Gene therapy constructs for treating Wilson disease

Abstract

The present application relates to an adeno-associated viral vector encoding a truncated but functional ATP7B for use in gene therapy for the treatment of Wilson's disease (WD). The truncated ATP7B described herein has several advantages over wild-type ATP7B, such as higher efficacy and improved production yield.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 788,324, filed on January 4, 2019, and U.S. Provisional Patent Application No. 62 / 834,830, filed on April 16, 2019, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on December 31, 2019, is named ULP-003WO_SL_ST25.txt, and is 49,846 bytes in size. Technical Field

[0005] The present application generally relates to adeno-associated viral vectors and methods for use thereof in gene therapy for treating Wilson's disease (WD). Background Art

[0006] Wilson disease (WD) is an autosomal recessive genetic disorder that causes copper to accumulate primarily in the liver and subsequently in the nervous system and other tissues. WD is a rare disorder, occurring in approximately 1 in 30,000 individuals, and is caused by mutations in the copper-transporting ATPase 2 (ATP7B) gene on chromosome 13. There are more than 600 unique ATP7B mutations. ATP7B is primarily expressed in hepatocytes and plays a role in the transport of copper across membranes. The absence or reduction of ATP7B protein function leads to decreased excretion of copper by hepatocytes into the bile, causing liver disease. Without appropriate treatment, high copper levels can lead to life-threatening organ damage over time.

[0007] Patients with hepatic WD typically present in late childhood or adolescence and present with features of acute hepatitis, fulminant hepatic failure, or progressive chronic liver disease. Neurologic manifestations of WD typically develop later than liver disease, most commonly in the second or third decade of life, and include extrapyramidal, cerebellar, and cerebral symptoms.

[0008] The goal of medical treatment of WD is to remove toxic copper deposits from the body and prevent their reaccumulation. The current treatment for WD is daily oral chelation (D-penicillamine, trientine and zinc salts). Drug therapy is effective in most but not all WD patients. For WD patients who develop fulminant or progressive liver failure, liver transplantation is a treatment option. However, transplant recipients need to maintain a constant immunosuppressive regimen to prevent rejection.

[0009] The present invention addresses the need for improved and sustainable treatment of WD by delivering a gene expressing a truncated but functional ATP7B to a patient using an adeno-associated virus vector. The truncated ATP7B of the present invention has improved efficacy in treating WD and has the advantages of easy production and high efficiency compared to wild-type and other truncated forms of ATP7B protein. Summary of the invention

[0010] The present invention provides compositions and methods for their use in gene therapy. An adeno-associated virus (AAV) vector for treating WD is provided herein. In one aspect, the present invention provides a recombinant nucleic acid construct comprising: a 5'-terminal inverted repeat (ITR) sequence; a promoter sequence; a nucleic acid sequence encoding a truncated human copper transporting ATPase 2 (ATP7B), wherein the truncated human copper transporting ATPase 2 lacks metal binding domains (MBD) 1-3, but there is a serine-rich loop containing two serine residues (S340 and S341) between MBD3 and MBD4; and a 3'-ITR sequence.

[0011] On the other hand, the present invention provides a recombinant adeno-associated virus (rAAV) for treating Wilson's disease, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, the vector genome comprising a 5'-terminal inverted repeat (ITR) sequence; a promoter sequence; a nucleic acid sequence encoding a truncated human copper-transporting ATPase 2 (ATP7B), wherein the truncated human copper-transporting ATPase 2 lacks metal binding domains (MBD) 1-3, but there is a serine-rich loop containing two serine residues (S340 and S341) between MBD3 and MBD4; and a 3'-ITR sequence.

[0012] These and other aspects and features of the present invention are described in the following section of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The invention may be more fully understood with reference to the following drawings.

[0014] Figure 1 Schematic diagram showing an exemplary vector genome construct comprising a nucleotide sequence encoding a truncated human copper-transporting ATPase 2 (ATP7B) in which metal binding domains (MBDs) 1-3 are missing, but a serine-rich loop containing two serine residues (S340 and S341) is present between MBD3 and MBD4 ("ATP7BΔ1-3-SS" or "native ATP7B dell-3"). Features of an exemplary vector genome construct are provided below:

[0015]

[0016]

[0017] Figure 2 is a schematic diagram of an exemplary AAV vector (DTC319) showing various key components.

[0018] Figure 3 is a schematic diagram of an exemplary plasmid, pAAV2 / 8.KanR (p2123FH) AAV Rep / Cap plasmid, which provides Rep and Cap functions in packaging rAAV when co-transfected with an AAV vector into host cells.

[0019] Figure 4 is a schematic diagram of an exemplary plasmid, pAdDeltaF6 (Kan), adenoviral helper plasmid, used for rAAV production when co-transfected with AAV vector and Rep / Cap plasmid into host cells.

[0020] Figure 5 Injection 10 9 , 10 10 or 10 11 Genome copies (GC) / kg ATP7BcoFL (codon-optimized full-length human ATP7B) C3He-Atp7b tx-j Female mice (indicated by circles) and injected with 10 10 or 10 11 GC / kg C3He-Atp7b of the same carrier tx-j Scatter plot of hepatic copper (μg / g) in male mice (indicated by squares) from age-matched uninjected male and female heterozygous (Het) and C3He-Atp7b tx-j Copper levels of the mice are also shown in this scatter plot.

[0021] Figure 6 It is a bar graph showing the total yield of rAAV (GC titer) produced by host cells after transfection of AAV vectors encoding the complete or partial coding sequence of human ATP7B (AAV vectors carrying a nucleotide sequence encoding full-length (FL) human ATP7B; AAV vectors carrying a nucleotide sequence encoding human ATP7B (in which MBD 1-3 is deleted, but there is a serine-rich loop (ATP7BΔ1-3-SS) containing two serine residues (S340 and S341) between MBD3 and MBD4; or AAV vectors carrying a nucleotide sequence encoding human ATP7B (ATP7BΔ1-4) in which MBD 1-4 is deleted).

[0022] Figure 7 C3He-Atp7b tx-jScatter plot of urinary copper and liver copper levels (μg / g) measured in mice injected with AAV8 carrying full-length human ATP7B (ATP7B FL), ATP7BΔ1-3-SS, or ATP7BΔ1-4, represented by squares and circles, respectively. tx-j Mice were used as control (vehicle).

[0023] Figure 8 is a bar graph showing the total yield of rAAV (GC titer) produced by host cells after transfection with an AAV vector (DTC319) encoding a truncated human ATP7B in which metal binding domains (MBDs) 1-3 are missing but a serine-rich loop containing two serine residues (S340 and S341) is present between MBD3 and MBD4, encoding AAV8 or AAV9 capsid.

[0024] Fig. 9 The animals were either intravenously injected with vehicle control (dilution buffer; WD) or infused with AAV8 carrying native ATP7BΔ1-3-SS (DelA) C3He-Atp7b tx-j Bar graph of hepatic copper accumulation levels (μg / g dry weight) in mice. The hepatic copper accumulation levels in non-injected wild-type mice (WT) represented in this bar graph were used as negative controls. Values ​​are expressed as mean ± SEM (standard error of the mean).

[0025] Fig.10 The animals were either intravenously injected with vehicle control (dilution buffer; WD) or infused with AAV8 carrying native ATP7BΔ1-3-SS (DelA) C3He-Atp7b tx-j Bar graph of ceruloplasmin activity in mice, as measured by an enzyme-based colorimetric activity assay. Ceruloplasmin activity in uninjected wild-type (WT) mice, as measured by the same enzyme-based colorimetric activity assay, is also represented in this bar graph.

[0026] Fig.11 Bar graph of mean scores after standard assessment of nuclear enlargement and hepatocyte hypertrophy, tissue destruction, inflammatory infiltrate, and hepatocyte necrosis on hematoxylin and eosin (H&E) sections.

[0027] Fig.12is a schematic diagram showing an exemplary vector genome construct DTC327, which comprises an AAV9 capsid with a PPIA poly A, an AAV2Rep / ITR with an intact p5 promoter containing a 145 bp ITR, and a nucleotide sequence encoding a truncated human copper-transporting ATPase 2 (ATP7B) in which metal-binding domains (MBDs) 1-3 are missing, but a serine-rich loop containing two serine residues (S340 and S341) is present between MBD3 and MBD4. DETAILED DESCRIPTION

[0028] The present invention provides agents and compositions for use in treating Wilson's disease (WD). The nucleic acid sequences, vectors, recombinant viruses and related compositions of the present invention as described herein can be used to improve, prevent or treat WD.

[0029] Unless otherwise indicated, technical terms are used conventionally. Definitions of commonly used terms in molecular biology can be found in: Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, 1995 (ISBN 1-56081-569-8).

