A gene therapy vector encoding glucose-6-phosphatase (G6Pase-a)

Abstract

The present invention relates to adeno-associated virus (AAV) vectors comprising a nucleic acid construct for expressing glucose-6-phosphatase-a (G6Pase-a) in cells, the nucleic acid construct comprising a nucleic acid sequence encoding G6Pase-a operably linked to the human alpha-1 antitrypsin (hAAT) promoter, cells transformed with the vectors of the invention, compositions comprising the vectors or cells of the invention, and uses thereof.

Description

[Technical field]

[0001] The present invention relates to an adeno-associated virus (AAV) useful for the treatment of glycogen storage disease Ia (GSD-Ia) comprising a nucleic acid construct for expressing glucose-6-phosphatase-a (G6Pase-a) in a cell, wherein the nucleic acid sequence encoding said G6Pase-a is operably linked to the human alpha-1 antitrypsin (hAAT) promoter. [Background technology]

[0002] Glycogen storage disease type Ia (GSD-Ia or von Gierke disease) is caused by a deficiency of glucose-6-phosphatase-a (G6Pase-a), an enzyme expressed primarily in the liver, kidney, and intestine. G6Pase-a, encoded by the G6PC gene, is a hydrophobic protein anchored in the endoplasmic reticulum (ER) by nine transmembrane helices. The enzyme catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate in the terminal steps of glycogenolysis and gluconeogenesis. Patients with GSD-Ia are unable to maintain glucose homeostasis and exhibit fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia.

[0003] In most cases, hypoglycemia can be managed with dietary therapy that allows patients to achieve near-normal growth and pubertal development. However, long-term clinical complications and the underlying pathological process remain unmodified. One of the most significant chronic risks is hepatocellular adenomas (HCAs), which occur in 70-80% of GSD-I patients over the age of 25 years. HCAs in GSD-Ia patients are multiple and nonencapsulated, with complications including local compression and intratumoral hemorrhage. In 10% of GSD-Ia patients, HCAs undergo malignant transformation into hepatocellular carcinoma (HCC).

[0004] Thus, there is a need for improved therapeutic vectors for the treatment of GSD-Ia and its associated complications.

[0005] To date, gene therapy studies have been conducted using recombinant adeno-associated viruses (AAV) carrying G6Pase-a in animal models of GSD-Ia. In particular, the prior art discloses the use of a nucleic acid construct comprising a nucleic acid sequence encoding G6Pase-a operably linked to the G6PC promoter / enhancer (GPE).

[0006] Treatment complications related to immune responses to the vector represent a significant obstacle to the use of AAV vectors in gene therapy. In general, administration of lower doses of AAV vectors results in less activation of the immune response and better stability of transgene expression.

[15] Therefore, in order to reduce the occurrence of side effects associated with the use of AAV vectors, it is preferable to administer the lowest amount of AAV vector sufficient to produce a therapeutically effective effect in the subject.

[0007] Thus, there is a need for improved nucleic acid constructs that can increase the expression and activity of G6Pase-a in gene therapy, thereby reducing the amount of AAV vector required to achieve a therapeutic effect in a subject. Summary of the Invention [Means for solving the problem]

[0008] The present inventors have surprisingly shown that, particularly in an AAV gene therapy setting, a nucleic acid encoding G6Pase-a under the control of the hAAT promoter increases G6Pase-a expression and activity compared to the hGPE promoter, thereby allowing phenotypic rescue in GSDIa mice, which was particularly unexpected in view of the prior art, which used various forms of the native G6Pase promoter (GPE) from various species to treat GSDIa.

[0009] The inventors also demonstrated that the nucleic acid encoding G6Pase-a under the control of the hAAT promoter reduces the risk of tumor formation, such as HCA and HCC, when compared with the hGPE promoter in gene therapy, which was also unexpected in view of the prior art, which predicts that promoters with higher activity are more likely to increase the risk of HCA and HCC after gene therapy [1].

[0010] Thus, in a first aspect, the present invention relates to an adeno-associated virus (AAV) vector comprising a nucleic acid construct for expressing glucose-6-phosphatase-a (G6Pase-a) in a cell, said construct comprising a nucleic acid sequence encoding said G6Pase-a, said nucleic acid sequence encoding said G6Pase-a being operably linked to a human alpha-1 antitrypsin (hAAT) promoter.

[0011] In a second aspect, the present invention relates to a cell transformed with a vector according to the invention.

[0012] In a third aspect, the present invention relates to a composition comprising a vector of the invention or a cell of the invention.

[0013] In a fourth aspect, the invention relates to a vector, a cell or a composition according to the invention for use as a medicament, in particular for use in the treatment of glycogen storage disease Ia (GSD-Ia). [Brief description of the drawings]

[0014] [Figure 1]Figure 1 shows the correction of liver phenotype in GSD-Ia and WT mice after 15 days of treatment with AAV vectors or PBS. A, scheme of the protocol. B, blood glucose measured after a 6-h fast at the end of the protocol. C, G6Pase activity measured in liver tissue. D, glycogen content in liver tissue. E, hepatomegaly reported as percentage of liver weight / body weight. F, vector genome copy number per diploid genome measured in livers of mice treated with AAV. Statistical analysis was performed by ANOVA in A–E (#, P<0.05 vs. L.G6pc+ / + mice injected with PBS; *, P<0.05 vs. L.G6pc- / - mice injected with PBS) and by t-test in F (ns: not significant). [Diagram 2] Figure 2 shows the long-term correction of the liver phenotype in GSD-Ia mice. A represents the scheme of the protocol. B shows blood glucose measured after a 6-h fast at the end of the protocol, i.e., 7 months after vector injection. C shows G6Pase activity measured in liver tissues collected at sacrifice. D shows glycogen content measured in liver tissues. E shows hepatomegaly reported as percentage of liver weight / body weight at sacrifice. F shows vector genome copy number per diploid genome measured in livers of mice treated with AAV. Statistical analysis was performed by ANOVA (#, ​​P<0.05 vs. L.G6pc+ / + mice injected with PBS; *, P<0.05 vs. L.G6pc- / - mice injected with PBS; †, P<0.05 as indicated) and in F by t-test (*P<0.05). [Diagram 3]Figure 3 shows that gene therapy directed against hGPE promotes liver tumor formation in L.G6pc- / - mice fed a high-fat, high-sucrose diet. A: Scheme of the protocol. B: Blood glucose measured after a 6-h fast at the end of the protocol, i.e., 8 months after vector injection. C: G6Pase-a activity assay performed on liver tissues collected at the time of sacrifice. D: Vector genome copy number per diploid genome measured in liver. E: Number of tumors >2 mm in size observed in L.G6pc- / - mice treated with PBS or AAV vectors 8 months after the start of the HF / HS feeding regimen. Statistical analysis was performed by ANOVA in B and C (#, P < 0.05 vs. PBS-injected L.G6pc+ / + mice; *, P < 0.05 vs. PBS-injected L.G6pc- / - mice), t test in D (ns: not significant), and nonparametric ANOVA in E (*, P < 0.05 vs. PBS-injected L.G6pc+ / + mice). [Figure 4] Figure 4 shows that in a larger cohort of L.G6pc- / - animals injected at 2.5x1011vg / mouse, higher G6Pase-a activity was achieved by AAV9 vector carrying the hAAT promoter compared to AAV9 vector expressing wild-type G6pc. Activity assays were performed on liver tissues harvested at the time of sacrifice. Statistical analysis was performed by ANOVA (#, ​​P<0.05 vs. L.G6pc+ / + mice injected with PBS; *, P<0.05 vs. L.G6pc- / - mice injected with PBS; X, P<0.05 as indicated). [Diagram 5]Figure 5 shows the G6Pase-a activity obtained by various AAV vectors encoding the human G6pc gene injected into L.G6pc- / - mice. PBS-injected L.G6pc+ / + and L.G6pc- / - mice were used as controls. Activity assays were performed on liver tissues harvested 15 days after vector injection. Statistical analysis was performed by ANOVA (#, ​​P<0.05 vs. PBS-injected L.G6pc+ / + mice; *, P<0.05 vs. PBS-injected L.G6pc- / - mice; +, P<0.05 as indicated). [Figure 6] Figure 6 shows a comparison of the efficacy of three different codon-optimized sequences. A shows the G6Pase-a activity assay (expressed as a percentage of L.G6pc+ / +G6Pase-a activity) of codon-optimized G6pc sequences 1 and 2 (G6pc co1 and G6pc co2) and wild-type G6pc sequence (G6pc wt) expressed with AAV8 in L.G6pc- / - mice injected with a dose of 1x1012 vg / kg and followed for 15 days. B shows the G6Pase-a activity assay (expressed as a percentage of L.G6pc+ / +G6Pase-a activity) of codon-optimized G6pc sequence 3 (G6pc co3) and wild-type G6pc sequence (G6pc wt) in L.G6pc- / - mice injected with AAV9 at a dose of 1x1011 vg / mouse and followed for 15 days. Statistical analysis was performed by ANOVA (#, ​​P < 0.05 vs. PBS-injected L.G6pc+ / + mice; *, P < 0.05 vs. PBS-injected L.G6pc- / - mice; +, P < 0.05 as indicated). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition The term "glucose-6-phosphatase alpha" or "G6Pase-a" relates to the enzyme encoded by the G6pc gene. This enzyme catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate in the terminal steps of glycogenolysis and gluconeogenesis. According to the present invention, G6Pase-a may be a wild-type G6Pase-a or a modified G6Pase-a, in particular a modified G6Pase-a with enhanced phosphohydrolase activity. Modified G6Pase-a are disclosed in WO2016106303 and

