present invention include those described by Rincon et al.

[18] , Chuah et al.

[19] , or Nair et al.

[20] . In another embodiment, the promoter is a hybrid promoter. For example, the hybrid promoter is composed of a liver-selective enhancer operably linked to a short muscle-selective promoter, such as the spC5-12 promoter, the CK6 promoter, the CK8 promoter, or the Acta1 promoter. The liver-selective enhancer can be selected from HS-CRM1, HS-CRM2, HS-CRM3, HS-CRM4, HS-CRM5, HS-CRM6, HS-CRM7, HS-CRM8, HS-CRM9, HS-CRM10, HS-CRM11, HS-CRM12, HS-CRM13, and HS-CRM14, which can be repeated.Other examples of hybrid promoters include a combination of the ApoE enhancer, the hAAT promoter, and the spC5.12 promoter (shown in SEQ ID NO: 55); or a combination of the ApoE enhancer, the hAAT promoter, and the Syn promoter; or a combination of the ApoE enhancer and the spC5.12 promoter.

[0046] In another specific embodiment, the nucleic acid construct comprises an intron, particularly an intron located between the promoter and the GAA coding sequence. Introns can be introduced to increase mRNA stability and protein production. In a further embodiment, the nucleic acid construct comprises a human beta globin b2 (or HBB2) intron, a coagulation factor IX (FIX) intron, an SV40 intron, or a chicken beta globin intron. In yet another embodiment, the nucleic acid construct of the present invention contains a modified intron (particularly a modified HBB2 or FIX intron) designed to reduce or even completely remove the number of alternative open reading frames (ARFs) found in the intron. Preferably, ARFs spanning 50 bp in length and having a stop codon in frame with the start codon are removed. ARFs may be removed by modifying the intron sequence. For example, modifications may be made by nucleotide substitution, insertion, or deletion, preferably nucleotide substitution. By way of example, one or more nucleotides, particularly one nucleotide, in the ATG or GTG start codon present in the intron sequence of interest may be replaced, resulting in a non-start codon. For example, ATG or GTG may be replaced by CTG, which is not a start codon, within the sequence of the intron of interest.

[0047] The classical HBB2 intron used in the nucleic acid construct is set forth in SEQ ID NO: 16. For example, this HBB2 intron may be modified by eliminating the start codons (ATG and GTG codons) within the intron. In a specific embodiment, the modified HBB2 intron included in the construct has the sequence set forth in SEQ ID NO: 17. The classical FIX intron used in the nucleic acid construct is derived from the first intron of human FIX and is set forth in SEQ ID NO: 18. The FIX intron may be modified by eliminating the start codons (ATG and GTG codons) within the intron. In a specific embodiment, the modified FIX intron included in the construct of the invention has the sequence set forth in SEQ ID NO: 19. The classical chicken beta globin intron used in the nucleic acid construct is set forth in SEQ ID NO: 20. The chicken beta globin intron may be modified by eliminating the start codons (ATG and GTG codons) within the intron. In a specific embodiment, the modified chicken beta globin intron included in the construct of the invention has the sequence set forth in SEQ ID NO: 21. In a preferred embodiment, the intron used in the nucleic acid construct of the present invention is a modified HBB2 intron (SEQ ID NO: 17).

[0048] In certain embodiments, a nucleic acid construct of the invention comprises, in a 5' to 3' orientation, a promoter optionally preceded by an enhancer, a nucleic acid molecule encoding a therapeutic GAA polypeptide, and a polyadenylation signal (e.g., a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or another naturally occurring or artificial polyadenylation signal). In certain embodiments, a nucleic acid construct of the invention comprises, in a 5' to 3' orientation, a promoter optionally preceded by an enhancer (e.g., an ApoE regulatory region), an intron (particularly an intron as defined above), a nucleic acid molecule encoding a therapeutic GAA polypeptide, and a polyadenylation signal. In further specific embodiments, a nucleic acid construct of the invention comprises, in a 5' to 3' orientation, an enhancer such as an ApoE regulatory region, a promoter, an intron (particularly an intron as defined above), a nucleic acid molecule encoding a therapeutic GAA polypeptide, and a polyadenylation signal. In a further specific embodiment of the invention, the nucleic acid construct comprises, in the 5' to 3' direction, an ApoE regulatory region, a hAAT liver-specific promoter, an HBB2 intron (particularly a modified HBB2 intron as defined above), a nucleic acid molecule encoding a therapeutic GAA polypeptide, and a bovine growth hormone polyadenylation signal.

[0049] According to the present invention, the nucleic acid construct may be inserted into a vector, preferably a viral vector. The term "vector" according to the present invention refers to a vector suitable for protein expression, preferably suitable for use in gene therapy. In one embodiment, the vector is a plasmid vector. In another embodiment, the vector is a nanoparticle containing a nucleic acid molecule of the present invention, particularly a messenger RNA encoding a GAA polypeptide of the present invention. In another embodiment, the vector is a transposon-based system that allows integration of the nucleic acid molecule or construct of the present invention into the genome of a target cell, such as the hyperactive Sleeping Beauty (SB100X) transposon system

[21] . In another embodiment, the vector is a viral vector suitable for gene therapy. The vector may target any cell of interest, such as liver tissue or cells, muscle cells, CNS cells (e.g., brain cells or spinal cord cells), or hematopoietic stem cells, such as cells of the erythroid lineage (e.g., red blood cells). In this case, the nucleic acid construct of the present invention also contains sequences suitable for producing efficient viral vectors, as is well known in the art. In a preferred embodiment, the nucleic acid construct is inserted into a retroviral vector, such as a lentiviral vector, or an AAV vector, such as a single-stranded or double-stranded self-complementary AAV vector.In a highly preferred embodiment of the present invention, the viral vector is an AAV vector, such as an AAV vector suitable for transducing liver tissue or cells, more particularly AAV-1, -2, and AAV-2 variants (e.g., the quadruple mutant capsid-optimized AAV-2 comprising an engineered capsid with Y44+500+730F+T491V changes as disclosed in Ling et al.

[22] ), -3 and AAV-3 variants (e.g., the AAV3-ST variant comprising an engineered AAV3 capsid with two amino acid changes S663V+T492V as disclosed in Vercauteren et al.

[23] , -3B and AAV-3B variants, -4, -5, -6, and AAV-6 variants). Vectors or retroviral vectors such as lentiviral vectors and alpharetroviruses, such as AAV6 variants (e.g., AAV6 variants including the triple-mutated AAV6 capsid Y731F / Y705F / T492V form disclosed in Rosario et al.

[24] , -7, -8, -9, -10, e.g., -cy10 and -rh10, -rh74, -dj, Anc80, LK03, AAV2i8, porcine AAV serotypes, e.g., AAVpo4 and AAVpo6). As is known in the art, depending on the particular viral vector to be used, additional suitable sequences are introduced into the nucleic acid construct of the invention to obtain a functional viral vector. Suitable sequences include AAV ITRs for AAV vectors or LTRs for lentiviral vectors. Accordingly, the present invention also relates to nucleic acid constructs as described above, flanked on each side by ITRs or LTRs.

[0050] In addition, other non-naturally occurring engineered variants and chimeric AAVs may also be useful. Conventional molecular biology techniques can be used to engineer AAV viruses, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, minimized immunogenicity, tunable stability and particle lifespan, efficient degradation, and precise delivery to the nucleus. Desirable AAV fragments for assembly into vectors include cap proteins, including vp1, vp2, vp3, and hypervariable regions; rep proteins, including rep78, rep68, rep52, and rep40; and sequences encoding these proteins. These fragments can be easily utilized in a variety of vector systems and host cells. AAV-based recombinant vectors lacking Rep proteins integrate into the host genome with low efficiency, exist primarily as stable, circular episomes, and can persist in target cells for many years. As an alternative to using natural AAV serotypes, artificial AAV serotypes may be used in the context of the present invention, including, but not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be generated by any suitable technique using selected AAV sequences (e.g., fragments of the vp1 capsid protein) in combination with heterologous sequences that may be obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, a non-AAV viral source, or a non-viral source. The artificial AAV serotype can be, but is not limited to, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid.

