Methods and compositions for treating glycogen storage disease

Abstract

This invention provides a variety of novel adeno-associated virus (AAV) vectors for gene therapy applications in the treatment of glycogen storage disease type Ia (GSD-Ia). This document discloses numerous recombinant nucleic acid molecules, vectors, and recombinant AAVs incorporating modified G6PC promoter / enhancer sequences. When expressed from various host cell platforms, the use of said modified G6PC promoter / enhancer sequences results in enhanced AAV yield and quality. This document also provides compositions comprising the novel AAVs of this invention and methods for treating GSD-Ia using said compositions.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 781,380, filed December 18, 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on December 16, 2019, and is named DIM-010WO_SL_ST25.txt, with a size of 42,290 bytes. Technical Field

[0005] This application generally relates to viral vectors for treating glycogen storage diseases such as type Ia glycogen storage disease, and more specifically to adeno-associated virus vectors. Background Technology

[0006] Type Ia glycogen storage disease (also known as GSD-Ia or von Gierke disease) is a genetic disorder caused by the accumulation of glycogen in the body's cells. The accumulation of glycogen in certain organs and tissues, particularly the liver, kidneys, and small intestine, impairs their ability to function properly. GSD-Ia typically appears in the first year of life and is characterized by severe hypoglycemia and hepatomegaly due to glycogen accumulation. Affected individuals exhibit growth retardation, delayed puberty, lactic acidosis, hyperlipidemia, hyperuricemia, and a high incidence of hepatic adenomas in adults. See Lei et al., 1993, Science 262:580-3.

[0007] GSD-Ia is a rare orphan genetic disorder caused by a deficiency of active glucose-6-phosphatase-α (G6P-α), a key enzyme involved in maintaining glucose homeostasis. Encoded by the G6PC gene, G6P-α catalyzes the hydrolysis of glucose-6-phosphate (G6P) into glucose and phosphate in the final step of glycogenolysis and gluconeogenesis. To date, more than 80 mutations have been identified that lead to G6P-α deficiency and the development of GSD-Ia. See Chou et al., 2010, Nat RevEndocrinol [Nature Reviews Endocrinology] 6(12): 676-88.

[0008] Currently, there is no cure for GSD-Ia, and the standard of care for patients is dietary supplementation. If strictly followed, dietary strategies can generally promote normal growth and puberty development, but dietary therapy cannot completely prevent hyperlipidemia, hyperuricemia, lactic acidosis, and hepatic steatosis. See Rake et al., 2002, Eur J Pediatr [European Journal of Pediatric Surgery] 161 Supplement 1: S20-34.

[0009] Gene therapy methods using recombinant adeno-associated virus (AAV) carrying G6P enzyme-α have been explored to manage GSD-Ia. See, for example, U.S. Patent No. 9,644,216 and U.S. Patent Publication No. 2017 / 0362670. However, for AAV vectors to be used in human gene therapy, developing robust, reliable, and scalable vector production methods is crucial. The inventors have discovered that modifying the promoter / enhancer region of the G6PC gene to remove certain sequences (described herein as "Alu elements") significantly improves rAAV yield and quality when expressed from various host cell platforms. Summary of the Invention

[0010] This invention provides methods and compositions for treating glycogen storage diseases. More specifically, this document provides recombinant nucleic acid molecules, adeno-associated virus (AAV) vectors, and recombinant adeno-associated virus (rAAV) that can be used in gene therapy applications to treat GSD-Ia.

[0011] On one hand, this application relates to recombinant nucleic acid molecules comprising a modified G6PC promoter / enhancer (GPE) sequence, wherein the modified GPE lacks one or more sequences that are at least 80% identical to the Alu element. In some embodiments, the Alu element is selected from the consecutive nucleotides 934-1127 (Alu-1), 1488-1823 (Alu-2), and 1995-2350 (Alu-3) of SEQ ID NO: 6. In some embodiments, the modified GPE has a sequence that is 80% (e.g., 80%, 85%, 90%, 95%, or 100%) identical to the consecutive nucleotides 146-2123 of SEQ ID NO: 1; or 80% (e.g., 80%, 85%, 90%, 95%, or 100%) identical to the sequence of SEQ ID NO: 7, 8, 9, 10, 11, or 1280% (e.g., 80%, 85%, 90%, 95%, or 100%).

[0012] On the other hand, this application relates to recombinant nucleic acid molecules comprising the modified G6PC promoter / enhancer (GPE) sequence and the G6P enzyme-α coding sequence described herein, wherein the modified GPE is capable of directing the expression of the G6P enzyme-α coding sequence. In some embodiments, the G6P enzyme-α coding sequence comprises at least 80% (e.g., 80%, 85%, 90%, 95%, or 100%) the same sequence as SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the recombinant nucleic acid molecule further comprises a polyadenylation (poly-A) signal sequence, such as the SV40 poly-A signal sequence (SEQ ID NO: 14). In some embodiments, the recombinant nucleic acid molecule further comprises an intron (SEQ ID NO: 13). In some embodiments, the recombinant nucleic acid molecule comprises SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] On the other hand, this application relates to recombinant vectors comprising the recombinant nucleic acid molecules described herein. In some embodiments, the vector is an adeno-associated virus (AAV) vector, such as AAV vectors of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or rh10 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or rh10). In an exemplary embodiment, the AAV vector is an AAV serotype 8 (AAV8) vector. Furthermore, host cells comprising the recombinant nucleic acid molecules or recombinant vectors disclosed herein are provided. In specific embodiments, the host cells may be adapted for AAV propagation.

[0014] On the other hand, this application relates to a method for increasing rAAV production, the method comprising delivering the AAV vector described herein to a host cell culture and harvesting rAAV from the cell culture. In some embodiments, the host cell culture is a eukaryotic host cell culture.

[0015] This document also provides rAAV comprising the recombinant nucleic acid molecules or AAV vectors disclosed herein. In some embodiments, this application relates to rAAV for the treatment of GSD-Ia, and to rAAV comprising an AAV capsid and an AAV vector genome packaged therein, said AAV vector genome comprising an AAV 5' inverted terminal repeat (ITR) sequence; a modified GPE sequence disclosed herein; a coding sequence encoding glucose-6-phosphatase α (G6P enzyme-α) or an active fragment or variant thereof; and an AAV 3' ITR sequence. In some exemplary embodiments, the AAV capsid is an AAV8 capsid. In some embodiments, the vector genome comprises the same 5' and 3' ITR sequences as SEQ ID NO: 15. In some embodiments, the vector genome comprises a modified GPE comprising consecutive nucleotides 146-2123 of SEQ ID NO: 1. In some embodiments, the vector genome further comprises a polyadenylation (polyA) signal sequence, for example, the SV40 polyA signal sequence (SEQ ID NO: 14). In some embodiments, the vector genome further comprises an intron (SEQ ID NO: 13). In some embodiments, G6P enzyme-α comprises at least 80% (e.g., 80%, 85%, 90%, 95%, or 100%) the same amino acid sequence as SEQ ID NO: 5. In some embodiments, the amino acid sequence of G6P enzyme-α comprises SEQ ID NO: 5. In some embodiments, the amino acid sequence of G6P enzyme-α consists of SEQ ID NO: 5. In some embodiments, the coding sequence of G6P enzyme-α is at least 80% (e.g., 80%, 85%, 90%, 95%, or 100%) the same as SEQ ID NO: 3 or SEQ ID NO: 4. In some exemplary embodiments, the vector genome comprises the same nucleic acid sequence as SEQ ID NO: 1 or 2.

[0016] This application further relates to pharmaceutical compositions comprising the rAAV of the present invention. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous administration. In an exemplary embodiment, the pharmaceutical composition is formulated for intravenous administration.

[0017] In another aspect, this application relates to a method of treating type Ia glycogen storage disease (GSD-Ia) in a human subject, the method comprising administering to the human subject a therapeutically effective amount of the rAAV disclosed herein. In some embodiments, the rAAV is administered subcutaneously, intramuscularly, intradermally, intraperitoneally, or intravenously. In an exemplary embodiment, the rAAV is administered intravenously. In some embodiments, the rAAV is administered at approximately 1 x 10 11 To approximately 1x1014 The dose is 1 x 10^6 genomic copies (GC) / kg. In a further embodiment, rAAV is administered at approximately 1 x 10^6 GC / kg. 12 To approximately 1x10 13 The dose is administered at 1 genomic copy (GC) / kg. In some embodiments, a single dose of rAAV is administered. In other embodiments, multiple doses of rAAV are administered.

[0018] These and other aspects and features of the invention are described in the following portions of this application. Attached Figure Description

[0019] The invention can be more fully understood by referring to the following figures.