[0030] To facilitate review of the various embodiments of the present disclosure, the following explanations of specific terms are provided:

[0031] Adeno-associated virus (AAV): A small, replication-defective, non-enveloped virus that infects humans and some other primates. AAV is known to not cause disease and elicits a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and dormant cells and can persist in an extrachromosomal state without integrating into the host cell's genome. These characteristics make AAV an attractive viral vector for gene therapy. There are currently 12 recognized AAV serotypes (AAV1-12).

[0032] Administration (Administration / Administer): Providing or giving an agent, such as a therapeutic agent (e.g., recombinant AAV), to a subject by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intratubular, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

[0033] ATP7BΔ1-3-SS: As used herein, ATP7BΔ1-3-SS refers to a truncated human copper-transporting ATPase 2 (ATP7B) in which metal-binding domains (MBDs) 1-3 are missing, but a serine-rich loop containing two serine residues (S340 and S341) is present between MBD3 and MBD4.

[0034] Codon optimized: A "codon optimized" nucleic acid refers to a nucleic acid sequence that has been altered so that the codons are optimal for expression in a particular system, such as a particular species or group of species. For example, a nucleic acid sequence can be optimized for expression in mammalian cells or a particular mammalian species, such as human cells. Codon optimization does not change the amino acid sequence of the encoded protein.

[0035] Enhancer: A nucleic acid sequence that increases the rate of transcription by increasing the activity of a promoter.

[0036] Intron: A section of DNA within a gene that does not contain protein-coding information. Introns are removed before the messenger RNA is translated.

[0037] Inverted Terminal Repeat (ITR): A symmetrical nucleic acid sequence in the adeno-associated virus genome required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as replication origins for viral DNA synthesis and are required for vector encapsidation.

[0038] Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, virus or cell) has been substantially separated or purified from other biological components in the cells or tissues of the organism or the organism itself (where the component naturally occurs, such as other chromosomal and extrachromosomal DNA and RNA, proteins and cells). Nucleic acid molecules and proteins that have been "isolated" include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acid molecules and proteins.

[0039] Operably linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0040] Pharmaceutically acceptable carriers: Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences (EW Martin, Mack Publishing Co., Easton, Pa., 15th ed. (1975)) describes compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.

[0041] In general, the property of the carrier will depend on the specific mode of administration adopted. For example, parenteral formulations generally include injectable fluids, which include pharmaceutically and physiologically acceptable fluids, such as water, saline, balanced salt solutions, aqueous glucose solutions, glycerol, etc. as vehicles. For solid compositions (e.g., powders, pills, tablets, or capsule forms), conventional non-toxic solid carriers may include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biological neutral carriers, the pharmaceutical composition to be used may contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0042] Prevent, treat, or ameliorate a disease: "Preventing" a disease (such as WD) means inhibiting the overall development of the disease. "Treatment" refers to therapeutic intervention to improve the signs or symptoms of a disease or pathological condition (such as WD) after it has begun to develop. "Amelioration" refers to a decrease in the number or severity of signs or symptoms of a disease (such as WD).

[0043] Promoter: A DNA region that directs / initiates transcription of a nucleic acid (e.g., a gene). A promoter includes essential nucleic acid sequences near the start site of transcription.

[0044] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, virus, or other active compound is one that is completely or partially separated from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture media, or other crude preparations and fractionated to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0045] Recombinant: A recombinant nucleic acid molecule is a nucleic acid molecule that has a sequence that does not occur naturally or that has been made by the artificial combination of two otherwise separate sequence fragments. This artificial combination can be achieved by chemical synthesis or by artificial manipulation of separate fragments of nucleic acid molecules, such as through genetic engineering techniques.

[0046] Similarly, a recombinant virus is a virus that contains a sequence (such as a genomic sequence) that is not naturally occurring or is made by the artificial combination of sequences from at least two different sources. The term "recombinant" also includes nucleic acids, proteins and viruses that are changed only by adding, replacing or deleting a portion of a natural nucleic acid molecule, protein or virus. As used herein, "recombinant AAV" refers to an AAV particle in which a recombinant nucleic acid molecule (such as a recombinant nucleic acid molecule encoding a truncated human ATP7B (e.g., SEQ ID NO: 1 or SEQ ID NO: 15)) is packaged.

[0047] Sequence Identity: Identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed as identity or similarity between the sequences. Sequence identity can be measured as a percentage of identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured as a percentage similarity (taking into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of 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) than from more distantly related species (such as human and nematode sequences).

[0048] Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene, 73:237-44, 1988; Higgins and Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Detailed considerations of sequence alignment methods and homology calculations are given in Biosciences [Application of Computers in Biological Sciences] 8, 155-65, 1992; and Pearson et al., Meth. Mol. Rio. [Mathematical Molecular Alignment] 24: 307-31, 1994; Altschul et al., J. Mol. Biol. [Journal of Molecular Biology] 215: 403-10, 1990.

[0049] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological 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.

[0050] Serotype: A group of closely related microorganisms (such as viruses) distinguished by characteristic sets of antigens.

[0051] Filler sequence: refers to a nucleotide sequence contained in a larger nucleic acid molecule (such as a vector), which is usually used to create a required spacing between two nucleic acid features (such as between a promoter and a coding sequence), or to extend a nucleic acid molecule to a desired length. The filler sequence does 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.

[0052] Subject: Living multicellular vertebrate organisms, this category includes humans and non-human mammals.

[0053] Synthetic: Produced artificially in a laboratory, e.g. synthetic nucleic acids can be chemically synthesized in a laboratory.

[0054] Therapeutically effective amount: An amount of a particular drug or therapeutic agent (e.g., recombinant AAV) sufficient to achieve a desired effect in a subject or cell treated with the agent. The effective amount 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.

[0055] Vector: A vector is a nucleic acid molecule that allows the insertion of exogenous nucleic acid without destroying the ability of the vector to replicate and / or integrate in 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 transcription and translation of one or more inserted genes. In some embodiments herein, the vector is an AAV vector.

[0056] Unless otherwise explained, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which this disclosure belongs. Unless the context clearly indicates otherwise, the singular terms "a", "an", "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 are provided for illustration. Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of this disclosure, suitable methods and materials are described below. All disclosures, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification (including the explanation of the terms) will prevail. In addition, materials, methods, and embodiments are only illustrative and are not intended to be restrictive.

[0057] Viral vectors:

[0058] In some aspects, the present disclosure provides a recombinant adeno-associated virus (AAV) vector containing a genome comprising an AAV 5'-terminal inverted repeat (ITR) sequence, a promoter sequence, a nucleic acid sequence encoding ATP7BΔ1-3-SS (e.g., SEQ ID NO: 1 or SEQ ID NO: 15), and an AAV 3'-terminal inverted repeat sequence (ITR).

[0059] In some embodiments, the genome may further comprise an enhancer, intron, consensus Kozak sequence and / or polyadenylation signal as described herein. In some embodiments, the recombinant vector may further comprise one or more filler nucleic acid sequences. In one embodiment, the filler nucleic acid sequence is located between an intron and a portion or complete coding sequence of ATP7B.

[0060] In various embodiments described herein, the recombinant viral vector is an adeno-associated virus (AAV) vector. The AAV vector can be an AAV vector of 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), as well as any of more than 100 variants isolated from human and non-human primate tissues (see, e.g., Choi et al., 2005, Curr Gene Ther. [Contemporary Gene Therapy] 5: 299-310, 2005 and Gao et al., 2005, Curr Gene Ther. [Contemporary Gene Therapy] 5: 285-297). AAV vectors of any serotype can be used in the present invention, and the choice of AAV serotype will depend in part on the cell type or cells targeted by the gene therapy. For the treatment of WD, the liver is one of the relevant target organs. In some embodiments, the AAV vector is selected from serotype 9 (AAV9), serotype 8 (AAV8), serotype 5 (AAV5), or a variant thereof. In an exemplary embodiment, the AAV vector is serotype 9 (AAV9) or a variant thereof.

[0061] In some embodiments, the recombinant AAV vector includes an AAV ITR sequence, which acts as both an origin of vector DNA replication and a packaging signal for the vector genome when AAV and adenovirus helper functions are provided in trans. In addition, the ITR is a target for single-stranded endonucleolytic cleavage by the large Rep protein, splitting individual genomes from replication intermediates.

[0062] In some embodiments, the 5'-ITR sequence is from AAV2. In some embodiments, the 3'-ITR sequence is from AAV2. In some embodiments, the 5'-ITR sequence and the 3'-ITR sequence are from AAV2. In some embodiments, the 5'-ITR sequence and / or the 3'-ITR sequence are from AAV2 and comprise or consist of SEQ ID NO: 2. In other embodiments, the 5'-ITR sequence and / or the 3'-ITR sequence are from a non-AAV2 source.

