Vectors encoding glucose-6-phosphatase (g6pase-a) for gene therapy

Abstract

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, the construct comprising a nucleic acid sequence encoding G6Pase-a, wherein the nucleic acid sequence encoding G6Pase-a is operably linked to a human alpha-1 antitrypsin (hAAT) promoter, a cell transformed with the vector of the invention, a composition comprising the vector or cell of the invention, and uses thereof.

Description

Invention Field

[0001] This invention relates to an adeno-associated virus (AAV) comprising a nucleic acid construct for expressing glucose-6-phosphatase-a (G6Pase-a) in cells for the treatment of glycogen storage disease Ia (GSD-Ia), wherein the nucleic acid sequence encoding G6Pase-a is operatively linked to a human α-1 antitrypsin (hAAT) promoter. Background Technology

[0002] Type Ia glycogen storage disease (GSD-Ia or von Gierke's disease) is caused by a deficiency of glucose-6-phosphatase-a (G6Pase-a), an enzyme primarily expressed in the liver, kidneys, and intestines. Encoded by the G6PC gene, G6Pase-a is a hydrophobic protein anchored in the endoplasmic reticulum (ER) via nine transmembrane helices. This enzyme catalyzes the hydrolysis of glucose-6-phosphate (G6P) into glucose and inorganic phosphate in the final steps of glycogenolysis and gluconeogenesis. Patients affected by GSD-Ia are unable to maintain glucose homeostasis and exhibit fasting hypoglycemia, growth retardation, hepatomegaly, nephromeletus, hyperlipidemia, hyperuricemia, and lactic acidosis.

[0003] In most cases, hypoglycemia can be controlled through dietary therapy, allowing patients to achieve near-normal growth and puberty development. However, long-term clinical complications and their underlying pathological processes remain uncorrected. One of the most significant chronic risks is hepatocellular adenoma (HCA), which occurs in 70-80% of GSD-I patients over 25 years of age. In GSD-Ia patients, HCAs are multiple and non-capsulated, accompanied by complications such as local compression and intratumoral hemorrhage. In 10% of GSD-Ia patients, HCAs undergo malignant transformation into hepatocellular carcinoma (HCC).

[0004] Therefore, improved therapeutic vectors are needed to treat GSD-1a and its associated complications.

[0005] Gene therapy studies using recombinant adeno-associated virus (AAV) carrying G6Pase-a have previously been conducted in GSD-Ia animal models. Specifically, the prior art discloses the use of nucleic acid constructs comprising a nucleic acid sequence encoding G6Pase-a, said nucleic acid sequence being operatively linked to a G6PC promoter / enhancer (GPE).

[0006] Treatment complications associated with immune responses to vectors represent a significant obstacle to the use of AAV vectors in gene therapy. Generally, administration of lower doses of AAV vectors is associated with lower immune response activation and better transgene expression stability

[15] . To reduce the occurrence of side effects associated with the use of AAV vectors, it is therefore preferable to administer the lowest possible amount of AAV vector sufficient to achieve a therapeutic effect in the subject.

[0007] Therefore, improved nucleic acid constructs are needed to increase G6Pase-a expression and activity in gene therapy, thereby allowing for a reduction in the amount of AAV vector necessary to achieve therapeutic effects in subjects. Invention Overview

[0009] The inventors have surprisingly demonstrated that, particularly in the AAV gene therapy setting, nucleic acids encoding G6Pase-a under the control of the hAAT promoter lead to increased G6Pase-a expression and activity, which allows for phenotypic rescue in GSDIa mice compared to the hGPE promoter. This is particularly unexpected considering existing techniques for treating GSDIa using different forms of natural G6Pase promoters (GPEs) derived from different species.

[0010] In addition, the inventors have demonstrated that, in gene therapy, the same nucleic acid encoding G6Pase-a under the control of the hAAT promoter reduces the risk of tumorigenesis (e.g., HCA and HCC) compared to the hGPE promoter. This is particularly surprising given that prior art predicts that promoters with higher activity are more likely to increase the risk of HCA and HCC after gene therapy [1].

[0011] Therefore, 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 cells, the construct comprising a nucleic acid sequence encoding G6Pase-a, wherein the nucleic acid sequence encoding G6Pase-a is operatively linked to a human α-1 antitrypsin (hAAT) promoter.

[0012] In a second aspect, the present invention relates to cells transformed using the vector of the present invention.

[0013] In a third aspect, the present invention relates to compositions comprising the carrier or cells of the present invention.

[0014] In a fourth aspect, the present invention relates to the use of the carrier, cell or composition of the present invention as a medicine, particularly for the treatment of glycogen storage disease Ia (GSD-Ia). Brief description of the attached diagram

[0016] Figure 1The corrected liver phenotypes in GSD-Ia and WT mice after 15 days of treatment with AAV vector or PBS are shown. A. Treatment regimen. B. Blood glucose measured 6 hours after fasting at the end of the treatment regimen. C. G6Pase activity measured in liver tissue. D. Glycogen content in liver tissue. E. Hepatomegaly reported as a percentage of liver / body weight. F. Vector genome copy number measured in the liver of AAV-treated mice. Compare with L.G6pc injected with PBS via analysis of variance in AE (#P<0.05). + / + Mice; *P<0.05 compared to L.G6pc mice injected with PBS - / - Statistical analysis was performed on mice using t-tests (ns, not significant) in F.

[0017] Figure 2 Long-term corrected liver phenotypes in GSD-Ia mice are shown. A. Represents the treatment regimen. B. Blood glucose measured 6 hours after fasting at the end of the treatment regimen (i.e., 7 months after vector injection), i.e., 7 months after vector injection. C. G6Pase activity measured in liver tissue collected at sacrifice. D. Glycogen content measured in liver tissue. E. Hepatomegaly reported as liver / body weight percentage at sacrifice. F. Vector genome copy number measured in the liver of AAV-treated mice. Comparison with L.G6pc injected with PBS via analysis of variance (#P<0.05). + / + Mice; *P<0.05 compared to L.G6pc mice injected with PBS - / - Mice; As shown in the figure, statistical analysis was performed in F by t-test (*P<0.05).

[0018] Figure 3 hGPE-directed gene therapy showed that feeding L.G6pc with a high-fat, high-sucrose diet promoted the growth of L.G6pc. - / - Hepatic tumor formation in mice. A. Represents the treatment regimen. B. Shows blood glucose measured 6 hours after fasting at the end of the treatment regimen (i.e., 8 months after vector injection). C. Shows G6Pase-a activity assay performed on liver tissue collected at sacrifice. D. Shows the vector genome copy number measured in the liver for each diploid genome. E. Shows L.G6pc treated with PBS or AAV vector 8 months after the start of the HF / HS regimen. - / - The number of tumors larger than 2 mm observed in mice. Analysis of variance was used to compare L.G6pc mice injected with PBS in B and C (#P<0.05). + / + Mice; *P<0.05 compared to L.G6pc mice injected with PBS - / -Mice), compared with L.G6pc injected with PBS in D by t-test (ns, not significant) and in E by nonparametric ANOVA (*P<0.05). + / + Statistical analysis was performed on mice.

[0019] Figure 4 It is shown in 2.5x10 11 Larger L.G6pcs injected into mice via vg / mice - / - In an animal cohort, the AAV9 vector with the hAAT promoter achieved higher G6Pase-a activity compared to the AAV9 vector expressing wild-type G6pc. Activity was determined from liver tissue collected at sacrifice. Analysis of variance (#P<0.05) compared to L. G6pc injected with PBS. + / + Mice; *P<0.05 compared to L.G6pc mice injected with PBS - / - Statistical analysis was performed on mice (XP < 0.05, as shown).

[0020] Figure 5 This demonstrates the use of different AAV vectors encoding the human G6pc gene in L.G6pc. - / - G6Pase-a activity obtained by injection in mice. L.G6pc injected with PBS. + / + and L.G6pc - / - Mice were used as controls. Liver tissue collected 15 days after vector injection was used for activity assay. Analysis of variance (#P<0.05) was used to compare L.G6pc injected with PBS. + / + Mice; *P<0.05 compared to L.G6pc mice injected with PBS - / - Statistical analysis was performed on mice (+P<0.05, as shown).

[0021] Figure 6 shows a comparison of the power of three different codon-optimized sequences. A. Shows the power of 1x10... 12 AAV8 was injected at a dose of vg / kg and L.G6pc was followed up for 15 days. - / - G6Pase-a activity assay of wild-type (G6pc wt), codon-optimized 1 and 2 (G6pc co1 and G6pc co2) G6pc sequences expressed in mice, denoted as L.G6pc + / + The percentage of G6Pase-a activity is shown in B. (This is based on a 1x10⁻¹) 11 L.G6pc mice were injected with AAV9 at a dose of vg / v and followed up for 15 days. - / - G6Pase-a activity assay of wild-type (G6pc wt) and codon-optimized 3(G6pc co3)G6pc sequences in mice, denoted as L.G6pc + / +The percentage of G6Pase-a activity was determined by analysis of variance (#P<0.05) and compared with L.G6pc injected with PBS. + / + Mice; *P<0.05 compared to L.G6pc mice injected with PBS - / - Statistical analysis was performed on mice (+P<0.05, as shown). Detailed Implementation

[0022] definition

[0023] The term “glucose-6-phosphatase α” or “G6Pase-a” refers to an enzyme encoded by the G6pc gene. This enzyme catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate in the final steps of glycogenolysis and gluconeogenesis. According to the invention, G6Pase-a can be wild-type G6Pase-a or modified G6Pase-a, particularly modified G6Pase-a with increased phosphorylase activity. Modified G6Pase-a is disclosed in WO2016106303 and

[16] . For example, G6Pase-a can 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.

[0024] According to the present invention, "identity" is calculated by comparing two aligned sequences within a comparison window. Sequence alignment determines the number of common positions (nucleotides or amino acids) between the two sequences within the comparison window. Therefore, the number of common positions is divided by the total number of positions within the comparison window, and then multiplied by 100 to obtain the identity percentage. The determination of the sequence identity percentage can be performed manually or by a well-known computer program. In a specific embodiment of the invention, identity or homology corresponds to at least one substitution of amino acid residues, such as 1, 2, 3, 4, or 5 substitutions, without a significant loss of interaction binding ability. Preferably, at least one substitution is a conserved amino acid substitution. A "conserved amino acid substitution" means that an amino acid can be replaced by another amino acid with a similar side chain. Families of amino acids with similar side chains have been defined in this 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0025] According to the present invention, the term "nucleic acid sequence" refers to DNA or RNA molecules in single-stranded or double-stranded form, especially DNA.

[0026] According to the present invention, the term "nucleic acid sequence encoding G6Pase-a" refers to a nucleic acid sequence encoding G6Pase-a (wild-type G6Pase-a or modified G6Pase-a). Modified nucleic acid sequences encoding modified G6Pase-a are disclosed in references

[16] and WO2016106303. For example, a nucleic acid sequence encoding wild-type G6Pase-a has nucleotide sequences 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 nucleotide sequences SEQ ID NO:4 or SEQ ID NO:5. For example, a nucleic acid sequence encoding G6Pase-a can therefore be 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 any nucleic acid sequence encoding modified G6Pase-a selected from SEQ ID NO:13 to SEQ ID NO:28.

[0027] The term "nucleic acid construct" refers to an artificially constructed nucleic acid fragment that will be transplanted into target cells to express a transgene, such as for expressing G6Pase-a in cells. A nucleic acid construct may contain one or more expression-controlling nucleic acid sequences and / or other nucleic acid sequences that improve G6Pase-a expression and / or enhance G6Pase-a secretion and / or enhance tissue uptake of G6Pase-a, said nucleic acid sequences being operatively linked to a sequence encoding a transgene (e.g., G6Pase-a). As used herein, the term "operatively linked" refers to the linking of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operatively linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or another transcriptionally regulatory nucleic acid sequence affects the transcription of a nucleic acid sequence encoding a transgene (e.g., G6Pase-a), then said promoter or other transcriptionally regulatory nucleic acid sequence is operatively linked to said nucleic acid sequence encoding the transgene (e.g., G6Pase-a). Such expression-controlling nucleic acid sequences are known in the art, such as promoters, enhancers (e.g., cis-regulatory modules (CRMs)), introns, polyA signals, etc.

[0028] The term "α-1 antitrypsin" or "AAT" refers to a protein belonging to the serine protease inhibitor (serpin) superfamily. In humans, it is encoded by the SERPINA1 gene. The term "hAAT" refers to human AAT.

[0029] The term "promoter" refers to a region of DNA that directs / initiates the transcription of a nucleic acid sequence (such as a gene). Promoters consist of the necessary nucleic acid sequence located near the transcription start site. Typically, promoters are located near the gene they transcribe.

[0030] The term "hAAT promoter" refers to the promoter of hAAT (wild-type hAAT promoter or modified hAAT promoter).

[0031] According to the present invention, the term "vector" refers to a vector suitable for transgenic expression in gene therapy (e.g., for the expression of G6Pase-a in gene therapy).

[0032] In the context of this invention, the term "gene therapy" refers to a treatment involving the delivery of genes / nucleic acids into the cells of an individual subject for the purpose of treating a disease.

[0033] As used herein, the terms “subject,” “patient,” or “individual” refer to a human or non-human mammal (e.g., rodents (mice, rats), felines, canines, or primates) that is affected by or may be affected by glycogen storage disease Ia (GSD-Ia). Preferably, the subject is a human, a man, or a woman.

[0034] The term "glycogen storage disease" or "GSD" refers to a metabolic disorder caused by defects in enzymes affecting glycogen synthesis, glycogenolysis, or glycolysis, typically occurring in muscle and / or hepatocytes. GSD is classified into different types, from GSD type 0 to GSD type XV. GSD-I consists of two autosomal recessive disorders, GSD-Ia and GSD-Ib. GSD-Ia results from a deficiency of glucose-6-phosphatase-a. A deficiency of glucose-6-phosphate transporter (G6PT) is the cause of GSD-Ib. According to the present invention, GSD is GSD-Ia (Von Gillespie's disease; OMIM#232240).

[0035] The term "treatment" or "curative therapy" refers to reversing, alleviating, or inhibiting the progression of a disease or condition to which the term applies, or preventing a disease or condition, or one or more symptoms of such a disease or condition. In particular, treatment of the condition may refer to the treatment of functional impairment in GSD, preferably improving the subject's glycemic control.

[0036] The term "pharmaceuticalally acceptable" means that a product is approved by a federal or state regulatory agency or listed in the U.S. or European Pharmacopoeia or other recognized pharmacopoeia for use in animals and humans.

[0037] "Pharmaceutical composition" refers to a composition comprising a pharmaceutically acceptable carrier. For example, the carrier can be a diluent, adjuvant, excipient, or transporter administered with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous glucose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, etc. When a pharmaceutical composition is suitable for oral administration, tablets or capsules may be prepared by conventional methods using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); 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 glycolate); or wetting agents (e.g., sodium dodecyl sulfate). Tablets may be coated using methods known in the art. Liquid formulations for oral administration may be in the form of, for example, solutions, syrups, or suspensions, or they may be present as dry products for reconstitution with water or other suitable carriers prior to use. Such liquid formulations can be prepared by conventional methods with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous carriers (e.g., almond oil, oily esters, ethanol, or fractionated vegetable oils); and preservatives (e.g., methylparaben, propylparaben, or sorbic acid). The formulation may also contain suitable buffer salts, flavoring agents, coloring agents, and sweeteners. The compositions according to the invention are preferably pharmaceutical compositions.