[16] . For example, G6Pase-a may be SEQ ID NO: 1, SEQ ID NO: 12, or any modified G6Pase-a having an amino acid sequence selected from SEQ ID NO: 29 to SEQ ID NO: 44.

[0016] According to the present invention, "identity" is calculated by comparing two aligned sequences in a comparison window. By aligning sequences, the number of positions (nucleotides or amino acids) common to the two sequences in the comparison window can be determined. Thus, the percentage of identity is obtained by dividing the number of common positions by the total number of positions in the comparison window and multiplying by 100. The percentage of sequence identity can be determined manually or by using well-known computer programs. In a particular embodiment of the present invention, the identity or homology corresponds to at least one substitution of an amino acid residue, for example, 1, 2, 3, 4, 5 substitutions, as long as it does not significantly lose the binding ability to interact. Preferably, the at least one substitution is a conservative amino acid substitution. By "conservative amino acid substitution" is meant that an amino acid can be replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0017] According to the present invention, the term "nucleic acid sequence" refers to a DNA or RNA molecule, in particular DNA, in single-stranded or double-stranded form.

[0018] According to the present invention, the term "nucleic acid sequence encoding G6Pase-a" refers to a nucleic acid sequence encoding G6Pase-a, i.e., either wild-type G6Pase-a or modified G6Pase-a. Modified nucleic acid sequences encoding modified G6Pase-a are disclosed in reference

[16] and WO2016106303. For example, a nucleic acid sequence encoding wild-type G6Pase-a has the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 45, or SEQ ID NO: 46. For example, a nucleic acid sequence encoding modified G6Pase-a has the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5. Thus, for example, a nucleic acid sequence encoding G6Pase-a may be any nucleic acid sequence encoding modified G6Pase-a selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 13 to SEQ ID NO: 28.

[0019] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that is implanted into a target cell to express a transgene, for example, to express G6Pase-a in the cell. The nucleic acid construct may contain one or more expression control nucleic acid sequences and / or other nucleic acid sequences that enhance the expression of G6Pase-a, and / or nucleic acid sequences that enhance the secretion of G6Pase-a, and / or nucleic acid sequences that enhance the uptake of G6Pase-a into tissues, and the nucleic acid sequences may be operably linked to a sequence encoding a transgene (e.g., G6Pase-a). As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or other transcriptional regulatory nucleic acid sequence is operably linked to a nucleic acid sequence encoding a transgene (e.g., G6Pase-a) if it affects the transcription of the nucleic acid sequence. Such expression control nucleic acid sequences are well known in the art and include, for example, promoters, enhancers (eg, cis-regulatory modules (CRMs)), introns, polyA signals, and the like.

[0020] The term "alpha-1 antitrypsin" or "AAT" refers to a protein belonging to the serpin superfamily. In humans, it is encoded by the SERPINA1 gene. The term "hAAT" refers to human AAT.

[0021] The term "promoter" refers to a region of DNA that directs / initiats the transcription of a nucleic acid sequence (e.g., a gene). A promoter contains necessary nucleic acid sequences near the start site of transcription. Typically, promoters are located near the gene they transcribe.

[0022] The term "hAAT promoter" relates to the promoter of hAAT, ie either the wild-type hAAT promoter or a modified hAAT promoter.

[0023] The term "vector" according to the present invention means a vector suitable for expressing a transgene in gene therapy, such as the expression of G6Pase-a in gene therapy.

[0024] In the context of the present invention, the term "gene therapy" refers to treatment of a subject involving the delivery of genes / nucleic acids to the cells of an individual for the purpose of treating a disease.

[0025] As used herein, the terms "subject," "patient," or "individual" refer to a human or non-human mammal (e.g., rodent (mouse, rat), cat, dog, or primate) suffering from or susceptible to glycogen storage disease Ia (GSD-Ia). Preferably, the subject is a human, male or female.

[0026] The term "glycogen storage disease" or "GSD" refers to a metabolic disorder resulting from an enzyme deficiency 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. GSD-I consists of two autosomal recessive disorders, GSD-Ia and GSD-Ib. GSD-Ia is caused by a deficiency of glucose-6-phosphatase-a. Deficiency of glucose-6-phosphate transporter (G6PT) is involved in GSD-Ib. According to the present invention, the GSD is GSD-Ia (von Gierke's disease; OMIM #232240).

[0027] The term "treat" or "treatment" means to reverse, reduce, inhibit the progression of, or prevent the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder. In particular, treating a disease may consist of treating a dysfunction in GSD, preferably improving glycemic control in a subject.

[0028] The term "pharmaceutical acceptable" means approved by a regulatory agency of the Federal or state government or listed in the United States or European Pharmacopoeia or other generally recognized pharmacopoeias for animal and human use.

[0029] "Pharmaceutical composition" refers to a composition that includes a pharma- ceutical acceptable carrier. For example, a carrier may be a diluent, adjuvant, excipient, or vehicle used in administering a therapeutic agent. Such pharmaceutical carriers may be sterile liquids, such as water and oils (of petroleum, animal, vegetable, or synthetic origin, e.g., 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, aqueous dextrose, and aqueous glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. When the pharmaceutical composition is adapted for oral administration, tablets or capsules can be prepared by conventional means using pharma- ceutically acceptable excipients such as binders (e.g., corn pregelatinized 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 may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or gum acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or vegetable fractionated oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates, or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents, as appropriate.The composition according to the invention is preferably a pharmaceutical composition.