[0063] In some exemplary embodiments, the AAV vector is an AAV serotype 9 (AAV9) vector, and the vector includes an enhancer, a promoter, an intron, a nucleic acid sequence encoding ATP7BΔ1-3-SS (e.g., SEQ ID NO: 1 or SEQ ID NO: 15), and a polyadenylation signal as described herein. In some embodiments, the AAV9 vector further comprises two AAV2, AAV8, or AAV9 terminal inverted repeat (ITR) sequences: one at the 5' of the enhancer and the other at the 3' of the polyadenylation signal. In an exemplary embodiment, the AAV9 vector comprises two AAV2 terminal inverted repeat (ITR) sequences: one at the 5' of the enhancer and the other at the 3' of the polyadenylation signal. In some embodiments, the AAV2 ITR sequence comprises SEQ ID NO: 2 or consists of SEQ ID NO: 2. In another exemplary embodiment, the AAV9 vector comprises two AAV9 terminal inverted repeat (ITR) sequences: one at the 5' of the enhancer and the other at the 3' of the polyadenylation signal.

[0064] In some exemplary embodiments, the present disclosure provides a recombinant nucleic acid comprising a vector genome comprising an AAV 5'-terminal inverted repeat (ITR) sequence, a promoter sequence, a nucleic acid sequence encoding a native ATP7BΔ1-3-SS represented by SEQ ID NO: 1, and an AAV 3'-terminal inverted repeat (ITR). In some exemplary embodiments, the present disclosure provides a recombinant nucleic acid comprising a vector genome comprising an AAV 5'-terminal inverted repeat (ITR) sequence, a promoter sequence, a nucleic acid sequence encoding a codon-optimized ATP7BΔ1-3-SS represented by SEQ ID NO: 15, and an AAV 3'-terminal inverted repeat (ITR). In some exemplary embodiments, the present disclosure provides a vector genome comprising SEQ ID NO: 14, comprising an AAV 5'-terminal inverted repeat (ITR) sequence, a promoter sequence, a nucleic acid sequence encoding a native ATP7BΔ1-3-SS represented by SEQ ID NO: 1, or an adeno-associated virus (AAV) vector comprising the same.

[0065] In other aspects, the present application provides a recombinant nucleic acid sequence corresponding to a vector genome that can be used to treat WD. In some embodiments, the present application provides a recombinant nucleic acid that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 14. Thus, the present application provides a recombinant nucleic acid that is at least 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) identical to SEQ ID NO: 14. In an exemplary embodiment, the present application provides a recombinant nucleic acid sequence corresponding to a vector genome, the vector genome comprising an AAV 5'-terminal inverted repeat (ITR) sequence, a promoter sequence, a nucleic acid sequence encoding a native ATP7BΔ1-3-SS represented by SEQ ID NO: 1, and an AAV 3'-terminal inverted repeat (ITR), wherein the vector genome comprises SEQ ID NO: 14 or consists of SEQ ID NO: 14. In an exemplary embodiment, the present application provides a recombinant nucleic acid sequence corresponding to a vector genome, the vector genome comprising an AAV 5'-terminal inverted repeat (ITR) sequence, a promoter sequence, a nucleic acid sequence encoding a codon-optimized ATP7BΔ1-3-SS represented by SEQ ID NO: 15, and an AAV 3'-terminal inverted repeat (ITR).

[0066] Opening edge:

[0067] In various aspects described herein, an AAV vector is provided that contains a promoter sequence that facilitates driving and regulating transgene expression, such as expression of ATP7BΔ1-3-SS (e.g., the amino acid sequence of ATP7BΔ1-3-SS represented by SEQ ID NO: 8). In an exemplary embodiment, the promoter sequence is located between the selected 5′-ITR sequence and the coding sequence of ATP7BΔ1-3-SS (e.g., SEQ ID NO: 1 or SEQ ID NO: 15). In some embodiments, the promoter sequence is located downstream of the enhancer sequence. In some embodiments, the promoter sequence is located upstream of the intron sequence. In some illustrative embodiments, the vectors described herein use a thyroxine transporter (TTR) promoter, which may optionally be located downstream of a thyroxine transporter enhancer (enTTR).

[0068] In some embodiments, the promoter is selected from the group consisting of a thyroxine transporter (TTR) promoter, a chicken β-actin (CBA) promoter, a cytomegalovirus immediate early gene (CMV) promoter, a thyroxine binding globulin (TBG) promoter, an alpha-1 antitrypsin (A1AT) promoter, and a CAG promoter (constructed using a CMV early enhancer element, a promoter, the first exon and the first intron of the CBA gene, and a splice acceptor of a rabbit β-globin gene). In an exemplary embodiment, the promoter is a TTR promoter. In one embodiment, the TTR promoter comprises or consists of SEQ ID NO: 12.

[0069] In addition to the promoter, the AAV vector may contain other suitable transcription initiation, termination, enhancer sequences and efficient RNA processing signals. As described in further detail below, such sequences include splicing and polyadenylation (poly A) signals, regulatory elements that enhance expression (i.e., WPRE), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequences), and sequences that enhance protein stability.

[0070] In some embodiments, the AAV vector contains a vector genome that further comprises a consensus Kozak sequence. In some embodiments, the consensus Kozak sequence is located downstream of the intron sequence. In one embodiment, the consensus Kozak sequence is GCCGCC (SEQ ID NO: 11). As will be appreciated by those skilled in the art, the consensus Kozak sequence is generally immediately upstream of the coding sequence; in this case, immediately upstream of the coding sequence of the truncated ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1 or SEQ ID NO: 15). As will be appreciated by those skilled in the art, the consensus Kozak sequence can be considered to share the ATG residue corresponding to the start codon of the therapeutic polypeptide (e.g., truncated ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1 or SEQ ID NO: 15)). For simplicity of disclosure, the consensus Kozak sequence as described herein comprises a hexanucleotide sequence corresponding to a region that is not shared with a nucleic acid encoding a therapeutic polypeptide (e.g., truncated ATP7B (ATP7BΔ1-3-SS encoded by SEQ ID NO: 1 or SEQ ID NO: 15)).

[0071] ATP7B peptide:

[0072] As described herein, aspects of the invention provide a recombinant vector comprising a genome comprising an AAV 5'-terminal inverted repeat (ITR), a promoter sequence, a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) having an amino acid sequence of SEQ ID NO: 8 (e.g., SEQ ID NO: 1 or SEQ ID NO: 15), and an AAV 3'-terminal inverted repeat (ITR). ATP7B has eight transmembrane domains that form a pathway across the cell membrane for copper translocation; and a large N-terminus having six metal binding domains (MBDs), each of which contains about 70 amino acids and a highly conserved metal binding motif GMxCxxC (where x is any amino acid). In addition to the canonical sequence (also referred to as isoform a, which is the longest isoform; NCBI reference sequence: NP_000044.2), four other isoforms are known: NCBI reference sequence NP_001005918.1, NP_001230111.1, NP_001317507.1, NP_001317508.1. The compositions and methods described herein can be used to treat subjects with disease-causing non-functional ATP7B variant proteins.

[0073] In one embodiment, the coding sequence of the truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1 or SEQ ID NO: 15) encodes a protein having amino acids as described in SEQ ID NO: 8. SEQ ID NO: 1 provides the cDNA of the native human ATP7B lacking MBD 1-3. SEQ ID NO: 8 represents the native DEL1-3 or ATP7BΔ1-3-SS protein, wherein there are two serine residues corresponding to positions 340 and 341 of the wild-type ATP7B full-length protein sequence.

[0074] In various embodiments described herein, vectors are provided that contain a genome comprising a coding sequence for a truncated ATP7B (ATP7BΔ1-3-SS) (eg, SEQ ID NO: 1 or SEQ ID NO: 15).

[0075] In some embodiments, vectors are provided containing a genome comprising an engineered cDNA of codon-optimized human ATP7B (eg, SEQ ID NO: 15). The polypeptides delivered using the vectors described herein encompass truncated ATP7B (ATP7BΔ1-3-SS) in which MBD1-3 is deleted, suitable for use in treating WD.

[0076] In some embodiments, the polypeptide expressed using the vectors described herein is a truncated human ATP7B (SEQ ID NO: 8).

[0077] Carrier element:

[0078] In some embodiments, the AAV vector contains a genome further comprising one or more enhancer sequences. In one embodiment, the enhancer is selected from the group consisting of transthyretin enhancer (enTTR), cytomegalovirus immediate early gene (CMV) enhancer, chicken beta-actin (CBA) enhancer, En34 enhancer, and apolipoprotein (ApoE) enhancer. In an exemplary embodiment, the enhancer is an enTTR enhancer. In one embodiment, the enTTR enhancer comprises or consists of SEQ ID NO: 3.