[0020] Figure 1A This is a schematic diagram of the G6PC expression cassette, which is surrounded by two AAV2 inverted terminal repeats (ITR, SEQ ID NO: 15) and contains GPE, introns, codon-optimized human G6PC gene (hG6PCco), and SV40 late polyadenylation tail. Abbreviations used: GPE – G6P enzyme promoter / enhancer region; hG6PCco – human glucose-6-phosphatase coding region (codon-optimized); ITR – inverted terminal repeat; SV40L pA – SV40 late polyadenylation signal; UTR – untranslated region. Figure 1B This is a schematic diagram of a G6PC expression cassette containing either a wild-type (DTC161) or a modified GPE (DTC175 containing a G6PC expression cassette, wherein the G6PC expression cassette contains a GPE in which the Alu-1 and Alu-2 sequences are missing; DTC176 containing a G6PC expression cassette, wherein the G6PC expression cassette contains a GPE in which all three Alu elements are present but the orientation of the Alu-1 and Alu-2 sequences is reversed; DTC177 containing a G6PC expression cassette, wherein the G6PC expression cassette contains a GPE in which the Alu-1, Alu-2, and Alu-3 sequences are missing; DTC178 containing a G6PC expression cassette, wherein the G6PC expression cassette contains a GPE in which the Alu-3 sequence is missing; and DTC179 containing a G6PC expression cassette, wherein the G6PC expression cassette contains a GPE in which all three Alu elements are present but the orientation of the Alu-3 sequence is reversed).

[0021] Figure 2 This is a schematic diagram of an exemplary AAV carrier (DTC161), showing various key components. The carrier is characterized as follows:

[0022]

[0023] Figure 3This is a schematic diagram of the pAAV2-8.KanR(p2123-FH)AAV Rep / Cap plasmid. When co-transfected into host cells with an AAV vector, the plasmid provides Rep and Cap functions within the packaged rAAV.

[0024] Figure 4 This is a schematic diagram of the pAdDeltaF6(Kan) adenovirus helper plasmid used for rAAV production when co-transfected into host cells with the AAV vector and Rep / Cap plasmid.

[0025] Figure 5 This is a bar graph showing the rAAV titers produced from host cells after transfection with various AAV vectors. Three tests were performed under each condition, and the standard deviation is shown. * indicates P < 0.05 compared to DTC161.

[0026] Figure 6 This is a graph showing the generated rAAV titers plotted as a function of vector genome size.

[0027] Figure 7 This is an agarose gel image taken during agarose gel electrophoresis of full-length DNA isolated from rAAVs of control viral vectors, DTC161, DTC175, DTC176, DTC177, DTC178, and DTC179. The image shows bands of released DNA, assessing the ability of the capsid to degrade and release packaged DNA. Full-length viral DNA ranges from 3.8 kb to 5 kb. "*" indicates the complete genome of full-length DNA isolated from control viral vectors after capsid degradation and treatment with sodium dodecyl sulfate (SDS).

[0028] Figure 8A This is a graph showing the particle density analysis ultracentrifugation trace of the DTC161 carrier formulation produced from HEK293 cells. Figure 8B This is a graph showing the analytical ultracentrifugation traces of the particle density of the DTC177 vector (represented by SEQ ID NO: 1) formulation produced from HEK293 cells. Abbreviation used: RI - Refractive index.

[0029] Figure 9A This is the dose-response curve of G6P enzyme-α expression induced after infection with rAAV from the DTC161 vector. Figure 9BThis is a dose-response curve of G6P enzyme-α expression induced after infection with rAAV derived from the DTC177 vector (represented by SEQ ID NO: 1). The X-axis represents the rAAV dose used to infect HuH7 hepatocytes. The Y-axis represents the induced G6PC mRNA expression in the cells. rAAV derived from the DTC161 vector, which was developed and mass-produced, was used as the reference standard. Abbreviations used: REF - reference standard; UNK - test sample. Detailed Implementation

[0030] This invention provides a series of novel pharmaceutical agents and compositions for therapeutic applications. The molecules and compositions of this invention can be used in subjects to improve, prevent, or treat diseases associated with type Ia glycogen storage disease (GSD-Ia) or to increase the presence or function of glucose-6-phosphatase-alpha (G6P enzyme-α).

[0031] Unless otherwise stated, technical terms are used as is. Definitions of commonly used terms in molecular biology can be found in: Benjamin Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0032] To facilitate review of the various implementation schemes disclosed herein, the following explanations of specific terms are provided:

[0033] Adeno-associated virus (AAV): A small, replication-defective, non-enveloped virus that infects humans and some other primates. AAV is known not to cause disease and elicits a very mild immune response. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the host cell's genome. These characteristics make AAV an attractive viral vector for gene therapy. Currently, there are 12 recognized AAV serotypes (AAV1-12).

[0034] Administration / Administrator: The administration or delivery of a drug, such as a therapeutic agent (e.g., recombinant AAV), to a subject via any effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intraluminal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0035] Codon-optimized: "Codon-optimized" nucleic acids refer to nucleic acid sequences that have been altered to make the codons optimal for expression in a specific system (e.g., a specific species or group of species). For example, nucleic acid sequences can be optimized for expression in mammalian cells or specific mammalian species (e.g., human cells). Codon optimization does not alter the amino acid sequence encoding the protein.

[0036] Enhancers: Nucleic acid sequences that increase the transcription rate by increasing promoter activity.

[0037] G6PC: A gene located on human chromosome 17q21 that encodes glucose-6-phosphatase-α (G6Pase-α). G6Pase-α is a 357-amino acid hydrophobic protein with nine helices anchoring it in the endoplasmic reticulum (Chou et al., Nat RevEndocrinol [Nature Reviews Endocrinology] 6: 676-688, 2010). G6Pase-α catalyzes the hydrolysis of glucose-6-phosphate into glucose and phosphate in the final stages of gluconeogenesis and glycogenolysis and is a key enzyme for glucose homeostasis. Mutations in the G6PC gene result in type Ia glycogen storage disease (GSD-Ia), a metabolic disorder characterized by severe fasting hypoglycemia associated with the accumulation of glycogen and fat in the liver and kidneys.

[0038] Glycogen storage diseases (GSDs) are a group of diseases caused by defects in the synthesis or breakdown of glycogen in muscles, liver, and other tissues. GSDs can be hereditary or acquired. Hereditary GSDs are caused by any innate metabolic errors involved in these processes. There are currently 11 recognized glycogen storage diseases (GSD types I, II, III, IV, V, VI, VII, IX, XI, XII, and XIII). GSD-I consists of two autosomal recessive disorders, GSD-Ia and GSD-Ib (Chou et al., Nat Rev Endocrinol [Nature Reviews Endocrinology] 6: 676-688, 2010). GSD-Ia is caused by a deficiency of glucose-6-phosphatase-α. A deficiency of glucose-6-phosphate transporter (G6PT) is the cause of GSD-Ib.

[0039] Type Ia glycogen storage disease (GSD-Ia): Also known as hepatic glycogen storage disease, GSD-Ia is the most common glycogen storage disease, with an incidence of approximately 1 in 100,000 live births. GSD-Ia is a genetic disorder caused by a deficiency in glucose-6-phosphatase-α (G6P-α). G6P-α deficiency impairs the liver's ability to produce free glucose from glycogen and gluconeogenesis. Patients affected by GSD-Ia are unable to maintain glucose homeostasis and exhibit fasting hypoglycemia, growth retardation, hepatomegaly, renal enlargement, hyperlipidemia, hyperuricemia, and lactic acidosis (Xhou et al., Nat Rev Endocrinol [Nature Reviews Endocrinology] 6: 676-688, 2010). There is currently no cure for GSD-Ia.

[0040] Intron: A segment of DNA in a gene that does not contain protein-coding information. Introns are removed before messenger RNA translation.

[0041] Inverted terminal repeats (ITRs): Symmetrical nucleic acid sequences in the adeno-associated virus (AAV) genome required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as the replication origin for viral DNA synthesis and are essential for vector capsid formation.

[0042] Isolated: "Isolated" biological components (e.g., nucleic acid molecules, proteins, viruses, or cells) have been substantially separated or purified from other biological components in the cells or tissues of an organism or from the organism itself (where the components are naturally present, such as other chromosomes and extrachromosomal DNA and RNA, proteins, and cells). "Isolated" nucleic acid molecules and proteins include those purified using standard purification methods. The term also includes nucleic acid molecules and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acid molecules and proteins.

[0043] Operable ligation: The first nucleic acid sequence is operatively ligated to the second nucleic acid sequence when the first nucleic acid sequence is positioned to have a functional relationship with the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operatively ligated to the coding sequence. Typically, the operatively ligated DNA sequences are contiguous and, when it is necessary to ligate two protein-coding regions, are within the same reading frame.

[0044] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (mediators) that can be used in this disclosure are conventional. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) describes compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.