[0051] In the context of the present invention, an AAV vector comprises an AAV capsid capable of transducing a target cell of interest, particularly a hepatocyte. In a further specific 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 may be a vector whose genome is derived from one of the above-mentioned AAV serotypes and whose capsid is derived from another serotype. For example, the genome of a pseudotyped vector may have a capsid derived from AAV8, AAV9, AAVrh74, or AAV2i8 serotype, or its genome may be derived from a different serotype. In a specific embodiment, the AAV vector has a capsid of AAV8, AAV9, or AAVrh74 serotype, particularly AAV8 or AAV9 serotype, more particularly AAV8 serotype.

[0052] In certain embodiments, where the vector is for use in delivering a transgene to a muscle cell, the AAV vector may be selected from the group consisting of AAV8, AAV9, and AAVrh74, among others.

[0053] In another specific embodiment, where the vector is for use in delivering a transgene to hepatocytes, the AAV vector may be selected from the group consisting of AAV5, AAV8, AAV9, AAV-LK03, AAV-Anc80, and AAV3B, among others.

[0054] In another embodiment, the capsid is a modified capsid. In the context of the present invention, a "modified capsid" may be a chimeric capsid or capsid comprising one or more variant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins. In certain embodiments, the AAV vector is a chimeric vector, i.e., its capsid comprises VP capsid proteins from at least two different AAV serotypes, or comprises at least one chimeric VP protein that combines VP protein regions or domains from at least two AAV serotypes. Examples of such chimeric AAV vectors useful for transducing hepatocytes are described in Shen et al.

[25] and Tenney et al.

[26] . For example, a chimeric AAV vector can be derived from a combination of AAV8 capsid sequences with sequences from an AAV serotype other than the AAV8 serotype, such as any of those specifically described above. In another embodiment, the capsid of the AAV vector comprises one or more mutant VP capsid proteins, such as those described in WO2015013313, particularly the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4 and RHM15-6 capsid variants, which exhibit enhanced liver tropism.

[0055] In another embodiment, modified capsids can also be derived from capsid modifications inserted by error-prone PCR and / or peptide insertion (e.g., as described in Bartel et al.

[27] or Michelfelder et al.

[28] ). In addition, capsid variants may contain single amino acid changes, such as tyrosine mutations (e.g., as described in Zhong et al.

[29] ). Another example is the fusion of anthopleurin-B to the N-terminus of the AAV VP2 capsid protein

[30] .

[0056] Additionally, the genome of an AAV vector can be either single-stranded or a self-complementary double-stranded genome.

[31] Self-complementary double-stranded AAV vectors are generated by deleting the terminal resolution site (trs) from one of the AAV terminal repeats. These modified vectors, whose replicating genome is half the length of the wild-type AAV genome, tend to package DNA dimers. In particular, the AVV vector has a capsid derived from AAV, such as AAV1, AAV2, variant AAV2, AAV3, variant AAV3, AAV3B, variant AAV3B, AAV4, AAV5, AAV6, variant AAV6, AAV7, AAV8, AAV9, AAV10, such as AAVcy10 and AAVrh10, AAVrh74, AAVdj, AAV-Anc80, AAV-LK03, AAV2i8, and porcine AAV, such as AAVpo4 and AAVpo6 capsids, or a chimeric capsid.

[0057] In a preferred embodiment, the AAV vector employed in the practice of the present invention has a single-stranded genome, and more preferably comprises an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly an AAV8, AAV9, or AAVrh74 capsid, e.g., an AAV8 or AAV9 capsid, more particularly an AAV8 capsid. In a particularly preferred embodiment, the present invention relates to an AAV vector comprising a nucleic acid construct of the present invention in a single-stranded or double-stranded self-complementary genome (e.g., a single-stranded genome). In one embodiment, the AAV vector comprises an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly an AAV8, AAV9, or AAVrh74 capsid, e.g., an AAV8 or AAV9 capsid, more particularly an AAV8 capsid.

[0058] Therapeutic GAA polypeptides, nucleic acid molecules encoding therapeutic GAA polypeptides, or nucleic acid constructs for expressing nucleic acid molecules can be prepared by methods known in the art. For example, WO2018 / 046774 and WO2018 / 04675 provide methods for preparing therapeutic GAA polypeptides, nucleic acid molecules encoding GAA polypeptides, and nucleic acid constructs for expressing nucleic acid molecules.

[0059] According to the present invention, the term "GAA uptake" or "uptake of a therapeutic GAA polypeptide" refers to the absorption of GAA by a cell or tissue. In one embodiment, the tissue is muscle, such as the heart, triceps, quadriceps, and diaphragm. In another embodiment, the tissue is a tissue of the nervous system. The term "nervous system tissue" refers to tissue containing nerve cells, such as motor neurons and glial cells. In other words, the nervous system consists of the central nervous system, including the brain and spinal cord, and the peripheral nervous system includes branched peripheral nerves; for example, a central nervous system tissue according to the present invention is the brain and / or spinal cord. GAA uptake can be measured by any means known in the art, such as Western blot as described in the Examples.

[0060] As used herein, the terms "subject," "patient," or "individual" refer to a human or non-human mammal (e.g., a rodent (mouse, rat), cat, dog, or primate) that is suffering from or may be suffering from GSD. Preferably, the subject is human, male or female.

[0061] The term "treat" or "treatment" means to reverse, alleviate, inhibit the progression of, or prevent the disease or condition to which the term applies, or one or more symptoms of the disease or condition. In particular, treating a disease may consist of treating central nervous system (CNS) disorders in GSD, preferably improving respiratory neuromuscular function and / or reducing respiratory dysfunction in a subject.

[0062] Kit of Parts The present invention relates to a kit-of-parts comprising (i) a pharmacological chaperone or a pharmaceutically acceptable salt thereof, and (ii) a therapeutic acid alpha-glucosidase (GAA) polypeptide or a nucleic acid molecule encoding a therapeutic GAA polypeptide, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof and ambroxol (ABX) or a derivative thereof. The kit of the present invention comprises: - As a medicine, - in the treatment of glycogen storage disease (GSD), - a method for improving respiratory neuromuscular function and / or reducing respiratory dysfunction, comprising: - in a method for treating central nervous system (CNS) disorders in GSD, and / or - may be used in a method for increasing uptake of GAA into the nervous system, preferably tissues of the central nervous system, most preferably the spinal cord, particularly in GSD, In this embodiment, the method may further stabilize GAA in a conformation suitable for transport of GAA to lysosomes.

[0063] The kit-of-parts may be used in different forms for the use of therapeutic GAA polypeptides (i.e., ERT) and for the use of nucleic acid molecules encoding therapeutic GAA polypeptides (i.e., gene therapy).

[0064] ERT using therapeutic GAA polypeptides ERT increases the amount of GAA polypeptide by exogenously introducing a therapeutic GAA polypeptide, preferably a therapeutic GAA polypeptide without a signal peptide, via injection. After injection, the exogenous therapeutic GAA polypeptide is expected to be taken up into tissues via non-specific or receptor-specific mechanisms.

[0065] According to the present invention, the pharmacological chaperone and the therapeutic GAA polypeptide may be administered simultaneously or separately. As described in more detail below, the pharmacological chaperone may be administered before, simultaneously with, and / or after the therapeutic GAA polypeptide.

[0066] Pharmacological chaperones increase the efficacy of therapeutic GAA polypeptides, for example, by increasing the stability of therapeutic GAA polypeptides in vivo in GSD patients and / or by increasing tissue uptake of therapeutic GAA polypeptides in vivo in GSD patients and in vitro in formulations or compositions. Pharmacological chaperones are useful for enhancing the therapeutic efficacy of conventional ERT, such as treatment with alglucosidase alfa.

[0067] The pharmacological chaperone may be administered orally, nasally, transdermally, or by parenteral injection, e.g., intravenous, subcutaneous, or intraperitoneal injection, preferably orally or intravenously, more preferably orally.