[0030] nucleic acid construct The present specification relates to a nucleic acid construct for expressing glucose-6-phosphatase (G6Pase-a) in a cell, the construct comprising a nucleic acid sequence encoding the G6Pase-a, the nucleic acid sequence encoding the G6Pase-a being operably linked to a human alpha-1 antitrypsin (hAAT) promoter.

[0031] According to the present specification, the nucleic acid sequence may encode a wild-type G6Pase-a, for example a G6Pase-a having the amino acid sequence of SEQ ID NO: 1, or a modified G6Pase-a, preferably a modified G6Pase-a with enhanced phosphohydrolase activity, for example a G6Pase-a that comprises or has an amino acid sequence with at least 90% identity to SEQ ID NO: 1. The modified G6Pase-a may include one or more amino acid mutations (such as substitutions or deletions), so long as the protein retains enzymatic activity. In some embodiments, the modified G6Pase-a includes a serine to cysteine ​​substitution at amino acid position 298 of human G6Pase-a (herein, the amino acid sequence of wild-type human G6Pase-a is set forth as SEQ ID NO: 1). The modified G6Pase-a may include mutations at other residues, so long as the protein retains enzymatic activity. For example, the modified G6Pase-a may include substitutions at other residues, including residues 3, 54, 139, 196, 199, 242, 247, 292, 301, 318, 324, 332, 347, 349, 350, and / or 353 of human G6Pase-a (listed as SEQ ID NO: 1). Modified G6Pase-a is disclosed in WO2016 / 106303 and

[16] . For example, G6Pase-a may be SEQ ID NO: 1, SEQ ID NO: 12, or any modified G6Pase-a having an amino acid sequence selected from SEQ ID NO: 29 to SEQ ID NO: 44.

[0032] The G6Pase-a may comprise or have an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1.

[0033] Therefore, the nucleic acid sequence encoding G6Pase-a may encode either wild-type G6Pase-a (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 45, or SEQ ID NO: 46, preferably SEQ ID NO: 45) or modified G6Pase-a. The nucleic acid sequence encoding modified G6Pase-a may be any nucleic acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, any of SEQ ID NOs: 13 to 28, SEQ ID NO: 52, or SEQ ID NO: 54.

[0034] The nucleic acid sequence encoding the G6Pase-a may comprise a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:2.

[0035] Therefore, for example, the nucleic acid sequence encoding the G6Pase-a may be any of the nucleic acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or numbers 6 to 7 of WO2016106303.

[0036] The nucleic acid sequence encoding the G6Pase-a may comprise a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:45.

[0037] The nucleic acid sequence encoding the G6Pase-a may comprise a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:46.

[0038] The nucleic acid sequence encoding G6Pase-a is operably linked to a human alpha-1 antitrypsin (hAAT) promoter.

[0039] The hAAT promoter may be a wild-type hAAT promoter, e.g., an hAAT promoter having the nucleic acid sequence of SEQ ID NO: 8, or a modified hAAT promoter, e.g., an hAAT promoter that comprises or has a nucleic acid sequence having at least 90% identity to SEQ ID NO: 8. The modified hAAT promoter may contain one or more nucleic acid mutations (such as substitutions or deletions), so long as the promoter still directs / initiats transcription of G6Pase-a.

[0040] The hAAT promoter may comprise or have a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:8.

[0041] The hAAT promoter is preferably preceded by an enhancer, such as the ApoE enhancer (e.g., SEQ ID NO: 9). For example, the nucleic acid construct comprises SEQ ID NO: 7.

[0042] The nucleic acid construct may comprise an intron, in particular an intron located between the hAAT promoter and the nucleic acid sequence encoding G6Pase-a. The intron may be introduced to enhance mRNA stability and G6Pase-a production. Advantageously, the nucleic acid construct comprises an intron from a human β-globin gene (e.g., HBB2) located between the hAAT promoter and the nucleic acid sequence encoding G6Pase-a, and preferably the intron contained in the nucleic acid construct has the sequence shown in SEQ ID NO: 47. An intron having the sequence of SEQ ID NO: 47 is disclosed in WO2015 / 162302. Alternatively, the intron is located after the 3' end of the nucleic acid sequence encoding G6Pase-a.

[0043] The nucleic acid construct of the present invention may comprise, in the 5' to 3' direction, a hAAT promoter, which may be preceded by an enhancer, such as the ApoE enhancer (e.g., SEQ ID NO: 9), an intron, such as the intron of the human β-globin gene (e.g., SEQ ID NO: 47), a nucleic acid sequence encoding G6Pase-a, and a polyadenylation signal (bovine growth hormone polyadenylation signal, HBB2 polyadenylation signal, SV40 polyadenylation signal, or other natural or artificial polyadenylation signal). Advantageously, the nucleic acid construct of the present invention comprises, in the 5' to 3' direction, a hAAT promoter, which may be preceded by an enhancer, such as the ApoE enhancer (e.g., SEQ ID NO: 9), an intron (particularly the intron defined above), a nucleic acid molecule encoding G6Pase-a, and a polyadenylation signal.

[0044] The nucleic acid construct may comprise or have a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 11, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, or SEQ ID NO: 56. In a preferred embodiment, the nucleic acid construct may comprise or have a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 48.

[0045] The term "nucleic acid construct" refers to a construct that is introduced into a target cell for expressing G6Pase-a in said cell. Preferably, said target cell is a liver cell, a kidney cell, or an intestinal cell.

[0046] vector The term "nucleic acid construct herein" or "nucleic acid construct according to the present specification" means a nucleic acid construct disclosed herein, in particular a nucleic acid construct disclosed above.

[0047] The present specification also relates to a vector comprising the nucleic acid construct of the present specification.

[0048] The vector may be a plasmid vector. The vector may also be a nanoparticle comprising the nucleic acid construct of the present invention. The vector may also be a transposon-based system that can integrate the nucleic acid construct of the present invention into the genome of a target cell, such as the hyperactive Sleeping Beauty (SB100X) transposon system [2]. The vector may also be a viral vector suitable for gene therapy. The vector may target any target cell, such as liver cells, kidney cells, or intestinal cells.

[0049] The vector may be a viral vector, such as a lentiviral vector or an adeno-associated viral (AAV) vector.

[0050] The present invention relates to an AAV vector comprising the nucleic acid construct of the present invention. In a particularly preferred embodiment, the AAV vector implemented in the practice of the present invention is AAV8 or AAV9, preferably AAV8.

[0051] Thus, the nucleic acid constructs herein may also contain suitable sequences for obtaining efficient viral vectors, as is well disclosed in the art.