[0038] Nucleic acid constructs

[0039] This specification relates to a nucleic acid construct for expressing glucose-6-phosphatase (G6Pase-a) in cells, the construct comprising a nucleic acid sequence encoding G6Pase-a, wherein the nucleic acid sequence encoding G6Pase-a is operatively linked to a human α-1 antitrypsin (hAAT) promoter.

[0040] According to the specification, the nucleic acid sequence can encode wild-type G6Pase-a, such as G6Pase-a having the amino acid sequence SEQ ID NO:1, or modified G6Pase-a, preferably modified G6Pase-a with increased phosphorylase activity, such as G6Pase-a containing or having an amino acid sequence that is at least 90% identical to SEQ ID NO:1. Modified G6Pase-a can include one or more amino acid modifications, such as substitutions or deletions, as long as the protein retains its enzymatic activity. In some embodiments, modified G6Pase-a includes a serine-to-cysteine ​​substitution at amino acid 298 of human G6Pase-a (the amino acid sequence of wild-type human G6Pase-a is shown herein as SEQ ID NO:1). Modified G6Pase-a can include modifications at other residues, as long as the protein retains its enzymatic activity. For example, modified G6Pase-a may include substitutions at other residues, such residues being positions 3, 54, 139, 196, 199, 242, 247, 292, 301, 318, 324, 332, 347, 349, 350, and / or 353 of human G6Pase-a (as shown in 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.

[0041] G6Pase-a may contain or have an amino acid sequence that is 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% identical to SEQ ID NO:1.

[0042] Therefore, the nucleic acid sequence encoding G6Pase-a can encode 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 can be any nucleic acid sequence selected from any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:13 to SEQ ID NO:28, SEQ ID NO:52 or SEQ ID NO:54.

[0043] The nucleic acid sequence encoding G6Pase-a may contain a nucleotide sequence that has 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 with SEQ ID NO:2.

[0044] For example, the nucleic acid sequence encoding G6Pase-a can therefore be any nucleic acid sequence n°6-7 of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or WO2016106303.

[0045] The nucleic acid sequence encoding G6Pase-a may contain a nucleotide sequence that has 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 with SEQ ID NO:45.

[0046] The nucleic acid sequence encoding G6Pase-a may contain a nucleotide sequence that has 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 with SEQ ID NO:46.

[0047] The nucleic acid sequence encoding G6Pase-a is operatively linked to the human alpha-1 antitrypsin (hAAT) promoter.

[0048] The hAAT promoter can be a wild-type hAAT promoter, such as the hAAT promoter having the nucleic acid sequence SEQ ID NO:8, or a modified hAAT promoter, such as an hAAT promoter containing or having a nucleic acid sequence that is at least 90% identical to SEQ ID NO:8. Modified hAAT promoters can include one or more nucleic acid modifications, such as substitutions or deletions, as long as the promoter still directs / initiates the transcription of G6Pase-a.

[0049] The hAAT promoter may contain or have a nucleotide sequence that is 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% identical to SEQ ID NO:8.

[0050] 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 contains SEQ ID NO:7.

[0051] The nucleic acid construct may contain introns, particularly introns located between the hAAT promoter and the nucleic acid sequence encoding G6Pase-a. Introns can be introduced to increase mRNA stability and G6Pase-a production. Advantageously, the nucleic acid construct contains an intron derived from a human β-globin gene (e.g., HBB2) located between the hAAT promoter and the nucleic acid sequence encoding G6Pase-a; preferably, the intron contained in the nucleic acid construct has the sequence shown in SEQ ID NO:47. An intron having the sequence SEQ ID NO:47 is disclosed in WO2015 / 162302. Alternatively, the intron may be located after the 3' end of the nucleic acid sequence encoding G6Pase-a.

[0052] The nucleic acid construct of this specification may include, in the 5' to 3' direction, an hAAT promoter, optionally preceded by an enhancer, such as an ApoE enhancer (e.g., SEQ ID NO:9), optionally an intron, such as an intron of the human β-globin gene (e.g., SEQ ID NO:47), a nucleic acid sequence encoding G6Pase-a, and a polyadenylation signal (e.g., bovine growth hormone polyadenylation signal, HBB2 polyadenylation signal, SV40 polyadenylation signal, or another naturally occurring or artificial polyadenylation signal). Advantageously, the nucleic acid construct of the present invention includes, in the 5' to 3' direction, an hAAT promoter, optionally preceded by an enhancer, such as an ApoE enhancer (e.g., SEQ ID NO:9), an intron (particularly an intron as defined above), a nucleic acid molecule encoding G6Pase-a, and a polyadenylation signal.

[0053] The nucleic acid construct may contain 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 with SEQ ID NO:11, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55, or SEQ ID NO:56. In a preferred embodiment, the nucleic acid construct may contain 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 with SEQ ID NO:48.

[0054] The nucleic acid construct will be transferred to target cells for the expression of G6Pase-a in said cells. Preferably, the target cells are hepatocytes, kidney cells, or intestinal cells.

[0055] carrier

[0056] The terms “nucleic acid construct according to the specification” or “nucleic acid construct according to the specification” refer to the nucleic acid construct disclosed in this specification, especially the nucleic acid construct disclosed above.

[0057] This specification also relates to vectors containing nucleic acid constructs contained herein.

[0058] The vector can be a plasmid vector. The vector can also be nanoparticles containing the nucleic acid constructs of the present invention. The vector can also be a transposon-based system that allows the nucleic acid constructs of the present invention to be integrated into the genome of a target cell, such as the hyperactive Sleeping Beauty (SB100X) transposon system [2]. The vector can be a viral vector suitable for gene therapy. The vector can target any target cell, such as hepatocytes, kidney cells or intestinal cells.

[0059] The vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.

[0060] This invention relates to AAV vectors comprising nucleic acid constructs described herein. In a particularly preferred embodiment, the AAV vector used in practice is AAV8 or AAV9, preferably AAV8.

[0061] Therefore, the nucleic acid constructs described herein may also contain sequences suitable for generating effective viral vectors, as well as those fully disclosed in the art.

[0062] Nucleic acid constructs can be inserted into vectors, such as lentiviral vectors according to the specification, or AAV vectors according to the invention, such as single-stranded or double-stranded self-complementary AAV vectors. In a more preferred embodiment of the invention, the AAV vector is an AAV vector suitable for transducing hepatocytes, more particularly AAV-1, AAV-2 and AAV-2 variants (e.g., AAV-2 with a quadruple mutant capsid optimized, comprising an engineered capsid with the Y44+500+730F+T491V variation, disclosed in [3]), AAV-3 and AAV-3 variants (e.g., AAV3-ST variant, comprising an engineered AAV-3 capsid with the two amino acid variations S663V+T492V, disclosed in Vercauteren et al. [4]), AAV-3B and AAV-3B variants, AAV-4, AAV-5, AAV-6 and AAV-3B variants, AAV-4, AAV-5, AAV-6 and AAV-3B variants, AAV-3B ... AV-6 variants (e.g., AAV-6 variants containing the triple mutant AAV-6 capsid Y731F / Y705F / T492V form disclosed in [5]), AAV-7, AAV-8, AAV-9, AAV-10, such as AAV-cy10 and AAV-rh10, AAV-rh74, AAV-dj, Anc80, LK03, AAV-2i8, porcine AAV serotypes such as AAV-po4 and AAV-po6, etc. As is known in the art, additional suitable sequences will be introduced into the nucleic acid constructs of this specification to obtain a functional viral vector, depending on the specific viral vector to be used. Suitable sequences include AAV ITRs for AAV vectors or LTRs for lentiviral vectors. Therefore, this specification also relates to nucleic acid constructs as described above, which are side-joined with ITRs or LTRs on each side.

[0063] In addition, other non-naturally engineered variants and chimeric AAVs may also be useful. AAV viruses can be engineered using conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, minimization of immunogenicity, regulation of stability and particle lifespan, efficient degradation, and precise delivery to the cell nucleus. Ideal 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 readily used in a variety of vector systems and host cells. AAV-based recombinant vectors lacking Rep proteins integrate inefficiently into the host genome and exist primarily as stable, round appendages that can persist in target cells for many years. As an alternative to using natural AAV serotypes, artificial AAV serotypes can be used in the context of this invention, including but not limited to AAVs with non-naturally present capsid proteins. This artificial capsid can be produced by any suitable technique using a combination of a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) and a heterologous sequence (which may be obtained from different selected AAV serotypes, discontinuous portions of the same AAV serotype, from a non-AAV viral source, or from 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.

[0064] In the context of this invention, the AAV vector comprises an AAV capsid capable of transducing target cells, particularly hepatocytes. 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 aforementioned AAV serotypes and whose capsid is derived from another serotype.

[0065] According to a specific implementation, the AAV capsid is selected from AAV-1, -2, AAV-2 variants (e.g., AAV-2 with a quadruple mutant capsid optimized containing an engineered capsid with the Y44+500+730F+T491V variation, disclosed in Ling et al., 2016), -3 and AAV-3 variants (e.g., the AAV3-ST variant containing an engineered AAV3 capsid with two amino acid variations S663V+T492V, disclosed in Vercauteren et al., 2016), -3B and AAV-3B variants, -4, -5, -6 and AAV-6 Variants (e.g., AAV6 variants containing triple-mutant AAV6 capsids in the form Y731F / Y705F / T492V, disclosed in Rosario et al. 2016), -7, -8, -9 and AAV-9 variants (e.g., AAVhu68), -2G9, -10, such as -cy10 and -rh10, -rh39, -rh43, -rh74, -dj, Anc80, LK03, AAV.PHP, AAV2i8, porcine AAV, such as AAVpo4 and AAVpo6, and tyrosine, lysine, and serine capsid mutants of AAV serotypes. Furthermore, AAV capsids are selected from other non-natural engineered variants (e.g., AAV-spark100), chimeric AAVs, or AAV serotypes obtained through shuffling, basic principle design, error-prone PCR, and machine learning techniques. In a particular embodiment, the Cap gene encodes a VP capsid protein derived from at least two different AAV serotypes, or encodes at least one chimeric VP protein that binds to VP protein regions or domains derived from at least two AAV serotypes. For example, the chimeric AAV capsid may be derived from a combination of an AAV8 capsid sequence and an AAV serotype sequence different from AAV8 (e.g., any of those specifically mentioned above). In another embodiment, the capsid of the AAV vector comprises one or more variant VP capsid proteins, such as those described in WO2015013313, particularly RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6 capsid variants. In a particular 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 can be the -rh74-9 serotype disclosed in WO2019 / 193119 (e.g., the Hybrid Cap rh74-9 serotype described in the examples of WO2019 / 193119; 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 / 19319; also referred herein as "-9-rh74", "AAV9-rh74", "AAV-9-rh74" or "rh74-AAV9"). For example, the capsid of an AAV vector is a peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins, as described in PCT / EP2019 / 076958, such as the AAV9-rh74 hybrid capsid modified with P1 peptide or the AAVrh74-9 hybrid capsid described in the examples of PCT / EP2019 / 076958, or another example, the AAV serotype may be the hybrid AAV2 / 13 disclosed in PCT / EP2020 / 061380.

[0066] For example, the genome of a pseudotyped vector may have a capsid derived from serotypes AAV8, AAV9, AAVrh74, or AAV2i8, and its genome may be derived from different serotypes. In a particular embodiment, the AAV vector has a capsid of serotype AAV8, AAV9, or AAVrh74, particularly a capsid of serotype AAV8 or AAV9, and more particularly a capsid of serotype AAV8.

[0067] In a specific implementation, the vector is called "AAV8mut5". The AAV8mut5 vector has the amino acid sequence SEQ ID NO:57 and is encoded by a polynucleotide of SEQ ID NO:58.

[0068] In a specific implementation scheme, the vector is used to deliver the transgene to muscle cells, wherein the AAV vector can be selected from the group consisting of AAV8, AAV9 and AAVrh74.

[0069] In another specific embodiment, the vector is used to deliver the transgene to hepatocytes, and the AAV vector may be selected from the group consisting of AAV5, AAV8, AAV9, AAV-LK03, AAV-Anc80 and AAV3B.

[0070] In another embodiment, the capsid is a modified capsid. In the context of this invention, a "modified capsid" can be a chimeric capsid or a capsid comprising one or more variant VP capsid proteins derived from one or more wild-type AAV VP capsid proteins.

[0071] In a particular embodiment, the AAV vector is a chimeric vector, i.e., its capsid contains a VP capsid protein derived from at least two different AAV serotypes, or contains at least one chimeric VP protein that binds to a VP protein region or domain derived from at least two AAV serotypes. Examples of such chimeric AAV vectors that can be used to transduce hepatocytes are described in [6] and [7]. For example, a chimeric AAV vector may be derived from a combination of an AAV8 or AAV9 capsid sequence with an AAV serotype sequence different from the AAV8 or AAV9 serotype (e.g., any of those specifically mentioned above). In another embodiment, the capsid of the AAV vector contains one or more variant 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 that exhibit high hepatic tropism.

[0072] In another embodiment, the modified capsid may also be derived from a capsid modification inserted via error-prone PCR and / or peptide insertion (e.g., as described in [8] or [9]). Furthermore, capsid variants may include single amino acid changes such as tyrosine mutants (e.g., as described in

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

[11] .

[0073] Furthermore, the genome of an AAV vector can be a single-stranded or a self-complementary double-stranded genome

[12] . Self-complementary double-stranded AAV vectors are generated by deleting the terminal dissociation site (trs) from one of the terminal repeat sequences of an AAV. These modified vectors, whose replicated genome is half the length of the wild-type AAV genome, have a tendency to package DNA dimers.

[0074] In a preferred embodiment, the AAV vector implemented in practice with respect to the present invention has a single-stranded genome and further preferably contains an AAV8, AAV9, AAVmut5, AAVrh74 or AAV2i8 capsid, particularly an AAV8, AAV9 or AAVrh74 capsid, such as an AAV8 or AAV9 capsid, and more particularly an AAV8 capsid.

[0075] cell

[0076] The terms “carrier of the specification” or “carrier according to the specification” refer to the carrier disclosed in this specification, and in particular the carrier disclosed above.

[0077] The instructions refer to the use of nucleic acid molecules specified in the instructions or cells transformed according to the vector specified in the instructions.

[0078] This invention relates to cells transformed using the AAV vector of this invention.

[0079] For example, the host cell can be a cell (or cell line) suitable for vector production (e.g., AAV production). In some instances, the host cell is a mammalian cell, such as HEK-293, BHK, Vero, RD, HT-1080, A549, Cos-7, ARPE-19, or MRC-5 cells.

[0080] The host cell can also be a cell that serves as a target for gene therapy, such as liver cells, kidney cells, or intestinal cells.

[0081] In some implementations, the cells are isolated cells.

[0082] Composition

[0083] The terms “cells of the specification” or “cells according to the specification” refer to the cells disclosed in this specification, and in particular the cells disclosed above.

[0084] This instruction manual relates to compositions comprising nucleic acid constructs, vectors, or cells containing the instruction manual. Preferably, the composition in the instruction manual is a pharmaceutical composition.

[0085] This invention relates to compositions comprising the carrier or cells of the present invention. The compositions of the present invention are preferably pharmaceutical compositions.