[0068] Therapeutic GAA polypeptides may be administered orally, nasally, transdermally, or by parenteral injection, e.g., intravenous, subcutaneous, or intraperitoneal injection, preferably intravenous or subcutaneous. More preferably, therapeutic GAA polypeptides are administered intravenously in a sterile solution for injection.

[0069] In one embodiment, the therapeutic GAA polypeptide and the pharmacological chaperone are formulated separately. In this embodiment, the pharmacological chaperone and the therapeutic GAA polypeptide may be administered by the same route, e.g., intravenous infusion, or preferably by different routes, e.g., intravenous infusion for the therapeutic GAA polypeptide and oral administration for the pharmacological chaperone. The therapeutic GAA polypeptide may be administered by any route, although preferably administration is parenteral.

[0070] In another embodiment, the pharmacological chaperone and the therapeutic GAA polypeptide are formulated into a single composition. Such a composition reduces costs and increases therapeutic efficacy by increasing the stability of the therapeutic GAA polypeptide during storage and in vivo administration. The formulation is preferably suitable for parenteral administration, including intravenous, subcutaneous, and intraperitoneal administration, although formulations suitable for other routes of administration, such as oral, intranasal, or transdermal, are also contemplated.

[0071] The pharmacological chaperones may be formulated in the same composition or separately, preferably separately. For example, one of the pharmacological chaperones (DNJ or ABX) and the therapeutic GAA polypeptide may be formulated in one composition, and the other chaperone (DNJ or ABX) may be formulated in another composition. In another example, the chaperones (ABX and DNJ) and the therapeutic GAA polypeptide are formulated in three separate compositions.

[0072] In some embodiments, DNJ is formulated into a separate composition, such as the composition marketed under the name Zavesca® (corresponding to miglustat (CAS number 72599-27-0)). Pharmaceutical compositions comprising DNJ are described in WO2006 / 125141 and WO2014 / 110270.

[0073] In some embodiments, ABX is formulated in a separate composition, such as those marketed under the names Ambrobene®, Aponova®, Mucoangin®, and Ambroxol Mylan®. Pharmaceutical compositions containing ABX are described in EP 1543826.

[0074] In some embodiments, the therapeutic GAA polypeptide is formulated in a separate composition, such as those commercially available under the name Lumizyme® or Myozyme®.

[0075] The timing of administration will vary based on several factors, including, but not limited to, the route of administration, the GSD being treated, or the age of the subject. One of ordinary skill in the art can readily determine the required timing of administration based on these factors and others, based on knowledge in the art.

[0076] When the therapeutic GAA polypeptide and the pharmacological chaperone are formulated separately, administration may be simultaneous, or the pharmacological chaperone may be administered before or after the therapeutic GAA polypeptide. For example, if the therapeutic GAA polypeptide is administered intravenously, the pharmacological chaperone may be administered between hours 0 and 6 hours later. Alternatively, the pharmacological chaperone may be administered between hours 0 and 6 hours before the therapeutic GAA polypeptide. In preferred embodiments where the pharmacological chaperone and the therapeutic GAA polypeptide are administered separately and the pharmacological chaperone has a short circulating half-life (e.g., a small molecule), the pharmacological chaperone may be administered orally continuously, e.g., daily, to maintain a constant level in the circulation. Such a constant level is determined to be optimal with respect to interaction with the therapeutic GAA polypeptide during the time of administration to provide a non-toxic, non-inhibitory therapeutic effect to the patient. In another embodiment, the pharmacological chaperone is administered during the period required for turnover of the therapeutic GAA polypeptide (which is extended by the administration of the pharmacological chaperone).

[0077] The dose of a therapeutic GAA polypeptide administered to a subject in need thereof varies based on several factors, including, but not limited to, the route of administration, the GSD being treated, and the age of the subject. Those skilled in the art can easily determine the required dosage range based on these factors and other factors, based on their knowledge in the art. According to current methods, the concentration of a therapeutic GAA polypeptide is generally about 0.05 to 50.0 mg / kg of body weight, and is usually administered weekly or biweekly. Therapeutic GAA polypeptides can be administered at doses ranging from 0.1 mg / kg to about 30 mg / kg, preferably about 0.1 mg / kg to about 20 mg / kg. Periodic repeated administration of the protein may be necessary throughout the patient's life. Subcutaneous injections maintain systemic exposure to the therapeutic GAA polypeptide for longer periods. Subcutaneous administration is preferably 0.1 to 5.0 mg of therapeutic GAA polypeptide per kg of body weight, administered biweekly or weekly. Therapeutic GAA polypeptides may also be administered intravenously, for example, by intravenous bolus injection, slow push intravenous injection, or continuous intravenous infusion. Continuous IV infusion (e.g., over 2-6 hours) allows for maintenance of specific levels in the blood. For example, a therapeutic GAA polypeptide lacking a signal peptide, called alglucosidase alfa (sold as Lumizyme® or Myozyme® by Genzyme, Inc.), currently approved for the treatment of Pompe disease, is administered by intravenous infusion at a dose of 20 mg / kg body weight every two weeks.

[0078] The dose of pharmacological chaperone administered to a subject in need thereof will vary based on several factors, including, but not limited to, the route of administration, the GSD being treated, the age of the subject, or the amount of therapeutic GAA polypeptide administered to the subject. One of ordinary skill in the art can readily determine the required dosage range based on these factors and others, based on knowledge in the art.

[0079] The dosage of a pharmacological chaperone effective in treating glycogen storage disease can be determined by standard clinical techniques. Additionally, in vivo and / or in vitro assays may be employed, if appropriate, to help predict optimal dosage ranges. The precise dosage employed in a 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 each patient's circumstances. The dosage of a pharmacological chaperone administered to a subject in need thereof will vary based on several factors, including, but not limited to, the route of administration, the particular disease being treated, and the age of the subject. Those skilled in the art can readily determine the required dosage range based on these and other factors, based on their knowledge in the art.

[0080] For treatments involving administration of DNJ or a derivative thereof, preferably DNJ, a typical dose of DNJ or a derivative thereof can be, for example, 1 gram (g), 2 g, 3 g, 4 g, 5 g, 6 g, or more for 1 to 10 days, preferably 3 to 7 days. Treatment can be interrupted by 1 to 10 days, preferably 3 to 7 days, without treatment. After the interruption, treatment can be administered according to the previous typical dose and duration as described above. For example, a typical dose is 2.5 g for 3 days and no treatment for 4 days, e.g., 5 g for 3 days and no treatment for 4 days, or, e.g., 5 g for 7 days and no treatment for 7 days (clinical trial identification number NCT00688597).

[0081] For treatments involving the administration of ABX or a derivative thereof, preferably ABX hydrochloride, a typical dose of ABX or a derivative thereof may be 50-500 mg, preferably 60-420 mg, repeated, for example, three times per day (clinical trial identification number NCT02941822).

[0082] Gene therapy using nucleic acid molecules encoding therapeutic GAA polypeptides The present invention also contemplates the use of pharmacological chaperones in combination with gene therapy in GSD, where such combinations enhance the efficacy of gene therapy by increasing the expression levels of therapeutic GAA polypeptides in vivo in GSD patients, by increasing the stability of therapeutic GAA polypeptides expressed in vivo in GSD patients, and / or by increasing tissue uptake of therapeutic GAA polypeptides expressed in vivo in GSD patients.

[0083] According to the present invention, the pharmacological chaperone and the nucleic acid molecule encoding a therapeutic GAA polypeptide may be administered simultaneously or separately. As described in more detail below, the pharmacological chaperone may be administered before, simultaneously with, and / or after the nucleic acid molecule encoding the therapeutic GAA polypeptide.