[0045] Generally, the properties of the carrier will depend on the specific route of administration. For example, parenteral formulations typically contain injectable fluids, including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, glucose solutions, glycerol, etc., as a medium. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitol monolaurate.

[0046] Prevention, treatment, or improvement of disease: "Prevention" of disease (e.g., GSD-Ia) refers to inhibiting the overall development of disease. "Treatment" refers to therapeutic interventions that improve the signs or symptoms of a disease or pathological condition after it has begun to develop. "Improvement" refers to a reduction in the number or severity of disease signs or symptoms.

[0047] Promoter: A DNA region that directs / initiates the transcription of nucleic acids (such as genes). Promoters include essential nucleic acid sequences located near the transcription start site.

[0048] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, purified peptides, proteins, viruses, or other active compounds are peptides, proteins, viruses, or other active compounds that have been completely or partially isolated from naturally related proteins and other contaminants. In some embodiments, the term "substantially purified" refers to peptides, proteins, viruses, or other active compounds that have been isolated from cells, cell culture media, or other crude formulations and fractionated to remove various components (e.g., proteins, cell debris, and other components) of the initial formulation.

[0049] Recombination: A recombinant nucleic acid molecule is a nucleic acid molecule that has a sequence that is not naturally occurring or that is created by artificially combining two sequence fragments that are otherwise separated. This artificial combination can be achieved through chemical synthesis or through artificial manipulation of separated fragments of the nucleic acid molecule, such as through genetic engineering techniques.

[0050] Similarly, recombinant viruses are viruses that contain sequences (e.g., genomic sequences) that are not naturally occurring or are artificially created by combining sequences from at least two different sources. The term "recombinant" also includes nucleic acids, proteins, and viruses that are altered solely by adding, substituting, or deleting a portion of a natural nucleic acid molecule, protein, or virus. As used herein, "recombinant AAV" refers to AAV particles that package a recombinant nucleic acid molecule, such as one encoding G6P enzyme-α.

[0051] Sequence identity: The similarity or identity between two or more nucleic acid sequences or two or more amino acid sequences is expressed as the similarity or identity between sequences. Sequence identity can be measured as a percentage of identity; the higher the percentage, the more identical the sequences. Sequence similarity can be measured as a percentage of similarity (taking into account conserved amino acid substitutions); the higher the percentage, the more similar the sequences. Homologous or orthologous nucleic acid or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. This homology is more pronounced when orthologous proteins or cDNAs are derived from more closely related species (such as human and mouse sequences) compared to more distantly related species (such as human and nematode sequences).

[0052] The sequence alignment methods used for comparison are well known in the art. Various procedures and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. [Advances in Applied Mathematics] 2: 482, 1981; Needleman & Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 48: 443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 85: 2444, 1988; Higgins & Sharp, Gene, 73: 237-44, 1988; Higgins & Sharp, CABIOS5: 151-3, 1989; Corpet et al., Nuc. Acids Res. [Nucleic Acid Research] 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences [Computer Applications in Biological Sciences] 8, 155-65, 1992; and Pearson et al., Meth. Mol. Rio. [Mathematical Molecular Biology] 24: 307-31, 1994. Altschul et al., J.. Mol. Biol. [Journal of Molecular Biology] 215: 403-10, 1990, gave detailed considerations on sequence alignment methods and homology calculations.

[0053] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. [Journal of Molecular Biology] 215: 403-10, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. More information can be found on the NCBI website.

[0054] Serotype: A group of closely related microorganisms (such as viruses) that are distinguished by their characteristic antigens.

[0055] Filler sequences are nucleotide sequences contained within larger nucleic acid molecules (such as vectors). They are typically used to create the desired spacing between two nucleic acid features (e.g., between a promoter and a coding sequence) or to lengthen nucleic acid molecules to a desired length. Filler sequences do not contain protein-coding information and may be of unknown / synthetic origin and / or unrelated to other nucleic acid sequences within the larger nucleic acid molecule.

[0056] Subjects: Living multicellular vertebrate organisms, including humans and non-human mammals.

[0057] Synthetic: Produced artificially in a laboratory, such as synthetic nucleic acids which can be chemically synthesized in a laboratory.

[0058] Therapeutic effective dose: A specific amount of a drug or therapeutic agent (e.g., recombinant AAV) is sufficient to achieve the desired effect in a subject or cell treated with the agent. The effective dose of the agent will depend on several factors, including but not limited to the subject or cell being treated, and the manner in which the therapeutic composition is administered.

[0059] Vector: A vector is a nucleic acid molecule that allows the insertion of a foreign nucleic acid without disrupting its ability to replicate and / or integrate into a host cell. A vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow the transcription and translation of one or more inserted genes. In some embodiments described herein, the vector is an AAV vector.

[0060] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Unless the context clearly indicates otherwise, the singular terms "a," "an," and "the" include plural references. "Comprising A or B" means including A, or B, or A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weights or molecular weight values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes. While methods and materials similar to or equivalent to those disclosed herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All disclosures, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including the interpretation of terms) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be restrictive.

[0061] I. Recombinant nucleic acids

[0062] One aspect of the invention provides a recombinant nucleic acid sequence comprising a modified G6PC promoter / enhancer (GPE) lacking one or more Alu elements compared to a wild-type GPE, wherein the modified GPE is capable of directing the expression of a gene encoding G6P enzyme-α (SEQ ID NO: 5). In some embodiments, the modified GPE is obtained by removing one or more Alu elements from the endogenous promoter of the human G6PC gene, for example, by removing one or more of the consecutive nucleotides 934-1127 (Alu-1), 1488-1823 (Alu-2), and 1995-2350 (Alu-3) of SEQ ID NO: 6. In some other embodiments, the modified GPE is obtained by removing one or more Alu elements from the endogenous promoter of the G6PC gene of other mammals, such as non-human primates, sheep, rodents, etc. In some embodiments, the modified GPE does not affect G6PC gene expression compared to the wild-type GPE. In some embodiments, the modified GPE enhances G6PC gene expression compared to wild-type GPE. In some embodiments, the modified GPE has comparable activity in driving G6PC gene expression in the liver to wild-type GPE, and very low activity in driving G6P enzyme-α expression in other tissues.

[0063] In some embodiments, the modified GPE includes a nucleic acid sequence lacking at least 80% (e.g., 80%, 80%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence of consecutive nucleotides 934-1127 (Alu-1) from SEQ ID NO: 6. In some embodiments, the modified GPE includes a nucleic acid sequence lacking at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence of consecutive nucleotides 1488-1823 (Alu-2) from SEQ ID NO: 6. In some embodiments, the modified GPE comprises a nucleic acid sequence lacking at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence of consecutive nucleotides 1995-2350 (Alu-3) from SEQ ID NO: 6.

[0064] In some embodiments, the modified GPE comprises a nucleic acid sequence lacking at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identical to Alu-1 and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identical to Alu-2 from SEQ ID NO: 6. In some embodiments, the modified GPE comprises a nucleic acid sequence lacking at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identical to Alu-1 and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identical to Alu-3 from SEQ ID NO: 6. In some embodiments, the modified GPE comprises a nucleic acid sequence lacking at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identical to Alu-2 and at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identical to Alu-3 from SEQ ID NO: 6.In some embodiments, the modified GPE comprises a nucleic acid sequence lacking at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identical to the Alu-1 sequence, at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the sequence identical to the Alu-2 sequence, and a sequence identical to that from SEQ ID NO. NO:6 has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical Alu-3 sequence.

[0065] In some embodiments of the invention, the recombinant nucleic acid sequence includes GPE having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical nucleic acid sequence to the consecutive nucleotides 146-2123 of SEQ ID NO: 1, wherein GPE is capable of directing the expression of the coding sequence encoding G6P enzyme-α. In some embodiments, the recombinant nucleic acid sequence includes GPE, which has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same nucleic acid sequence as SEQ ID NO: 7, wherein GPE is capable of directing the expression of the coding sequence encoding G6P enzyme-α. In some embodiments, the recombinant nucleic acid sequence includes GPE, which has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same nucleic acid sequence as SEQ ID NO: 8, wherein GPE is capable of directing the expression of the coding sequence encoding G6P enzyme-α. In some embodiments, the recombinant nucleic acid sequence includes GPE, which has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same nucleic acid sequence as SEQ ID NO: 9, wherein GPE is capable of directing the expression of the coding sequence encoding G6P enzyme-α. In some embodiments, the recombinant nucleic acid sequence includes GPE, which has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same nucleic acid sequence as SEQ ID NO: 10, wherein GPE is capable of directing the expression of the coding sequence encoding G6P enzyme-α. In some embodiments, the recombinant nucleic acid sequence includes GPE, which has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same nucleic acid sequence as SEQ ID NO: 11, wherein GPE is capable of directing the expression of the coding sequence encoding G6P enzyme-α.In some embodiments, the recombinant nucleic acid sequence includes GPE, which has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same nucleic acid sequence as SEQ ID NO: 12, wherein GPE is capable of directing the expression of the coding sequence encoding G6P enzyme-α.