[0052] Said nucleic acid construct may be inserted into a vector, such as a lentiviral vector according to the present invention, or an AAV vector according to the present invention, such as a single-stranded or double-stranded self-complementary AAV vector. In a further preferred embodiment of the present invention, said AAV vector is an AAV vector suitable for transducing liver cells, more specifically AAV-1, AAV-2 and AAV-2 variants (such as the quadruple mutant capsid-optimized AAV-2 comprising an engineered capsid with the Y44+500+730F+T491V changes disclosed in [3]), AAV-3 and AAV-3 variants (Vercauteren et al. (e.g., AAV3-ST mutants comprising an engineered AAV-3 capsid with two amino acid alterations S663V+T492V as disclosed in [4]), AAV-3B and AAV-3B mutants, AAV-4, AAV-5, AAV-6 and AAV-6 mutants (e.g., AAV-6 mutants comprising a triple mutant AAV-6 capsid Y731F / Y705F / T492V form as disclosed in [5]), AAV-7, AAV-8, AAV-9, AAV-10 (e.g., AAV-cy10 and AAV-rh10), AAV-rh74, AAV-dj, Anc80, LK03, AAV-2i8, porcine AAV serotypes (e.g., AAV-po4 and AAV-po6), etc. Depending on the particular viral vector contemplated for use, as known in the art, additional suitable sequences will be introduced into the nucleic acid constructs herein to obtain a functional viral vector. Preferred sequences include AAV ITRs for AAV vectors or LTRs for lentiviral vectors.Thus, the present specification also relates to a nucleic acid construct as described above flanked on each side by an ITR or LTR.

[0053] Other non-natural genetically engineered mutants and chimeric AAVs may also be useful. AAV viruses may be genetically engineered using conventional molecular biology techniques that allow these particles to be optimized for cell-specific delivery of nucleic acid sequences, minimizing immunogenicity, regulating stability and particle life span, 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 are readily available in a variety of vector systems and host cells. AAV-based recombinant vectors that lack Rep proteins exist primarily as stable circular episomes that can integrate into the host genome with low efficiency and persist in target cells for years. Instead of using AAV natural serotypes, artificial AAV serotypes may be used in the context of the present invention, including but not limited to AAVs with non-natural capsid proteins. Such artificial capsids can be generated by any suitable technique by combining selected AAV sequences (e.g., fragments of the vp1 capsid protein) with heterologous sequences that can be obtained from a selected different AAV serotype, non-contiguous portions of the same AAV serotype, non-AAV viral sources, or non-viral sources. The artificial AAV serotype can be, but is not limited to, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid.

[0054] In the context of the present invention, said AAV vector comprises AAV capsid that can transduce target cells of subject, particularly hepatocytes.Moreover, in a particular embodiment, said AAV vector is a pseudotype vector.That is, its genome and capsid are derived from different serotypes of AAV.For example, said pseudotype AAV vector can be a vector whose genome is derived from one of the above-mentioned AAV serotypes and whose capsid is derived from another serotype.

[0055] According to a particular embodiment, the AAV capsid is selected from the group consisting of AAV-1, -2, AAV-2 variants (such as the quadruple mutant capsid-optimized AAV-2, which comprises an engineered capsid with the Y44+500+730F+T491V alterations disclosed in Ling et al., 2016), -3 and AAV-3 variants (such as the AAV3-ST variant, which comprises an engineered AAV3 capsid with the two amino acid alterations S663V+T492V, as disclosed in Vercauteren et al., 2016), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 variants (such as the AAV3-ST variant, which comprises an engineered AAV3 capsid with the two amino acid alterations S663V+T492V, as disclosed in Rosario et al., 2016). The AAV capsids are selected from AAV6 mutants including the triple mutant AAV6 capsid Y731F / Y705F / T492V form disclosed in et al., 2016), -7, -8, -9 and AAV-9 mutants (such as AAVhu68), -2G9, -10 (e.g., -cy10 and -rh10), -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP, AAV2i8, porcine AAV (e.g., AAVpo4 and AAVpo6), and tyrosine, lysine, and serine capsid mutants of AAV serotypes. The AAV capsids are also selected from other non-naturally engineered mutants (such as AAV-spark100), chimeric AAV, or AAV serotypes obtained by shuffling, rational design, error-prone PCR, and machine learning techniques. In a particular embodiment, the Cap gene encodes VP capsid proteins from at least two different AAV serotypes, or encodes at least one chimeric VP protein that combines VP protein regions or domains from at least two AAV serotypes. For example, a chimeric AAV capsid can be obtained by combining an AAV8 capsid sequence with a sequence from an AAV serotype other than the AAV8 serotype (e.g., any of those specifically mentioned above). In another embodiment, the capsid of the AAV vector comprises one or more mutant VP capsid proteins, such as those described in WO2015013313, in particular the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid mutants.In one specific embodiment, the capsid of the AAV vector is a hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins. For example, the AAV serotype may be the -rh74-9 serotype disclosed in WO2019 / 193119 (e.g., the hybrid Cap rh74-9 serotype described in the Examples of WO2019 / 193119; the rh74-9 serotype is also referred to herein as "-rh74-9", "AAVrh74-9", or "AAV-rh74-9"), or the -9-rh74 serotype disclosed in WO2019 / 193119 (e.g., the hybrid Cap 9-rh74 serotype described in the Examples of WO2019 / 193119; the -9-rh74 serotype is also referred to herein as "-9-rh74", "AAV9-rh74", "AAV-9-rh74", or "rh74-AAV9"). For example, the capsid of the AAV vector is a peptide-modified hybrid of AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins as described in PCT / EP2019 / 076958, such as the P1 peptide-modified AAV9-rh74 hybrid capsid or AAVrh74-9 hybrid capsid described in the Examples of PCT / EP2019 / 076958F. In another example, the AAV serotype may be a hybrid AAV2 / 13 as disclosed in PCT / EP2020 / 061380.

[0056] For example, the genome of the pseudotype vector may have a capsid derived from an AAV8, AAV9, AAVrh74, or AAV2i8 serotype, or the genome may be derived from a different serotype. In a specific embodiment, the AAV vector has a capsid of an AAV8, AAV9, or AAVrh74 serotype, particularly a capsid of an AAV8 or AAV9 serotype, more particularly a capsid of an AAV8 serotype.

[0057] In one particular embodiment, the vector is a vector referred to as "AAV8mut5". The AAV8mut5 vector has the amino acid sequence of SEQ ID NO:57 and is encoded by the polynucleotide of SEQ ID NO:58.

[0058] In one particular embodiment, in which the vector is used to deliver a transgene to muscle cells, the AAV vector may be selected from the group consisting of AAV8, AAV9, and AAVrh74, among others.

[0059] In other specific embodiments in which the vector is used to deliver a transgene to liver cells, the AAV vector may be selected from the group consisting of AAV5, AAV8, AAV9, AAV-LK03, AAV-Anc80, and AAV3B, among others.

[0060] In other embodiments, the capsid is a modified capsid. In the context of the present invention, a "modified capsid" may be a chimeric capsid or a capsid comprising one or more mutant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0061] In a particular embodiment, the AAV vector is a chimeric vector. That is, 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 liver cells are described in [6] and [7]. For example, a chimeric AAV vector can be obtained by combining AAV8 or AAV9 capsid sequences with sequences of AAV serotypes (e.g., any of those specifically mentioned above) that are different from AAV8 or AAV9 serotypes. In other embodiments, the capsid of the AAV vector comprises one or more mutant VP capsid proteins, such as those described in WO2015013313, in particular the RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid mutants (which exhibit increased liver tropism).

[0062] In other embodiments, the modified capsid may be derived from capsid mutations inserted by error-prone PCR and / or peptide insertion (e.g., as described in [8] or [9]). Capsid mutants may also contain single amino acid changes, such as tyrosine mutations (e.g., as described in

[10] ). Another example is the fusion of anthopleurin-B to the N-terminus of the AAV VP2 capsid protein, as described in

[11] .

[0063] Additionally, the genome of the AAV vector may be either single-stranded or a self-complementary double-stranded genome.

[12] Self-complementary double-stranded AAV vectors are generated by deleting the terminal release site (trs) from one of the AAV terminal repeats. These modified vectors have replicating genomes that are half the length of the wild-type AAV genome, but tend to package DNA dimers.