[0086] The composition can be in the form of solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0087] Such compositions will contain a therapeutically effective amount of the nucleic acid construct, vector, or cell of the present invention, preferably in purified form, together with an appropriate amount of the vector, to provide a form suitable for administration to a subject. In a particular embodiment, the nucleic acid construct, vector, or cell of the present 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 present invention is formulated in a composition comprising 0.01-0.0001% by weight of Ringer's lactate and a nonionic surfactant such as pluronic F68 at a final concentration, for example, 0.001%. The formulation may further comprise serum albumin, particularly human serum albumin, for example, 0.25% human serum albumin. Other suitable formulations for storage or administration are known in the art, particularly from WO2005 / 118792.

[0088] In a preferred embodiment, the composition is formulated according to conventional procedures to be suitable for intravenous or intrathecal administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to reduce pain at the injection site.

[0089] Treatment

[0090] The terms “composition of the specification” or “composition according to the specification” refer to the compositions disclosed in this specification, and in particular the compositions disclosed above.

[0091] The instructions pertain to nucleic acid constructs, vectors, cells, or compositions thereof, which are used as pharmaceuticals.

[0092] This specification also relates to nucleic acid constructs, vectors, cells, or compositions thereof for the treatment of glycogen storage disease Ia (GSD-Ia).

[0093] This invention relates to the carrier, the cell, or the composition thereof, which are used as pharmaceuticals.

[0094] This invention also relates to the carrier of this invention, the cell of this invention, or the composition of this invention for the treatment of glycogen storage disease Ia (GSD-Ia).

[0095] The nucleic acid constructs, vectors, cells, or compositions of the present invention can be administered to subjects via any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, and intravenous), oral, intracatheter, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0096] Nucleic acid constructs, vectors, cells, or compositions may be administered to a subject in a therapeutically effective amount, i.e., an amount sufficient to achieve the desired effect in the treated subject or cells. The effective amount of a nucleic acid construct, vector, cell, or composition will depend on several factors, including but not limited to the treated subject or cells, and the method of administration.

[0097] The amount of therapeutic agent (i.e., nucleic acid construct, vector, or cell) that can effectively treat a disease (e.g., GSD-1a) can be determined using standard clinical techniques. Additionally, in vivo and / or in vitro assays may be optionally employed to help predict the optimal dose range. The precise dose used in the composition will also depend on the route of administration and the severity of the disease, and should be determined based on the physician's judgment and each patient's individual circumstances. The dose of nucleic acid, vector, or cell administered to the recipient subject will vary depending on several factors, including but not limited to the route of administration, the specific disease being treated, the subject's age, or the expression level necessary for the desired therapeutic effect. Those skilled in the art can readily determine the required dose range based on these and other factors, according to their knowledge in the art. In cases involving treatments that include administration of a viral vector (e.g., an AAV vector) to the subject, a typical dose of the vector is at least 1 x 10⁻⁶. 8 Vector genome per kilogram of body weight (vg / kg), for example, at least 1 x 10-1 9 vg / kg, at least 1x10 10 vg / kg, at least 1x10 11 vg / kg, at least 1x10 12 vg / kg, at least 1x10 13 vg / kg, or at least 1x10 14 vg / kg.

[0098] Depending on the desired therapeutic effect, the therapeutic agents (i.e., nucleic acid constructs, vectors, or cells) of the present invention can be administered in single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses).

[0099] G6Pase-a codon optimization

[0100] Nucleic acid sequences encoding G6Pase-a can be optimized for in vivo expression of G6Pase-a. Sequence optimization can include many changes to the nucleic acid sequence, including codon optimization, increased GC content, reduced CG dimers, reduced CpG islands, reduced number of alternative open reading frames (ARFs), and / or reduced number of splice donor and acceptor sites. Due to the degeneracy of the genetic code, different nucleic acid molecules may encode the same protein. It is also well known that the genetic codes of different organisms often favor the use of one of several codons encoding the same amino acid. By introducing changes into the nucleotide sequence through codon optimization, the codon bias present in a given cellular environment is utilized, making the resulting codon-optimized nucleotide sequence more likely to be expressed at a relatively higher level in such a given cellular environment 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 if at least one of the above parameters is improved while one or more of the other parameters are not improved, the optimized sequence can be considered optimized as long as it leads to improved transgene expression, such as improved expression and / or reduced immune response to the transgene in vivo.

[0101] Compared to non-codon-optimized nucleotide sequences encoding the same G6Pase-a, nucleic acid sequences encoding G6Pase-a can be codon-optimized to improve its expression in human cells. Wild-type nucleic acid sequences encoding wild-type G6Pase-a are shown in SEQ ID NO:2. Examples of codon-optimized sequences encoding the same G6Pase-a are 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 the modified G6Pase of SEQ ID NO:12 and have been optimized for one of the aforementioned parameters compared to the nucleic acid sequence of SEQ ID NO:4.

[0102] Compared to wild-type nucleotide sequences encoding the same G6Pase-a amino acid sequence (e.g., G6Pase-a of SEQ ID NO:1), the nucleic acid sequences of the present invention encoding G6Pase may be codon-optimized and / or may have reduced GC content and / or reduced CG dimer number. Compared to the nucleotide sequence of SEQ ID NO:2, the nucleic acid sequences encoding G6Pase may also be codon-optimized and / or may have reduced GC content and / or reduced CG dimer number. Alternatively, such nucleic acid sequences encoding G6Pase may have SEQ ID NO:45 or SEQ ID NO:46, preferably the sequence of SEQ ID NO:45.

[0103] This specification also discloses a nucleic acid sequence encoding G6Pase-a, which contains 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 with SEQ ID NO:45.

[0104] This specification also discloses a nucleic acid construct for expressing G6Pase-a in cells, the construct comprising a nucleic acid sequence encoding G6Pase-a, wherein the nucleic acid sequence encoding G6Pase-a comprises 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 with SEQ ID NO:45. The nucleic acid sequence encoding G6Pase-a is preferably operatively ligated to a promoter.

[0105] This specification also discloses vectors containing the nucleic acid constructs disclosed in this section (i.e., the "G6Pase-Codon Optimization" section). The vector may be a lentiviral vector or an adeno-associated virus (AAV) vector, such as AAV8, AAV9, or AAVmut5, with AAV8 being preferred.

[0106] This specification also discloses cells transformed using the nucleic acid molecules or vectors disclosed in this section. The cells may be hepatocytes, intestinal cells, or kidney cells.

[0107] This specification also discloses compositions comprising the nucleic acid constructs, vectors, or cells disclosed in this section.

[0108] This specification also discloses the nucleic acid constructs, vectors, cells, or compositions disclosed in this section, which are used as pharmaceuticals.

[0109] This specification also discloses the nucleic acid constructs, vectors, cells, or compositions disclosed in this section for the treatment of glycogen storage disease Ia (GSD-Ia).

[0110] Example

[0111] Materials and methods

[0112] In Examples 1, 2, and 3, we prepared two nucleic acid constructs (hereinafter referred to as "transgenic expression cassettes") containing the human G6pc gene wt (SEQ ID NO:2) encoding G6Pase-a under the control of different promoters:

[0113] -α-1-Antitrypsin (hAAT) promoter. The nucleic acid sequence of the expression cassette is SEQ ID NO:11.

[0114] - The endogenous promoter of the human G6pc gene (hGPE). The nucleic acid sequence of the expression cassette is SEQ ID NO:10.

[0115] Transgenic expression cassettes, such as Figure 1 As shown in Figure A.

[0116] Two transgenic expression cassettes were pseudotyped in AAV9 to obtain two different AAV vectors, namely AAV9-hAAT-hG6PC and AAV9-hGPE-hG6PC.

[0117] The two vectors or PBS (negative control) were independently tested in a liver-specific G6pc knockout mouse model (L.G6pc- / -,

[13] ), namely GSD-Ia mice. As a positive control, PBS was injected into WT mice (L.G6pc+ / +).

[0118] In Example 4, we prepared a nucleic acid construct (hereinafter referred to as the “transgenic expression cassette”) containing the human G6pc gene wt (SEQ ID NO:2) encoding G6Pase-a under the control of the α-1-antitrypsin (hAAT) promoter. The nucleic acid sequence of the expression cassette is SEQ ID NO:56.

[0119] The expression cassette of the human G6pc gene wt is pseudotyped in AAV9, AAV8, and AAVmut5.

[0120] The three vectors or PBS (negative control) were independently tested in a liver-specific G6pc knockout mouse model (L.G6pc- / -,

[13] ), namely GSD-Ia mice. As a positive control, PBS was injected into WT mice (L.G6pc+ / +).

[0121] In Example 5, we prepared four nucleic acid constructs (hereinafter referred to as “transgenic expression cassettes”) containing a human G6pc gene wt (SEQ ID NO:2) encoding G6Pase-a under the control of the α-1-antitrypsin (hAAT) promoter 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).

[0122] The nucleic acid sequence of the human G6pc gene wt expression cassette is SEQ ID NO:11 or SEQ ID NO:56. The nucleic acid sequences of the human G6pc gene co1, human G6pc gene co2, and human G6pc gene co3 expression cassettes are SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively.

[0123] The expression cassette of the human G6pc gene wt was pseudotyped in AAV8 (SEQ ID NO:56) and AAV9 (SEQ ID NO:11).

[0124] The expression cassettes of human G6pc gene co1 and G6pc gene co2 are pseudotyped in AAV8.

[0125] The expression cassette of human G6pc gene co3 is pseudotyped in AAV9.

[0126] Five vectors or PBS (negative control) were independently tested in a liver-specific G6pc knockout mouse model (L.G6pc- / -,

[13] ), namely GSD-Ia mice. PBS was injected into WT mice (L.G6pc+ / +) as a positive control.

[0127] In vivo studies

[0128] Mice were fed a standard diet (A04 diet, safe) or a high-fat / high-sucrose diet (manufactured by INRAE, Jouyen Josas) (which is known to accelerate L.G6pc). - / - Development of liver tumors in mice). By feeding mice a modified diet consisting of 36% fat (INRA)

[14] , a tendency to form tumors can be induced in mice.

[0129] The AAV vector was administered intravenously via the tail vein of L.G6pc- / - mice. Wild-type mice (C57Bl / 6J mice, Charles Rivers) were injected with PBS via the tail vein. Peripheral blood glucose was measured using a Roche Diagnostic glucometer after a 6-hour fast. At sacrifice, the liver was harvested and weighed to determine the liver-to-body weight ratio and then rapidly frozen for further evaluation.

[0130] Tumors were visually assessed at the time of GSDIa mouse sacrifice. Only tumors larger than 2 mm were considered in the count.

[0131] AAV production

[0132] HEK293T cells were grown in suspension in 250 mL of serum-free medium. Cells were transfected with three plasmids: i) a transgenic plasmid containing AAV2 ITRs flanking the expression cassette, ii) the helper plasmid pXX6 containing the adenovirus sequence necessary for AAV production, and iii) a plasmid containing the AAV Rep and Cap genes, which define the AAV serotype. Two days post-transfection, cells were lysed to release AAV particles.

[0133] Viral lysates were purified by affinity chromatography. The viral genome was quantified by TaqMan real-time PCR using primers and probes corresponding to the ITR of the AAV vector genome

[17] .

[0134] G6Pase enzyme activity measurement

[0135] As reported in

[13] , G6Pase activity was measured in homogenates from frozen-forced livers. Briefly, the tissue homogenate was incubated with glucose-6-phosphate (Sigma) at 37°C for 15 min. The reaction was terminated by the addition of trichloroacetic acid, and the released phosphate was measured by complexation with ammonium molybdate and citrate arsenite. The absorbance was measured at 700 nm.

[0136] Measurement of glycogen content

[0137] The glycogen content in tissue homogenates was indirectly measured as glucose released after complete digestion with Aspergillus niger amylolucosidase (Sigma). Samples were incubated at 95°C for 20 minutes in the presence of 0.3M NaOH, followed by cooling at 4°C. Amylolucosidase was then added to the samples, and the mixture was incubated at 37°C for 90 minutes. The released glucose was determined using a commercial glucose assay kit.

[0138] Vector genome copy number (VGCN) quantification

[0139] For vector genome copy number (VGCN) quantification in samples, DNA was extracted from the samples using King Fisher (Thermo Fisher Scientific). Real-time PCR was performed on 1 μL of DNA using the AAV vector titration protocol described above. Mex5 exon of the titin gene was used as a control for loading genomic DNA.

[0140] Example 1: Liver phenotype of GSD-1a mice 15 days after intravenous injection of AAV vector expressing human G6Pase-a. Correction

[0141] Two AAV9 vectors were independently injected into L.G6pcs fed a standard diet. - / - In mice, the dose was 1x10 11 vg / mouse( Figure 1 A).

[0142] Fifteen days after vector injection, blood glucose concentrations were measured after a 6-hour fast. Blood glucose levels in mice injected with both vectors were completely normalized. Figure 1 B).

[0143] Then, G6PC activity in the liver was measured. Compared with the AAV9-hGPE-hG6PC vector, the AAV9-hAAT-hG6PC vector achieved supraphysiological activity and showed the highest activity. Figure 1 C).

[0144] Glycogen concentration was also measured. Full corrections for glycogen accumulation and hepatomegaly were obtained using AAV9-hAAT-hG6PC instead of a vector with the hGPE promoter (respectively...). Figure 1 (D, E) reflects the higher G6Pase activity achieved in mice treated with the AAV9 vector carrying the hAAT promoter. The number of similar vector genome copies per diploid genome measured in mice injected with both AAV vectors ( Figure 1 F) indicates that it has similar transduction effects in the liver.

[0145] Example 2: Following intravenous injection of an AAV vector expressing human G6Pase-a, the liver phenotype of GSD-1a mice was lengthened. Periodic correction

[0146] To evaluate the long-term efficacy of these two vectors, we conducted a 7-month study. The vectors were injected into L.G6pcs fed a standard diet. - / - In mice, the dose was 2.5 x 10⁻⁶. 11 vg / mouse( Figure 2 A). Seven months after vector injection, blood glucose concentrations were measured after a 6-hour fast. Glycemic levels were corrected in all animals that received the G6PC expression vector. Figure 2 B). Importantly, L.G6pc processed with AAV9-hAAT-hG6PC- / - Mice exhibited supraphysiological hepatic G6Pase-a activity, which was significantly higher than that of L. G6pc treated with AAV9-hGPE-hG6PC. - / - Activity measured in mice ( Figure 2 C).

[0147] In animals injected with both vectors, glycogen accumulation and hepatomegaly were completely saved. Figure 2 D, E). Higher vector genome copy numbers were measured per diploid genome in mice injected with the AAV9-hGPE-hG6PC vector. Figure 2 F), which may reflect slightly lower liver transduction achieved using the AAV9-hAAT-hG6PC vector.

[0148] In summary, these results indicate that G6Pase-a expression increases with the hAAT promoter. Therefore, the hAAT promoter is suitable for AAV gene therapy of GSD-1a and is more potent than the hGPE promoter.

[0149] Example 3: hGPE-targeted gene therapy promotes L.G6pc fed a high-fat / high-sucrose diet - / - Hepatomegaly in mice Tumor formation.

[0150] Adenoma formation is one of the hallmarks of human GSD-Ia, and has been reported in most affected individuals during the second and third decades of life. In L.G6pc - / - In mice, almost all mice fed a standard diet developed hepatic adenomas at 18 months of age

[13] . A high-fat / high-sucrose (HF / HS) diet can accelerate this slow process. Approximately 85% of L.G6pc mice fed an HF / HS diet developed hepatic adenomas. - / - Multiple liver tumors were developed in mice at 9 months of age

[14] . Therefore, L.G6pc mice fed with a HF / HS diet... - / - Mice represent a robust model for evaluating the efficacy and safety of gene substitution strategies in preventing tumor formation in GSDIa.