[0066] Another aspect of the present invention provides a recombinant nucleic acid sequence comprising the modified GPE disclosed herein and a coding sequence encoding G6P enzyme-α. In some embodiments, G6P enzyme-α comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same amino acid sequence as SEQ ID NO: 5. In some embodiments, G6P enzyme-α comprises SEQ ID NO: 5 or an active fragment or variant thereof. In exemplary embodiments, G6P enzyme-α comprises or consists of SEQ ID NO: 5.

[0067] In some embodiments, the coding sequence encoding G6P enzyme-α is incorporated with at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the same nucleic acid sequence as SEQ ID NO: 4.

[0068] In some implementations, the coding sequence for human G6P enzyme-α is a codon optimized for expression in human cells. OptimumGene can be used. TMCodon optimization technology (GenScript, Piscataway, NJ) is used to codon-optimize human G6PC cDNA. The optimized G6PC cDNA sequence can be examined and further modified to eliminate potential alternative reading frames (ARFs) from internal non-frame ATG sequences that could theoretically encode peptides of nine or more amino acids in length. For example, the codon-optimized G6PC cDNA sequence can be further modified to avoid potential cytotoxic T lymphocyte responses to ARF-generated transgenic products (Li et al., 2009, PNAS 106: 10770-4). In some embodiments, the codon-optimized coding sequence of human G6P enzyme-α is incorporated with at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the same nucleic acid sequence as SEQ ID NO: 3.

[0069] In some embodiments, the recombinant nucleic acid sequence comprising the modified GPE as described herein and the coding sequence encoding G6P enzyme-α further includes introns and / or polyadenylation signals. In some embodiments, the intron is located between the GPE and the G6P enzyme-α coding sequence. In some embodiments, the intron is a chimeric intron that increases G6P enzyme-α transgene expression. The intron may consist of a 5'-donor site from the first intron of the human β-globin gene and a branch and 3'-receptor site from the variable region of the heavy chain of the immunoglobulin gene, wherein the sequences of the donor and recipient sites, as well as the branch site, have been altered to match a common sequence for splicing. In some embodiments, the introns comprise at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the same nucleic acid sequence as SEQ ID NO: 13.

[0070] The polyadenylation signal can be placed downstream of the coding sequence encoding G6P enzyme-α to effectively polyadenylate G6PC mRNA. Various polyadenylation signals can be used, such as the late polyadenylation signal of simian virus 40 (SV40), the hGH polyadenylation signal, the BGH polyadenylation signal, or the rbGlob polyadenylation signal. In some embodiments, the polyadenylation signal is the late polyadenylation signal of SV40. In some embodiments, the polyadenylation signal comprises at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the same nucleic acid sequence as SEQ ID NO: 14.

[0071] Another aspect of the present invention provides a recombinant vector comprising a modified GPE and a coding sequence encoding the G6P enzyme-α disclosed herein. In some embodiments, the recombinant vector further comprises the introns and / or polyadenylation signals described herein. The vector may be a mammalian expression vector, a bacterial expression vector, a yeast expression vector, a lentiviral vector, a retroviral vector, an adenovirus vector, an adeno-associated virus (AAV) vector, an RNAi vector, a Cre-Lox expression vector, a CRISPR expression vector, a TALEN expression vector, etc. The vector may further comprise the introns and / or polyadenylation signals as described herein. In some embodiments, the recombinant vector further comprises a filler nucleic acid sequence located between the GPE and the intron and / or between the intron and the G6P enzyme-α coding sequence.

[0072] In some embodiments, the recombinant vector is an AAV vector. The AAV vector can be serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12) and any of more than 100 variants isolated from human and non-human primate tissues (see, for example, Choi et al., Curr Gene Ther. [Gene Therapy Today], 5:299-310, 2005; and Gao et al., Curr Gene Ther. [Gene Therapy Today], 5:285-297, 2005). Any serotype of AAV vector can be used in this invention, and the choice of AAV serotype will depend in part on one or more cell types targeted by the gene therapy. For the treatment of GSD-Ia, the liver is one of the relevant target organs.

[0073] In some implementations, the recombinant AAV vector includes an AAV ITR sequence, which, when AAV and adenovirus helper functions are provided in trans form, acts as both the vector DNA replication initiation point and a packaging signal for the vector genome. Furthermore, the ITR is a target for single-stranded endonuclease restriction of the large Rep protein, splitting the individual genome from the replication intermediate.

[0074] In some exemplary embodiments, the AAV vector is an AAV serotype 8 (AAV8) vector, and the vector comprises a modified GPE, introns, a coding sequence encoding G6P enzyme-α, and the SV40 late polyadenylation signal described herein. In some embodiments, the vector further comprises two AAV2 inverted terminal repeat (ITR) sequences (SEQ ID NO: 15): one at the 5' end of the GPE and one at the 3' end of the polyadenylation signal. In some specific non-limiting embodiments, the recombinant vector comprises at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) the same nucleic acid sequence as SEQ ID NO: 1 or SEQ ID NO: 2.

[0075] II. Host cells containing recombinant nucleic acids

[0076] Furthermore, isolated host cells containing the recombinant nucleic acid molecules or vectors disclosed herein are provided. A variety of host cells can be used, such as bacterial, yeast, insect, and mammalian cells. In some embodiments, the host cell can be a cell (or cell line) suitable for producing recombinant AAV (rAAV), such as HeLa, Cos-7, HEK293, A549, BHK, Vero, RD, HT-1080, ARPE-19, or MRC-5 cells.

[0077] Recombinant nucleic acid molecules or vectors can be delivered into host cell cultures using any suitable method known in the art. In some embodiments, a stable host cell line is generated having a recombinant nucleic acid molecule or vector inserted into its genome. In some embodiments, a stable host cell line is generated containing the AAV vector described herein. After transfection of the AAV vector into the host culture, integration of rAAV into the host genome can be determined by a variety of methods, such as antibiotic selection, fluorescence-activated cell sorting, Western blotting, PCR-based detection, and fluorescence in situ hybridization, as described in Nakai et al., Nature Genetics (2003) 34, 297-302; Philpott et al., Journal of Virology (2002) 76(11): 5411-5421; and Howden et al., J Gene Med (2008); 10: 42-50. In addition, stable cell lines can be established according to protocols well known in the art, such as those described in Clark, Kidney International, Vol. 61 (2002): S9-S15, and Yuan et al., Human Gene Therapy, May 2011; 22(5): 613-24.

[0078] III. Recombinant AAV

[0079] This invention also provides rAAV comprising the AAV capsid and AAV vector genome described herein. The AAV capsid may be serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or rh10). In some embodiments, the capsid is an AAV8 capsid.

[0080] rAAV can be produced by host cells that possess the AAV vector and AAV Rep and Cap gene functions disclosed herein, as well as additional auxiliary functions. The Rep and Cap gene functions can be provided to the host cell in various ways, such as via plasmids or any type of vector containing wild-type AAV Rep and Cap genes, and via electroporation of Rep and Cap mRNA. Additional auxiliary functions can be provided by, for example, adenovirus (AV) infection, plasmids carrying all the required AV auxiliary function genes, or other viruses such as herpes simplex virus (HSV) or baculovirus. Any genes, gene functions, or other genetic material necessary for rAAV production by the host cell may be transiently present within the host cell or stably inserted into the host cell genome. The rAAV production methods applicable to the methods of this invention include those disclosed in: Clark et al., Human Gene Therapy 6:1329-1341 (1995), Martin et al., Human Gene Therapy Methods 24:253-269 (2013), Thorne et al., Human Gene Therapy 20:707-714 (2009), Fraser Wright, Human Gene Therapy 20:698-706 (2009), and Virag et al., Human Gene Therapy 20:807-817 (2009).

[0081] In an exemplary embodiment, HEK293 cells are transfected with: an AAV vector containing the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; a plasmid encoding four wild-type AAV2 viral replication (Rep) proteins and three wild-type AAV capsid (cap) proteins from serotype 8; and a plasmid containing adenovirus genomic regions (i.e., E2A, E4, and VA RNA) important for AAV replication. rAAV containing an AAV8 capsid can then be generated and isolated from the host cells. In some embodiments, a modified GPE lacking one or more Alu elements in the AAV vector enhances the packaging of rAAV generated from the host cells. In some embodiments, a modified GPE lacking one or more Alu elements in the AAV vector affects the formation of self-complementary structures and thus increases the yield of rAAV generated from the host cells.