[0079] In some embodiments, the AAV vector contains a genome that further comprises one or more intron sequences. In one embodiment, the intron is selected from the SV40 small T intron, the rabbit hemoglobin subunit β (rHBB) intron, the human β-globin IVS2 intron, the β-globin / IgG chimeric intron (Promega chimeric intron) or the hFIX intron. In an exemplary embodiment, the intron is the SV40 small T intron. In one embodiment, the SV40 small T intron sequence comprises SEQ ID NO: 4 or consists of SEQ ID NO: 4. In another exemplary embodiment, the intron is the rHBB intron. In one embodiment, the rHBB intron sequence comprises SEQ ID NO: 5 or consists of SEQ ID NO: 5.

[0080] In some embodiments, the AAV vector contains a genome further comprising a polyadenylation signal sequence. In one embodiment, the polyadenylation signal sequence is selected from the group consisting of an SV40 polyadenylation signal sequence, a bovine growth hormone (BGH) polyadenylation signal sequence, and a rabbit beta globin polyadenylation signal sequence. In an exemplary embodiment, the polyadenylation signal sequence is a bovine growth hormone (BGH) polyadenylation signal sequence. In one embodiment, the BGH polyadenylation signal sequence comprises SEQ ID NO: 6 or consists of SEQ ID NO: 6. In another exemplary embodiment, the polyadenylation signal sequence is an SV40 polyadenylation signal sequence. In one embodiment, the SV40 polyadenylation signal sequence comprises SEQ ID NO: 7 or consists of SEQ ID NO: 7.

[0081] AAV capsid:

[0082] On the other hand, the present application provides a recombinant adeno-associated virus (rAAV) that can be used as a medicament for gene therapy for treating WD, wherein the rAAV comprises an AAV capsid and a vector genome as described herein. In some embodiments, the AAV capsid is from an AAV of serotype 9, 8, 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, rh10 or hu37 (i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh10 or AAVhu37). In an exemplary embodiment, the AAV vector is an AAV serotype 9 (AAV9) vector, an AAV9 variant vector, an AAV serotype 8 (AAV8) vector, an AAV serotype 5 (AAV5) vector or an AAV serotype 2 (AAV2) vector. In certain embodiments, the AAV capsid and vector are from an AAV9 serotype. In certain embodiments, the AAV capsid and vector are from the AAV8 serotype.

[0083] The AAV9 capsid is a self-assembling AAV capsid composed of a variety of AAV9 VP proteins. The AAV9 VP protein is generally expressed as an alternative splicing variant encoded by a nucleic acid sequence of SEQ ID NO: 9 or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% of a sequence thereof, which encodes the capsid protein VP1 amino acid sequence of SEQ ID NO: 10 (GenBank Accession No.: AAS99264). These splicing variants produce proteins of different lengths of SEQ ID NO: 10. In certain embodiments, the AAV9 capsid includes an AAV9 capsid protein having an amino acid sequence that is 99% identical to AAS99264 or 99% identical to SEQ ID NO: 10. See also U.S. Patent No. 7,906,111 and International Publication No. WO / 2005 / 033321. As used herein, AAV9 variants include those described in, for example, International Publication No. WO / 2016 / 049230, U.S. Pat. No. 8,927,514, U.S. Patent Publication No. 2015 / 0344911, and U.S. Pat. No. 8,734,809.

[0084] As indicated herein, AAV9 capsid sequences and capsid proteins encoded by the sequences (e.g., nucleic acid sequences of SEQ ID NO: 9 or amino acid sequences of SEQ ID NO: 10 encoding AAV9 capsid protein VP1) can be used to produce rAAV. However, in other embodiments, another AAV capsid is selected. Tissue specificity is determined by the capsid type. AAV serotypes that transduce suitable targets (e.g., liver, muscle, lung, or CNS) can be selected as sources of AAV viral vector capsids, including, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAVrh64R1, AAVrh64R2, AAVrh8. See, e.g., U.S. Patent Publication No. 2007 / 0036760; U.S. Patent Publication No. 2009 / 0197338; and EP 1310571. See also International Application No. WO 2003 / 042397 (AAV7 and other simian AAVs), U.S. Pat. Nos. 7,282,199 and 7,790,449 (AAV8). In addition, AAVs that have not yet been discovered or recombinant AAVs based thereon can be used as sources of AAV capsids. These documents also describe other AAVs that can be selected for producing AAVs and are incorporated herein by reference. In some embodiments, the AAV capsids for use in viral vectors can be produced by mutagenesis (i.e., by insertion, deletion or substitution) of one of the above-mentioned AAV capsids or its encoding nucleic acid.

[0085] Host cells containing recombinant nucleic acid molecules:

[0086] In some aspects, provided herein are host cells comprising a recombinant nucleic acid molecule, a viral vector (eg, an AAV vector) or an rAAV disclosed herein. In specific embodiments, the host cell may be suitable for the propagation of AAV.

[0087] A variety of host cells can be used, such as bacteria, yeast, insects, mammalian cells, etc. 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. In certain embodiments, the host cell line of the present invention is a HeLa cell line (e.g., HeLa S3). In another embodiment, the host cell line of the present invention is a HEK293 cell line.

[0088] The recombinant nucleic acid molecule or vector can be delivered to the host cell culture using any suitable method known in the art. In some embodiments, a stable host cell line having a recombinant nucleic acid molecule or vector inserted into its genome is generated. In some embodiments, a stable host cell line is generated that contains the AAV vector described herein. After transfection of the AAV vector into the host culture, the integration of the rAAV into the host genome can be determined by a variety of methods (such as antibiotic selection, fluorescence activated cell sorting, protein blotting, PCR-based detection, fluorescence in situ hybridization), such as Nakai et al., Nature Genetics [Natural Genetics] (2003) 34: 297-302; Philpott et al., Journal of Virology [Journal of Virology] (2002) 76 (11): 5411-5421 and Howden et al., J Gene Med [Journal of Gene Medicine] 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 61 (2002): S9-S15 and Yuan et al., Human Gene Therapy (2011) 22(5): 613-24.

[0089] Recombinant AAV for gene therapy:

[0090] Adeno-associated virus (AAV) belongs to the Parvoviridae family and the Dependovirus genus. AAV is a small non-enveloped virus that packages a linear single-stranded DNA genome. Both the sense and antisense strands of AAV DNA are packaged into the AAV capsid at the same frequency.

[0091] The AAV genome is characterized by two inverted terminal repeats (ITRs) that flank two open reading frames (ORFs). The AAV2 genome, for example, the first 125 nucleotides of the ITR are palindromes that fold on themselves to maximize base pairing and form a T-shaped hairpin structure. The other 20 bases of the ITR, called the D sequence, remain unpaired. The ITR is a cis-acting sequence that is important for AAV DNA replication; the ITR is the origin of replication and serves as a primer for DNA polymerase to synthesize the second strand. The double-stranded DNA formed during this synthesis process, called a replicative monomer, is used for a second round of self-priming replication and forms a replicative dimer. These double-stranded intermediates are processed by a strand displacement mechanism to produce single-stranded DNA for packaging and double-stranded DNA for transcription. Located within the ITR are the Rep binding elements and the terminal dissociation site (TRS). During AAV replication, the viral regulatory protein Rep uses these features to process double-stranded intermediates. In addition to their role in AAV replication, ITRs are essential for AAV genome packaging, transcription, negative regulation under nonpermissive conditions, and site-specific integration (Days and Berns, Clin Microbiol Rev (2008) 21(4): 583-593).

[0092] 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, which are arranged in icosahedral symmetry. VP1, VP2 and VP3 are present in a molar ratio of 1:1:10 (Daya and Berns, Clin Microbiol Rev [Clinical Microbiology Review] (2008) 21 (4): 583-593).

[0093] AAV is one of the most commonly used viruses in gene therapy today. Although AAV infects humans and some other primates, it is known not to cause disease and to elicit a very mild immune response. Gene therapy vectors utilizing AAV can infect dividing and dormant cells and persist in an extrachromosomal state without integrating into the host cell's genome. Due to the favorable characteristics of AAV, the present disclosure contemplates the use of AAV for the recombinant nucleic acid molecules and methods disclosed herein.

[0094] AAV has several desirable features for gene therapy vectors, including the ability to bind to and enter target cells, enter the nucleus, be expressed in the nucleus for extended periods of time, and have low toxicity. However, the small size of the AAV genome limits the size of heterologous DNA that can be incorporated. To minimize this problem, AAV vectors have been constructed that do not encode Rep and the integration efficiency element (IEE). The ITRs are retained because they are cis signals required for packaging (Daya and Berns, Clin Microbiol Rev [Clinical Microbiology Review], (2008) 21(4): 583-593).