[0151] Therefore, we fed L.G6pc with a HF / HS diet - / - The AAV9-hAAT-hG6PC and AAV9-hGPE-hG6PC vectors were tested in mice. Figure 3 A). After eight months of treatment, only vectors carrying the hAAT promoter could completely salvage glycogenemia. Figure 3 B). Under a HF / HS diet, the G6Pase activity level in AAV-treated animals was significantly lower than that in L.G6pc. + / +Levels measured in animals. Although animals treated with AAV9-hAAT-hG6PC showed higher levels of G6Pase activity, compared with L. G6pc treated with the AAV9-hGPE-hG6PC vector... - / - Compared with mice, the difference did not reach statistical significance ( Figure 3 C). Similar vector genome copies were measured for each diploid genome in AAV-treated mice. Figure 3 D).

[0152] Interestingly, autopsies of mice showed that mice treated with an AAV vector carrying hGPE had a higher number of tumors in their livers compared to mice that received AAV9-hAAT-hG6PC. Figure 3 E). These results suggest that, compared with AAV9-hGPE-hG6PC, the use of AAV9-hAAT-hG6PC may reduce the transformation rate of hepatocytes, thereby leading to a decrease in the frequency of adenomas.

[0153] Finish Figure 3 A obtained data to increase the number of mice in each group ( Figure 4 (and Table 1). Figure 4 The results showed that, in a larger cohort, the hAAT-driven vector achieved significantly higher G6Pase activity. Data provided in Table 1 confirm that the use of AAV9-hAAT-hG6PC resulted in a lower frequency of adenomas compared to AAV9-hGPE-hG6PC.

[0154]

[0155] Table 1: Frequency of adenomas

[0156] Example 4: Expression of G6Pase wt in the livers of mice with different AAV serotypes

[0157] We produced three different AAV vectors, namely AAV9, AAV8, and AAVmut5, which express the wild-type human G6pc gene wt (SEQ ID NO:2) under the control of the hAAT promoter.

[0158] Three vectors or PBS (negative control) in a liver-specific G6pc knockout mouse model (L.G6pc - / -

[13] , that is, independently tested in GSD-Ia mice at a dose of 1x10 12 vg / kg. As a positive control, PBS was injected into WT mice (L.G6pc). + / + Fifteen days after vector infusion, G6Pase activity in the liver for each vector was assessed. The three AAV vector injection groups showed similar G6Pase activity, with levels higher than those measured in wild-type animals. Figure 5This confirmed the possibility of expressing transgenes in mouse livers via different AAV vectors.

[0159] Example 5: Expression of G6Pase in mouse livers with G6pc sequences having different codon optimizations

[0160] We conducted two independent experiments to evaluate the efficacy of codon optimization with reduced CG dimer in G6Pase expression in mouse liver.

[0161] Codon optimization was performed on the G6pc wt sequence (SEQ ID NO:2) using different methods. G6pcco1 showed the highest similarity to the wt sequence and had a low CG dimer content. Compared to G6pc co1, G6pc co2 had the highest GC content and an increased CG dimer content. G6pc co3 showed less similarity to the wt sequence and had the highest CG dimer content (Table 2).

[0162]

[0163]

[0164] 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).

[0165] a Predict splice donors (SD) and splice acceptors (SA) using an online tool (www.fruitfly.org) with a minimum score of 0.8.

[0166] b GC content was calculated using an online molecular biology tool (www.genscript.com).

[0167] c Use the online tool MethPrimerDB (www.urogene.org) to predict CpG islands smaller than 100 bp with a GC content threshold of 60%.

[0168] In the first experiment, we produced three AAV8 vectors that expressed the following transgenes under the control of the hAAT promoter:

[0169] -hG6pc: Human G6pc gene wt (SEQ ID NO:2),

[0170] -hG6pc Co1: codon-optimized human G6pc gene (SEQ ID NO:45), and

[0171] -hG6pc Co2: Codon-optimized human G6pc gene (SEQ ID NO:46).

[0172] Then, in a liver-specific G6pc knockout mouse model (L.G6pc... - / - Three AAV8 vectors were tested in GSD-Ia mice at a dose of 1x10⁻¹,

[13] (GSD-Ia mice). 12 vg / kg. As a positive control, PBS was injected into WT mice (L.G6pc). + / + Fifteen days after vector infusion, G6Pase activity in the liver was assessed. The three AAV8 vector injection groups showed supraphysiological G6Pase activity at levels higher than those measured in wild-type animals. Figure 6A Importantly, compared with the same vector expressing hG6pc Co2, the AAV8 vector expressing hG6pc Co1 showed a significantly increased level of G6Pase activity. Figure 6A ).

[0173] For the second experiment, we produced two AAV9 vectors that expressed the following transgene under the control of the hAAT promoter:

[0174] -hG6pc: Human G6pc gene wt (SEQ ID NO:2),

[0175] -hG6pc Co3: Codon-optimized human G6pc gene (SEQ ID NO:3).

[0176] Then, in a liver-specific G6pc knockout mouse model (L.G6pc... - / - Two AAV9 vectors were tested in GSD-Ia mice at a dose of 1x10⁻¹,

[13] (GSD-Ia mice). 11 vg / mouse. As a positive control, PBS was injected into WT mice (L.G6pc). + / + Fifteen days after vector infusion, G6Pase activity in the liver was assessed. Importantly, only the group of animals injected with the AAV9 vector carrying the hG6pc wild-type sequence showed supraphysiological G6Pase activity at levels higher than those measured in wild-type animals. Figure 6B Compared with animals injected with the wild-type version of the AAV9 vector expressing hG6pc, animals injected with the AAV9 vector expressing hG6pc Co3 showed a trend of decreased G6Pase activity (p = 0.06). Figure 6B ).

[0177] References cited in the form of "[reference number]"

[0178] [1]Chandler et al.,Vector design influences hepatic genotoxicityafter,The Journal of Clinical Investigation,2015

[0179] [2]Mates et al.Nat Genet.2009Jun;41(6):753-61.doi:10.1038 / ng.343

[0180] [3]Ling et al.,2016 Jul 18,Hum Gene Ther Methods.

[0181] [4]Vercauteren et al.,2016,Mol.Ther.Vol.24(6),p.1042

[0182] [5]Rosario et al.,2016,Mol Ther Methods Clin Dev.3,p.16026

[0183] [6]Shen et al.,2007 Molecular Therapy,volume 15,issue 11,pages 1955-1962

[0184] [7]Tenney et al.,Virology,volumes 454–455,April 2014,pages 227-236

[0185] [8]Bartel et al.,Front.Microbiol.,04October 2011 https: / / doi.org / 10.3389 / fmicb.2011.00204

[0186] [9]Michelfelder et al.(PLoS ONE,2009,4,e5122

[0187]

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

[0188]

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

[0189]

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

[0190]

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

[0191]

[14] Gjorgjieva M.et al,Dietary exacerbation of metabolic stress leadsto 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 2018Sep 5.

[0192]

[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.

[0193]

[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

[0194]