[0082] Lysis of AAV-infected cells can be accomplished by chemically or enzymatically treating the cells to release infectious viral particles. These methods include using nucleases (such as benzonase or DNase), proteases (such as trypsin), or detergents or surfactants. Physical disruption, such as homogenization or grinding, or applying pressure via a microfluidic pressure unit, or freeze-thaw cycling, can also be used. Alternatively, the supernatant can be collected from AAV-infected cells without cell lysis.

[0083] It may be necessary to purify samples containing rAAV and helper virus particles to remove, for example, cell debris from cell lysis. Minimal purification methods for helper viruses and AAV particles are known in the art, and any suitable method can be used to prepare samples containing AAV and helper virus particles for use in the methods of this invention. Two exemplary purification methods are cesium chloride (CsCl)-based and iodixanol-based density gradient purification. These two methods are described in Strobel et al., Human Gene Therapy Methods, 26(4): 147-157 (2015). Affinity chromatography can also be used with, for example, AVB agarose gel affinity resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) or POROS. TM CaptureSelect TM Minimal purification was performed using AAV8, AAV9, or AAVX affinity resins (Thermo Fisher Scientific, Millersburg, PA). AAV purification methods using AVB agarose gel affinity resins are described, for example, Wang et al., Mol Ther Methods Clin Dev. [Molecular Therapy - Methods & Clinical Development], 2:15040 (2015).

[0084] The auxiliary virus may need to be inactivated by heating. Thermal inactivation techniques are based on the different thermal stability of AAV and auxiliary virus particles. For example, AAV particles can be heated to temperatures up to 56°C and remain intact, while AV particles become inactivated. Conway et al., Gene Therapy, 6, 986-993, 1999, describe the differential thermal inactivation of HSV in samples containing AAV. Thermal inactivation can be accomplished by any known method. In the examples described below, thermal inactivation is achieved using a thermal cycler to rapidly heat and cool sample volumes of 300 μL or less. This system was chosen because it relies on primarily conductive heat transfer, making it a viable model for continuous flow systems and larger-volume systems employing active mixing. Examples of continuous flow systems include passing samples through continuous flow heat exchangers, such as the DHX used in biotherapeutic fabrication. TM Disposable heat exchanger (Thermo Fisher Scientific, Millersburg, Pennsylvania). Such a system allows the operator to control the thermal inactivation process by controlling the flow rate of the sample through the heat exchanger, thereby controlling the duration of the heating process and the temperature of the heat exchanger, and thus the temperature of the thermal inactivation.

[0085] Alternatively, thermal inactivation can be achieved using batch systems of various sizes. For example, thermal inactivation can be accomplished on a 1L scale by placing a sample containing AAV in a 1L PETG bottle and immersing the bottle in a water bath set to the desired inactivation temperature for the desired time, followed by mixing; for example, the sample can be heated to 47°C for 20 minutes. On a larger scale, thermal inactivation can be achieved by placing a sample containing rAAV in a 5L bioprocessing bag and immersing it on a temperature-controlled rocking platform set to the desired inactivation temperature for the desired time. For example, the rocking platform can be set to 49°C, a rocking speed of 30 RPM, a mixing angle of 12°, for 40 minutes.

[0086] Heat inactivation can occur at any temperature where there is a sufficient difference in stability between rAAV particles and helper virus particles to substantially inactivate the helper virus particles while retaining the active rAAV particles. Those skilled in the art will understand that higher temperatures may be required to achieve a greater level of AV reduction. In some embodiments, the heat inactivation step includes using a buffer containing a hydrophilic salt and / or a divalent or trivalent cation. A method for heat inactivation in the presence of a buffer containing a hydrophilic salt and / or a divalent or trivalent cation is described in WO / 2017 / 172772.

[0087] Once heat inactivation is complete, it may be necessary or desirable to determine the inactivation efficiency. The effectiveness of the inactivation protocol is determined by assays that detect the presence of assisting viruses in detecting replication, such as plaque assays. Plaque assays for assisting viruses are well known to those skilled in the art and include plaque assays for AV, HSV, baculoviruses, etc. Adenovirus plaque assays can be performed using any suitable cell type, such as HeLa or HEK293 cells. Standard plaque assay protocols are described, for example, in Current Protocols in Human Genetics, 2003. Alternative assays for measuring adenovirus titers include methods that allow identification of infected cells in a culture by detecting viral proteins (such as hexagonal proteins) using immunocytochemical staining. Such assays include QuickTiter. TM Adenovirus titer immunoassay kit (CellBiolabs, San Diego, CA). Inactivation efficiency is typically reported as a log reduction in viral load (LRV).

[0088] Because AAV infection does not induce cytopathic effects in vitro, quantification of rAAV particles becomes complex, and therefore plaque assays cannot be used to determine infection titers. However, AAV particles can be quantified using various methods, including quantitative polymerase chain reaction (qPCR) (Clark et al., Hum. Gene Ther. [Human Gene Therapy] 10, 1031-1039 (1999)) or dot blot hybridization (Samulski et al., J. Virol. [Journal of Virology] 63, 3822-3828 (1989)), or by optical density analysis of highly purified carrier formulations (Sommer et al., Mol. Ther. [Molecular Therapy] 7, 122-128 (2003)). Quantification can also be achieved using a thermal cycler (e.g., iCycleriQ). Quantitative real-time polymerase chain reaction (qPCR) (DRP-qPCR) was performed on a 96-well modular thermal cycler (Bio-Rad, Hercules, CA). Samples containing AAV particles were incubated at 37°C for 60 min in the presence of DNase I (100 U / ml; Promega, Madison, Wisconsin), followed by digestion with proteinase K (10 U / ml) at 50°C for 60 min (Invitrogen, Carlsbad, CA) and then denatured at 95°C for 30 min. The primer-probe set used should be specific to non-natural portions of the AAV vector genome, such as the poly(A) sequence of the target protein. Based on the length and composition of the primers, probes, and amplified sequences, any suitable set of cycling parameters can be used to amplify the PCR products. Alternatives are disclosed, for example, in Lock et al., Human Gene Therapy. Methods [Human Gene Therapy Methods] 25(2): 115-125 (2014).

[0089] TCID can be used 50 The infectivity of rAAV particles was determined by a (50% tissue culture infection dose) assay, as described, for example, in Zhen et al., Human Gene Therapy 15:709-715 (2004). In this assay, AAV vector particles were serially diluted and used to co-infect Rep / Cap-expressing cell lines with AV particles in 96-well plates. Total cellular DNA was extracted from infected and control wells 48 hours post-infection. AAV vector replication was then measured using qPCR with transgene-specific probes and primers. TCID 50 Infectivity / mL (TCID) 50 / m1) use The equation calculates the ratio of AAV positive wells using 10-fold serial dilutions.

[0090] IV. Recombinant AAV for Gene Therapy

[0091] AAV belongs to the family Parvoviridae and the genus Dependovirus. AAV is a small, non-enveloped virus that packages a linear, single-stranded DNA genome. Both the sense and antisense strands of the AAV DNA are packaged into the AAV capsid at the same frequency.

[0092] The AAV genome is characterized by two inverted terminal repeats (ITRs) located flanking two open reading frames (ORBs). For example, in the AAV2 genome, the first 125 nucleotides of the ITR are palindromic, folding themselves to maximize base pairing and form a T-shaped hairpin structure. The remaining 20 bases of the ITR, called the D sequence, remain unpaired. The ITR is a cis-acting sequence crucial for AAV DNA replication; it is the origin of replication and acts as a primer for DNA polymerase to synthesize the second strand. The double-stranded DNA formed during this synthesis, called replicative monomers, is used for a second round of self-initiated replication and forms replicative dimers. These double-stranded intermediates are processed through strand displacement mechanisms, producing single-stranded DNA for packaging and double-stranded DNA for transcription. Located within the ITR are Rep binding elements and terminal dissociation sites (TRS). During AAV replication, the viral regulatory protein Rep uses these features to process the double-stranded intermediates. In addition to its role in AAV replication, the ITR is also essential for AAV genome packaging, transcription, negative regulation under non-permissioned conditions, and site-specific integration (Days and Berns, Clin Microbiol Rev 21(4): 583-593, 2008).

[0093] The left ORF of AAV contains the Rep gene, which encodes four proteins—Rep78, Rep68, Rep52, and Rep40. The right ORF contains the Cap gene, which produces three viral capsid proteins (VP1, VP2, and VP3). The AAV capsid contains 60 viral capsid proteins arranged in an icosahedral symmetry. VP1, VP2, and VP3 are present in a molar ratio of 1:1:10 (Daya and Berns, Clin Microbiol Rev. 21(4): 583-593, 2008).