[0084] In a preferred embodiment, the pharmacological chaperone is administered after the nucleic acid molecule encoding the therapeutic GAA polypeptide. For example, the pharmacological chaperone can be administered one month, two months or more after the administration of the nucleic acid molecule encoding the therapeutic GAA polypeptide. According to this embodiment, the pharmacological chaperone and the nucleic acid molecule encoding the therapeutic GAA polypeptide are formulated separately. Delivery of nucleic acid molecules to a patient can be direct, in which case the patient is directly exposed to the nucleic acid molecule, nucleic acid construct, or vector, e.g., a viral vector, or indirect, in which cells are first transformed in vitro with the nucleic acid molecule, nucleic acid construct, or vector, e.g., a viral vector, and then transplanted into the patient. These two approaches are known as in vivo and ex vivo gene therapy, respectively. In the case of delivery of hepatocytes, the cells may be cells previously obtained from the subject and engineered by introducing into them a nucleic acid molecule or nucleic acid construct encoding a therapeutic GAA polypeptide, thereby enabling them to produce the therapeutic GAA polypeptide.

[0085] In some embodiments, the nucleic acid molecule is inserted into a nucleic acid construct for expressing the nucleic acid molecule, and the nucleic acid construct is inserted into a viral vector selected from a retroviral vector, e.g., a lentiviral vector, or an AAV vector, e.g., a single-stranded or double-stranded self-complementary AAV vector, wherein the viral vector is preferably an AAV vector having an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly an AAV8, AAV9, or AAVrh74 capsid, more particularly an AAV8 capsid.

[0086] Administration of a nucleic acid molecule, nucleic acid construct, or viral vector encoding a therapeutic GAA polypeptide can be via, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. In certain embodiments, administration is via the intravenous or intramuscular route. A nucleic acid molecule encoding a therapeutic GAA polypeptide, whether vectorized or not, can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can also be administered in conjunction with other biologically active agents. Administration can be systemic or local. In specific embodiments, it may be desirable to administer a nucleic acid molecule, nucleic acid construct, or viral vector encoding a therapeutic GAA polypeptide locally to an area requiring treatment, e.g., the liver. This can be achieved, for example, by means of an implant, which can be a porous, non-porous, or gelatinous material, such as a membrane, e.g., a sialastic membrane, or a fiber.

[0087] The amount of a nucleic acid construct, vector molecule, nucleic acid construct, or viral vector encoding a therapeutic GAA polypeptide that will be effective in treating glycogen storage disease can be determined by standard clinical techniques. Additionally, in vivo and / or in vitro assays may be employed, in some cases, to help predict optimal dosage ranges. The precise dosage employed in a 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 physician and each patient's circumstances. The dosage of a nucleic acid construct, vector molecule, nucleic acid construct, or viral vector encoding a therapeutic GAA polypeptide administered to a subject in need thereof will vary based 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 level required to achieve a therapeutic effect. Those skilled in the art can readily determine the required dosage range based on these factors and other factors, based on their knowledge in the art. For treatments involving administering a viral vector, e.g., an AAV vector, to a subject, a typical dosage of the vector is at least 1 x 10 per kilogram of body weight. 8 of vector genome (vg / kg), e.g., at least 1 × 10 9 vg / kg, at least 1 × 10 10 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. By virtue of the pharmacological chaperone, the dose of a nucleic acid construct, vector molecule, nucleic acid construct or viral vector encoding a therapeutic GAA polypeptide of the present invention can be reduced compared to typical doses.

[0088] In one aspect of the present invention, a subject receives repeated administrations of a nucleic acid molecule encoding a therapeutic GAA polypeptide. In this aspect, the administration may be repeated at least once or more, and may even be considered to be administered according to a periodic schedule, such as once a year. A periodic schedule may also include administration every 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, or more than every 10 years. In another specific embodiment, each administration of the viral vector of the present invention is administered using a different virus for each successive administration, thereby avoiding a decrease in efficacy, because an immune response to the previously administered viral vector is possible. For example, a first administration of a viral vector containing an AAV8 capsid may be followed by administration of a vector containing an AAV9 capsid, or even administration of a virus unrelated to AAV, such as a retroviral or lentiviral vector. Alternatively, transient immunosuppression can be used to avoid an immune response to the capsid.

[0089] Methods of gene therapy are known in the art, and some embodiments are also detailed in the definition section above.

[0090] The pharmacological chaperone may be administered orally, nasally, transdermally, or by parenteral injection, e.g., intravenous, subcutaneous, or intraperitoneal injection, preferably orally or intravenously, more preferably orally.

[0091] The pharmacological chaperones may be formulated in the same composition or separately, preferably separately. DNJ may be formulated in a separate pharmaceutical composition, such as the one marketed under the name Zavesca® (corresponding to miglustat (CAS number 72599-27-0)). Pharmaceutical compositions containing DNJ are described in WO2006 / 125141 and WO2014 / 110270. ABX may be formulated in a separate pharmaceutical composition, such as the one marketed under the name Ambrobene®, Aponova®, Mucoangin®, Ambroxol Mylan®. Pharmaceutical compositions containing ABX are described in EP1543826.

[0092] The pharmacological chaperone may be administered orally continuously, e.g., daily, to maintain a constant level in the circulation, which is determined to be optimal with respect to interaction with the therapeutic GAA polypeptide expressed during the time of administration to provide a non-toxic, non-inhibitory therapeutic effect to the patient.

[0093] Doses of pharmacological chaperones that may be effective in treating glycogen storage diseases according to the present invention are described above under "ERT using therapeutic GAA polypeptides."

[0094] In a preferred embodiment, the kit of parts comprises (i) duvoglustat and ambroxol hydrochloride, and (ii) a viral vector into which a nucleic acid construct for expressing a nucleic acid molecule encoding a therapeutic GAA polypeptide has been inserted. In an even more preferred embodiment, the kit-of-parts comprises (i) duvoglustat and ambroxol hydrochloride, and (ii) a viral vector into which a nucleic acid construct for expressing the nucleic acid molecule of SEQ ID NO: 25 or SEQ ID NO: 26 has been inserted.

[0095] composition The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in treating glycogen storage disease (GSD) in a subject receiving therapeutic acid alpha-glucosidase (GAA) treatment for treating said GSD, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof and ambroxol (ABX) or a derivative thereof.

[0096] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for increasing GAA uptake into nervous system tissue in a subject receiving therapeutic GAA treatment for treating GSD, wherein the pharmacological chaperone is DNJ or a derivative thereof and ABX or a derivative thereof. In some embodiments, the nervous system tissue is, for example, a central nervous system tissue, preferably the spinal cord. In some embodiments, the method further stabilizes GAA in a conformation suitable for transport to lysosomes.

[0097] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for improving respiratory neuromuscular function and / or reducing respiratory dysfunction in a subject undergoing therapeutic GAA treatment for treating GSD, wherein the pharmacological chaperone is DNJ or a derivative thereof and ABX or a derivative thereof.

[0098] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for treating central nervous system (CNS) disorders in a subject undergoing therapeutic GAA treatment for treating GSD, wherein the pharmacological chaperone is DNJ or a derivative thereof and ABX or a derivative thereof.

[0099] According to the present invention, a composition is administered to a subject undergoing therapeutic GAA treatment. As described above, the therapeutic GAA treatment may be a therapeutic GAA polypeptide (ERT), preferably a therapeutic GAA polypeptide without a signal peptide, or a nucleic acid molecule encoding a therapeutic GAA polypeptide (gene therapy). Preferably, the composition is administered to a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide. In some embodiments, the nucleic acid molecule is inserted into a nucleic acid construct for expressing the nucleic acid molecule, and the nucleic acid construct is inserted into a viral vector selected from a retroviral vector, e.g., a lentiviral vector, or an AAV vector, e.g., a single-stranded or double-stranded self-complementary AAV vector, and the viral vector is preferably an AAV vector having an AAV8, AAV9, AAVrh74, or AAV2i8 capsid, particularly an AAV8, AAV9, or AAVrh74 capsid, more particularly an AAV8 capsid.