[0095] Methods for producing rAAV suitable for gene therapy are well known in the art (see, e.g., U.S. Patent Application Nos. 2012 / 0100606; 2012 / 0135515; 2011 / 0229971; and 2013 / 0072548; and Ghosh et al., Gene Ther. (2006) 13(4):321-329) and can be used with the recombinant nucleic acid molecules and methods disclosed herein.

[0096] In some aspects, the present application relates to the use of the rAAV disclosed herein for treating Wilson's disease (WD), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein. In some embodiments, the vector contains a genome comprising, in 5' to 3' order, operably linked components: a 5'-terminal inverted repeat sequence (ITR), a promoter sequence, a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1 or SEQ ID NO: 15), and a 3'-terminal inverted repeat sequence (ITR). In an exemplary embodiment, the vector genome further comprises an enhancer sequence upstream of the promoter sequence, an intron downstream of the promoter, and a polyadenylation sequence upstream of the 3'-ITR. Thus, in another exemplary embodiment, the vector genome comprises, in 5' to 3' order, operably linked components: 5'-terminal inverted repeat sequence (ITR), enhancer sequence, promoter sequence, intron sequence, coding sequence of truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1 or SEQ ID NO: 15), polyadenylation signal sequence, and 3'-terminal inverted repeat sequence (ITR). In another exemplary embodiment, the vector genome comprises, in 5' to 3' order, operably linked components: AAV2 5'-ITR sequence, enTTR enhancer, TTR promoter, SV40 small T intron, coding sequence of truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1 or SEQ ID NO: 15), SV40 polyadenylation signal sequence, and AAV2 3'-ITR. In some embodiments, the vector genome further comprises a consensus Kozak sequence located downstream of the intron sequence. In some embodiments, the capsid is an AAV9 capsid.

[0097] In some aspects, the present application relates to the use of rAAV disclosed herein for treating Wilson's disease (WD), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein. In some embodiments, the vector genome comprises operably linked components in 5' to 3' order: a 5'-terminal inverted repeat sequence (ITR), a promoter sequence, a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1), and a 3'-terminal inverted repeat sequence (ITR). In an exemplary embodiment, the vector genome further comprises an enhancer sequence upstream of the promoter sequence, an intron downstream of the promoter, and a polyadenylation sequence upstream of the 3'-ITR. Thus, in another exemplary embodiment, the vector contains a genome comprising, in 5' to 3' order, operably linked components: 5'-terminal inverted repeat sequence (ITR), enhancer sequence, promoter sequence, intron sequence, coding sequence of truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1), polyadenylation signal sequence, and 3'-terminal inverted repeat sequence (ITR). In another exemplary embodiment, the vector contains a genome comprising, in 5' to 3' order, operably linked components: AAV2 5'-ITR sequence, enTTR enhancer, TTR promoter, SV40 small T intron, coding sequence of truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1), SV40 polyadenylation signal sequence, and AAV2 3'-ITR. In some embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the intron sequence. In some embodiments, the capsid is an AAV9 capsid.

[0098] In some aspects, the present application relates to the use of rAAV disclosed herein for treating Wilson's disease (WD), wherein the rAAV comprises an AAV capsid and a packaged vector genome. In some embodiments, the vector contains a packaged genome, which comprises operably linked components in 5' to 3' order: a 5'-terminal inverted repeat sequence (ITR), a promoter sequence, a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 15) and a 3'-terminal inverted repeat sequence (ITR). In an exemplary embodiment, the packaged genome also comprises an enhancer sequence upstream of the promoter sequence, an intron downstream of the promoter, and a polyadenylation sequence upstream of the 3'-ITR. Thus, in another exemplary embodiment, the vector contains a packaged genome comprising, in 5' to 3' order, operably linked components: 5'-terminal inverted repeat sequence (ITR), enhancer sequence, promoter sequence, intron sequence, coding sequence of truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 15), polyadenylation signal sequence, and 3'-terminal inverted repeat sequence (ITR). In another exemplary embodiment, the vector contains a packaged genome comprising, in 5' to 3' order, operably linked components: AAV2 5'-ITR sequence, enTTR enhancer, TTR promoter, SV40 small T intron, coding sequence of truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 15), SV40 polyadenylation signal sequence, and AAV2 3'-ITR. In some embodiments, the packaged genome further comprises a consensus Kozak sequence located downstream of the intron sequence. In some embodiments, the capsid is an AAV9 capsid.

[0099] Figure 1 A schematic diagram of a vector genome construct showing exemplary packaging for expressing a truncated ATP7B that retains MBD4, 5, and 6 is provided. The 5'-ITR is represented by nucleotides 1-145; the enTTR enhancer is represented by nucleotides 146-245; the TTR promoter is represented by nucleotides 246-435; the SV40 small T intron is represented by nucleotides 436-530; the consensus Kozak sequence is represented by nucleotides 531-536; the truncated ATP7B coding sequence is represented by nucleotides 540-4142; the SV40 polyadenylation signal sequence is represented by nucleotides 4143-4340; and the 3'-ITR is represented by nucleotides 4341-4485.

[0100] In certain embodiments, the nucleic acid sequence encoding ATP7BΔ1-3-SS is a native human sequence (represented by SEQ ID NO: 1). Alternatively, in some embodiments, the nucleic acid sequence encoding ATP7BΔ1-3-SS is a codon-optimized human sequence (represented by SEQ ID NO: 15).

[0101] Improved efficacy in treating WD:

[0102] In certain embodiments, the truncated human ATP7B (ATP7BΔ1-3-SS) described herein and encoded by SEQ ID NO: 1 or SEQ ID NO: 15 is more effective than full-length or other truncated forms of ATP7B (e.g., ATP7BΔ1-4, SEQ ID NO: 13). In some aspects, the ATP7BΔ1-3-SS of the present disclosure is localized to the trans-Golgi network (TGN). In certain embodiments, the rAAV comprising the nucleic acid sequence of SEQ ID NO: 1 encoding ATP7BΔ1-3-SS reduces copper levels in the liver and urine of a mammal diagnosed with a copper metabolism disorder (e.g., Wilson's disease) after injection into the mammal.

[0103] Improving the yield of AAV vectors containing truncated ATP7B:

[0104] In one aspect, rAAV comprising a nucleic acid sequence encoding ATP7B Δ1-3-SS packaged in AAV8 or AAV9 as described herein has a production yield that is about 1.1 to about 10 times higher (e.g., about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times) than full-length ATP7B or ATP7B Δ1-4.

[0105] Improving the production of AAV vectors containing AAV9 capsids:

[0106] In one aspect, rAAV comprising an AAV9 capsid has a titer yield that is about 1.1 to about 10 times higher (e.g., about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times) than rAAV comprising an AAV8 capsid.

[0107] Pharmaceutical composition:

[0108] The present disclosure provides compositions comprising 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. Pharmaceutically acceptable carriers (vehicles) useful for the present disclosure are conventional. Remington's Pharmaceutical Sciences (EW Martin, Mack Publishing Co., Easton, Pa., 15th ed. (1975)) describes compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.

[0109] As described in the preceding paragraphs, the present application relates in some aspects to a pharmaceutical composition comprising a 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.

[0110] In some embodiments, rAAV is formulated in a buffer / carrier suitable for infusion into a human subject. The buffer / carrier should include components that prevent rAAV from adhering to the infusion tube but do not interfere with the in vivo binding activity of rAAV. Various suitable solutions may include one or more of: buffered saline, a surfactant, and a physiologically compatible salt or salt mixture (whose ionic strength is adjusted to be equal to about 100 mM sodium chloride (NaCl) to about 250 mM NaCl), or a physiologically compatible salt adjusted to an equivalent ionic concentration. The pH value can 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 poloxamers, i.e. nonionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene 10 (poly(propylene oxide)) and two flanking hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS15 (Macrogol-15 hydroxystearate), LABRASOL (polyoxycaprylylglyceride), polyoxyl 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.

[0111] Treatment for Wilson disease:

[0112] In yet another aspect, the present application relates to a method of treating WD in a human subject, the method comprising administering to the human subject a therapeutically effective amount of a rAAV comprising SEQ ID NO: 1 or SEQ ID NO: 15 encoding a truncated ATP7B (ATP7BΔ1-3-SS) disclosed herein.

[0113] In one embodiment, the present application provides a method for treating WD, which comprises administering a rAAV comprising an AAV capsid and a packaged vector genome, wherein the vector genome comprises a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (eg, SEQ ID NO: 1 or SEQ ID NO: 15).

[0114] In yet another aspect, the present application relates to a method for treating WD in a human subject, the method comprising administering a therapeutically effective amount of at least one rAAV to a human subject diagnosed with at least one mutation in ATP7B, the rAAV comprising a vector genome, the vector genome comprising a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1 or SEQ ID NO: 15). In one embodiment, the present application provides a method for treating WD in a human subject diagnosed with at least one mutation in ATP7B, the method comprising administering a rAAV comprising an AAV capsid and a packaged vector genome, wherein the vector genome comprises a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1 or SEQ ID NO: 15). The coding sequence represented by SEQ ID NO: 1 encodes a truncated ATP7B represented by SEQ ID NO: 8. In some embodiments, the capsid is an AAV9 capsid.