[0094] AAV is one of the most commonly used viruses in gene therapy. Although AAV can infect humans and some other primates, it is known not to cause disease and to elicit a very mild immune response. Gene therapy vectors using AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the host cell's genome. Due to the favorable characteristics of AAV, this disclosure considers the use of AAV in the recombinant nucleic acid molecules and methods disclosed herein.

[0095] AAV possesses several desirable characteristics for gene therapy vectors, including the ability to bind and enter target cells, the ability to enter the nucleus, the ability to be expressed in the nucleus for extended periods, and low toxicity. However, the small size of the AAV genome limits the size of the heterologous DNA that can be incorporated. To minimize this problem, AAV vectors that do not encode Rep and integration efficiency elements (IEE) have been constructed. ITRs have been preserved because they are the cis signals required for packaging (Daya and Betrns, Clin Microbiol Rev [Clinical Microbiology Review], 21(4): 583-593, 2008).

[0096] Methods for producing rAAV suitable for gene therapy are well known in the art (see, for example, U.S. Patent Application Nos. 2012 / 0100606; 2012 / 0135515; 2011 / 0229971; and 2013 / 0072548; and Ghosh et al., GeneTher [Gene Therapy] 13(4): 321-329, 2006), and can be used in conjunction with the recombinant nucleic acid molecules and methods disclosed herein.

[0097] This disclosure provides compositions comprising the rAAV disclosed herein and a pharmaceutically acceptable carrier. Suitable pharmaceutical formulations for administering rAAV can be found, for example, in U.S. Patent Application Publication No. 2012 / 0219528. The pharmaceutically acceptable carriers (mediators) that can be used in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975) describes compositions and formulations suitable for the drug delivery of one or more therapeutic compounds, molecules, or pharmaceutical agents.

[0098] In some implementations, rAAV is formulated in a buffer / carrier suitable for infusion into a human subject. The buffer / carrier should include components that prevent rAAV adhesion to the infusion tubing without interfering with rAAV's in vivo binding activity. Various suitable solutions may include one or more of the following: buffered saline, surfactants, and physiologically compatible salts or mixtures of salts (with their ionic strength adjusted to be equivalent to about 100 mM sodium chloride (NaCl) to about 250 mM sodium chloride), or physiologically compatible salts adjusted to equivalent ionic concentrations. The pH may be in the range of 6.5 to 8.5, or 7 to 8.5, or 7.5 to 8. Suitable surfactants or combinations of surfactants may be selected from poloxamer, which is a nonionic triblock copolymer consisting of a central hydrophobic chain of poly(propylene oxide) and two flanking hydrophilic chains of poly(ethylene oxide), SOLUTOL HS 15 (Macrogol-15 hydroxystearate), LABRASOL (polyoxyethyl octanoate), polyoxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid ester), ethanol, and polyethylene glycol.

[0099] The present invention also provides a method for treating a subject diagnosed with glycogen storage disease type 1a (GSD-Ia) and administering a therapeutically effective amount of the rAAV disclosed herein (or a composition comprising rAAV) to the subject.

[0100] The rAAV or rAAV-containing compositions described herein can be administered using any suitable method or route. Routes of administration include, for example, systemic, oral, inhalation, intranasal, intratracheal, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes. In some embodiments, rAAV or rAAV-containing compositions are administered intravenously.

[0101] The specific dose administered can be a uniform dose for each patient, for example, 1.0 x 10⁻⁶. 13 -1.0x10 15One viral genome copy (GC) per patient. Alternatively, the patient's dose may be adjusted based on the patient's approximate weight or surface area. Other factors in determining the appropriate dose may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and medical condition. Further refinement of the calculations required to determine the appropriate therapeutic dose is routinely performed by those skilled in the art, particularly based on the dosage information and assays disclosed herein. The dose may also be determined by using known assays for determining the dose in conjunction with appropriate dose-response data. For example, the optimal biological dose of rAAV may be determined by assessing the time (in minutes) at which the first hypoglycemic event occurs (defined as controlling glucose <60 mg / dL (<3.33 mmol / L) during a fasting period (which will end when hypoglycemia occurs or reaches 15 hours). The individual patient's dose may also be adjusted as the progression of the disease is monitored.

[0102] In some implementations, rAAV is, for example, about 1.0 x 10⁻⁶. 11 One genome copy per kilogram of patient weight (GC / kg) to approximately 1 x 10-1 14 GC / kg, approximately 5 x 10 11 One genome copy per kilogram of patient weight (GC / kg) is approximately 5 x 10^6 13 GC / kg, or approximately 1x10 12 To approximately 1x10 13 Administered at a dose of Gc / kg, as measured by qPCR or digital droplet PCR (ddPCR). In some embodiments, rAAV is administered at approximately 2 × 10⁻⁶. 12 Administered at a dose of GC / kg. In some embodiments, rAAV is administered at approximately 6 × 10⁻⁶. 12 Administered at a dose of GC / kg. In some embodiments, rAAV is administered at approximately 1 × 10⁻⁶. 13 Administered at a dose of GC / kg. rAAV can be administered in single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses) as needed to achieve the desired treatment outcome.

[0103] Dosage can be administered weekly, monthly, or annually, or even every 2 to 20 years. For example, each dose can be administered at least 1 week, 2 weeks, 3 weeks, 1 month, 3 months, 6 months, or 1 year apart. Those skilled in the art can readily estimate the repetition rate of administration based on measured residence time and the concentration of the targetable construct or complex in body fluids or tissues.

[0104] V. Methods to increase recombinant virus yield and gene therapy efficacy

[0105] The present invention also provides a method for increasing the yield of recombinant viruses in host cells, wherein the method includes removing one or more Alu elements or Alu element-related sequences from a recombinant viral vector. In some embodiments, the Alu element-related sequences are at least 50% identical to Alu elements (e.g., consecutive nucleotides 934-1127 (Alu-1), 1488-1823 (Alu-2), and 1995-2350 (Alu-3) selected from SEQ ID NO: 6). The recombinant viral vector may be, for example, a lentiviral vector, a retroviral vector, an adenovirus vector, or an adeno-associated virus (AAV) vector. In some embodiments, one or more Alu elements or Alu element-related sequences are removed from promoter / enhancer regions within the viral vector. In some embodiments, one or more Alu elements or Alu element-related sequences are removed from intron regions within the viral vector. In some embodiments, removing one or more Alu elements or Alu element-related sequences reduces the self-complementary structures formed during the packaging of recombinant viral particles, thereby increasing the viral yield from host cells.

[0106] Throughout this specification, where compositions are described as having, including, or containing specific compounds, or where processes and methods are described as having, including, or containing specific steps, it is understood that there are compositions of the invention that consist substantially of or comprise of the described compounds, and processes and methods according to the invention that consist substantially of or comprise the described processing steps.

[0107] In this application, when an element or component is referred to as being included in and / or selected from the list of enumerated elements or components, it should be understood that the element or component can be any one of the enumerated elements or components, or the element or component can be selected from a group consisting of two or more of the enumerated elements or components.

[0108] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways without departing from the spirit and scope of the invention, whether explicitly stated or implicit herein. For example, when a particular compound is referred to, it is understood from the context otherwise to be usable in various embodiments of the compositions of the invention and / or methods of the invention. In other words, embodiments have been described and depicted in this application in a manner that allows for clear and concise writing and drawing, but it is intended and will be understood that embodiments can be combined or separated in various ways without departing from the teachings of this application and one or more inventions. For example, it should be understood that all features described and depicted herein are applicable to all aspects of one or more inventions described and depicted herein.

[0109] It should be understood that, unless otherwise understood from the context and use, the expression "at least one" individually includes each of the objects listed after the expression, as well as various combinations of two or more of the listed objects. Unless otherwise understood from the context, the expressions "and / or" related to three or more listed objects should be understood to have the same meaning.

[0110] The use of the terms "include", "includes", "have", "has", "having", "contain", "contains", or "containing" should be understood, including their syntactic equivalents, which are generally open-ended and non-restrictive, for example, not excluding additional unlisted elements or steps unless otherwise expressly stated or understood from the context.

[0111] If the term "about" is used before a quantity value, the invention also includes the specific quantity value itself, unless otherwise specified. As used herein, unless otherwise stated or inferred, the term "about" means ±10% variation of an index value.

[0112] It should be understood that the order of steps or the sequence of actions is irrelevant as long as the invention remains operable. Furthermore, two or more steps or actions can be performed simultaneously.

[0113] Any and all examples or exemplary language used herein, such as "e.g." or "including," are intended only to better illustrate the invention and, unless otherwise stated, do not constitute a limitation on the scope of the invention. The language in this specification should not be construed as indicating that any unclaimed element is necessary for practicing the invention.