[0100] The compositions of the present invention may be administered before, simultaneously with, and / or after a therapeutic GAA treatment, preferably before, simultaneously with, and / or after a nucleic acid molecule encoding a therapeutic GAA polypeptide. For example, if a therapeutic GAA polypeptide is administered intravenously, the compositions may be administered during the period from 0 to 6 hours after treatment. In preferred embodiments where the compositions of the present invention and the therapeutic GAA polypeptide are administered separately and the pharmacological chaperone has a short circulating half-life (e.g., a small molecule), the compositions may be administered orally continuously, e.g., daily, to maintain a constant level in the circulation. Such a constant level is determined to be optimal with respect to interaction with the therapeutic GAA polypeptide during the time of administration to provide a non-toxic, non-inhibitory therapeutic effect to the patient. In another embodiment, the compositions are administered during the period required for turnover of the therapeutic GAA polypeptide (which is extended by administration of a pharmacological chaperone). For example, if a nucleic acid molecule encoding a therapeutic GAA polypeptide is administered intravenously or intramuscularly, the compositions of the present invention may be administered two months after treatment and may be administered orally continuously, e.g., daily, to maintain a constant level in the circulation. The compositions of the present invention may be administered orally, nasally, transdermally, or by parenteral injection, for example, intravenous, subcutaneous, or intraperitoneal injection, preferably orally or intravenously, more preferably orally. Each pharmacological chaperone of the composition may be formulated separately or in the same composition, for example, one pharmacological chaperone, DNJ, may be formulated in one composition and the other chaperone, ABX, may be formulated in another composition.

[0101] In one embodiment, ABX and DNJ are formulated separately. In this embodiment, the separate compositions may be administered by the same route or by different routes, preferably by the same route, more preferably by oral administration.

[0102] In another embodiment, ABX and DNJ are formulated in the same composition, in which the composition is suitable for oral, intranasal, or transdermal administration, preferably oral administration.

[0103] DNJ may be formulated into a separate pharmaceutical composition, such as the one marketed under the name Zavesca® (corresponding to miglustat (CAS number 72599-27-0)). Pharmaceutical compositions containing DNJ are described in WO2006 / 125141 and WO2014 / 110270.

[0104] ABX may be formulated in a separate pharmaceutical composition, such as those marketed under the names Ambrobene®, Aponova®, Mucoangin®, Ambroxol Mylan®. Pharmaceutical compositions containing ABX are described in EP 1543826.

[0105] The dose of the pharmacological chaperone in the composition administered to a subject in need thereof will vary based on several factors, including, but not limited to, the route of administration, the GSD being treated, the age of the subject, or the therapeutic GAA treatment (e.g., ERT or gene therapy). One of ordinary skill in the art can easily determine the required dosage range based on these factors and other factors, based on knowledge in the art. For example, the composition of the present invention may contain 1 g, 2 g, 3 g, 5 g, 6 g, or more of DNJ or a derivative thereof and 50 to 500 mg, e.g., 60 to 420 mg, of ABX or a derivative thereof.

[0106] In a preferred embodiment, the composition comprises duvoglustat and ambroxol hydrochloride for use in treating GSD in a subject receiving a therapeutic GAA treatment, wherein the therapeutic GAA treatment is a viral vector into which a nucleic acid molecule encoding a therapeutic GAA polypeptide has been inserted for treating said GSD.

[0107] In a further preferred embodiment, the composition comprises duvoglustat and ambroxol hydrochloride for use in the treatment of GSDII in a subject receiving a viral vector into which a nucleic acid construct for expressing a nucleic acid molecule of SEQ ID NO: 25 or SEQ ID NO: 26 for treating said GSDII has been inserted.

[0108] Treatment method The present invention also relates to a method for treating glycogen storage disease (GSD) in a subject undergoing therapeutic GAA treatment for treating said GSD, comprising administering to the subject a composition comprising pharmacological chaperones, wherein the pharmacological chaperones are DNJ or a derivative thereof and ABX or a derivative thereof.

[0109] The present invention also relates to a method for increasing GAA uptake into tissues of the nervous system in a subject undergoing therapeutic GAA treatment for treating GSD, the method comprising administering to the subject a composition comprising pharmacological chaperones, wherein the pharmacological chaperones are DNJ or a derivative thereof and ABX or a derivative thereof.

[0110] The present invention also relates to a method for improving respiratory neuromuscular function and / or reducing respiratory dysfunction in a subject undergoing therapeutic GAA treatment for treating GSD, the method comprising administering to the subject a composition comprising a pharmacological chaperone, wherein the pharmacological chaperone is DNJ or a derivative thereof and ABX or a derivative thereof.

[0111] The present invention also relates to a method for treating central nervous system (CNS) disorders of GSD in a subject receiving therapeutic GAA treatment for treating GSD, the method comprising administering to the subject a composition comprising pharmacological chaperones, wherein the pharmacological chaperones are DNJ or a derivative thereof and ABX or a derivative thereof.

[0112] Definitions and embodiments regarding the administration of pharmacological chaperones and therapeutic GAA treatments are described herein above, particularly in the "Kit of Parts" section.

[0113] Other purposes The present inventors also demonstrated that the pharmacological chaperones DNJ, ABX, or NAC increased GAA uptake into tissues, particularly in GSD.

[0114] Nucleic acid molecules encoding DNJ and therapeutic GAA polypeptides The present invention relates to a kit-of-parts comprising (i) a pharmacological chaperone or a pharmaceutically acceptable salt thereof, and (ii) a nucleic acid molecule encoding a therapeutic GAA polypeptide, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof. - As a medicine, in the treatment of glycogen storage diseases (GSD), and / or - in a method for increasing GAA uptake into tissues of the nervous system, particularly in GSD; may be used.

[0115] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in treating glycogen storage disease (GSD) in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating said GSD, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof.

[0116] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for increasing GAA uptake into tissues of the nervous system in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating GSD, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof.

[0117] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for increasing uptake of GAA into the nervous system, preferably tissue of the central nervous system, more preferably the spinal cord, in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating GSD, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof.

[0118] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for treating a central nervous system (CNS) disorder of GSD in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating GSD, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof.

[0119] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for improving respiratory neuromuscular function and / or reducing respiratory dysfunction in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating GSD, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof.

[0120] The present invention also relates to a method for treating glycogen storage disease (GSD) in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating said GSD, comprising administering to the subject a composition comprising a pharmacological chaperone, wherein the pharmacological chaperone is DNJ or a derivative thereof.

[0121] The present invention also relates to a method for increasing GAA uptake into tissues of the nervous system in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating GSD, the method comprising administering to the subject a composition comprising a pharmacological chaperone, wherein the pharmacological chaperone is DNJ or a derivative thereof.

[0122] The present invention also relates to a method for improving respiratory neuromuscular function and / or reducing respiratory dysfunction in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating GSD, the method comprising administering to the subject a composition comprising a pharmacological chaperone, wherein the pharmacological chaperone is DNJ or a derivative thereof.

[0123] The present invention also relates to a method for treating a central nervous system (CNS) disorder of a GSD in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating the GSD, the method comprising administering to the subject a composition comprising a pharmacological chaperone, wherein the pharmacological chaperone is DNJ or a derivative thereof.

[0124] Nucleic acid molecules encoding NAC and therapeutic GAA polypeptides The present invention also relates to a kit-of-parts comprising (i) a pharmacological chaperone or a pharmaceutically acceptable salt thereof and (ii) a nucleic acid molecule encoding a therapeutic GAA polypeptide, wherein the pharmacological chaperone is N-acetylcysteine ​​(NAC) or a derivative thereof, which kit-of-parts may be used as a medicine, particularly in the treatment of glycogen storage disease (GSD).

[0125] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in treating glycogen storage disease (GSD) in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating said GSD, wherein the pharmacological chaperone is N-acetylcysteine ​​(NAC) or a derivative thereof.

[0126] The present invention also relates to a method for treating glycogen storage disease (GSD) in a subject receiving a nucleic acid molecule encoding a therapeutic GAA polypeptide for treating said GSD, comprising administering to the subject a composition comprising a pharmacological chaperone, wherein the pharmacological chaperone is NAC or a derivative thereof.