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

[0195] Sequence Listing

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[0223] sequence list <110> Genius Corporation French National Institute of Health and Medical Research Evri-Wald-Isson University Claude Bernard - Lyon 1 University <120> Vectors encoding glucose-6-phosphatase (G6Pase-a) for gene therapy <130> 1H274440_CAS 4 <150> EP20 305 540.5 <151> 2020-05-22 <160> 58 <170> BiSSAP 1.3.6 <210> 1 <211> 357 <212> PRT <213> Homo sapiens <220> <223> hG6Pase-a wt (amino acid) <400> 1 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 2 <211> 1074 <212> DNA <213> Homo sapiens <220> <223> hG6pc wt (nucleic acid) (encoding SEQ ID NO:1) <400> 2 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 3 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> hG6pc co 3 (Nucleic acid) (Codon - optimized - encoding SEQ ID NO:1 - for Example 5) <400> 3 atggaggagg gaatgaatgt gctgcacgac ttcgggattc agtctaccca ctatctccaa 60 gtcaattatc aagacagtca agattggttc atcctcgtct ccgttatagc tgacctcagg 120 aatgcttttt atgtactgtt cccaatatgg tttcatctgc aagaagcagt tggaattaaa 180 ctgctgtggg tggccgtaat cggagactgg ttgaacctgg tgttcaaatg gatccttttt 240 ggtcagaggc catattggtg ggtcttggac accgactatt atagcaacac atctgtaccc 300 ctgataaagc aattccctgt aacgtgtgaa actgggcctg ggtcacccag tggacacgcg 360 atggggactg ccggggttta ctacgtaatg gttactagca cgcttagcat tttccagggc 420 aagatcaaac cgacttatcg cttccgatgc ctcaatgtga tcctctggct gggattctgg 480 gcggttcaac tgaacgtatg cctctcacgg atttatctgg ctgcacattt cccgcatcaa 540 gtagtggctg gtgtgttgtc tggcatagca gtggctgaaa cattctcaca cattcattct 600 atttacaacg cttcattgaa aaaatacttt ctgattactt tttttctttt cagctttgca 660 attggctttt acttgctgct taaaggtctt ggcgtcgacc tgctctggac tcttgagaaa 720 gcgcaacgct ggtgcgaaca acccgagtgg gtccacatag atacgacccc gttcgcgtct 780 cttctgaaaa atctcggtac cctcttcgga ctgggacttg ctttgaattc ttcaatgtat 840 cgggaatcct gcaagggcaa actgtccaag tggttgccct ttcgcctttc aagcattgtc 900 gcttcccttg tgctcctgca cgtcttcgac tcactgaagc cgccgagcca agtcgagctc 960 gtgttttacg tgttgtcttt ctgtaaaagt gccgtcgtgc cactcgccag tgtgagcgtg 1020 ataccttatt gcctcgcaca ggtgcttggg cagcctcata agaagtctct gtaa 1074 <210> 4 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> hG6pc mut (Nucleic acid) (Same as SEQ ID NO: 2 except for the c>g mutation at position 893) (Encoding SEQ ID NO: 12) <400> 4 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctgtattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 5 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> hG6pc mut co (Nucleic Acid) (Codon Optimized – Encoding SEQ ID NO : 12) <400> 5 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctgtattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 6 <211> 2700 <212> DNA <213> Artificial sequence <220> <223> hGPE promoter (with 4 nucleotide differences from the hGPE promoter in NG011808.1) <400> 6 tggaggaagc agaaaggggc tggcaggtgg aaagatgagg accagctcat cgtctcatga 60 ctatgaggtt gctctgatcc agaggtgccc cctgcctggt ggcccaccgc caggaagact 120 cccactgtcc ctggatgccc agagtgggat gtcaactcca tcacttatca actccttatc 180 cataggggta ttcttcctga ggcgtctcag aaaacagggc cctccccata tgctgaccac 240 ataatagaac ccctcccaac tcagagaccc tggctgctag ctgccctggc atgacccaga 300 cagtggcctt tgtatatgtt tttagactca ccttgactca cctctgacca tagaaactct 360 catcccagag gtcactgcaa tagttactcc acaacagagg cttatctggg tagagggagg 420 ctccctacct atggcccagc agccctgaca gtgcagatca catatacccc acgccccagc 480 actgcctgcc acgcatgggc ttactttaca cccacccaca gtcaccaaca cattacctgc 540 tctccaaggt taggcgtggc aggagaagtt tgcttggacc agcagaaacc atgcagtcaa 600 ggacaactgg agtcagcatg ggctgggtgc gagcccttgg tggggtgggg aggagactcc 660 aggtcatacc tcctggagga tgttttaatc atttccagca tggaatgctg tcaacttttg ccacagattc attagctctg agtttctttt ttctgtcccc agctacccct tacatgtcaa 780 840. 840. 840. 840. 840. 840. 840. 840. 840. 840. 840 tatcctcaaa aaatgcatga atttggaggc agtggctcat gcctgtaatc ccaatgcttt gctaggttga ggcgggagga tcacttgaag ccaggaattt gagaccagcc tgggccgcat agtgagaccc cgtttctaca aaaataata aataataat aaaatatagt gatatgaagc 1020. atgattaat agccctattt tttaaaatgc atgagttcgt tacctgattc attccctggt tcctttcaca gtcctccgtg acccaagtgt tagggttttg gtctctctac tatttgtag 1140 ctgatatata gtatacacac acacacacac acacatatac acacacacag tgtatcttga gctttctttt gtatatctac acacatatgt ataagaaagc tcaagatata gaagcccttt ttcaaaaata actgaaagtt tcaaactctt tagtctcca gttaccattt tgctggtatt cttatttgga accatacatt catcatattg ttgcacagta agactataca ttcattattt 1380 tgcttaaacg tatgagttaa aacacttggc caggcatggt gttcacacc tgtaatccca 1440 gagctttggg aagccagac tggcagtact cttgagctca ggaattcaag accagcctgg 1500 gcacatgga aaaacccat ctctacaaaa gatagaaaaa ttagccaggc atggtggcgt 1560 gtgcctgtgg gatcacatta gcccaggagg 1620 ttgaggctgc agtgagccgt gattatgcca ctgcactcca gcctgggaga cagagtgaga 1680 ccctgtttca aaaaaaagag agaaaatt taaaaaaagaaaaacacca agggctgtaa 1740 ctttaaggtc atttaatgaa ttaatcactg cattcaaaa cgattactt ctggccctaa 1800 gagacatgag gccaatacca ggaagggggt tgatctccca aaccagaggc agaccctaga 1860 ctctaataca gttaaggaa gaccagcaag atgatagtcc ccaatacaat agagttact 1920 atattttatt tgttgtttt cttttgtttt gttttgtttt gttttgtttt gttttagaga 1980 ctggggtctt gctcgattgc ccaggctgta gtgcagcggt gggacaatag ctcactgcag 2040 actccaactc ctgggctcaa gcaatcctcc tgcctcagcc tcctgaatag ctgggactac 2100 aagggtacac catcacacac accaaaacaa ttttttaaat ttttgtgtag aaacgagggt 2160 cttgctttgt tgcccaggct ggtctccaac tcctggcttc aagggatcct cccacctcag 2220 cctcccaaat tgctgggatt acaggtgtga gccaccacaa ccagccagaa ctttactaat 2280 tttaaaatta agaacttaaa acttgaatag ctagagcacc aagatttttc tttgtcccca 2340 aataagtgca gttgcaggca tagaaaatct gacatctttg caagaatcat cgtggatgta 2400 gactctgtcc tgtgtctctg gcctggtttc ggggaccagg agggcagacc cttgcactgc 2460 caagaagcat gccaaagtta atcattggcc ctgctgagta catggccgat caggctgttt 2520 ttgtgtgcct gtttttctat tttacgtaaa tcaccctgaa catgtttgca tcaacctact 2580 ggtgatgcac ctttgatcaa tacattttag acaaacgtgg tttttgagtc caaagatcag 2640 ggctgggttg acctgaatac tggatacagg gcatataaaa caggggcaag gcacagactc 2700 <210> 7 <211> 727 <212> DNA <213> Artificial Sequence <220> <223> ApoE + hAAT <400> 7 aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60 ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120 tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180 cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240 tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300 ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360 gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420 acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480 cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540 gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600 gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat ccactgctta 660 aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac 720 agtgaat 727 <210> 8 <211> 406 <212> DNA <213> Artificial sequence <220> <223> hAAT promoter <400> 8 gtacccgggg atcttgctac cagtggaaca gccactaagg attctgcagt gagagcagag 60 ggccagctaa gtggtactct cccagagact gtctgactca cgccaccccc tccaccttgg 120 acacaggacg ctgtggtttc tgagccaggt acaatgactc ctttcggtaa gtgcagtgga 180 agctgtacac tgcccaggca aagcgtccgg gcagcgtagg cgggcgactc agatcccagc 240 cagtggactt agcccctgtt tgctcctccg ataactgggg tgaccttggt taatattcac 300 cagcagcctc ccccgttgcc cctctggatc cactgcttaa atacggacga ggacagggcc 360 ctgtctcctc agcttcaggc accaccactg acctgggaca gtgaat 406 <210> 9 <211> 321 <212> DNA <213> Artificial sequence <220> <223> ApoE enhancer <400> 9 aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60 ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120 tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180 cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240 tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300 ggtttaggta gtgtgagagg g 321 <210> 10 <211> 4492 <212> DNA <213> Artificial Sequence <220> <223> hGPE_HBB2_hG6pcwt_bGH (Expression cassette used in Examples 1, 2 and 3) <400> 10 tggaggaagc agaaaggggc tggcaggtgg aaagatgagg accagctcat cgtctcatga 60 ctatgaggtt gctctgatcc agaggtgccc cctgcctggt ggcccaccgc caggaagact 120 cccactgtcc ctggatgccc agagtgggat gtcaactcca tcacttatca actccttatc 180 cataggggta ttcttcctga ggcgtctcag aaaacagggc cctccccata tgctgaccac 240 father ccctcccaac tcagagaccc tggctgctag ctgccctggc atgacccaga cagtggcctt tgtatatgtt tttagactca ccttgactca cctctgacca tagaaactct catcccagag gtcactgcaa tagttactcc acaacagagg cttatctggg taggggagg ctccctacct atggcccagc agccctgaca gtgcagatca catatacccc acgccccagc 480 actgcctgcc acgcatgggc ttactttaca cccacccaca gtcaccaaca cattacctgc 540 tctccaaggt taggcgtggc aggagaagtt tgcttggacc agcagaacc atgcagtcaa ggacaactgg agtcagcatg ggctgggtgc gagcccttgg tggggtgggg aggagactcc 660 aggtcatacc tcctggagga tgttttaatc atttccagca tggaatgctg tcaacttttg ccacagattc attagctctg agtttctttt ttctgtcccc agctacccct tacatgtcaa 780 840. 840. 840. 840. 840. 840. 840. 840. 840. 840. 840 tatcctcaaa aaatgcatga atttggaggc agtggctcat gcctgtaatc ccaatgcttt gctaggttga ggcgggagga tcacttgaag ccaggaattt gagaccagcc tgggccgcat agtgagaccc cgtttctaca aaaataaata aataaataat aaataatagt gatatgaagc 1020 atgattaaat agccctattt tttaaaatgc atgagttcgt tacctgattc attccctggt 1080 tcctttcaca gtcctccgtg acccaagtgt tagggttttg gtctctctac tatttgtagg 1140 ctgatatata gtatacacac acacacacac acacatatac acacacacag tgtatcttga 1200 gctttcttt gtatatctac acacatatgt ataagaaagc tcaagatata gaagcccttt 1260 ttcaaaaata actgaaagtt tcaaactctt taagtctcca gttaccattt tgctggtatt 1320 cttatttgga accatacatt catcatattg ttgcacagta agactataca ttcattattt 1380 tgcttaaacg tatgagttaa aacttggc caggcatggt ggttcacacc tgtaatccca 1440 gagctttggg aagccaagac tggcagtact cttgagctca ggaattcaag accagcctgg 1500 gcaacatgga aaaaccccat ctctacaaaa gatagaaaaa ttagccaggc atggtggcgt 1560 gtgcctgtgg tcccagctac tcaggaggct gaggtgggag gatcacatta gcccaggagg 1620 ttgaggctgc agtgagccgt gattatgcca ctgcactcca gcctgggaga cagagtgaga 1680 ccctgtttca aaaaaaagag agaaaatt taaaaaaagaaaaacacca agggctgtaa 1740 ctttaaggtc atttaatgaa ttaatcactg cattcaaaa cgattactt ctggccctaa 1800 gagacatgag gccaatacca ggaagggggt tgatctccca aaccagaggc agaccctaga 1860 ctctaataca gttaaggaa gaccagcaag atgatagtcc ccaatacaat agagttact 1920 atattttatt tgttgtttt cttttgtttt gttttgtttt gttttgtttt gttttagaga 1980 ctggggtctt gctcgattgc ccaggctgta gtgcagcggt gggacaatag ctcactgcag 2040 actccaactc ctgggctca gcaatcctcc tgctcagcc tcctgaatag ctgggactac 2100 aagggtacac catcacacac accaaaaaa ttttaaat tttgtgtag aaacgagggt 2160 cttgctttgt gcccaggct gtctccac tcctggctc aagggatcct cccacctcag 2220 cctccaaat tgctgggatt acaggtgtga gccaccacaa ccagccagaa ctttactaat 2280 tttaaaatta agaacttaaa acttgaatag ctagagcacc aagattttc ttgtcccca 2340 aaagtgca gttgcaggca tagaaatct gatactttg caagaatcat cgtggatgta 2400 gactctgtcc tgtgtctctg gcctggtttc ggggaccagg agggcagacc cttgcactgc 2460 caagaagcat gccaaagtta atcattggcc ctgctgagta catggccgat caggctgttt 2520 ttgtgtgcct gttttcttat tttacgtaaa tcaccctgaa catgtttgca tcaacctact 2580 ggtgatgcac ctttgatcaa tacattttag acaaacgtgg tttttgagtc caaagatcag 2640 ggctgggttg acctgaatac tggatacagg gcatataaaa caggggcaag gcacagactc 2700 gggacccttg atgttttctt tccctcttt ttctatggtt aagttcatgt cataggaagg 2760 ggagaagtaa cagggtacac atattgacca aatcagggta atttgcatt tgtaatttta 2820 aaaaatgctt tcttctttta atatacttttt ttgtttatct tatttctaat actttccta 2880 atctctttct ttcagggcaa tattgataca atgtatcttg cctctttgca ccattctaaa 2940 gaataacagt gataatttct gggttaaggc aatagcaata tttctgcata taatatttc 3000 tgcatataaa ttgtaactga tgtaagaggt ttcatattgc tatagcagc tacaatccag 3060 ctaccattct gcttttattt tctggttggg ataaggctgg attattctga gtccaagcta 3120 ggcccttttg ctaatcttgt tcatacctct tatcttcctc ccacagctcc tgggcaacct 3180 gctggtctct ctgctggccc atcactttgg caaagcacgc gtgccaccat ggaggaagga 3240 atgaatgttc tccatgactt tgggatccag tcaacacatt acctccaggt gaattaccaa 3300 gactcccagg actggttcat cttggtgtcc gtgatcgcag acctcaggaa tgccttctac 3360 gtcctcttcc ccatctggtt ccatcttcag gaagctgtgg gcattaaact cctttgggta 3420 gctgtgattg gagactggct caacctcgtc tttaagtgga ttctctttgg acagcgtcca 3480 tactggtggg ttttggatac tgactactac agcaacactt ccgtgcccct gataaagcag 3540 ttccctgtaa cctgtgagac tggaccaggg agcccctctg gccatgccat gggcacagca 3600 ggtgtatact acgtgatggt cacatctact ctttccatct ttcagggaaa gataaagccg 3660 acctacagat ttcggtgctt gaatgtcatt ttgtggttgg gattctgggc tgtgcagctg 3720 aatgtctgtc tgtcacgaat ctaccttgct gctcattttc ctcatcaagt tgttgctgga 3780 gtcctgtcag gcattgctgt tgcagaaact ttcagccaca tccacagcat ctataatgcc 3840 agcctcaaga aatattttct cattaccttc ttcctgttca gcttcgccat cggattttat 3900 ctgctgctca agggactggg tgtagacctc ctgtggactc tggagaaagc ccagaggtgg 3960 tgcgagcagc cagaatgggt ccacattgac accacaccct ttgccagcct cctcaagaac 4020 ctgggcacgc tctttggcct ggggctggct ctcaactcca gcatgtacag ggagagctgc 4080 aaggggaaac tcagcaagtg gctcccattc cgcctcagct ctattgtagc ctccctcgtc 4140 ctcctgcacg tctttgactc cttgaaaccc ccatcccaag tcgagctggt cttctacgtc 4200 ttgtccttct gcaagagtgc ggtagtgccc ctggcatccg tcagtgtcat cccctactgc 4260 ctcgcccagg tcctgggcca gccgcacaag aagtcgttgt aaagatctga attcaccgcg 4320 ggtttaaact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 4380 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 4440 gcattgtctg agtaggtgtc attctattct ggggggtggg gtgggggcta gc 4492 <210> 11 <211> 3153 <212> DNA <213> Artificial Sequence <220> <223> hAAT_HBB2_hG6pc wt (Expression cassette used in Examples 1, 2, 3, 4 and 5) <400> 11 ggatcaaggc tcagaggcac acaggagttt ctgggctcac cctgccccct tccaacccct 60 cagttcccat cctccagcag ctgtttgtgt gctgcctctg aagtccacac tgaacaaact 120 tcagcctact catgtcccta aaatgggcaa acattgcaag cagcaaacag caaacacaca 180 gccctccctg cctgctgacc ttggagctgg ggcagaggtc agagacctct ctgggcccat 240 gccacctcca acatccactc gaccccttgg aatttcggtg gagaggagca gaggttgtcc 300 tggcgtggtt taggtagtgt gagaggggta cccggggatc ttgctaccag tggaacagcc 360 actaaggatt ctgcagtgag agcagagggc cagctaagtg gtactctccc agagactgtc 420 tgactcacgc caccccctcc accttggaca caggacgctg tggtttctga gccaggtaca 480 atgactcctt tcggtaagtg cagtggaagc tgtacactgc ccaggcaaag cgtccgggca 540 gcgtaggcgg gcgactcaga tcccagccag tggacttagc ccctgtttgc tcctccgata 600 actggggtga ccttggttaa tattcaccag cagcctcccc cgttgcccct ctggatccac 660 tgcttaaata cggacgagga cagggccctg tctcctcagc ttcaggcacc accactgacc 720 tgggacagtg aatagatcct gagaacttca gggtgagtct atgggaccct tgatgttttc 780 tttccccttc ttttctatgg ttaagttcat gtcataggaa ggggagaagt aacagggtac 840 acatattgac caaatcaggg taattttgca tttgtaattt taaaaaatgc tttcttcttt 900 taatatactt ttttgtttat cttatttcta atactttccc taatctcttt ctttcagggc 960 aatattgata caatgtatct tgcctctttg caccattcta aagaataaca gtgataattt 1020 ctgggttaag gcaatagcaa tatttctgca tataaatatt tctgcatata aattgtaact 1080 gatgtaagag gtttcatatt gctaatagca gctacaatcc agctaccatt ctgcttttat 1140 tttctggttg ggataaggct ggattattct gagtccaagc taggcccttt tgctaatctt 1200 gttcatacct cttatcttcc tcccacagct cctgggcaac ctgctggtct ctctgctggc 1260 ccatcacttt ggcaaagcac gcgtgccacc atggaggaag gaatgaatgt tctccatgac 1320 tttgggatcc agtcaacaca ttacctccag gtgaattacc aagactccca ggactggttc 1380 atcttggtgt ccgtgatcgc agacctcagg aatgccttct acgtcctctt ccccatctgg 1440 ttccatcttc aggaagctgt gggcattaaa ctcctttggg tagctgtgat tggagactgg 1500 ctcaacctcg tctttaagtg gattctcttt ggacagcgtc catactggtg ggttttggat 1560 actgactact acagcaacac ttccgtgccc ctgataaagc agttccctgt aacctgtgag 1620 actggaccag ggagcccctc tggccatgcc atgggcacag caggtgtata ctacgtgatg 1680 gtcacatcta ctctttccat ctttcaggga aagataaagc cgacctacag atttcggtgc 1740 ttgaatgtca ttttgtggtt gggattctgg gctgtgcagc tgaatgtctg tctgtcacga 1800 atctaccttg ctgctcattt tcctcatcaa gttgttgctg gagtcctgtc aggcattgct 1860 gttgcagaaa ctttcagcca catccacagc atctataatg ccagcctcaa gaaatatttt 1920 ctcattacct tcttcctgtt cagcttcgcc atcggatttt atctgctgct caagggactg 1980 ggtgtagacc tcctgtggac tctggagaaa gcccagaggt ggtgcgagca gccagaatgg 2040 gtccacattg acaccacacc ctttgccagc ctcctcaaga acctgggcac gctctttggc 2100 ctggggctgg ctctcaactc cagcatgtac agggagagct gcaaggggaa actcagcaag 2160 tggctcccat tccgcctcag ctctattgta gcctccctcg tcctcctgca cgtctttgac 2220 tccttgaaac ccccatccca agtcgagctg gtcttctacg tcttgtcctt ctgcaagagt 2280 gcggtagtgc ccctggcatc cgtcagtgtc atcccctact gcctcgccca ggtcctgggc 2340 cagccgcaca agaagtcgtt gtaaagatct gaattcaccc caccagtgca ggctgcctat 2400 cagaaagtgg tggctggtgt ggctaatgcc ctggcccaca agtatcacta agctcgcttt 2460 cttgctgtcc aatttctatt aaaggttcct ttgttcccta agtccaacta ctaaactggg 2520 ggatattatg aagggccttg agcatctgga ttctgcctaa taaaaaacat ttattttcat 2580 tgcaatgatg tatttaaatt atttctgaat atttactaa aaagggaatg tgggaggtca 2640 gtgcatttaa aacataaaga aatgaagagc tagttcaaac cttgggaaaa tacactatat 2700 cttaaactcc atgaaagaag gtgaggctgc aaacagctaa tgcacattgg caacagcccc 2760 tgatgcctat gccttattca tccctcagaa aggattcaa gtagaggctt gatttggagg 2820 ttaaagtttg gctatgctgt attttacatt acttattgtt ttagctgtcc tcatgaatgt 2880 cttttcacta cccatttgct tatcctgcat ctctcagcct tgactccact cagttctctt 2940 gcttagagat accacctttc ccctgaagtg ttccttccat gttttacggc gagatggttt 3000 ctcctcgcct ggccactcag ccttagttgt ctctgttgtc ttatagaggt ctacttgaag 3060 aaggaaaaac agggggcatg gtttgactgt cctgtgagcc cttcttccct gcctccccca 3120 ctcacagtga cccggaatcc ctcgacatgg cag 3153 <210> 12 <211> 357 <212> PRT <213> Synthetic Sequence <220> <223> hG6Pase-a mut (amino acid) <400> 12 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Cys Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 13 <211> 1074 <212> Ms <213> artificial sequence <220> <223>Nucleic acids encoding modified G6Pase-a (R3K) <400>13 atggagaaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 14 <211> 1074 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid encoding modified G6Pase-a (Q54R) <400> 14 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc gtgaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 15 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (Q139R) <400> 15 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcgggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 [[ID=We have detected that the original text contains Chinese characters. Please provide an English text for translation.]] <211> 1074 <212> DNA <213> Synthetic Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (I142K) <400> 16 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagaaaaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600<​​atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 17 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (S196R) <400> 17 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttccgcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 18 <211> 1074 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid encoding modified G6Pase-a (H199Q) <400> 18 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccagagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 19 <211> 1074 <212> DNA <213> Synthetic Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (Q242R) <400> 19 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gccaggaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 20 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (Q247R) <400> 20 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagcg gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 21 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (L292F) <400> 21 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggttcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 22 <211> 1074 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid encoding modified G6Pase-a (S298C) <400> 22 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctgcattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 23 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (A301V) <400> 23 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gtgtccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 24 <211> 1074 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid encoding modified G6Pase-a (V318T) <400> 24 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca aactgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 25 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (V324T) <400> 25 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 <223> 编码修饰的 G6Pase-a (V324T) 的核酸aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 It should be noted that there seems to be an error in the repeated display of in the original text. The translation is adjusted according to the overall content. If there are specific requirements for handling such situations, please let me know.atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctaca ccttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 26 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (V332A) <400> 26 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggcagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 27 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid encoding modified G6Pase-a (Q347R) <400> 27 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgcccg ggtcctgggc cagccgcaca agaagtcgtt gtaa 1074 <210> 28 <211> 1074 <212> DNA[[ID=2​​​ <223> Nucleic acid encoding modified G6Pase-a (L349F) <400> 28 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ccgtgatcgc agacctcagg 120 aatgccttct acgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctcg tctttaagtg gattctcttt 240 ggacagcgtc catactggtg ggttttggat actgactact acagcaacac ttccgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctacgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc cgacctacag atttcggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcacga atctaccttg ctgctcattt tcctcatcaa 540 ]gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagcttcgcc 660 atcggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgcgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac gctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tccgcctcag ctctattgta 900 gcctccctcg tcctcctgca cgtctttgac tccttgaaac ccccatccca agtcgagctg 960 gtcttctacg tcttgtcctt ctgcaagagt gcggtagtgc ccctggcatc cgtcagtgtc 1020 atcccctact gcctcgccca ggtcttcggc cagccgcaca agaagtcgtt gtaa 1074 <210> 29 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> Modified G6Pase-a (R3K) <400> 29 Met Glu Lys Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 30 <211> 357 <212> PRT <213> Artificial sequence <220> <223> Modified G6Pase-a (Q54R) <400> 30 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Arg Glu Ala Val Gly Ile Lys Leu Leu Trp Val [[ID=2,7]]50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 31 <211> 357 <212> PRT <213> Artificial sequence <220> <{223}> Modified G6Pase-a (Q139R) <400> 31 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Arg Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 32 <211> 357 <212> PRT <213> artificial sequence <220> Modified G6Pase-a (I142K) <400> 32 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Lys Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 33 <211> 357 <212> PRT <213> Artificial sequence <220> <223> Modified G6Pase-a (S196R) <400> 33 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Arg His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 [[ID=二十九]] His Lys Lys Ser Leu 355 <210> 34 <211> 357 [[ID=三十八]]<212> PRT <213> Artificial sequence <220> <223> Modified G6Pase-a (H199Q) <400> 34 It should be noted that in the above translation, for the tags like , etc., they are preserved as they are because they are likely specific identifiers in a particular context and not meant to be translated in a traditional sense. Also, the translation of "人工序列" as "Artificial sequence" is a direct translation for the purpose of maintaining the integrity of the text in the context of patent translation.Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile Gln Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 35 <211> 357 <212> PRT <213> Artificial sequence <220> <223> Modified G6Pase-a (Q242R) <400> 35 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Arg Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 36 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> Modified G6Pase-a (Q247R) <400> 36 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Arg Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 37 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> Modified G6Pase-a (L292F) <400> 37 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Phe Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 38 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> Modified G6Pase-a (S298C) <400> 38 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 ​ 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Cys Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 39 <211> 357 <212> PRT <213> Artificial sequence <220> <223> Modified G6Pase-a (A301V) <400> 39 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Val Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 40 <211> 357 <212> PRT <213> Artificial sequence <220> <223> Modified G6Pase-a (V318T) <400> 40 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Thr Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 41 <211> 357 <212> PRT <213> artificial sequence <220> <223> Modified G6Pase-a (V324T) <400> 41 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Thr Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 42 <211> 357 <212> PRT <213> Artificial sequence <220> <223> Modified G6Pase-a (V332A) <400> 42 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Ala Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 43 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> Modified G6Pase-a (Q347R) <400> 43 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305 310 315 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Arg Val Leu Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 44 <211> 357 <212> PRT <213> Artificial Sequence <220> <223> Modified G6Pase-a (L349F) <400> 44 Met Glu Glu Gly Met Asn Val Leu His Asp Phe Gly Ile Gln Ser Thr 1 5 10 15 His Tyr Leu Gln Val Asn Tyr Gln Asp Ser Gln Asp Trp Phe Ile Leu 20 25 30 Val Ser Val Ile Ala Asp Leu Arg Asn Ala Phe Tyr Val Leu Phe Pro 35 40 45 Ile Trp Phe His Leu Gln Glu Ala Val Gly Ile Lys Leu Leu Trp Val 50 55 60 Ala Val Ile Gly Asp Trp Leu Asn Leu Val Phe Lys Trp Ile Leu Phe 65 70 75 80 Gly Gln Arg Pro Tyr Trp Trp Val Leu Asp Thr Asp Tyr Tyr Ser Asn 85 90 95 Thr Ser Val Pro Leu Ile Lys Gln Phe Pro Val Thr Cys Glu Thr Gly 100 105 110 Pro Gly Ser Pro Ser Gly His Ala Met Gly Thr Ala Gly Val Tyr Tyr 115 120 125 Val Met Val Thr Ser Thr Leu Ser Ile Phe Gln Gly Lys Ile Lys Pro 130 135 140 Thr Tyr Arg Phe Arg Cys Leu Asn Val Ile Leu Trp Leu Gly Phe Trp 145 150 155 160 Ala Val Gln Leu Asn Val Cys Leu Ser Arg Ile Tyr Leu Ala Ala His 165 170 175 Phe Pro His Gln Val Val Ala Gly Val Leu Ser Gly Ile Ala Val Ala 180 185 190 Glu Thr Phe Ser His Ile His Ser Ile Tyr Asn Ala Ser Leu Lys Lys 195 200 205 Tyr Phe Leu Ile Thr Phe Phe Leu Phe Ser Phe Ala Ile Gly Phe Tyr 210 215 220 Leu Leu Leu Lys Gly Leu Gly Val Asp Leu Leu Trp Thr Leu Glu Lys 225 230 235 240 Ala Gln Arg Trp Cys Glu Gln Pro Glu Trp Val His Ile Asp Thr Thr 245 250 255 Pro Phe Ala Ser Leu Leu Lys Asn Leu Gly Thr Leu Phe Gly Leu Gly 260 265 270 Leu Ala Leu Asn Ser Ser Met Tyr Arg Glu Ser Cys Lys Gly Lys Leu 275 280 285 Ser Lys Trp Leu Pro Phe Arg Leu Ser Ser Ile Val Ala Ser Leu Val 290 295 300 Leu Leu His Val Phe Asp Ser Leu Lys Pro Pro Ser Gln Val Glu Leu 305, 310, 315, 320 Val Phe Tyr Val Leu Ser Phe Cys Lys Ser Ala Val Val Pro Leu Ala 325 330 335 Ser Val Ser Val Ile Pro Tyr Cys Leu Ala Gln Val Phe Gly Gln Pro 340 345 350 His Lys Lys Ser Leu 355 <210> 45 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized G6pc (co1) - for Example 5 <400> 45 atggaggaag gaatgaatgt tctccatgac tttgggatcc agtcaacaca ttacctccag 60 gtgaattacc aagactccca ggactggttc atcttggtgt ctgtgattgc agacctcagg 120 aatgccttct atgtcctctt ccccatctgg ttccatcttc aggaagctgt gggcattaaa 180 ctcctttggg tagctgtgat tggagactgg ctcaacctgg tctttaagtg gattctcttt 240 ggacagaggc catactggtg ggttttggat actgactact acagcaacac ttctgtgccc 300 ctgataaagc agttccctgt aacctgtgag actggaccag ggagcccctc tggccatgcc 360 atgggcacag caggtgtata ctatgtgatg gtcacatcta ctctttccat ctttcaggga 420 aagataaagc ccacctacag atttaggtgc ttgaatgtca ttttgtggtt gggattctgg 480 gctgtgcagc tgaatgtctg tctgtcaagg atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagctttgcc 660 attggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgtgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac cctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tcaggctcag ctctattgta 900 gcctccctgg tcctcctgca tgtctttgac tccttgaaac ccccatccca agtggagctg 960 gtcttctatg tcttgtcctt ctgcaagagt gctgtagtgc ccctggcatc tgtcagtgtc 1020 atcccctact gcctggccca ggtcctgggc cagccccaca agaagtcctt gtaa 1074 <210> 46 <211> 1074 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized G6pc (co2) – for Example 5 <400> 46 atggaggagg ggatgaacgt gctgcacgac tttgggatcc agagcaccca ctacctgcag 60 gtgaactacc aggacagcca ggactggttt atcctggtgt ctgtgattgc tgacctgagg 120 aacgccttct acgtgctgtt ccctatctgg ttccacctgc aggaggctgt ggggatcaag 180 ctgctgtggg tggctgtgat tggggactgg ctgaacctgg tgttcaagtg gatcctgttt 240 ggccagaggc cctactggtg ggtgctggac acagactact acagcaacac ctctgtgccc 300 ctgatcaagc agttccctgt gacctgtgag acaggccctg gcagcccctc tggccacgct 360 atgggcacag ctggggtgta ctacgtgatg gtgaccagca ccctgtctat cttccagggc 420 aagatcaagc ccacctacag gttcaggtgc ctgaacgtga tcctgtggct gggcttctgg 480 gctgtgcagc tgaacgtgtg cctgagcagg atctacctgg ctgcccactt cccccaccag 540 gtggtggctg gggtgctgtc tgggattgct gtggctgaga ccttcagcca catccactct 600 atctacaacg ccagcctgaa gaagtacttc ctgatcacct tcttcctgtt cagctttgct 660 attggcttct acctgctgct gaagggcctg ggggtggacc tgctgtggac cctggagaag 720 gcccagaggt ggtgtgagca gcctgagtgg gtgcacattg acaccacccc ctttgccagc 780 ctgctgaaga acctgggcac cctgtttggc ctgggcctgg ccctgaacag ctctatgtac 840 agggagagct gcaagggcaa gctgagcaag tggctgccct tcaggctgag ctctattgtg 900 gccagcctgg tgctgctgca cgtgtttgac agcctgaagc cccccagcca ggtggagctg 960 gtgttctacg tgctgagctt ctgcaagtct gctgtggtgc ccctggcctc tgtgtctgtg 1020 atcccctact gcctggccca ggtgctgggc cagccccaca agaagagcct gtag 1074 <210> 47 <211> 441 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized intron (HBB2 co) of the β-globin gene – used in Examples 4 and 5 <400> 47 gtacacatat tgaccaaatc agggtaattt tgcatttgta attttaaaaa atgctttctt 60 cttttaatat acttttttgt ttatcttatt tctaatactt tccctaatct ctttctttca 120 gggcaataat gatacaatgt atcatgcctc tttgcaccat tctaaagaat aacagtgata 180 atttctgggt taaggcaata gcaatatttc tgcatataaa tatttctgca tataaattgt 240 aactgatgta agaggtttca tattgctaat agcagctaca atccagctac cattctgctt 300 ttattttctg gttgggataa ggctggatta ttctgagtcc aagctaggcc cttttgctaa 360 tcttgttcat acctcttatc ttcctcccac agctcctggg caacctgctg gtctctctgc 420 tggcccatca ctttggcaaa g 441 <210> 48 <211> 3134 <212> DNA <213> Artificial sequence <220> <223> hAAT_HBB2Co_hG6pc wt co1 (Expression cassette used in Example 5) <400> 48 aaggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60 cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120 ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180 ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240 ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300 tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360 ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420 cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480 tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540 ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600 ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660 aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720 cagtgaatag atcctgagaa cttcagggtg agtctatggg acccttgatg ttttctttcc 780 cctttttttc tatggttaag ttcatgtcat aggaagggga ggaataacag ggtacacata 840 ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata 900 tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc agggcaataa 960 tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg 1020 ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt 1080 aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct tttatttct 1140 ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta atcttgttca 1200 tacctcttat cttctcccca cagctcctgg gcaacctgct ggtctctctg ctggcccatc 1260 actttggcaa agcacgcgtg ccaccatgga ggaaggaatg aatgttctcc atgactttgg 1320 gatccagtca acacattacc tccaggtgaa ttaccaagac tcccaggact ggttcatctt 1380 ggtgtctgtg attgcagacc tcaggaatgc cttctatgtc ctcttcccca tctggttcca 1440 tcttcaggaa gctgtgggca ttaaactcct ttgggtagct gtgattggag actggctcaa 1500 cctggtcttt aagtggattc tctttggaca gaggccatac tggtgggttt tggatactga 1560 ctactacagc aacacttctg tgcccctgat aaagcagttc cctgtaacct gtgagactgg 1620 accagggagc ccctctggcc atgccatggg cacagcaggt gtatactatg tgatggtcac 1680 atctactctt tccatctttc agggaagat aaagcccacc tacagattta ggtgcttgaa 1740 tgtcattttg tggttgggat tctgggctgt gcagctgaat gtctgtctgt caaggatcta 1800 ccttgctggt cattttcctc atcaagttgt tgctggagtc ctgtcaggca ttgctgttgc 1860 agaaactttc agccacatcc acagcatcta taatgccagc ctcaagaaat attttctcat 1920 taccttcttc ctgttcagct ttgccattgg attttatctg ctgctcaagg gactgggtgt 1980 agacctcctg tggactctgg agaaagccca gaggtggtgt gagcagccag aatgggtcca 2040 cattgacacc acaccctttg ccagcctcct caagaacctg ggcaccctct ttggcctggg 2100 gctggctctc aactccagca tgtacaggga gagctgcaag gggaactca gcaagtggct 2160 cccattcagg ctcagctcta ttgtagcctc cctggtcctc ctgcatgtct ttgactcctt 2220 gaaaccccca tcccaagtgg agctggtctt ctatgtcttg tccttctgca agagtgctgt 2280 agtgcccctg gcatctgtca gtgtcatccc ctactgcctg gcccaggtcc tgggccagcc 2340 ccacaagaag tccttgtaac tcgaggaatt caccccacca gtgcaggctg cctatcagaa 2400 agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc gctttcttgc 2460 tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata 2520 ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 2580 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 2640 tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 2700 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gcccctgatg 2760 cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt ggaggttaaa 2820 gttttgctat gctgtatttt acattactta ttgttttagc tgtcctcatg aatgtctttt 2880 cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt ctcttgctta 2940 gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat ggtttctcct 3000 cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact tgaagaagga 3060 aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc ccccactcac 3120 agtgacccgg aatc 3134 <210> 49 <211> 3134 <212> DNA <213> Artificial Sequence <220> <223> hAAT_HBB2Co_hG6pc wt co2 (Expression cassette used in Example 5) <400> 49 aaggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60 cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120 ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180 ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240 ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300 tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360 ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420 cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480 tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540 ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600 ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660 aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720 cagtgaatag atcctgagaa cttcagggtg agtctatggg acccttgatg ttttctttcc 780 ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag ggtacacata 840 ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata 900 tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc agggcaataa 960 tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg 1020 ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt 1080 aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct tttatttct 1140 ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta atcttgttca 1200 tacctcttat cttctcccca cagctcctgg gcaacctgct ggtctctctg ctggcccatc 1260 actttggcaa agcacgcgtg ccaccatgga ggaggggatg aacgtgctgc acgactttgg 1320 gatccagagc accacactacc tgcaggtgaa ctaccaggac agccaggact ggtttatcct 1380 ggtgtctgtg attgctgacc tgaggaacgc cttctacgtg ctgttcccta tctggttcca 1440 cctgcaggag gctgtggga tcaagctgct gtgggtggct gtgattgggg actggctgaa 1500 cctggtgttc aagtggatcc tgtttggcca gaggccctac tggtgggtgc tggacacaga 1560 ctactacagc aacacctctg tgcccctgat caagcagttc cctgtgacct gtgagacagg 1620 ccctggcagc ccctctggcc acgctatggg cacagctgggg gtgtactacg tgatggtgac 1680 cagcaccctg tctatcttcc agggcaagat caagcccacc tacaggttca ggtgcctgaa 1740 cgtgatcctg tggctggggct tctgggctgt gcagctgaac gtgtgcctga gcaggatcta 1800 cctggctgcc cacttccccc accaggtggt ggctgggggtg ctgtctggga ttgctgtggc 1860 tgagaccttc agccacatcc actctatcta caacgccagc ctgaagaagt acttcctgat 1920 caccttcttc ctgttcagct ttgctattgg cttctacctg ctgctgaagg gcctggggt 1980 ggacctgctg tggaccctgg agaaggccca gaggtggtgt gagcagcctg agtgggtgca 2040 cattgacacc accccctttg ccagcctgct gaagaacctg ggcaccctgt ttggcctggg 2100 cctggccctg aacagctcta tgtacaggga gagctgcaag ggcaagctga gcaagtggct 2160 gcccttcagg ctgagctcta ttgtggccag cctggtgctg ctgcacgtgt ttgacagcct 2220 gaagcccccc agccaggtgg agctggtgtt ctacgtgctg agcttctgca agtctgctgt 2280 ggtgcccctg gcctctgtgt ctgtgatccc ctactgcctg gcccaggtgc tgggccagcc 2340 ccacaagaag agcctgtagc tcgaggaatt cacccacca gtgcaggctg cctatcagaa 2400 agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc gctttcttgc 2460 tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata 2520 tttgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 2580 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 2640 tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 2700 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gcccctgatg 2760 cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt ggaggttaaa 2820 gttttgctat gctgtatttt acattactta ttgttttagc tgtcctcatg aatgtctttt 2880 cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt ctcttgctta 2940 gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat ggtttctcct 3000 cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact tgaagaagga 3060 aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc ccccactcac 3120 agtgacccgg aatc 3134 <210> 50 <211> 3134 <212> DNA <213> Artificial sequence <220> <223> hAAT_HBB2Co_hG6pc wt co3 (Expression cassette used in Example 5) - Comprising SEQ ID NO: 3 <400> 50 aaggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60 cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120 ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180 ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240 ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300 tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360 ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420 cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480 tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 54t ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600 It should be noted that there seems to be a typo in the original text where "54t" should probably be "540" in line 27 of the English translation.ggtgaccttg gttaatattc accagcagcc tccccccgttg cccctctgga tccactgctt 660 720 cagtgaatag atcctgagaa cttcagggtg agtctatggg acccttgatg ttttctttcc 780 cctttttttc tatggttaag ttcatgtcat aggaagggga ggaataacag ggtacacata 840 ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata 900 tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc agggcaatat 960 tgatacaatg tatcttgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg 1020 ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt 1080 aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct tttatttct 1140 ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta atcttgttca 1200 tacctcttat cttctcccca cagctcctgg gcaacctgct ggtctctctg ctggcccatc 1260 actttggcaa agcacgcgtg ccaccatgga ggagggaatg aatgtgctgc acgacttcgg 1320 gattcagtct acccactatc tccaagtcaa ttatcaagac agtcaagatt ggttcatcct 1380 cgtctccgtt atagctgacc tcaggaatgc tttttatgta ctgttcccaa tatggtttca 1440 tctgcaagaa gcagttggaa ttaaactgct gtgggtggcc gtaatcggag actggttgaa 1500 cctggtgttc aaatggatcc tttttggtca gaggccatat tggtgggtct tggacaccga 1560 ctattatagc aacacatctg tacccctgat aaagcaattc cctgtaacgt gtgaaactgg 1620 gcctgggtca cccagtggac acgcgatggg gactgccggg gtttactacg taatggttac 1680 tagcacgctt agcattttcc agggcaagat caaaccgact tatcgcttcc gatgcctcaa 1740 tgtgatcctc tggctgggat tctgggcggt tcaactgaac gtatgcctct cacggattta 1800 tctggctgca catttcccgc atcaagtagt ggctggtgtg ttgtctggca tagcagtggc 1860 tgaaacattc tcacacattc attctattta caacgcttca ttgaaaaaat actttctgat 1920 tacttttttt cttttcagct ttgcaattgg cttttacttg ctgcttaaag gtcttggcgt 1980 cgacctgctc tggactcttg agaaagcgca acgctggtgc gaacaacccg agtgggtcca 2040 catagatacg accccgttcg cgtctcttct gaaaaatctc ggtaccctct tcggactggg 2100 acttgctttg aattcttcaa tgtatcggga atcctgcaag ggcaaactgt ccaagtggtt 2160 gccctttcgc ctttcaagca ttgtcgcttc ccttgtgctc ctgcacgtct tcgactcact 2220 gaagccgccg agccaagtcg agctcgtgtt ttacgtgttg tctttctgta aaagtgccgt 2280 cgtgccactc gccagtgtga gcgtgatacc ttattgcctc gcacaggtgc ttgggcagcc 2340 tcataagaag tctctgtaaa gatctgaatt caccccacca gtgcaggctg cctatcagaa 2400 agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc gctttcttgc 2460 tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata 2520 ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 2580 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 2640 tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 2700 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gcccctgatg 2760 cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt ggaggttaaa 2820 gtttggctat gctgtatttt acattactta ttgttttagc tgtcctcatg aatgtctttt 2880 cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt ctcttgctta 2940 gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat ggtttctcct 3000 cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact tgaagaagga 3060 aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc ccccactcac 3120 agtgacccgg aatc 3134 <210> 51 <211> 3134 <212> DNA <213> Artificial sequence <220> <223> hAAT_HBB2 co_hG6pc mut (Expression cassette) contains SEQ ID NO: 5 <400> 51 aaggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60 cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120 ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180 ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240 ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300 tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360 ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420 cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480 tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540 ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600 ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660 aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720 cagtgaatag atcctgagaa cttcagggtg agtctatggg acccttgatg ttttctttcc 780 ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag ggtacacata 840 ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata 900 tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc agggcaataa 960 tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg 1020 ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt 1080 aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct tttatttct 1140 ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta atcttgttca 1200 tacctcttat cttctcccca cagctcctgg gcaacctgct ggtctctctg ctggcccatc 1260 actttggcaa agcacgcgtg ccaccatgga ggaaggaatg aatgttctcc atgactttgg 1320 gatccagtca acacattacc tccaggtgaa ttaccaagac tcccaggact ggttcatctt 1380 ggtgtccgtg atcgcagacc tcaggaatgc cttctacgtc ctcttcccca tctggttcca 1440 tcttcaggaa gctgtgggca ttaaactcct ttgggtagct gtgattggag actggctcaa 1500 cctcgtcttt aagtggattc tctttggaca gcgtccatac tggtgggttt tggatactga 1560 ctactacagc aacacttccg tgcccctgat aaagcagttc cctgtaacct gtgagactgg 1620 accagggagc ccctctggcc atgccatggg cacagcaggt gtatactacg tgatggtcac 1680 atctactctt tccatctttc agggaaagat aaagccgacc tacagatttc ggtgcttgaa 1740 tgtcattttg tggttgggat tctgggctgt gcagctgaat gtctgtctgt cacgaatcta 1800 ccttgctgct cattttcctc atcaagttgt tgctggagtc ctgtcaggca ttgctgttgc 1860 agaaactttc agccacatcc acagcatcta taatgccagc ctcaagaaat attttctcat 1920 taccttcttc ctgttcagct tcgccatcgg attttatctg ctgctcaagg gactgggtgt 1980 agacctcctg tggactctgg agaaagccca gaggtggtgc gagcagccag aatgggtcca 2040 cattgacacc acaccctttg ccagcctcct caagaacctg ggcacgctct ttggcctggg 2100 gctggctctc aactccagca tgtacaggga gagctgcaag gggaaactca gcaagtggct 2160 cccattccgc ctcagctgta ttgtagcctc cctcgtcctc ctgcacgtct ttgactcctt 2220 gaaaccccca tcccaagtcg agctggtctt ctacgtcttg tccttctgca agagtgcggt 2280 agtgcccctg gcatccgtca gtgtcatccc ctactgcctc gcccaggtcc tgggccagcc 2340 gcacaagaag tcgttgtaac tcgaggaatt caccccacca gtgcaggctg cctatcagaa 2400 agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc gctttcttgc 2460 tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata 2520 tttgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 2580 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 2640 tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 2700 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gcccctgatg 2760 cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt ggaggttaaa 2820 gttttgctat gctgtatttt acattactta ttgttttagc tgtcctcatg aatgtctttt 2880 cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt ctcttgctta 2940 gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat ggtttctcct 3000 cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact tgaagaagga 3060 aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc ccccactcac 3120 agtgacccgg aatc 3134 <210> 52 <211> 1074 <​​​​​​​​​​​​​​​​​​​​​​​​​gctgtgcagc tgaatgtctg tctgtcaagg atctaccttg ctgctcattt tcctcatcaa 540 gttgttgctg gagtcctgtc aggcattgct gttgcagaaa ctttcagcca catccacagc 600 atctataatg ccagcctcaa gaaatatttt ctcattacct tcttcctgtt cagctttgcc 660 attggatttt atctgctgct caagggactg ggtgtagacc tcctgtggac tctggagaaa 720 gcccagaggt ggtgtgagca gccagaatgg gtccacattg acaccacacc ctttgccagc 780 ctcctcaaga acctgggcac cctctttggc ctggggctgg ctctcaactc cagcatgtac 840 agggagagct gcaaggggaa actcagcaag tggctcccat tcaggctcag ctgtattgta 900 gcctccctgg tcctcctgca tgtctttgac tccttgaaac ccccatccca agtggagctg 960 gtcttctatg tcttgtcctt ctgcaagagt gctgtagtgc ccctggcatc tgtcagtgtc 1020 atcccctact gcctggccca ggtcctgggc cagccccaca agaagtcctt gtaa 1074 <210> 53 <211> 3134 <212> DNA <213> Artificial Sequence <220> <223> hAAT_HBB2 co_hG6pc mut co1 (Expression Cassette) <400> 53 aaggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60 cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120 ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180 ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240 ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300 tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360 ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420 cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480 tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540 ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600 ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660 aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720 cagtgaatag atcctgagaa cttcagggtg agtctatggg acccttgatg ttttctttcc 780 cctttttttc tatggttaag ttcatgtcat aggaagggga ggaataacag ggtacacata 840 ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata 900 tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc agggcaataa 960 tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg 1020 ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt 1080 aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct tttatttct 1140 ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta atcttgttca 1200 tacctcttat cttctcccca cagctcctgg gcaacctgct ggtctctctg ctggcccatc 1260 actttggcaa agcacgcgtg ccaccatgga ggaaggaatg aatgttctcc atgactttgg 1320 gatccagtca acacattacc tccaggtgaa ttaccaagac tcccaggact ggttcatctt 1380 ggtgtctgtg attgcagacc tcaggaatgc cttctatgtc ctcttcccca tctggttcca 1440 tcttcaggaa gctgtgggca ttaaactcct ttgggtagct gtgattggag actggctcaa 1500 cctggtcttt aagtggattc tctttggaca gaggccatac tggtgggttt tggatactga 1560 ctactacagc aacacttctg tgcccctgat aaagcagttc cctgtaacct gtgagactgg 1620 accagggagc ccctctggcc atgccatggg cacagcaggt gtatactatg tgatggtcac 1680 atctactctt tccatctttc agggaagat aaagcccacc tacagattta ggtgcttgaa 1740 tgtcattttg tggttgggat tctgggctgt gcagctgaat gtctgtctgt caaggatcta 1800 ccttgctggt cattttcctc atcaagttgt tgctggagtc ctgtcaggca ttgctgttgc 1860 agaaactttc agccacatcc acagcatcta taatgccagc ctcaagaaat attttctcat 1920 taccttcttc ctgttcagct ttgccattgg attttatctg ctgctcaagg gactgggtgt 1980 agacctcctg tggactctgg agaaagccca gaggtggtgt gagcagccag aatgggtcca 2040 cattgacacc acaccctttg ccagcctcct caagaacctg ggcaccctct ttggcctggg 2100 gctggctctc aactccagca tgtacaggga gagctgcaag gggaactca gcaagtggct 2160 cccattcagg ctcagctgta ttgtagcctc cctggtcctc ctgcatgtct ttgactcctt 2220 gaaaccccca tcccaagtgg agctggtctt ctatgtcttg tccttctgca agagtgctgt 2280 agtgcccctg gcatctgtca gtgtcatccc ctactgcctg gcccaggtcc tgggccagcc 2340 ccacaagaag tccttgtaac tcgaggaatt caccccacca gtgcaggctg cctatcagaa 2400 agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc gctttcttgc 2460 tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata 2520 ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 2580 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 2640 tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 2700 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gcccctgatg 2760 cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt ggaggttaaa 2820 gttttgctat gctgtatttt acattactta ttgttttagc tgtcctcatg aatgtctttt 2880 cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt ctcttgctta 2940 gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat ggtttctcct 3000 cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact tgaagaagga 3060 aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc ccccactcac 3120 agtgacccgg aatc 3134 <210> 54 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized hG6pc mut (co2) <400> 54 atggaggagg ggatgaacgt gctgcacgac tttgggatcc agagcaccca ctacctgcag 60 gtgaactacc aggacagcca ggactggttt atcctggtgt ctgtgattgc tgacctgagg 120 aacgccttct acgtgctgtt ccctatctgg ttccacctgc aggaggctgt ggggatcaag 180 ctgctgtggg tggctgtgat tggggactgg ctgaacctgg tgttcaagtg gatcctgttt 240 ggccagaggc cctactggtg ggtgctggac acagactact acagcaacac ctctgtgccc 300 ctgatcaagc agttccctgt gacctgtgag acaggccctg gcagcccctc tggccacgct 360 atgggcacag ctggggtgta ctacgtgatg gtgaccagca ccctgtctat cttccagggc 420 aagatcaagc ccacctacag gttcaggtgc ctgaacgtga tcctgtggct gggcttctgg 480 gctgtgcagc tgaacgtgtg cctgagcagg atctacctgg ctgcccactt cccccaccag 540 gtggtggctg gggtgctgtc tgggattgct gtggctgaga ccttcagcca catccactct 600 atctacaacg ccagcctgaa gaagtacttc ctgatcacct tcttcctgtt cagctttgct 660 attggcttct acctgctgct gaagggcctg ggggtggacc tgctgtggac cctggagaag 720 gcccagaggt ggtgtgagca gcctgagtgg gtgcacattg acaccacccc ctttgccagc 780 ctgctgaaga acctgggcac cctgtttggc ctgggcctgg ccctgaacag ctctatgtac 840 agggagagct gcaagggcaa gctgagcaag tggctgccct tcaggctgag ctgtattgtg 900 gccagcctgg tgctgctgca cgtgtttgac agcctgaagc cccccagcca ggtggagctg 960 gtgttctacg tgctgagctt ctgcaagtct gctgtggtgc ccctggcctc tgtgtctgtg 1020 atcccctact gcctggccca ggtgctgggc cagccccaca agaagagcct gtag 1074 <210> 55 <211> 3134 <212> DNA <213> Artificial Sequence <220> <223> hAAT_HBB2 co_hG6pc mut co2 (Expression Cassette) <400> 55 aaggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60 cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120 ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180 ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240 ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300 tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360 ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420 cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480 tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540 ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600 ggtgaccttg gttaatattc accagcagcc tccccccgttg cccctctgga tccactgctt 660 720 cagtgaatag atcctgagaa cttcagggtg agtctatggg acccttgatg ttttctttcc 780 cctttttttc tatggttaag ttcatgtcat aggaagggga ggaataacag ggtacacata 840 ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata 900 tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc agggcaataa 960 tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg 1020 ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt 1080 aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct tttatttct 1140 ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta atcttgttca 1200 tacctcttat cttctcccca cagctcctgg gcaacctgct ggtctctctg ctggcccatc 1260 actttggcaa agcacgcgtg ccaccatgga ggaggggatg aacgtgctgc acgactttgg 1320 gatccagagc acccactacc tgcaggtgaa ctaccaggac agccaggact ggtttatcct 1380 ggtgtctgtg attgctgacc tgaggaacgc cttctacgtg ctgttcccta tctggttcca 1440 cctgcaggag gctgtgggga tcaagctgct gtgggtggct gtgattgggg actggctgaa 1500 cctggtgttc aagtggatcc tgtttggcca gaggccctac tggtgggtgc tggacacaga 1560 ctactacagc aacacctctg tgcccctgat caagcagttc cctgtgacct gtgagacagg 1620 ccctggcagc ccctctggcc acgctatggg cacagctggg gtgtactacg tgatggtgac 1680 cagcaccctg tctatcttcc agggcaagat caagcccacc tacaggttca ggtgcctgaa 1740 cgtgatcctg tggctgggct tctgggctgt gcagctgaac gtgtgcctga gcaggatcta 1800 cctggctgcc cacttccccc accaggtggt ggctggggtg ctgtctggga ttgctgtggc 1860 tgagaccttc agccacatcc actctatcta caacgccagc ctgaagaagt acttcctgat 1920 caccttcttc ctgttcagct ttgctattgg cttctacctg ctgctgaagg gcctgggggt 1980 ggacctgctg tggaccctgg agaaggccca gaggtggtgt gagcagcctg agtgggtgca 2040 cattgacacc accccctttg ccagcctgct gaagaacctg ggcaccctgt ttggcctggg 2100 cctggccctg aacagctcta tgtacaggga gagctgcaag ggcaagctga gcaagtggct 2160 gcccttcagg ctgagctgta ttgtggccag cctggtgctg ctgcacgtgt ttgacagcct 2220 gaagcccccc agccaggtgg agctggtgtt ctacgtgctg agcttctgca agtctgctgt 2280 ggtgcccctg gcctctgtgt ctgtgatccc ctactgcctg gcccaggtgc tgggccagcc 2340 ccacaagaag agcctgtagc tcgaggaatt cacccacca gtgcaggctg cctatcagaa 2400 agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc gctttcttgc 2460 tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata 2520 ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 2580 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 2640 2700 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gcccctgatg 2760 cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt ggaggttaaa 2820 gttttgctat gctgtatttt acattactta ttgttttagc tgtcctcatg aatgtctttt 2880 cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt ctcttgctta 2940 gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat ggtttctcct 3000 cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact tgaagaagga 3060 aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc ccccactcac 3120 agtgacccgg aatc 3134 <210> 56 <211> 3134 <212> RNA <213> Artificial Sequence <220> <223> hAAT_HBB2 co_hG6pc wt (Expression cassette used in Examples 4 and 5) – contains SEQ ID NO: 2 <400> 56 aaggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60 cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120 ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180 ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240 ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300 tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360 ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420 cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480 tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540 ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600 ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660 aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720 cagtgaatag atcctgagaa cttcagggtg agtctatggg acccttgatg ttttctttcc 780 ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag ggtacacata 840 ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata 900 tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc agggcaataa 960 tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg 1020 ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt 1080 aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct tttatttct 1140 ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta atcttgttca 1200 tacctcttat cttctcccca cagctcctgg gcaacctgct ggtctctctg ctggcccatc 1260 actttggcaa agcacgcgtg ccaccatgga ggaaggaatg aatgttctcc atgactttgg 1320 gatccagtca acacattacc tccaggtgaa ttaccaagac tcccaggact ggttcatctt 1380 ggtgtccgtg atcgcagacc tcaggaatgc cttctacgtc ctcttcccca tctggttcca 1440 tcttcaggaa gctgtgggca ttaaactcct ttgggtagct gtgattggag actggctcaa 1500 cctcgtcttt aagtggattc tctttggaca gcgtccatac tggtgggttt tggatactga 1560 ctactacagc aacacttccg tgcccctgat aaagcagttc cctgtaacct gtgagactgg 1620 accagggagc ccctctggcc atgccatggg cacagcaggt gtatactacg tgatggtcac 1680 atctactctt tccatctttc agggaaagat aaagccgacc tacagatttc ggtgcttgaa 1740 tgtcattttg tggttgggat tctgggctgt gcagctgaat gtctgtctgt cacgaatcta 1800 ccttgctgct cattttcctc atcaagttgt tgctggagtc ctgtcaggca ttgctgttgc 1860 agaaactttc agccacatcc acagcatcta taatgccagc ctcaagaaat attttctcat 1920 taccttcttc ctgttcagct tcgccatcgg attttatctg ctgctcaagg gactgggtgt 1980 agacctcctg tggactctgg agaaagccca gaggtggtgc gagcagccag aatgggtcca 2040 cattgacacc acaccctttg ccagcctcct caagaacctg ggcacgctct ttggcctggg 2100 gctggctctc aactccagca tgtacaggga gagctgcaag gggaaactca gcaagtggct 2160 cccattccgc ctcagctcta ttgtagcctc cctcgtcctc ctgcacgtct ttgactcctt 2220 gaaaccccca tcccaagtcg agctggtctt ctacgtcttg tccttctgca agagtgcggt 2280 agtgcccctg gcatccgtca gtgtcatccc ctactgcctc gcccaggtc tgggccagcc 2340 gcacaagaag tcgttgtaaa gatctgaatt caccccacca gtgcaggctg cctatcagaa 2400 agtggtggct ggtgtggcta atgccctggc ccacaagtat cactaagctc gctttcttgc 2460 tgtccaattt ctattaaagg ttcctttgtt ccctaagtcc aactactaaa ctgggggata 2520 tttgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 2580 tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 2640 tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 2700 actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gcccctgatg 2760 cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt ggaggttaaa 2820 gttttgctat gctgtatttt acattactta ttgttttagc tgtcctcatg aatgtctttt 2880 cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt ctcttgctta 2940 gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat ggtttctcct 3000 cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact tgaagaagga 3060 aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc ccccactcac 3120 agtgacccgg aatc 3134 <210> 57 <211> 2217 <212> DNA <213> Artificial Sequence <220> <223> Synthetic polynucleotide encoding AAVMut5 <400> 57 atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60 gagtggtggg cgctgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120 gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180 aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240 cagcagctgc aggcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300<​​​​480. ggaagaga gaccggtaga gccatcaccc cagcgttctc cagactcctc tacgggcatc ggcaagaaag gccaacagcc cgccagaaaa agactcaatt ttggtcagac tggcgactca 540 gagtcagttc cagaccctca acctctcgga gaacctccag cagcgccctc tggtgtggga cctaacaa tggctgcagg cggtggcgca ccaatggcag acaataacga aggcgccgac ggagtgggta gttcctcggg aaattggcat tgcgattcca catggctggg cgacagagtc 720 atcaccacca gcacccgac ctgggccctg cccacctaca acaaccacct ctacaagcaa atctccaacg ggacatcggg aggagccacc aacgacaaca cctacttcgg ctacagcacc ccctgggggt attttgactt taacagattc cactgccact tttcaccacg tgactggcag cgactcatca acaacaactg gggattccgg cccaagagac tcagcttcaa gctcttcaac atccaggtca aggaggtcac gcagaatgaa ggcaccaaga ccatcgccaa taacctcacc agcaccatcc aggtgtttac ggactcggag taccagctgc cgtacgttct cggctctgcc 1080. caccagggct gcctgcctcc gttcccggcg gacgtgttca tgattcccca gtacggctac 1140 ctaacactca acaacggtag tcaggccgtg ggacgctcct ccttctactg cctggaatac 1200 tttccttgc agatgctgag aaccggcaac aacttccagt ttacttacac cttcgaggac 1260 gtgcctttcc acagcagcta cgcccacagc cagagcttgg accggctgat gaatcctctg 1320 attgaccagt acctgtacta tctgaacaag acacaatcaa atagtggaac tcttcagcag 1380 tctcggctac tgtttagtca agctggaccc accagcatgt ctcttcaagc taaaaactgg 1440 ctgcctggac cttgctaccg ccaacaacgc gtctcaacga caaccgggca aaacaacaat 1500 agcaactttg cctggactgc tgggaccaaa taccatctga atggaagaaa ttcattggct 1560 aatcctggca tcgctatggc aacacacaaa gacgacgagg agcgtttttt tcccagtaac 1620 gggatcctga tttttggcaa acaaaatgct gccagagaca atgcggatta cagcgatgtc 1680 atgctcacca gcgaggaaga aatcaaaacc actaaccctg tggctacaga ggaatacggt 1740 atcgtggcag ataacttgca gcagcaaaac acggctcctc aaattggaac tgtcaacagc 1800 caggggcct tacccggtat ggtctggcag aaccgggacg tgtacctgca gggtcccatc 1860 tgggccaaga ttcctcacac ggacggcaac ttccacccgt ctccgctgat gggcggcttt 1920 ggcctgaaac atcctccgcc tcagatcctg atcaagaaca cgcctgtacc tgcggatcct 1980 ccgaccacct tcaaccagtc aaagctgaac tctttcatca cgcaatacag caccggacag 2040 gtcagcgtgg aaattgaatg ggagctgcag aaggaaaaca gcaagcgctg gaaccccgag 2100 atccagtaca cctccaacta ctacaaatct acaagtgtgg actttgctgt taatacagaa 2160 ggcgtgtact ctgaaccccg ccccattggc acccgttacc tcacccgtaa tctgtaa 2217 <210> 58 <211> 738 <212> PRT <213> Artificial Sequence <220> <223> AAVmut5 (amino acid) <400> 58 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile 145 150 155 160 Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln 165 170 175 Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro 180 185 190 Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly 195 200 205 Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser 210 215 220 Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val 225 230 235 240 Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 245 250 255 Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp 260 265 270 Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285 Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300 Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn 305 310 315 320 Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala 325 330 335 Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln 340 345 350 Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe 355 360 365 Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn 370 375 380 Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr 385 390 395 400 Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr 405 410 415 Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430 Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440 445 Asn Lys Thr Gln Ser Asn Ser Gly Thr Leu Gln Gln Ser Arg Leu Leu 450 455 460 Phe Ser Gln Ala Gly Pro Thr Ser Met Ser Leu Gln Ala Lys Asn Trp 465 470 475 480 Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly 485 490 495 Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His 500 505 510 Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr 515 520 525 His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile 530 535 540 Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val 545 550 555 560 Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr 565 570 575 Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala 580 585 590 Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val 595 600 605 Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile 610 615 620 Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe 625 630 635 640 Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655 Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe 660 665 670 Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu 675 680 685 Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr 690 695 700 Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu 705 710 715 720 Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg 725 730 735 Asn Leu