[0064] In a preferred embodiment, the AAV vector embodied in the practice of the invention has a single-stranded genome, and more preferably comprises an AAV8, AAV9, AAVmut5, AAVrh74, or AAV2i8 capsid, particularly an AAV8, AAV9, or AAVrh74 capsid, e.g., an AAV8 or AAV9 capsid, more particularly an AAV8 capsid.

[0065] cell The term "vector of the invention" or "vector according to the invention" refers to a vector disclosed in the present invention, in particular a vector disclosed above.

[0066] The present specification relates to a cell transformed with a nucleic acid molecule herein or a vector according to the present specification.

[0067] The present invention relates to cells transformed with the AAV vector of the invention.

[0068] For example, the host cell can be a cell (or cell line) suitable for producing a vector, such as for producing an AAV. In some embodiments, the host cell is a mammalian cell, such as a HEK-293, BHK, Vero, RD, HT-1080, A549, Cos-7, ARPE-19, or MRC-5 cell.

[0069] The host cell can also be a cell that is a target of gene therapy, such as a liver cell, a kidney cell, or an intestinal cell.

[0070] In some embodiments, the cell is an isolated cell.

[0071] composition The term "cells herein" or "cells according to the present specification" refers to cells disclosed herein, in particular cells disclosed above.

[0072] The present disclosure relates to a composition comprising a nucleic acid construct as described herein, a vector as described herein, or a cell as described herein. The composition as described herein is preferably a pharmaceutical composition.

[0073] The present invention relates to a composition comprising a vector of the invention or a cell of the invention. The composition of the invention is preferably a pharmaceutical composition.

[0074] The compositions can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulations and the like.

[0075] Such compositions will contain a therapeutically effective amount of the nucleic acid construct of the invention, the vector of the invention, or the cell of the invention, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration to a subject. In a particular embodiment, the nucleic acid construct, vector, or cell of the invention is formulated in a composition comprising phosphate buffered saline supplemented with 0.25% human serum albumin. In another particular embodiment, the nucleic acid construct, vector, or cell of the invention is formulated in a composition comprising lactated Ringer's solution and a non-ionic surfactant (such as Pluronic® F68) at a final concentration of 0.01-0.0001% by weight (e.g., at a concentration of 0.001% by weight) of the total composition. The above formulations may further comprise serum albumin, particularly human serum albumin, for example 0.25% human serum albumin. Other formulations suitable for either storage or administration are known in the art, particularly WO2005 / 118792.

[0076] In a preferred embodiment, the composition is formulated according to normal procedures as a pharmaceutical composition suitable for intravenous or intrathecal administration to humans.Typically, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer.If necessary, the composition may also contain a solubilizing agent and a local anesthetic (such as lignocaine) to ease the pain at the injection site.

[0077] treatment method The term "composition herein" or "composition according to the present invention" refers to the compositions disclosed herein, in particular those disclosed above.

[0078] The present specification relates to a nucleic acid construct herein, a vector herein, a cell herein, or a composition herein for use as a medicament.

[0079] The present specification also relates to a nucleic acid construct herein, a vector herein, a cell herein, or a composition herein for use in treating glycogen storage disease Ia (GSD-Ia).

[0080] The present invention relates to a vector of the invention, a cell of the invention, or a composition of the invention for use as a medicament.

[0081] The present invention also relates to the vector of the invention, the cell of the invention, or the composition of the invention for use in the treatment of glycogen storage disease Ia (GSD-Ia).

[0082] The nucleic acid construct, vector, cell, or composition of the present invention can be administered to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, and intravenous injection), oral, intraductal, sublingual, rectal, transdermal, nasal, vaginal, and inhalation routes.

[0083] The nucleic acid construct, the vector, the cell, or the composition can be administered to a subject in a therapeutically effective amount, i.e., an amount sufficient to achieve the desired effect in the subject or cell being treated. The effective amount of the nucleic acid construct, the vector, the cell, or the composition will depend on several factors, including, but not limited to, the subject or cell being treated and the method of administration.

[0084] The amount of therapeutic agent (i.e., nucleic acid construct, vector, or cell) that will be effective in treating a disease such as GSD-Ia can be determined by standard clinical techniques. In vivo and / or in vitro assays may also be used, if necessary, to help predict optimal dosage ranges. The exact dosage used in the composition will also depend on the route of administration and the severity of the disease, and must be determined according to the judgment of the physician and each patient's circumstances. The dosage of the nucleic acid, vector, or cell administered to a subject in need thereof will vary based on several factors, including, but not limited to, the route of administration, the specific disease being treated, the age of the subject, or the amount of expression required to achieve a therapeutic effect. Those skilled in the art can readily determine the required dosage range based on these factors and others, based on their knowledge in the art. For treatments involving administration of a viral vector, such as an AAV vector, to a subject, a typical dosage of the vector is at least 1×10 per kilogram of body weight. 8 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.

[0085] Therapeutic agents of the invention (i.e., nucleic acid constructs, vectors, or cells) can be administered in a single dose or in multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as appropriate for the desired therapeutic result.

[0086] Codon-optimized G6Pase-a The nucleic acid sequence encoding G6Pase-a may be optimized for the expression of G6Pase-a in vivo. Sequence optimization may include a number of modifications of the nucleic acid sequence, including codon optimization, increasing the GC content, reducing CG dimers, reducing the number of CpG islands, reducing the number of alternative open reading frames (ARFs), and / or reducing the number of splice donor and splice acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules may code for the same protein. It is also well known that the genetic code of various organisms is often biased to use one of several codons that code for the same amino acid over others. Codon optimization takes advantage of the codon bias present in a given cellular context to introduce modifications into the nucleotide sequence that make the resulting codon-optimized nucleotide sequence more likely to be expressed at a relatively high level in such a given cellular context compared to a non-codon-optimized sequence. Of course, as is well known to those skilled in the art, sequence optimization is a balance between all these parameters, meaning that a sequence can be considered optimized even if at least one of the above parameters is improved and one or more of the other parameters are not, so long as the optimized sequence results in an improved transgene (e.g., improved expression and / or a reduced immune response to the transgene in vivo).

[0087] The nucleic acid sequence encoding G6Pase-a may be codon-optimized to enhance expression in human cells compared to a non-codon-optimized nucleotide sequence encoding the same G6Pase-a. A wild-type nucleic acid sequence encoding wild-type G6Pase-a is as shown in SEQ ID NO:2. Examples of codon-optimized sequences encoding the same G6Pase-a are as shown in SEQ ID NO:3, SEQ ID NO:45, or SEQ ID NO:46. Other examples of sequence-optimized nucleic acids encoding modified G6Pase-a are SEQ ID NO:5, SEQ ID NO:52, and SEQ ID NO:54, which encode modified G6Pase of SEQ ID NO:12, all of which are optimized for one of the above parameters compared to the nucleic acid sequence of SEQ ID NO:4.

[0088] The nucleic acid sequence of the invention encoding G6Pase may be codon-optimized and / or may have a reduced GC content and / or a reduced number of CG dimers compared to a wild-type nucleotide sequence encoding the same G6Pase-a amino acid sequence, such as G6Pase-a of SEQ ID NO: 1. Said nucleic acid sequence encoding G6Pase may also be codon-optimized and / or may have a reduced GC content and / or a reduced number of CG dimers compared to the nucleotide sequence of SEQ ID NO: 2. Alternatively, such a nucleic acid sequence encoding G6Pase may have the sequence of SEQ ID NO: 45 or SEQ ID NO: 46, preferably SEQ ID NO: 45.