[0127] ABX and a therapeutic GAA polypeptide or a nucleic acid molecule encoding a therapeutic GAA polypeptide The present invention also relates to a kit-of-parts comprising (i) a pharmacological chaperone or a pharmaceutically acceptable salt thereof, and (ii) a therapeutic GAA polypeptide or a nucleic acid molecule encoding a therapeutic GAA polypeptide, wherein the pharmacological chaperone is ambroxol (ABX) or a derivative thereof, which kit-of-parts may be used as a medicine, particularly in the treatment of glycogen storage disease (GSD).

[0128] The present invention also relates to a composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in treating glycogen storage disease (GSD) in a subject receiving therapeutic acid alpha-glucosidase (GAA) treatment for treating said GSD, wherein the pharmacological chaperone is ambroxol (ABX) or a derivative thereof.

[0129] The present invention also relates to a method for treating glycogen storage disease (GSD) in a subject receiving therapeutic GAA treatment for treating said GSD, comprising administering to the subject a composition comprising a pharmacological chaperone, wherein the pharmacological chaperone is ABX or a derivative thereof.

[0130] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way.

[0131] Example Example 1: Studies on WT mice Materials and Methods Construction of AAV8-sp7-Δ8-coGAA vector expressing therapeutic human GAA polypeptide (hGAA)

[0132] AAV vector production AAV8 vectors were produced using an adenovirus-free transient transfection method (Matsushita et al.,

[32] ) and purified as previously described (Ayuso et al.,

[33] ). The titer of the AAV vector stock was determined using quantitative real-time PCR (qPCR) and confirmed by SDS-PAGE followed by SYPRO® Ruby protein gel staining and band densitometry. The nucleic acid construct was inserted into an AAV vector, specifically an expression cassette sequence containing two ITRs and a bGH polyA. From 5' to 3', the nucleic acid construct contains the ApoE regulatory region SEQ ID NO: 15, the hAAT liver-specific promoter SEQ ID NO: 14, the modified HBB2 intron SEQ ID NO: 17, and the nucleic acid molecule SEQ ID NO: 25. The resulting AAV8 vector is referred to as the AAV8-sp7-Δ8-co1GAA vector.

[0133] In vivo studies Standard animal care and husbandry followed national guidelines. Animal experiments were approved by the Ethics Committee of CERFE in accordance with European Directive 2010 / 63 / EU (approval number: 2015008D). Six- to eight-week-old C57Bl / 6 male mice were divided into nine groups of five mice. On day 0, awake, restrained animals were injected with 5 × 10 11 The AAV8-sp7-Δ8-co1GAA vector was administered intravenously at 1000 mg / kg. Two months after AAV treatment, mice were orally administered seven pharmacological chaperone molecules or their combinations using a "3 on / 4 off" regimen (3 consecutive days of treatment followed by 4 consecutive days of drinking water only) for 4 weeks. At the end of this 4-week period, all mice were sacrificed and different organs and muscles (liver, heart, brain, spinal cord, diaphragm, quadriceps, and triceps) were harvested. C57Bl / 6 mice were treated with either PBS (CTRL) or 5 × 10 11 vg / kg of either an AAV8 vector expressing a secreted therapeutic hGAA polypeptide (AAV-GAA).

[0134] Two months after vector injection, mice were treated with 100 mg / kg / day (die) duvoglustat (DNJ, AX61Q1, Interchim, San Diego, CA), 100 mg / kg / day duvoglustat in combination with 25 mg / kg / day ambroxol (DNJ-ABX), or 25 mg / kg / day ambroxol hydrochloride (ABX, A9797, Sigma, Saint-Joseph, CA) dissolved in drinking water. Mice were treated with 2 mg / kg / day of voglibose (VOGLIBOSE, S4101, Selleckchem, Houston, TX), 20 mg / kg / day of acarbose (ACARBOSE, S1271, Selleckchem, Houston, TX), 2 mg / kg / day of miglitol (MIGLITOL, S2589, Selleckchem, Houston, TX), or 4200 mg / kg / day of N-acetyl-cysteine ​​(NAC, A7250, Sigma, Saint Louis, MO) for 4 weeks (Figure 1). One group received regular drinking water.

[0135] Three months after vector injection, mice were sacrificed and the levels of human GAA (hGAA) in blood and tissues were analyzed by Western blot.

[0136] Plasma collection Blood samples were collected 3 months after vector injection by retroorbital blood collection into heparinized capillary tubes followed by plasma separation.

[0137] Tissue collection At the end of the study (3 months after injection), animals were sacrificed by CO2 inhalation. Liver, heart, brain, spinal cord, diaphragm, quadriceps, and triceps muscles were harvested and snap-frozen in liquid nitrogen for biochemical analysis. Frozen samples were stored at -80°C until processing.

[0138] Western blot analysis Mouse tissues were collected at the time of sacrifice and homogenized in PBS. Protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, Waltham, MA). Plasma and tissue samples were diluted 1:20 in water (except for brain, which was diluted 1:200). SDS-page electrophoresis was performed on a 4-12% gradient polyacrylamide gel. After transfer, the membrane was blocked with Odyssey buffer (Li-Cor Biosciences, Lincoln, NE) and incubated with anti-human GAA antibody (rabbit monoclonal antibody, Abcam, Cambridge, UK) and anti-GAPDH antibody (rabbit polyclonal antibody, PA1-988, Life Technologies, Carlsbad, CA). The membranes were washed, incubated with the appropriate secondary antibody (Li-Cor Biosciences, Lincoln, NE), and visualized using an Odyssey imaging system (925-32213, Li-Cor Biosciences, Lincoln, NE). To measure GAA uptake into tissues, the 70 kDa band (mature form) visualized with anti-GAA antibody was quantified and normalized to the expression level of GAPDH, which was used as a loading control. Animals that exhibited low plasma GAA levels before the initiation of pharmacological chaperone treatment were excluded from the analysis.

[0139] result We observed a significant increase in circulating hGAA levels in mice treated with DNJ and NAC, as measured by Western blot (Figure 2). In tissues, after uptake, the immature form of hGAA secreted by the liver was taken up and modified by proteolytic cleavage to the 70 kDa form. The 70 kDa form (mature form) was obtained only after proteolytic digestion of the precursor in lysosomes. Therefore, this form is intracellular, and its quantification allows estimation of uptake into tissues. Quantification of the mature lysosomal form of hGAA in tissues (by measuring band density on Western blot) showed increased enzyme levels in the diaphragm, triceps, and spinal cord of mice treated with DNJ and DNJ-ABX, which reached significance only in the triceps for the DNJ-treated group (Figure 3).

[0140] No differences were observed in heart, quadriceps, and brain.

[0141] Interestingly, the combination of DNJ and ABX led to a significant increase in hGAA levels in the spinal cord compared with the levels measured in mice receiving DNJ alone or water (Figure 3). These data support the synergistic effect of combined administration of DNJ and ABX to enhance the efficacy of gene therapy.

[0142] Example 2: Studies on GAA KO (knockout or deficient) mice Materials and Methods AAV vector production AAV8 vectors were produced as described above in Example 1.

[0143] In vivo studies Standard animal care and husbandry followed national guidelines. Animal experiments were approved by the Ethics Committee of CERFE in accordance with European Directive 2010 / 63 / EU (approval number: 2017-11-B #13643). GAA-deficient male mice (3–4 months old) were divided into six groups, including one control group consisting of wild-type littermates. Each group consisted of up to eight mice in two cages to facilitate administration of drugs dissolved in drinking water. Starting from day 0, GAA-deficient (KO) mice (mouse strain B6; 129-Gaa tm1Rabn / J) in combination with different pharmacological chaperones (PC) dissolved in drinking water, via tail vein injection. 11 The mice were injected with AAV-hGAA at a dose of 1000 mg / kg. In parallel, two groups of untreated GAA wild-type mice and GAA-deficient mice injected with the AAV-hGAA vector or PBS were used as controls. As shown in Figure 4, the PC molecule was orally administered to the mice using a "3-day on / 4-day off" regimen consisting of 3 consecutive days of treatment followed by 4 consecutive days of drinking water alone. Mice received 100 mg / kg / day duvoglustat (DNJ, AX61Q1, Interchim, San Diego, CA) dissolved in drinking water, 100 mg / kg / day duvoglustat in combination with 25 mg / kg / day ambroxol hydrochloride (DNJ-ABX), 25 mg / kg / day ambroxol hydrochloride (ABX, A9797, Sigma, Saint Louis, MO), 4200 mg / kg / day N-acetyl-cysteine ​​(NAC, A7250, Sigma, Saint Louis, MO) (Figure 4), or regular drinking water. Circulating hGAA activity and levels were monitored over a two-month period. Mice were sacrificed and different organs and muscles (heart, diaphragm, quadriceps, and triceps) were harvested to measure tissue GAA uptake and glycogen clearance.