[0114] Example

[0115] The invention described in general will now be more readily understood by referring to the following embodiments, which are included for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0116] Example 1 - AAV vector and rAAV generated from the vector

[0117] AAV carrier

[0118] AAV vectors containing G6PC expression cassettes surrounded by two AAV2 inverted terminal repeats (ITR, SEQ ID NO: 15) were constructed. The G6PC expression cassettes were delimited at the 5' end of their G6PC promoter / enhancer (GPE) by the primer sequence "1S" and its associated KpnI restriction endonuclease site listed in Yiu et al., 2010, Molecular Therapy 18(6): 1076-84. The G6PC expression cassettes were delimited at the 3' end by their SV40 late polyadenylation signal, by alignment with the SV40 genome and associated SalI restriction endonuclease site. All G6PC expression cassettes contained the GPE, introns, codon-optimized human G6PC gene, and SV40 late polyA tail, as shown below. Figure 1A As shown. Different versions of the G6PC expression box were generated, each containing either a wild-type GPE or a modified GPE, such as... Figure 1B As shown. The components of the G6PC expression box are described below.

[0119] The wild-type G6PC promoter / enhancer (GPE, SEQ ID NO: 6) is derived from Homo sapiens and defined by RefSeq NG_011808. This sequence is the endogenous promoter of the human G6PC gene, exhibiting almost exclusive activity in the liver and very low activity in the kidney. The wild-type GPE contains three Alu elements located at nucleotides 934-1127 (Alu-1), 1488-1823 (Alu-2), and 1995-2350 (Alu-3) of SEQ ID NO: 6. AAV vector DTC161 contains a G6PC expression cassette containing the wild-type GPE. AAV vector DTC175 contains a G6PC expression cassette containing a GPE in which the Alu-1 and Alu-2 sequences are deleted. AAV vector DTC176 contains a G6PC expression cassette containing a GPE in which the orientations of Alu-1 and Alu-2 are reversed. AAV vector DTC177 (represented by SEQ ID NO: 1) contains a G6PC expression cassette, which includes a GPE containing the Alu-1, Alu-2, and Alu-3 sequences deleted therein. AAV vector DTC178 contains a G6PC expression cassette, which includes a GPE containing the Alu-3 sequence deleted therein. AAV vector DTC179 contains a G6PC expression cassette, which includes a GPE containing the Alu-3 sequence with the orientation reversed.

[0120] The chimeric intron (SEQ ID NO: 13) consists of a 5'-donor site from the first intron of the human β-globin gene and a branch and 3'-receptor site from the variable region of the heavy chain of the immunoglobulin gene. The sequences of the donor and recipient sites, as well as the branch site, have been modified to match the shared sequence used for splicing (CI-neo Mammalian Expression Vector Technical Bulletin TB215, Promega Life Sciences Corporation). The purpose of the chimeric intron is to enhance gene expression.

[0121] The G6PC cDNA (SEQ ID NO: 4) is derived from Homo sapiens and has been codon-optimized for expression in human cells. It can be expressed using the proprietary OptimumGene. TM Codon optimization technology (GenScript, Piscataway, New Jersey) is used to codon-optimize human G6PC cDNA. The optimized cDNA sequence can be examined and further modified to eliminate potential alternative reading frames (ARFs) from the internal non-frame ATG sequence, which could theoretically encode peptides of nine or more amino acids in length. For example, codon-optimized G6PC cDNA is represented by SEQ ID NO: 3.

[0122] The late polyadenylation signal of simian virus 40 (SV40) (Genbank#J02400, SEQ ID NO: 14) provides a cis sequence for efficient polyadenylation of G6PC mRNA. This element serves as a signal for a specific 3' end cleavage event of the nascent transcript and the addition of a long polyadenylated tail.

[0123] Each G6PC expression cassette was cloned into an AAV vector. All AAV vectors contained a backbone encoding the kanamycin resistance gene. The AAV vector DTC161 (pDTX.hG6PCco.401) is shown below. Figure 2 Take China as an example.

[0124] rAAV virus particles

[0125] The AAV vector genome is a single-stranded DNA genome. Only sequences between and containing ITR sequences are packaged into AAV viral particles. Viral particles are produced by transfecting human embryonic kidney 293 (HEK293) cells with three plasmids, which provide the E1a and E1b gene products. The first plasmid is the AAV vector described herein. The second plasmid is pAAV2-8.KanR (p2123-FH), a packaging plasmid containing the wild-type AAV2 rep and AAV8 cap genes. The third plasmid is pAdDeltaF6 (Kan), a helper adenovirus plasmid.

[0126] The adeno-associated receptor Rep / Cap plasmid pAAV2 / 8.KanR(p2123-FH) (8354 bp) encodes four wild-type AAV2 viral replication (Rep) proteins and three wild-type AAV VP capsid (cap) proteins from serotype 8. A diagram of the pAAV2 / 8.KanR(p2123-FH) plasmid is shown in... Figure 3 In the plasmid, the AAV p5 promoter, which normally drives Rep gene expression, has been moved from the 5' end of the Rep region to the 3' end of the AAV8 cap region. This arrangement introduces a spacer region (i.e., the plasmid backbone) between the promoter and the Rep gene, leading to downregulation of Rep expression and an increased ability to support high-titer rAAV production. The kanamycin resistance gene and the MB1 origin were both included for plasmid production in *E. coli*.

[0127] The plasmid pAdDeltaF6(Kan) contains adenovirus genomic regions important for AAV replication, namely E2A, E4, and VARNA ( Figure 4 Adenovirus E1 function is also required, but is provided by the HEK293 host cell. The plasmid does not contain other adenovirus replication, structural genes, or cis-elements crucial for adenovirus replication, such as the adenovirus ITR; therefore, no infectious adenovirus is expected to be produced. The kanamycin resistance gene and MB1 origin are both included for plasmid production in *E. coli*.

[0128] Example 2 - Alu Component Missing to Improve rAAV Yield

[0129] AAV vectors containing wild-type GPE or modified GPE were used to transfect HEK293 cells with the above-mentioned Rep / Cap plasmid and helper plasmid.

[0130] The modified GPEs have missing Alu elements (in DTC175, DTC177 and DTC178 carriers) or the Alu elements are reversed in orientation (in DTC176 and DTC179 carriers). The DTC175 viral vector contains a G6PC expression cassette containing a GPE that lacks the Alu-1 and Alu-2 sequences; the DTC176 viral vector contains a G6PC expression cassette containing a GPE in which all three Alu elements are present, but the orientation of the Alu-1 and Alu-2 sequences is reversed; the DTC177 viral vector contains a G6PC expression cassette containing a GPE that lacks the Alu-1, Alu-2, and Alu-3 sequences; the DTC178 viral vector contains a G6PC expression cassette containing a GPE that lacks the Alu-3 sequence; and the DTC179 viral vector contains a G6PC expression cassette containing a GPE in which all three Alu elements are present, but the orientation of the Alu-3 sequence is reversed.

[0131] On day 5 post-co-transfection, infected cells were placed in a solution containing sodium deoxycholate and The sample was lysed in lysis buffer at 37°C for 2 hours. The supernatant was then digested sequentially with DNase I and proteinase K to release the rAAV genomic DNA. The BGH-polyA coding region of the AAV vector was then amplified using TaqMan qPCR to determine the rAAV genomic copy number (GC) based on the rAAV plasmid standard curve. Figure 5 This is a bar graph showing the rAAV titers produced from host cells after transfection with various AAV vectors. For example... Figure 5 The rAAV titers measured by qPCR are shown, and it is demonstrated that the deletion of one or more Alu elements from GPE (in vectors DTC175, DTC177, and DTC178) significantly increased rAAV yield compared to DTC161 containing wild-type GPE. However, reversing the deletion of one or more Alu elements (in vectors DTC176 and DTC179) did not increase rAAV yield compared to DTC161. A quantitative summary of viral yield is presented in Table 1. As shown in Table 1, the deletion of one or more Alu elements from GPE significantly increased rAAV yield. A second analysis showing the resulting rAAV titers as a function of vector genome size is presented. Figure 6 The image in the image. (As shown in the image) Figure 6 The vector genome DTC177 (represented by SEQ ID NO: 1), which has the smallest size, produced the highest titer.

[0132] Table 1: Quantitative summary of viral yield produced in HEK293 cells after transfection with AAV vectors (DTC161, DTC175, DTC176, DTC177, DTC178 or DTC179).

[0133]

[0134] Example 3 - Improved rAAV packaging for missing Alu components

[0135] To assess whether the absence of one or more Alu elements affects rAAV packaging, rAAV generated as described in Example 2 was harvested and total DNA was isolated from each rAAV (generated from control viral vectors DTC161, DTC175, DTC176, DTC177, DTC178, or DTC179). Approximately 7.12 x 10⁻⁶ 10 Each rAAV GC was subjected to agarose gel electrophoresis and then stained with SYBR Gold. The control viral vector used in this experiment was AAV8-LSP-hFIXco3-WPRE-pA (produced at Virovek, custom purified, catalog / lot number 061015 150282), which provided known DNA-size migration on the gel and confirmed that the experimental method was able to disrupt the integrity of the AAV capsid and release the packaged DNA.