[0115] In yet another aspect, the present application relates to a method for treating WD in a human subject, the method comprising administering a therapeutically effective amount of at least one rAAV to a human subject diagnosed with at least one mutation in ATP7B, the rAAV comprising a vector genome, the vector genome comprising a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1). In one embodiment, the present application provides a method for treating WD in a human subject diagnosed with at least one mutation in ATP7B, the method comprising administering a rAAV comprising an AAV capsid and a packaged vector genome, wherein the vector genome comprises a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 1). The coding sequence represented by SEQ ID NO: 1 encodes a truncated ATP7B represented by SEQ ID NO: 8. In some embodiments, the capsid is an AAV9 capsid.

[0116] In yet another aspect, the present application relates to a method for treating WD in a human subject, the method comprising administering a therapeutically effective amount of at least one rAAV to a human subject diagnosed with at least one mutation in ATP7B, the rAAV comprising a vector genome, the vector genome comprising a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 15). In one embodiment, the present application provides a method for treating WD in a human subject diagnosed with at least one mutation in ATP7B, the method comprising administering a rAAV comprising an AAV capsid and a packaged vector genome, wherein the vector genome comprises a coding sequence of a truncated human ATP7B (ATP7BΔ1-3-SS) (e.g., SEQ ID NO: 15). In some embodiments, the capsid is an AAV9 capsid.

[0117] Any suitable method or route can be used to administer the rAAV or rAAV-containing compositions described herein. Routes of administration include, for example, systemic, oral, inhaled, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. In some embodiments, rAAV or rAAV-containing compositions are administered intravenously.

[0118] The specific dose administered may be a uniform dose for each patient, for example 1.0 x 10 11 -1.0x10 14 Viral genomes / kilogram patient body weight (vg) / kg. Alternatively, the patient's dosage can be adjusted according to the patient's approximate body weight or surface area. Other factors determining a suitable dosage 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 a suitable therapeutic dose is routinely performed by those skilled in the art, especially according to the dosage information and assays disclosed herein. Dosage can also be determined by using known assays for determining dosage in conjunction with appropriate dose-response data. When monitoring the progression of the disease, the dosage of individual patients can also be adjusted.

[0119] In some embodiments, rAAV is administered at a concentration of, for example, about 1.0 x 1011 vg / kg to about 1 x 10 14 vg / kg, about 5x10 11 vg / kg to about 5x10 13 vg / kg, or about 1x10 12 About 1x10 13 vg / kg, as measured by qPCR or digital droplet PCR (ddPCR). In some embodiments, rAAV is administered at a dose of about 2×10 12In some embodiments, rAAV is administered at a dose of about 5×10 12 In some embodiments, rAAV is administered at a dose of about 6×10 12 In some embodiments, rAAV is administered at a dose of about 1×10 13 In some embodiments, rAAV is administered at a dose of about 7×10 13 vg / kg. rAAV can be administered in a single dose or multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses) as required for the desired therapeutic outcome. In some exemplary embodiments, only a single dose of a particular rAAV is administered.

[0120] Throughout the specification, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that additionally there are compositions of the invention that consist essentially of, or consist of, the recited components, and there are processes and methods according to the invention that consist essentially of, or consist of, the recited processing steps.

[0121] In the present application, when an element or component is referred to as being included in and / or selected from a list of listed elements or components, it should be understood that the element or component can be any one of the listed elements or components, or the element or component can be selected from a group consisting of two or more of the elements or components.

[0122] In addition, 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 present invention, whether it is clear or implicit herein. For example, when referring to a specific compound, unless otherwise understood from the context, the compound can be used in various embodiments of the compositions of the present invention and / or the inventive method. In other words, in the present application, embodiments have been described and depicted in a manner that enables clear and concise applications to be written and drawn, but it is intended and will be understood that embodiments can be combined or separated in various ways without departing from the teachings of the present application and one or more inventions. For example, it should be understood that all features described and depicted here can be applicable to all aspects of one or more inventions described and depicted here.

[0123] It should be understood that, unless otherwise understood from the context and usage, the expression "at least one" includes individually each of the listed objects after the expression and various combinations of two or more of the listed objects. Unless otherwise understood from the context, the expression "and / or" related to three or more listed objects should be understood to have the same meaning.

[0124] It should be understood that use of the terms "include, includes, including," "have, has, having," "contain, contains or containing," including their grammatical equivalents, is generally intended to be open-ended and non-limiting, e.g., does not exclude additional, unrecited elements or steps unless expressly stated otherwise or understood from the context.

[0125] If the term "about" is used before a numerical value, the present invention also includes the specific numerical value itself unless otherwise specifically stated. As used herein, unless otherwise stated or inferred, the term "about" refers to a ±10% variation of the nominal value.

[0126] It should be understood that the order of steps or the order in which certain actions are performed is immaterial as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0127] Any and all examples or exemplary language used herein, such as "such as" or "including", are intended only to better illustrate the present invention and are not intended to limit the scope of the present invention unless otherwise stated. Language in the specification should not be construed as indicating that any unclaimed element is essential to the practice of the present invention.

[0128] Example

[0129] The invention, as generally described, will now be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0130] Example 1 - AAV vectors and rAAV produced therefrom

[0131] AAV vectors

[0132] This example describes the construction of an AAV vector having a nucleic acid sequence represented by SEQ ID NO: 1, which is restricted by two AAV2 inverted terminal repeats (ITRs, SEQ ID NO: 2). SEQ ID NO: 1 represents the cDNA of native human ATP7B lacking MBDs 1-3. Nucleotides 223-225 in SEQ ID NO: 1 encode serine residue S340, and nucleotides 226-228 in SEQ ID NO: 1 encode serine residue S341 (numbering based on the wild-type full-length ATP7B protein sequence).

[0133] like Figure 1As shown, in the AAV vector, the ATP7B expression cassette contains an enhancer (EnTTR), a promoter (TTR), an intron (SV40 small T intron), the nucleotide sequence of SEQ ID NO: 1 (which encodes a truncated human ATP7B (ATP7BΔ1-3-SS)) and an SV40 poly (A) signal. Figure 2 A circular diagram of the vector illustrating the various components is shown.

[0134] AAV vector DTC319 contains a truncated human ATP7B sequence, in which metal binding domains 4, 5 and 6 are retained. The truncated human ATP7B sequence encodes a protein represented by SEQ ID NO: 8 comprising two serine residues S340 and S341 (according to NCBI reference sequence number: NP_000044.2).

[0135] The Simian Virus 40 (SV40) late polyadenylation signal (Genbank Accession No. J02400 (SEQ ID NO: 7) provides a cis sequence for efficient polyadenylation of ATP7B mRNA. This element serves as a signal for a specific cleavage event at the 3' end of the nascent transcript and the addition of a long poly(A) tail.

[0136] Each truncated ATP7B expression cassette was cloned into an AAV vector. All AAV vectors had a backbone encoding a kanamycin resistance gene. Figure 2 An exemplary AAV vector DTC319 is shown. Figure 1 An expression cassette for DTC319 for expressing ATP7B (ATP7BΔ1-3-SS) is described.

[0137] rAAV virus particles

[0138] The AAV vector genome is a single-stranded DNA genome. Only the sequences between and including the ITR sequences are packaged into AAV virions. Virions are produced by transfecting three plasmids into human embryonic kidney 293 (HEK293) cells, which provide E1a and E1b gene products. The first plasmid can be the AAV vector described herein. The second plasmid can be a packaging plasmid, which contains wild-type AAV2 rep and AAV8 or AAV9 cap genes. The third plasmid is a helper adenovirus plasmid.

[0139] A diagram of an exemplary packaging plasmid, pAAV2 / 8.KanR (p2123FH) plasmid, is shown in Figure 3In this plasmid, the adeno-associated Rep / Cap plasmid pAAV2 / 8.KanR (p2123FH) (8354bp) encodes four wild-type AAV2 viral replication (Rep) proteins and three wild-type AAVVP capsid (cap) proteins from serotype 8. In this plasmid, the AAVp5 promoter that 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 (i.e., a plasmid backbone) between the promoter and the Rep gene, resulting in downregulation of Rep expression and an increase in the ability to support high-titer rAAV production. Both the kanamycin resistance gene and the MB1 origin are included for plasmid production in E. coli.