[0089] The present specification also discloses a nucleic acid sequence encoding G6Pase-a comprising a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:45.

[0090] The present specification also discloses a nucleic acid construct for expressing G6Pase-a in a cell, the construct comprising a nucleic acid sequence encoding G6Pase-a, the nucleic acid sequence encoding G6Pase-a comprising a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 45. The nucleic acid sequence encoding G6Pase-a is preferably operably linked to a promoter.

[0091] The present specification also discloses a vector comprising the nucleic acid construct disclosed in this section (i.e., in the section "codon-optimized G6Pase-a").The vector may be a lentivirus vector or an adeno-associated virus (AAV) vector, such as AAV8, AAV9, or AAVmut5, preferably AAV8.

[0092] The present specification also discloses a cell transformed with a nucleic acid molecule disclosed in this section or a vector disclosed in this section. The cell may be a liver cell, an intestinal cell, or a kidney cell.

[0093] The present specification also discloses a composition comprising a nucleic acid construct, a vector, or a cell disclosed in this section.

[0094] The present specification also discloses a nucleic acid construct, a vector, a cell, or a composition disclosed in this section for use as a medicament.

[0095] The present specification also discloses a nucleic acid construct, a vector, a cell, or a composition disclosed in this section for use in treating Glycogen Storage Disease Ia (GSD-Ia). EXAMPLES

[0096] material and method In Examples 1, 2, and 3, two types of nucleic acid constructs (hereinafter, "transgene expression cassettes") were prepared, which contain the human G6pc gene wt (SEQ ID NO: 2) encoding G6Pase-a under the control of various promoters below. - alpha-1-antitrypsin (hAAT) promoter. The nucleic acid sequence of this expression cassette is SEQ ID NO:11. The endogenous promoter of the human G6pc gene (hGPE). The nucleic acid sequence of this expression cassette is SEQ ID NO: 10.

[0097] These transgene expression cassettes are shown in FIG. 1A.

[0098] These two transgene expression cassettes were pseudotyped in AAV9 to obtain two different AAV vectors, namely AAV9-hAAT-hG6PC and AAV9-hGPE-hG6PC.

[0099] Liver-specific G6pc knockout mouse model (L.G6pc - / -We tested these two vectors or PBS (negative control) independently in GSD-Ia mice (L.G6pc + / + ) was injected with PBS.

[0100] In Example 4, a nucleic acid construct (hereinafter, "transgene expression cassette") was generated that contains the human G6pc gene wt (SEQ ID NO: 2) encoding G6Pase-a under the control of the alpha-1-antitrypsin (hAAT) promoter. The nucleic acid sequence of this expression cassette was SEQ ID NO: 56.

[0101] An expression cassette for the human G6pc gene wt was pseudotyped in AAV9, AAV8, and AAVmut5.

[0102] Liver-specific G6pc knockout mouse model (L.G6pc - / - We tested these three vectors or PBS (negative control) independently in GSD-Ia mice (L.G6pc + / + ) was injected with PBS.

[0103] In Example 5, four nucleic acid constructs (hereinafter "transgene expression cassettes") were generated containing the human G6pc gene wt (SEQ ID NO: 2) and two codon-optimized human G6pc genes (co1, co2, and co3, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 3, respectively), each encoding G6Pase-a, under the control of the alpha-1-antitrypsin (hAAT) promoter.

[0104] The nucleic acid sequence of the expression cassette of human G6pc gene wt was SEQ ID NO: 11 or SEQ ID NO: 56. The nucleic acid sequences of the expression cassettes of human G6pc gene co1, human G6pc gene co2, and human G6pc gene co3 were SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively.

[0105] The expression cassette for the human G6pc gene wt was pseudotyped in AAV8 (SEQ ID NO:56) and AAV9 (SEQ ID NO:11).

[0106] The expression cassettes of human G6pc gene co1 and G6pc gene co2 were pseudotyped in AAV8.

[0107] The expression cassette of the human G6pc gene co3 was pseudotyped in AAV9.

[0108] Liver-specific G6pc knockout mouse model (L.G6pc - / -

[13] ), i.e., GSD-Ia mice, were independently tested with the five vectors or PBS (negative control). As a positive control, WT mice (L.G6pc + / + ) was injected with PBS.

[0109] In vivo studies Mice were fed either a standard chow diet (A04 diet, Safe) or a high-fat, high-sucrose diet (INRAE, Jouy-en-Josas) known to promote liver tumor formation in L.G6pc- / - mice. A predisposition to tumor formation was induced in mice by feeding them a modified chow diet (INRA) consisting of 36% fat

[14] .

[0110] AAV vector L.G6pc - / - Mice were administered intravenously via the tail vein. Wild-type mice (C57Bl / 6J mice, Charles Rivers) were injected with PBS via the tail vein. Peripheral blood glucose was measured after a 6-h fast using a blood glucose meter (Roche Diagnostic). At the time of sacrifice, livers were harvested, weighed to determine liver weight / body weight ratio, and snap frozen for further evaluation.

[0111] At the time of sacrifice, the tumors in the GSDIa mice were evaluated by visual inspection. Only tumors larger than 2 mm were counted.

[0112] Production of AAV HEK293T cells were grown in suspension in 250 mL of serum-free medium. The cells were transduced with three plasmids: i) a transgene plasmid containing the AAV2 ITRs flanking the expression cassette, ii) a helper plasmid, pXX6, containing the adenoviral sequences required for AAV production, and iii) a plasmid containing the AAV Rep and Cap genes that define the AAV serotype. Two days after transfection, the cells were lysed to release AAV particles.

[0113] Viral lysates were purified by affinity chromatography, and viral genomes were quantified by TaqMan real-time PCR assays using primers and probes corresponding to the ITRs of the AAV vector genome

[17] .

[0114] Measurement of G6Pase enzyme activity G6Pase enzyme activity was measured in freeze-fixed liver homogenates as previously reported

[13] . Briefly, tissue homogenates were incubated with glucose-6-phosphate (Sigma) for 15 min at 37°C. The reaction was stopped by adding trichloroacetic acid and the released phosphate was measured by complexing with ammonium molybdate and citrate arsenite. The resulting absorbance was measured at 700 nm.

[0115] Measurement of glycogen content Glycogen content in tissue homogenates was measured indirectly as glucose released after total digestion with Aspergillus niger amyloglucosidase (Sigma). Samples were incubated at 95°C for 20 min in the presence of 0.3 M NaOH and then cooled at 4°C. Amyloglucosidase was then added to the samples and incubated at 37°C for 90 min. Released glucose was measured with a commercially available glucose assay kit.

[0116] Quantification of Vector Genome Copy Number (VGCN) For quantification of vector genome copy number (VGCN) in samples, DNA was extracted from samples using KingFisher (Thermo Fisher Scientific). Real-time PCR was performed on 1 μL of DNA using the protocol for AAV vector titration described above. Exon Mex5 of the titin gene was used as a genomic DNA loading control.

[0117] Example 1: Correction of the liver phenotype in GSD-Ia mice 15 days after intravenous injection of an AAV vector expressing human G6Pase-a L.G6pc fed standard diet - / - Mice were independently inoculated with the above two AAV9 vectors at 1 × 10 11 vg / mouse (Fig. 1A).

[0118] On day 15 after vector injection, blood glucose levels were measured after a 6-hour fast, and blood glucose was completely normalized in mice injected with the two vectors (FIG. 1B).