[0144] Plasma collection Blood samples were collected by retroorbital bleeding into heparinized capillary tubes followed by plasma separation.

[0145] Tissue collection At the end of the study (2 months after injection), animals were sacrificed by CO2 inhalation. Heart, diaphragm, quadriceps, and triceps muscles were harvested and snap-frozen in liquid nitrogen for biochemical analysis. Frozen samples were stored at -80°C until processing.

[0146] Measurement of GAA activity GAA activity in plasma and tissues was assessed by measuring 4-methylumbelliferyl-α-d-glucoside (4-MU, Sigma, St. Louis, MO) cleavage at pH 4.3. For this purpose, mouse tissues collected at the time of sacrifice were homogenized in PBS. Insoluble proteins were removed by centrifugation. The protein content of the resulting lysates was quantified using the BCA protein assay (Thermo Fisher Scientific, Waltham, MA). Plasma and tissue samples were diluted with water, and 10 μL of each sample was incubated with 20 μL of reconstituted substrate for 1 h at 37°C. After the reaction was stopped, emitted fluorescence was measured using an EN-SPIRE® fluorometer (Perkin Elmer, Waltham, MA). GAA activity was normalized to total protein content or plasma volume.

[0147] Western blot analysis At the time of sacrifice, mouse tissues were collected and homogenized in PBS. Western blot analysis was performed according to the method described in Example 1.

[0148] Analysis of glycogen content Glycogen content was measured indirectly in tissue homogenates as glucose released after complete digestion with Aspergillus Niger amyloglucosidase (Sigma-Aldrich). Samples were incubated at 95°C for 5 minutes and then cooled to 4°C. Next, 25 μl of amyloglucosidase diluted 1:50 in 0.1 M potassium acetate, pH 5.5, was added to each sample. A control reaction without amyloglucosidase was prepared for each sample. Both sample and control reactions were incubated at 37°C for 90 minutes. The reactions were stopped by incubating the samples at 95°C for 5 minutes. Released glucose was determined using a glucose assay kit (Sigma-Aldrich) and measuring the resulting absorbance at 540 nm on an EnSpire Alpha plate reader (Perkin-Elmer).

[0149] result We observed a significant increase in circulating hGAA levels in mice treated with DNJ and the combination of DNJ-ABX, as measured by Western blot (Figure 5). These results are consistent with the results of circulating GAA activity (Figure 6). In tissues, GAA activity measurements showed increased enzyme activity in the heart, diaphragm, triceps, and quadriceps of mice treated with DNJ and DNJ-ABX, which only reached significance in the group of mice treated with both DNJ and ABX (Figures 7A-7D). No significant differences were observed in mice injected with ABX alone or NAC.

[0150] These data obtained in GAA-deficient mice confirm the data previously obtained in wild-type mice and support the synergistic effect of combined administration of DNJ and ABX to enhance the efficacy of gene therapy.

[0151] GAA activity resulted in a decrease in glycogen accumulation in tissues, particularly in the heart, where glycogen levels were similar to those observed in wild-type mice (Figure 8A). Partial normalization of glycogen content was observed in other tissues (Figures 8B-D). Although mice treated with DNJ-ABX exhibited lower glycogen levels than mice treated with AAV alone, the difference did not reach significance.

[0152] Example 3: Study of the effect of pharmacological chaperones in combination with alglucosidase alfa (ERT) Materials and Methods

[0153] In vivo studies Standard animal care and husbandry followed national guidelines. Animal experiments were approved by the Ethics Committee of CERFE in accordance with European Directive 2010 / 63 / EU (approval number: 2017-11-B #13643). GAA-deficient male mice aged 3–4 months were divided into three groups, and one control group consisted of wild-type littermates. Starting on day -2, one group of GAA-deficient (KO) mice (mouse strain B6; 129-Gaa tm1Rabn Mice (J / J) received pharmacological chaperone (PC) treatment dissolved in drinking water, as shown in Figure 9. Mice received a combination of 100 mg / kg / day of duvoglustat (DNJ, AX61Q1, Interchim, San Diego, CA) and 25 mg / kg / day of ambroxol hydrochloride (ABX, A9797, Sigma, Saint Louis, MO). In parallel, two groups of GAA wild-type and GAA-deficient mice served as controls receiving regular drinking water. On day 0, PC-treated GAA-deficient mice and one control group of GAA-deficient mice were injected with alglucosidase alfa (ERT, Myozyme, Genzyme, Cambridge, MA) at 20 mg / kg via tail vein injection. Circulating GAA activity and levels were monitored 3 hours after ERT.

[0154] Plasma collection Blood samples were collected by retroorbital bleeding into heparinized capillary tubes followed by plasma separation.

[0155] Measurement of GAA activity GAA activity in plasma and tissues was assessed by measuring 4-methylumbelliferyl-α-d-glucoside (4-MU, Sigma, St. Louis, MO) cleavage at pH 4.3. For this purpose, mouse tissues collected at the time of sacrifice were homogenized in PBS. Insoluble proteins were removed by centrifugation. The protein content of the resulting lysates was quantified using the BCA protein assay (Thermo Fisher Scientific, Waltham, MA). Plasma and tissue samples were diluted with water, and 10 μL of each sample was incubated with 20 μL of reconstituted substrate for 1 h at 37°C. After the reaction was stopped, emitted fluorescence was measured using an EN-SPIRE® fluorometer (Perkin Elmer, Waltham, MA). GAA activity was normalized to total protein content or plasma volume.

[0156] Western blot analysis Western blot analysis was performed according to the method described in Example 1.

[0157] result The half-life of recombinant hGAA (ERT) is relatively short. Therefore, measurements of levels and activity were performed 3 hours after injection. Compared with mice treated with ERT alone, we observed increased circulating GAA levels and activity in mice co-treated with the DNJ-ABX combination (Figure 10). These data support the synergistic effect of co-administration of DNJ and ABX to enhance the bioavailability of GAA, and further demonstrate that the chaperones of the present invention are useful for enhancing the therapeutic efficacy of conventional ERT, such as treatment with alglucosidase alfa.