Claims

1. An adeno-associated virus (AAV) vector comprising a nucleic acid construct for expressing glucose-6-phosphatase-a (G6Pase-a) in hepatocytes, the construct comprising a nucleic acid sequence encoding G6Pase-a, wherein the nucleic acid sequence encoding G6Pase-a is operatively linked to a human α-1 antitrypsin (hAAT) promoter, wherein the hAAT promoter is preceded by an ApoE enhancer.

2. The AAV vector according to claim 1, wherein the G6Pase-a has the amino acid sequence of SEQ ID NO:

1.

3. The AAV vector according to any one of claims 1 or 2, wherein the nucleic acid sequence encoding G6Pase-a comprises the nucleotide sequence of SEQ ID NO:

2.

4. The AAV vector according to claim 1, wherein the nucleic acid sequence encoding G6Pase-a is codon-optimized.

5. The AAV vector according to claim 1, wherein the nucleic acid sequence encoding G6Pase-a is codon-optimized by reducing GC content and GC dimer in the nucleic acid sequence encoding G6Pase-a.

6. The AAV vector according to claim 1, wherein the hAAT promoter comprises the nucleotide sequence of SEQ ID NO:

8.

7. The AAV vector according to claim 1, wherein the hAAT promoter is preceded by the ApoE enhancer of SEQ ID NO:

9.

8. The AAV vector according to claim 1, wherein the nucleic acid construct comprises the nucleotide sequence of SEQ ID NO:

7.

9. The AAV vector according to claim 1, wherein the nucleic acid construct comprises the nucleotide sequence of SEQ ID NO:11, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, or SEQ ID NO:55 or SEQ ID NO:

56.

10. The AAV vector according to claim 1, wherein the nucleic acid construct comprises the nucleotide sequence of SEQ ID NO:

48.

11. The AAV vector according to claim 1, wherein the nucleic acid construct comprises, in the 5' to 3' direction: (i) hAAT promoter, which is preceded by ApoE enhancer; (ii) Introns can be selected arbitrarily; (iii) The nucleic acid sequence encoding G6Pase-a; and (iv) Polyadenylation signal.

12. The AAV vector according to claim 1, wherein the nucleic acid construct comprises an intron of the human β-globin gene.

13. The AAV vector according to claim 1, wherein the nucleic acid construct comprises an intron of the human β-globin gene of SEQ ID NO:

47.

14. The AAV vector according to claim 1, wherein the nucleic acid construct comprises bovine growth hormone polyadenylation signal, HBB2 polyadenylation signal or SV40 polyadenylation signal.

15. The vector according to claim 1, wherein it is an AAV serotype 8 (AAV8) vector, an AAV9 vector, an AAVrh74 vector, an AAV2i8 vector, or an AAV8mut5 vector, wherein the AAV8mut5 vector has the amino acid sequence SEQ ID NO: 57 and is encoded by a polynucleotide of SEQ ID NO:

58.

16. The vector according to claim 1, wherein it is an AAV serotype 8 (AAV8) vector.

17. Hepatocytes, transformed with the vector according to any one of claims 1 to 16.

18. A composition comprising the carrier of any one of claims 1 to 16 or the cell of claim 17.

19. Use of the carrier according to any one of claims 1 to 16, the cell according to claim 17, or the composition according to claim 18 in the preparation of a medicament for treating glycogen storage disease Ia (GSD-Ia).

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