[0119] G6PC activity was then measured in the liver. Compared with the AAV9-hGPE-hG6PC vector, the AAV9-hAAT-hG6PC vector achieved supraphysiological activity and showed the highest activity (Figure 1C).

[0120] Glycogen concentrations were also measured. A complete correction of glycogen accumulation and hepatomegaly was obtained with AAV9-hAAT-hG6PC but not with the vector carrying the hGPE promoter (Figure 1D and Figure 1E, respectively). This reflects the higher G6Pase activity achieved in mice treated with the AAV9 vector carrying the hAAT promoter. Similar vector genome copy numbers per diploid genome measured in mice injected with the two AAV vectors (Figure 1F) indicate similar transduction efficacies in the liver.

[0121] Example 2: Long-term correction of liver phenotype in GSD-Ia mice following intravenous injection of an AAV vector expressing human G6Pase-a To evaluate the long-term efficacy of the two vectors, a 7-month study was conducted. - / -2.5 x 10 on mouse 11 The vector was injected at a dose of 10 ... - / - Mice were treated with AAV9-hGPE-hG6PC L.G6pc - / - They showed supraphysiological hepatic G6Pase-a activity that was significantly higher than the activity measured in mice ( Fig. 2C ).

[0122] Glycogen accumulation and hepatomegaly were completely rescued in animals injected with the two vectors (Fig. 2D, E). A higher vector genome copy number per diploid genome was measured in mice injected with the AAV9-hGPE-hG6PC vector (Fig. 2F). This likely reflects the slightly lower hepatic transduction achieved with the AAV9-hAAT-hG6PC vector.

[0123] Taken together, these results demonstrated that the expression of G6Pase-a was increased by the hAAT promoter, which is therefore more efficient than the hGPE promoter and is suitable for AAV gene therapy of GSD-Ia.

[0124] Example 3: hGPE-directed gene therapy enhances L.G6pc mice fed a high-fat, high-sucrose diet - / - Promotes liver tumor formation in mice Adenoma formation is a hallmark of human GSD-Ia, reported in most affected individuals in their teens and twenties. - / - In mice, almost all mice fed a standard diet developed hepatic adenomas by 18 months of age.

[13] This slow process can be accelerated by a high-fat / high-sucrose (HF / HS) diet. - / - Approximately 85% of mice developed multiple liver tumors by the age of 9 months

[14] . - / -Mice represent a robust model to evaluate the efficacy and safety of gene replacement strategies in preventing tumor formation in GSDIa.

[0125] Therefore, L.G6pc fed the HF / HS diet - / - We tested AAV9-hAAT-hG6PC and AAV9-hGPE-hG6PC vectors in mice (Figure 3A). At 8 months after treatment, only the vector carrying the hAAT promoter completely rescued glycemia (Figure 3B). Under HF / HS diet, G6Pase activity levels in AAV-treated animals were significantly higher than that in L.G6pc + / + The levels of G6Pase activity in animals treated with AAV9-hAAT-hG6PC were significantly lower than those measured in L.G6pc animals. Animals treated with AAV9-hAAT-hG6PC showed high G6Pase activity levels, whereas animals treated with AAV9-hGPE-hG6PC vectors showed high G6Pase activity levels. - / - This did not reach statistical significance when compared with mice (Fig. 3C). Similar vector genome copy numbers per diploid genome were measured in AAV-treated mice (Fig. 3D).

[0126] Interestingly, autopsy of the mice revealed a higher number of tumors in the livers of mice treated with hGPE-carrying AAV vectors compared to mice receiving AAV9-hAAT-hG6PC (Figure 3E). These results suggest that the use of AAV9-hAAT-hG6PC may reduce the transformation rate of hepatocytes compared to AAV9-hGPE-hG6PC, thereby decreasing the frequency of adenomas.

[0127] The data obtained for Figure 3A was completed using an increased number of mice per group (Figure 4 and Table 1). Figure 4 shows that in the larger cohort, the hAAT-driven vector achieved significantly higher G6Pase activity. The data presented in Table 1 confirm that adenoma frequency was reduced with AAV9-hAAT-hG6PC compared to AAV9-hGPE-hG6PC.

[0128] [Table 1] Table 1: Frequency of adenomas

[0129] Example 4: Expression of G6Pase wt in mouse liver with various AAV serotypes Three different AAV vectors were generated, namely AAV9, AAV8, and AAVmut5, expressing the wild-type human G6pc gene wt (SEQ ID NO:2) under the control of the hAAT promoter.

[0130] Liver-specific G6pc knockout mouse model (L.G6pc - / -

[13] ), i.e., 1 × 10 12 These three vectors or PBS (negative control) were tested independently at a dose of 1000 ng / kg. As a positive control, WT mice (L.G6pc + / + ) were injected with PBS. 15 days after vector injection, G6Pase activity of each vector was evaluated in the liver. Groups injected with the three AAV vectors showed similar G6Pase activity, but at higher levels than that measured in wild-type animals (Figure 5). This confirmed the feasibility of expressing transgenes in mouse liver by various AAV vectors.

[0131] Example 5: Expression of G6Pase in mouse liver with various codon-optimized G6pc sequences Two independent experiments were performed to evaluate the effectiveness of codon optimization along with the reduction of CG dimers in the expression of G6Pase in mouse liver.

[0132] The G6pc wt sequence (SEQ ID NO:2) was codon-optimized using various methods. G6pc co1 has the highest similarity to the wt sequence and the lowest CG dimer content. G6pc co2 has the highest GC content and higher CG dimer content compared to G6pc co1. G6pc co3 has the lowest similarity to the wt sequence and the highest CG dimer content (Table 2).

[0133] [Table 2] Table 2. Comparative analysis of codon-optimized sequences. Sequence analysis was performed on the wild-type hG6pc sequence (wt) and three codon-optimized sequences (co1, co2, and co3). a Splicing donors (SD) and splicing acceptors (SA) were predicted using an online tool (www.fruitfly.org) with a minimum score of 0.8. b GC content was calculated using an online molecular biology tool (www.genscript.com). c CpG islands smaller than 100 bp with a GC content threshold of 60% were predicted using the online tool MethPrimerDB ( www.urogene.org ).

[0134] In the first experiment, three AAV8 vectors were generated expressing the following transgenes under the control of the hAAT promoter: hG6pc: human G6pc gene wt (SEQ ID NO: 2) hG6pc Co1: codon-optimized human G6pc gene (SEQ ID NO: 45), and hG6pc Co2: codon-optimized human G6pc gene (SEQ ID NO: 46)

[0135] We then developed a liver-specific G6pc knockout mouse model (L.G6pc - / -

[13] ), i.e., 1 × 10 12 These three AAV8 vectors were tested at a dose of 1000 ng / kg. As a positive control, WT mice (L.G6pc + / + ) were injected with PBS. G6Pase activity was assessed in the liver 15 days after vector injection. Groups injected with the three AAV8 vectors showed supraphysiological G6Pase activity, levels higher than those measured in wild-type animals (Fig. 6A). Importantly, AAV8 vectors expressing hG6pc Co1 induced significantly higher levels of G6Pase activity when compared to the same vectors expressing hG6pc Co2 (Fig. 6A).