[0136] like Figure 7 As shown, the full-length viral DNA ranges from 3.8kb to 5kb. "*" indicates the complete genome of the full-length DNA isolated from the control viral vector after capsid degradation and treatment with sodium dodecyl sulfate (SDS).

[0137] Full-length DNA isolated from rAAVs of DTC177, DTC175, and DTC178, which lack one or more Alu elements in GPE, showed higher staining intensity (see [link to relevant documentation]). Figure 7 This result indicates that the deletion of at least one Alu element improves rAAV packaging of the full-length viral genome. Furthermore, ultracentrifuge trace analysis of DTC161 and DTC177 particles showed that empty particles (appearing at approximately 60 s) and particles containing the full vector DNA (appearing at approximately 100 s) produced a higher percentage of intact particles with the Alu element deletion (see [link to documentation]). Figures 8A-8B ). Figure 8A This is a graph showing the particle density analysis ultracentrifugation trace of the DTC161 carrier formulation produced from HEK293 cells. Figure 8B This is a graph showing the analytical ultracentrifugation trace of the particle density of the DTC177 vector (represented by SEQ ID NO: 1) formulation produced from HEK293 cells.

[0138] These data indicate enhanced packaging in vectors lacking the Alu sequence.

[0139] In Example 4-GPE, the absence of the Alu element does not affect promoter effectiveness.

[0140] To assess whether the deletion of one or more Alu elements affects the efficacy of GPE in directing G6PC gene expression in vivo, HuH7 hepatocytes were infected with rAAVs derived from different vectors (as shown in Table 2). Forty-eight hours after infection, cells were lysed and total mRNA was harvested. G6PC mRNA expression was analyzed by quantitative RT-PCR. Figures 9A-9B An exemplary dose-response curve is shown between rAAV dose and G6PC mRNA expression level in HuH7 hepatocytes for rAAV derived from DTC161 and DTC177 (represented by SEQ ID NO: 1). Figure 9A This is the dose-response curve of G6P enzyme-α expression induced after infection with rAAV containing DTC161. Figure 9B This is a dose-response curve of G6P enzyme-α expression induced after infection with rAAV containing DTC177 (represented by SEQ ID NO: 1). To calculate the relative potency of the test samples, the RNA values ​​(genomic copy number / μg total RNA) of the reference standard (rAAV derived from the DTC161 vector produced in the development batch) and the test samples at each multiplicity of infection (MOI) were added to a GraphPad Prism file template. The template used a log-log transformation and linear fit of the data, which was constrained so that the curves had a shared slope. The Y-axis corresponds to the RNA value, and the X-axis corresponds to the MOI of the sample. The Y-intercept and slope data generated by the GraphPadPrism template were used to determine the X-intercept of the reference standard and the test sample by implementing the following formula: Xint = (0 - [Y-intercept]) / slope. The final relative potency value was determined by comparing the X-intercepts using the following formula: 10-1{[X-intercept reference] - [X-intercept sample]}.

[0141] Table 2. Summary of the relative potency of GPE in each rAAV sample.

[0142] sample Relative effectiveness DTC-161 80％ DTC-175 83％ DTC-176 79％ DTC-177 89％ DTC-178 75％ DTC-179 85％

[0143] These results indicate that the absence of one or more Alu elements in GPE (in the DTC175, DTC177, and DTC178 vectors) does not impair the efficacy of GPE in driving G6PC gene expression in vivo.

[0144] All publications, patents and documents specifically mentioned herein are incorporated herein by reference for all purposes.

[0145] It should be understood that the present invention is not limited to the specific methods, schemes, materials, and reagents described, as they can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which will be covered solely by the appended claims.

[0146] It must be noted that, unless the context clearly specifies otherwise, the singular forms "a / an" and "the" as used herein and in the appended claims include plural indicators. Similarly, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein. It should also be noted that the terms "comprises," "containing," "including," and "having" are used interchangeably.

[0147] Without further elaboration, it is believed that those skilled in the art can utilize the invention to its fullest extent based on the foregoing description. Therefore, specific embodiments are to be construed as illustrative only and are in no way intended to limit the remainder of this disclosure.

[0148] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be used as a substitute feature for the same, equivalent, or similar purpose.

Claims

1. A recombinant nucleic acid molecule comprising a modified G6PC promoter / enhancer (GPE) sequence and a G6P enzyme-α coding sequence, wherein the modified GPE sequence comprises the following: (i) SEQ ID NO:1, consecutive nucleotides 146-2123, (ii)SEQ ID NO:9, or (ⅲ)SEQ ID NO:

10.

2. The recombinant nucleic acid molecule according to claim 1, wherein the G6P enzyme-α coding sequence comprises the same sequence as SEQ ID NO:3 or SEQ ID NO:

4.

3. The recombinant nucleic acid molecule according to claim 1, wherein the recombinant nucleic acid molecule further comprises a polyadenylation (polyA) signal sequence.

4. The recombinant nucleic acid molecule according to claim 3, wherein the polyadenylation (polyA) signal sequence is the SV40 polyA signal sequence.

5. The recombinant nucleic acid molecule according to claim 1, wherein the recombinant nucleic acid molecule further comprises an intron sequence.

6. The recombinant nucleic acid molecule according to claim 1, wherein the recombinant nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:

2.

7. A recombinant vector comprising the recombinant nucleic acid molecule according to claim 1.

8. The recombinant vector according to claim 7, wherein the recombinant vector is an adeno-associated virus (AAV) vector.

9. The recombinant vector according to claim 8, wherein the AAV vector is an AAV serotype 8 (AAV8) vector.

10. An isolated host cell comprising the recombinant nucleic acid molecule according to claim 1.

11. An isolated host cell, said isolated host cell comprising a recombinant vector according to any one of claims 7-9.

12. A method for increasing the yield of rAAV in the preparation of recombinant AAV (rAAV) containing a G6P enzyme-α coding sequence, the method comprising delivering a recombinant vector according to claim 8 or 9 to a eukaryotic host cell culture and harvesting the rAAV from the eukaryotic cell culture.

13. A recombinant AAV (rAAV), said recombinant AAV comprising the recombinant nucleic acid molecule according to claim 1.

14. The rAAV of claim 13, wherein the rAAV is a recombinant AAV serotype 8 vector (rAAV8).

15. A composition comprising rAAV as described in claim 13 or 14, and a pharmaceutically acceptable carrier.

16. Use of the rAAV of claim 13 or the composition of claim 15 in the preparation of a medicament for treating type Ia glycogen storage disease (GSD-Ia) in a human subject, the treatment comprising administering a therapeutically effective amount of the rAAV or the composition to the human subject.

17. The use according to claim 16, wherein the drug is for intravenous administration.

18. A recombinant adeno-associated virus (rAAV) for treating type Ia glycogen storage disease (GSD-Ia), said rAAV comprising an AAV capsid and a vector genome packaged therein, said vector genome comprising: (a) AAV 5' inverted terminal repeat (ITR) sequence; (b) The G6PC promoter / enhancer (GPE) sequence, which consists of consecutive nucleotides 146-2123 of SEQ ID NO:1; (c) The coding sequence for glucose-6-phosphatase α (G6P enzyme-α); and (d)AAV 3'ITR sequence.

19. The rAAV of claim 18, wherein the amino acid sequence of the G6P enzyme-α comprises at least 90% the same amino acid sequence as SEQ ID NO:

5.

20. The rAAV according to claim 18, wherein the amino acid sequence of the G6P enzyme-α is identical to that of SEQ ID NO:

5.

21. The rAAV according to claim 18, wherein the coding sequence encoding G6P enzyme-α is at least 90% identical to SEQ ID NO:3 or SEQ ID NO:

4.

22. The rAAV of claim 18, wherein the AAV capsid is an AAV serotype 8 (AAV8) capsid.

23. The rAAV of claim 18, wherein the vector genome further comprises a polyadenylation (polyA) signal sequence.

24. The rAAV of claim 23, wherein the polyadenylation (polyA) signal sequence is an SV40 polyA signal sequence.

25. The rAAV of claim 18, wherein the vector genome further comprises intron sequences.

26. A composition comprising rAAV according to any one of claims 18-25, and a pharmaceutically acceptable carrier.

27. Use of rAAV according to any one of claims 18-25 in the preparation of a medicament for treating type Ia glycogen storage disease (GSD-Ia) in a human subject, the treatment comprising administering a therapeutically effective amount of the rAAV to the human subject.

28. The use according to claim 27, wherein the drug is for intravenous administration.

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