[0158] References cited in the format "[reference number]": 1. Hirschhorn, R. and Reuser, AJ 2001 In The Metabolic and Molecular Basis for Inherited Disease (Scriver, CR, Beaudet, AL, Sly, WS & Valle, D. Eds.), pp. 3389-3419. McGraw-Hill, New York, pp. 3403-3405. 2. Van der Ploeg and Reuser, 2008 Lancet 372:1342~1351, 3.Van den Hout et al., 2003, Pediatrics;112:332~340[PubMed:12897283] 4. DeRuisseau et al., 2009; Proc Natl Acad Sci USA, 106:9419~9424. [PubMed:19474295] 5.Amalfitano, A. et al. 2001 Genet.In Med.3:132~138 6.Kikuchi et al., 1998, Clinical and metabolic correction of Pompe disease by enzyme therapy in acid maltase-deficient quail. The Journal of Clinical Investigation;101:827~833 [PubMed:9466978] 7.Raben et al., 2003, Molecular Genetics and Metabolism;80:159~169[PubMed:14567965] 8. Xu et al., JCI Insight 2019;4(5):e125358 9. Lukas et al., The American Society of Gene and Cell Therapy 2015, Vol. 23, No. 3, pp. 456-464 10. Hoefsloot et al., 1988 EMBO J.7:1697; 11.Martiniuk et al., 1990 DNA and Cell Biology 9:85) 12.Hoefsloot et al. 1990 Biochem.J.272:485; 13.Wisselaar et al., 1993 J.Biol.Chem.268:2223; 14. Hermans et al., 1993 Biochem. J. 289:681). 15. Van Hove et al., 1996 Proc. Natl. Acad. Sci. USA 93:65 (Human) 16. Kunita et al., 1997 Biochemica et Biophysica Acta 1362:269; 17 Ill, CR et al. (1997). Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A.Blood Coag.Fibrinol.8:S23~S30 18. Rincon et al., Mol Ther. 2015 January;23(1):43-52 19. Chuah et al., Mol Ther. 2014 Sep;22(9):1605-13 20. Nair et al., Blood. 2014 May 15;123(20):3195-9. 21. Mates et al. 2009 Nat Genet. 2009 June;41(6):753–61. doi:10.1038 / ng.343 22. Ling et al., 2016 July 18, Hum Gene Ther Methods. 23. Vercauteren et al., 2016, Mol. Ther., 24(6), 1042 24. Rosario et al., 2016, Mol Ther Methods Clin Dev.3, 16026 pages 25. Shen et al., 2007 Molecular Therapy, Vol. 15, No. 11, pp. 1955-1962 26. Tenney et al., 2014, Virology, Vol. 454-455, April 2014, pp. 227-236 27. Bartel et al., 2011, Front. Microbiol., October 4, 2011, https: / / doi.org / 10.3389 / fmicb.2011.00204 28. Michelfelder et al. (PLoS ONE, 2009, 4, e5122 29. Zhong et al., 2008 PNAS June 3, 2008, 2008 105 (22) 7827~7832;https: / / doi.org / 10.1073 / pnas.0802866105 30.Finet et al., Virology, 2018, 513, 43~51). 31. McCarty et al., 2003 Gene Therapy December;10(26):2112-8. 32. Matsushita et al., Gene Therapy, 1998, 5, 938-945 33. Ayuso et al., Gene Therapy, 2010, 17, 503-510 34. Zhang et al., Hum Gen Ther, 2012, Vol. 23, No. 5: 460-472.

[0159] Sequence Listing [Table 2-1]

[0160] [Table 2-2]

Claims

1. A kit of parts comprising (i) a pharmacological chaperone or a pharmaceutically acceptable salt thereof, and (ii) a therapeutic acid alpha-glucosidase (GAA) polypeptide or a nucleic acid molecule encoding a therapeutic GAA polypeptide, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof and ambroxol (ABX) or a derivative thereof.

2. 2. A kit of parts according to claim 1 for use as a medicine, preferably for use in the treatment of glycogen storage diseases (GSD).

3. 3. A kit of parts according to claim 1 or 2 for use in a method for increasing the uptake of GAA into tissues of the nervous system, preferably tissues of the central nervous system, more preferably the spinal cord.

4. 10. The kit of parts of claim 1 for use in a method for treating central nervous system (CNS) disorders in GSD.

5. 5. The kit of parts or kit of parts for use according to any one of claims 1 to 4, comprising (i) duvoglustat and ambroxol hydrochloride, and (ii) a viral vector into which is inserted a nucleic acid construct for expressing a nucleic acid molecule encoding a therapeutic GAA polypeptide.

6. 1. A composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in treating glycogen storage disease (GSD) in a subject receiving therapeutic acid alpha-glucosidase (GAA) treatment for treating the GSD, wherein the pharmacological chaperone is 1-deoxynojirimycin (DNJ) or a derivative thereof and ambroxol (ABX) or a derivative thereof.

7. A composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for increasing uptake of GAA into tissues of the nervous system, preferably the spinal cord, in a subject undergoing therapeutic GAA treatment to treat GSD, wherein the pharmacological chaperone is DNJ or a derivative thereof and ABX or a derivative thereof.

8. A composition comprising a pharmacological chaperone or a pharmaceutically acceptable salt thereof for use in a method for treating central nervous system (CNS) disorders in a subject undergoing therapeutic GAA treatment for treating GSD, wherein the pharmacological chaperone is DNJ or a derivative thereof and ABX or a derivative thereof.

9. 9. The composition for use of any one of claims 6 to 8, wherein the therapeutic GAA treatment is a nucleic acid molecule encoding a therapeutic GAA polypeptide.

10. 10. The composition for use of any one of claims 6 to 9, wherein the therapeutic GAA treatment is a viral vector into which a nucleic acid construct for expressing a nucleic acid molecule encoding a therapeutic GAA polypeptide has been inserted, and the pharmacological chaperones are duvoglustat and ambroxol hydrochloride.

11. the DNJ derivative is selected from N-methyl-DNJ, N-butyl-DNJ, N-cyclopropylmethyl-DNJ, N-(2-(N,N-dimethylamido)ethyloxy-DNJ, N-4-t-butyloxycarbonyl-piperidinylmethyl-DNJ, N-2-R-tetrahydrofuranylmethyl-DNJ, N-2-R-tetrahydrofuranylmethyl-DNJ, N-(2-(2,2,2-trifluoroethoxy)ethyl-DNJ, N-2-methoxyethyl-DNJ, N-2-ethoxyethyl-DNJ, N-4-trifluoromethylbenzyl-DNJ, N-alpha-cyano-4-trifluoromethylbenzyl-DNJ, N-4-trifluoromethoxybenzyl-DNJ, N-4-n-pentoxybenzyl-DNJ, N-4-n-butoxybenzyl-DNJ, and Cl-nonyl DNJ; - the DNJ is duvoglustat (CAS number 19130-96-2), duvoglustat hydrochloride (CAS number 73285-50-4), miglustat (CAS number 72599-27-0) or miglustat hydrochloride (CAS number 210110-90-0), and / or - A kit of parts, a kit of parts for use or a composition for use according to any one of claims 1 to 4 and claims 6 to 9, wherein said ABX is ambroxol hydrochloride (CAS number 23828-92-4).

12. 12. The kit of parts for use or composition for use according to any one of claims 2 to 11, wherein the GSD is selected from GSDI (von Gierke disease), GSDII (Pompe disease), GSDIII (Cawley disease), GSDIV, GSDV, GSDVI, GSDVII, GSDVIII or fatal congenital glycogen storage diseases of the heart, preferably GSDI, GSDII or GSDIII, more preferably GSDII or GSDIII, most preferably GSDII.

13. 13. The kit of parts, kit of parts for use, or composition for use of any one of claims 1 to 5 and claims 9 to 12, wherein the pharmacological chaperone is administered before, simultaneously with, and / or after a nucleic acid molecule encoding a therapeutic GAA polypeptide.

14. 14. The kit of parts, kit of parts for use or composition for use according to any one of claims 1 to 5 and claims 9 to 13, wherein the nucleic acid molecule is inserted into a nucleic acid construct for expressing the nucleic acid molecule, and the nucleic acid construct is inserted into a viral vector selected from a retroviral vector, such as a lentiviral vector, or an AAV vector, such as a single-stranded or double-stranded self-complementary AAV vector, and the viral vector is preferably an AAV vector having an AAV8, AAV9, AAVrh74 or AAV2i8 capsid, particularly an AAV8, AAV9 or AAVrh74 capsid, more particularly an AAV8 capsid.

15. 15. A kit of parts, kit of parts for use or composition for use according to any one of claims 1 to 5 and claims 9 to 14, wherein the therapeutic GAA polypeptide encoded by the nucleic acid molecule comprises a GAA polypeptide portion and a signal peptide portion fused to the N-terminus of the GAA polypeptide portion, wherein the signal peptide portion fused to the N-terminus of the GAA polypeptide portion is selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, preferably SEQ ID NO: 3, and the GAA polypeptide portion is selected from SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36, preferably SEQ ID NO:

27.

16. 16. The kit of parts, kit of parts for use or composition for use according to any one of claims 1 to 5 and claims 9 to 15, wherein the nucleic acid molecule is selected from SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46 or SEQ ID NO:47, preferably SEQ ID NO:25.

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