[0136] In a second experiment, two AAV9 vectors were generated expressing the following transgenes under the control of the hAAT promoter: hG6pc: human G6pc gene wt (SEQ ID NO: 2) hG6pc Co3: codon-optimized human G6pc gene (SEQ ID NO:3)

[0137] We then developed a liver-specific G6pc knockout mouse model (L.G6pc - / -

[13] ), i.e., 1 × 10 11 These two AAV9 vectors were tested at a dose of 10 ... + / + ) were injected with PBS. G6Pase activity was assessed in the liver 15 days after vector injection. Importantly, only animals injected with AAV9 vectors carrying the hG6pc wild-type sequence exhibited supraphysiological G6Pase activity, levels higher than those measured in wild-type animals (Figure 6B). Animals injected with AAV9 vectors expressing hG6pc Co3 showed a trend toward reduced G6Pase activity when compared to animals injected with AAV9 vectors expressing the wild-type version of hG6pc (p=0.06, Figure 6B).

[0138] References cited in the form "[reference number]" [1]Chandler et al., Vector design influences hepatic genotoxicity after,The Journal of Clinical Investigation,2015 [2]Mates et al.Nat Genet.2009 Jun;41(6):753-61.doi:10.1038 / ng.343 [3]Ling et al.,2016 Jul 18,Hum Gene Ther Methods. [4]Vercauteren et al.,2016,Mol.Ther.Vol.24(6),p.1042 [5]Rosario et al.,2016,Mol Ther Methods Clin Dev.3,p.16026 [6]Shen et al.,2007 Molecular Therapy,volume 15,issue 11,pages 1955-1962 [7]Tenney et al.,Virology,volumes 454-455,April 2014,pages 227-236 [8]Bartel et al.,Front.Microbiol.,04 October 2011 https: / / doi.org / 10.3389 / fmicb.2011.00204 [9]Michelfelder et al.(PLoS ONE,2009,4,e5122

[10] Zhong et al.,PNAS June 3,2008 105(22)7827-7832;https: / / doi.org / 10.1073 / pnas.0802866105

[11] Finet et al.,Virology,2018,513,43-51.

[12] McCarty et al.,2003 Gene Therapy Dec;10(26):2112-8.

[13] Mutel et al.,Targeted deletion of liver glucose-6 phosphatase mimics glycogen storage disease type 1a including development of multiple adenomas,Journal of Hepatology,2011 Mar;54(3):529-37.doi:10.1016 / j.jhep.2010.08.014.Epub 2010 Oct 1.

[14] Gjorgjieva M. et al, Dietary exacerbation of metabolic stress leads to accelerated hepatic carcinogenesis in glycogen storage disease type Ia, J. Hepatol. 2018; Nov; 69(5): 1074 - 1087. doi: 10.1016 / j.jhep.2018.07.017. Epub 2018 Sep 5.

[15] George L. et al, Hemophilia B Gene Therapy with a High - Specific - Activity Factor IX Variant. N Engl J Med. 2017 Dec 7; 377(23): 2215 - 2227. doi: 10.1056 / NEJMoa1708538.

[16] Zhang L. et al, An evolutionary approach to optimizing glucose - 6 - phosphatase - α enzymatic activity for gene therapy of glycogen storage disease type Ia. J Inherit Metab Dis. 2019 May; 42(3): 470 - 479. doi: 10.1002 / jimd.12069. Epub 2019 Feb 22

[17] Rohr et al., J.Virol.Methods, 2002, 106, 81 - 88)

[0139] Array List

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

Claims

1. An adeno-associated virus (AAV) vector comprising a nucleic acid construct for expressing glucose-6-phosphatase-a (G6Pase-a) in liver cells, said construct comprising a nucleic acid sequence encoding said G6Pase-a, said nucleic acid sequence encoding said G6Pase-a being operably linked to a human alpha-1 antitrypsin (hAAT) promoter.

2. The vector of claim 1, which is one or more selected from the group consisting of an AAV serotype 8 (AAV8) vector, an AAV9 vector, an AAVrh74 vector, an AAV2i8 vector, an AAVmut5 vector, an AAV-3 vector, an AAV-3 mutant vector, an AAV-3B vector, an AAV-3B mutant vector, an LK03 vector, an AAV-5 vector, a RHM4-1 vector, a RHM15-1 vector, a RHM15-2 vector, a RHM15-3 / RHM15-5 vector, a RHM15-4 vector, and a RHM15-6 vector.

3. 3. The vector of claim 1 or 2, which is one or more selected from the group consisting of an AAV serotype 8 (AAV8) vector, an AAV9 vector, an AAVrh74 vector, an AAV2i8 vector, and an AAVmut5 vector.

4. The AAV vector according to any one of claims 1 to 3, wherein the G6Pase-a has an amino acid sequence having at least 90% identity with SEQ ID NO:

1.

5. The AAV vector according to any one of claims 1 to 4, wherein the nucleic acid sequence encoding G6Pase-a comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:

2.

6. The AAV vector of any one of claims 1 to 5, wherein the nucleic acid sequence encoding the G6Pase-a is codon-optimized.

7. The AAV vector according to any one of claims 1 to 6, wherein the nucleic acid sequence encoding the G6Pase-a is codon-optimized by reducing the GC content and GC dimers in the nucleic acid sequence encoding the G6Pase-a.

8. The AAV vector of any one of claims 1 to 7, wherein the hAAT promoter comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:

8.

9. The AAV vector according to any one of claims 1 to 8, wherein an enhancer is arranged in front of the hAAT promoter.

10. The AAV vector according to any one of claims 1 to 9, wherein the hAAT promoter is preceded by an ApoE enhancer.

11. The AAV vector according to any one of claims 1 to 10, wherein an ApoE enhancer having the base sequence shown in SEQ ID NO: 9 is arranged in front of the hAAT promoter.

12. The AAV vector of any one of claims 1 to 11, wherein the nucleic acid construct comprises SEQ ID NO:

7.

13. The AAV vector of any one of claims 1 to 12, wherein the nucleic acid construct comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:11, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, or SEQ ID NO:

56.

14. The AAV vector of any one of claims 1 to 13, wherein the nucleic acid construct comprises a nucleotide sequence having at least 90% identity to SEQ ID NO:

48.

15. The nucleic acid construct is (i) a hAAT promoter preceded by an enhancer; (ii) an intron, (iii) a nucleic acid sequence encoding the G6Pase-a; and (iv) polyadenylation signal The AAV vector according to any one of claims 1 to 14, comprising:

16. The AAV vector of claim 15, wherein the enhancer is an ApoE enhancer.

17. The AAV vector of claim 16, wherein the ApoE enhancer has the base sequence shown in SEQ ID NO:

9.

18. The AAV vector according to any one of claims 15 to 17, wherein the intron is an intron of a human β-globin gene.

19. The AAV vector according to any one of claims 15 to 18, wherein the intron has a base sequence shown in SEQ ID NO:

47.

20. The AAV vector according to any one of claims 15 to 19, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, an HBB2 polyadenylation signal, an SV40 polyadenylation signal, or any other natural or artificial polyadenylation signal.

21. The AAV vector according to any one of claims 1 to 20, which is an AAV8 vector.

22. A liver cell transformed with the vector according to any one of claims 1 to 21.

23. A composition comprising a vector according to any one of claims 1 to 21 or a liver cell according to claim 22.

24. A vector according to any one of claims 1 to 21, a liver cell according to claim 22 or a composition according to claim 23 for use as a medicament.

25. A vector according to any one of claims 1 to 21, a liver cell according to claim 22, or a composition according to claim 23 for use in the treatment of glycogen storage disease (GSD).

26. 26. The vector, liver cell, or composition of claim 25, wherein the GSD is GSD-Ia.

Images