[0140] A schematic diagram of an exemplary helper plasmid, pAdDeltaF6 (Kan), is shown in Figure 4 In this plasmid, the adenoviral genomic regions important for AAV replication, namely E2A, E4 and VA RNA, are provided. Adenoviral E1 function is also required but is provided by the HEK293 host cells. Figure 4 The plasmid shown in does not contain other adenoviral replication, structural genes or cis elements essential for adenoviral replication, such as adenoviral ITRs, and therefore, is not expected to produce infectious adenovirus. A kanamycin resistance gene and MB1 origin are included for plasmid production in E. coli.

[0141] Example 2 - Deletion of metal binding domains (MBD) 1-3 in human ATP7B improves production yield

[0142] This example describes experiments demonstrating that ATP7BΔ1-3-SS has higher production than full-length ATP7B or the truncated form ATP7BΔ1-4.

[0143] The lack of functional ATP7B leads to the accumulation of copper in the liver and other tissues, which manifests as liver disease with neurological or psychiatric symptoms. WD can be treated by reducing copper absorption or removing excess copper from the body. C3He-Atp7b tx-j Mice do not express functional Atp7b and are therefore used as a mouse model for WD. An AAV vector containing a codon-optimized full-length human ATP7B sequence was used to transfect HEK293 cells with a Rep / Cap plasmid (which encodes four wild-type AAV2 viral replication (Rep) proteins and three wild-type AAV VP capsid (cap) proteins from serotype 8) and a helper plasmid to obtain ATP7BcoFL viral particles.

[0144] C3He-Atp7b tx-j Mice were injected intravenously (iv) for 10 10 or 10 11GC / kg of ATP7BcoFL (codon-optimized full-length human ATP7B). tx-j Mice were injected intravenously for 10 9 , 10 10 Hepatic copper levels in males (indicated by squares) and females (indicated by circles) were assessed by inductively coupled plasma-mass spectrometry (ICP-MS) and compared with those from age-matched uninjected males and females heterozygous (Het) and C3He-Atp7b tx-j The copper levels of mice were compared. The mice were autopsied at approximately 9 months of age and the livers were harvested. The data are shown in Figure 5 middle.

[0145] Gene therapy using AAV vectors can be used to treat WD. However, there is a limit to the size of cDNA that can be packaged in the AAV vector capsid. The wild-type AAV genome is 4.7 kb, and packaging a larger genome can potentially reduce the yield and integrity of the DNA sequence encapsulated in the AAV capsid. Therefore, the nucleotide sequence encoding ATP7BΔ1-3-SS was packaged in the AAV8 capsid and the production yield of ATP7BΔ1-3-SS was tested. AAV vectors encoding full-length (FL) human ATP7B, human ATP7B (ATP7BΔ1-3-SS) in which MBD 1-3 is deleted but a serine-rich loop containing two serine residues (S340 and S341) is present between MBD3 and MBD4, or human ATP7B (ATP7BΔ1-4) in which MBD 1-4 is deleted were transfected into HEK293 cells. Rep / Cap plasmids encoding four wild-type AAV2 viral replication (Rep) proteins and three wild-type AAV VP capsid (cap) proteins from serotype 8 and helper plasmids were co-transfected with AAV vectors expressing various ATP7B proteins. Figure 6 is a bar graph showing the rAAV titers produced by host cells after transfection with various AAV vectors. The Y axis indicates the total yield of each rAAV titer in genome copies (GC). The data show that ATP7BΔ1-3-SS has higher yields than full-length or truncated forms of ATP7BΔ1-4.

[0146] Example 3 - ATP7B Δ1-3-SS is more effective than ATP7B FL in restoring copper metabolism

[0147] This example describes experiments demonstrating that ATP7BΔ1-3-SS plays an important role in restoring C3He-Atp7b tx-j The mice metabolized copper more efficiently than ATP7B full-length (ATP7B FL) or ATP7BΔ1-4.

[0148] As described in Example 2, packaging large amounts of cDNA sequences within AAV vector capsids can reduce the integrity of the DNA sequence and have potential quality issues. Therefore, a truncated form of human ATP7B was packaged in an AAV8 capsid and tested for its efficacy in restoring copper metabolism. tx-j Mice were administered 1.0 x 10 13 GC / kg of AAV8 vector. Liver copper and urine copper levels were assessed by inductively coupled plasma-mass spectrometry. Figure 7 C3He-Atp7b tx-j Scatter plot of urinary copper and liver copper levels (μg / g) measured in mice injected with AAV8 carrying full-length human ATP7B (ATP7B FL), ATP7BΔ1-3-SS, or ATP7BΔ1-4, represented by squares and circles, respectively. Figure 7 showed that ATP7BΔ1-3-SS restored C3He-Atp7b tx-j C3He-Atp7b was more efficient than full-length ATP7B (ATP7B FL) or ATP7BΔ1-4 in copper metabolism in mice. tx-j Mice were used as control (vehicle).

[0149] Example 4 - AAV vectors containing AAV9 capsids show higher virus production

[0150] This example describes experiments showing that production of AAV vectors containing AAV9 capsids has higher yields compared to production of AAV vectors containing AAV8 capsids.Different AAV vectors were titrated by quantification of DNase-resistant particles (DRPs) by qPCR. Figure 8 Shown is the total yield of rAAV (titer of genome copies (GC)) produced from host cells after transfection of an AAV vector (DTC319) encoding a truncated human ATP7B, in which metal binding domains (MBDs) 1-3 are deleted, but a serine-rich loop containing two serine residues (S340 and S341) is present between MBD3 and MBD4, and co-transfection of plasmids encoding AAV8 capsid or AAV9 capsid.

[0151] Example 5 - Therapeutic properties of ATP7B Δ1-3-SS

[0152] This example describes animal studies demonstrating that ATP7BΔ1-3-SS (e.g., DTC319, an rAAV vector encoding a truncated human ATP7B lacking metal binding domains (MBDs) 1-3 but with a serine-rich loop containing two serine residues (S340 and S341) between MBD3 and MBD4) improves mouse models (C3He-Atp7b tx-j ) and for the treatment of Wilson's disease (WD). In this example, three groups of male mice were evaluated: WD mice (C3He-Atp7b) administered AAV infusions of AAV8 vectors encoding ATP7BΔ1-3-SS (e.g., DTC319) or intravenous injections of vehicle control (dilution buffer). tx-j Mice), and wild-type (WT) mice used as negative controls. For infusion, rAAV was produced by three transient transfections of adherent HEK cells and purified by cesium chloride gradient ultracentrifugation (a purification method well known in the art). At the study endpoint (4 weeks after infusion), liver copper accumulation, ceruloplasmin activity, and liver pathology were evaluated in each group of mice.

[0153] Hepatic copper accumulation was measured by inductively coupled plasma-mass spectrometry (ICP-MS) and demonstrated that hepatic copper levels were significantly reduced in WD mice administered ATP7BΔ1-3-SS (eg, DTC319) compared to vehicle controls (see Fig. 9 , bar graph of DelA). Fig. 9 Display of C3He-Atp7b tx-j Hepatic copper accumulation levels (μg / g) in mice after administration of intravenous vehicle control (bar graph of dilution buffer, WD) or infusion of AAV8 carrying native ATP7BΔ1-3-SS (bar graph of DelA). Hepatic copper accumulation levels in uninjected wild-type mice (WT) represented in this bar graph were used as negative controls. Values ​​are expressed as mean ± SEM.

[0154] After administration of ATP7BΔ1-3-SS (eg, DTC319), ceruloplasmin activity was significantly increased in WD mice (see Fig.10 , bar graph of DelA). Ceruloplasmin activity was detected using a colorimetric activity assay based on an enzyme reaction well known in the art (see Schosinsky et al., Clin Chem. 1974; 20(12): 1556-63). Fig.10 C3He-Atp7b after intravenous injection of vehicle control (bar graph for dilution buffer, WD) or AAV infusion of AAV8 vector encoding ATP7BΔ1-3-SS (bar graph for DelA) is shown. tx-jThe activity of ceruloplasmin in mice, as measured by a colorimetric activity assay based on an enzyme reaction. The activity of ceruloplasmin in uninjected wild-type (WT) mice, as measured by the same colorimetric activity assay based on an enzyme reaction, is also shown in this bar graph. The figure shows the activity of ceruloplasmin, as measured by the optical density OD read at 540 nm. Values ​​are expressed as mean ± SEM.

[0155] The livers were harvested from all animals in each group and stained with H&E (hematoxylin and eosin). H&E sections were evaluated for nuclear enlargement and hepatocyte hypertrophy, tissue destruction, inflammatory infiltration, and hepatocyte necrosis using a 0-4 scoring system by a board-certified pathologist. The scores for each mouse in a group were averaged. Fig.11 Shown are the mean scores obtained after standard evaluation of H&E sections from each group of animals.

[0156] Example 6 - AAV9 gene therapy as a viable treatment for Wilson's disease (WD)

[0157] This example describes the use of rAAV particles comprising ATP7BΔ1-3-SS to treat WD in a subject. An AAV vector containing a nucleotide sequence encoding a truncated human ATP7B (ATP7BΔ1-3-SS) is introduced into a DTC319 ( Figure 2 ), a Rep / Cap plasmid encoding four wild-type AAV2 viral replication (Rep) proteins and three wild-type AAVVP capsid (cap) proteins from serotype 9 (AAV9), and a helper plasmid as described in Example 1 are co-transfected into host cells. The harvested rAAV particles are then administered intravenously to a subject in need of WD treatment. Alternatively, the subject is administered with Fig.12 rAAV particles harvested from host cells transfected with the indicated vectors are used to treat WD.

[0158] Incorporated by Reference

[0159] The entire disclosure of each patent document and scientific article mentioned herein is incorporated by reference for all purposes.

[0160] Equivalent

[0161] Without departing from the spirit or essential features of the present disclosure, the present disclosure may be implemented in other specific forms. Therefore, the foregoing embodiments are considered to be illustrative in all aspects rather than limiting the disclosure described herein. The various structural elements of different embodiments and the various disclosed method steps may be used in various combinations and arrangements, and all such variations are considered to be forms of the present disclosure. Therefore, the scope of the present disclosure is indicated by the appended claims rather than by the foregoing description, and all changes within the equivalent meaning and scope of the claims are intended to be included therein.

Claims

1. A recombinant nucleic acid construct comprising: (a) 5'-inverted terminal repeat (ITR) sequence; (b) promoter sequence; (c) a nucleic acid sequence encoding a truncated human copper-transporting ATPase 2 (ATP7B) having an amino acid sequence as shown in SEQ ID NO: 8, wherein the metal binding domains (MBD) 1-3 are missing from the truncated human copper-transporting ATPase 2, but a serine-rich loop containing two serine residues S340 and S341 is present between MBD3 and MBD4; and (d) 3'-ITR sequence.

2. The recombinant nucleic acid construct according to claim 1, wherein the promoter sequence is selected from the group consisting of a thyroxine transporter (TTR) promoter sequence, a chicken β-actin (CBA) promoter sequence, a cytomegalovirus immediate early gene (CMV) promoter sequence, a thyroxine binding globulin (TBG) promoter sequence, an alpha-1 antitrypsin (A1AT) promoter sequence, and a CAG promoter sequence.

3. The recombinant nucleic acid construct according to claim 2, wherein the promoter sequence is a TTR promoter sequence.

4. The recombinant nucleic acid construct according to any one of claims 1 to 3, wherein the 5'-ITR sequence is from AAV2.

5. The recombinant nucleic acid construct according to any one of claims 1 to 3, wherein the 3'-ITR sequence is from AAV2.

6. The recombinant nucleic acid construct according to any one of claims 1 to 3, wherein the 5'-ITR sequence and the 3'-ITR sequence are from AAV2. The recombinant nucleic acid construct according to claim 6 , wherein the 5′-ITR sequence and the 3′-ITR sequence comprise SEQ ID NO:

2.

8. The recombinant nucleic acid construct according to any one of claims 1 to 3, wherein the 5'-ITR sequence and / or the 3'-ITR sequence is from a non-AAV2 source.

9. The recombinant nucleic acid construct according to any one of claims 1 to 3, wherein the recombinant nucleic acid construct further comprises one or more enhancer sequences.

10. The recombinant nucleic acid construct according to claim 9, wherein the enhancer sequence is selected from the group consisting of a thyroxine transporter enhancer (enTTR) sequence, a cytomegalovirus immediate early gene (CMV) enhancer sequence, a chicken β-actin (CBA) enhancer sequence, an En34 enhancer sequence, and an apolipoprotein ApoE enhancer sequence.

11. The recombinant nucleic acid construct according to claim 10, wherein the enhancer sequence is an enTTR enhancer sequence.

12. The recombinant nucleic acid construct according to claim 11, wherein the enhancer sequence comprises SEQ ID NO:

3.

13. The recombinant nucleic acid construct according to any one of claims 10-12, wherein the enhancer sequence is located upstream of the promoter sequence.

14. The recombinant nucleic acid construct according to any one of claims 1 to 3, wherein the recombinant nucleic acid construct further comprises one or more intron sequences.

15. The recombinant nucleic acid construct according to claim 14, wherein the intron sequence is selected from the group consisting of SV40 small T intron sequence, rabbit hemoglobin subunit β (rHBB) intron sequence, human β-globin IVS2 intron sequence, Promega chimeric intron sequence and hFIX intron sequence.

16. The recombinant nucleic acid construct according to claim 15, wherein the intron sequence is the SV40 small T intron sequence.

17. The recombinant nucleic acid construct according to claim 16, wherein the intron sequence comprises SEQ ID NO:

4.

18. The recombinant nucleic acid construct according to claim 15, wherein the intron sequence is a rHBB intron sequence.

19. The recombinant nucleic acid construct according to claim 18, wherein the intron sequence comprises SEQ ID NO:

5.

20. The recombinant nucleic acid construct according to any one of claims 1-3, wherein the recombinant nucleic acid construct further comprises a polyadenylation signal sequence.

21. The recombinant nucleic acid construct according to claim 20, wherein the polyadenylation signal sequence is selected from the group consisting of SV40 polyadenylation signal sequence, bovine growth hormone (BGH) polyadenylation signal sequence and rabbit β-globin polyadenylation signal sequence.

22. The recombinant nucleic acid construct according to claim 21, wherein the polyadenylation signal sequence is a bovine growth hormone (BGH) polyadenylation signal sequence.

23. The recombinant nucleic acid construct of claim 22, wherein the polyadenylation signal sequence comprises SEQ ID NO:

6.

24. The recombinant nucleic acid construct of claim 21, wherein the polyadenylation signal sequence is the SV40 polyadenylation signal sequence.

25. The recombinant nucleic acid construct of claim 24, wherein the polyadenylation signal sequence comprises SEQ ID NO:

7.

26. A recombinant adeno-associated virus (rAAV) for treating Wilson's disease, the rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising: a. AAV 5'-inverted terminal repeat (ITR) sequence; b. Promoter sequence; c. A nucleic acid sequence encoding a truncated human copper-transporting ATPase 2 (ATP7B) having an amino acid sequence as shown in SEQ ID NO: 8, wherein the metal binding domains (MBD) 1-3 are missing from the truncated human copper-transporting ATPase 2, but a serine-rich loop containing two serine residues S340 and S341 is present between MBD3 and MBD4; and d. AAV 3'-ITR sequence.

27. The rAAV of claim 26, wherein the AAV capsid is from an AAV serotype 9, 8, 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, rh10 or hu37.

28. The rAAV of claim 27, wherein the AAV capsid is from AAV9.

29. The rAAV of claim 27, wherein the AAV capsid is from AAV8.

30. The rAAV of claim 28, wherein the AAV capsid is an AAV9 variant capsid.

31. The rAAV according to any one of claims 26-30, wherein the promoter sequence is selected from the group consisting of a thyroxine transporter (TTR) promoter sequence, a chicken β-actin (CBA) promoter sequence, a cytomegalovirus immediate early gene (CMV) promoter sequence, a thyroxine binding globulin (TBG) promoter sequence, an alpha-1 antitrypsin (A1AT) promoter sequence, and a CAG promoter sequence.

32. The rAAV of claim 31 , wherein the promoter sequence is a TTR promoter sequence.

33. The rAAV according to any one of claims 26-30, wherein the 5'-ITR sequence is from AAV2.

34. The rAAV according to any one of claims 26-30, wherein the 3'-ITR sequence is from AAV2.

35. The rAAV according to any one of claims 26-30, wherein the 5'-ITR sequence and the 3'-ITR sequence are from AAV2.

36. The rAAV of any one of claims 26-30, wherein the 5'-ITR sequence and the 3'-ITR sequence comprise SEQ ID NO:

2.

37. The rAAV according to any one of claims 26-30, wherein the 5'-ITR sequence and / or the 3'-ITR sequence is from a non-AAV2 source.

38. The rAAV of any one of claims 26-30, wherein the packaged vector genome further comprises one or more enhancer sequences.

39. The rAAV of claim 38, wherein the packaged vector genome further comprises an enhancer sequence, and wherein the enhancer sequence is selected from the group consisting of a thyroxine transporter enhancer (enTTR) sequence, a cytomegalovirus immediate early gene (CMV) enhancer sequence, a chicken β-actin (CBA) enhancer sequence, an En34 enhancer sequence, and an apolipoprotein ApoE enhancer sequence.

40. The rAAV of claim 39, wherein the enhancer sequence is an enTTR enhancer sequence.

41. The rAAV of claim 40, wherein the enhancer sequence comprises SEQ ID NO:

3.

42. The rAAV according to any one of claims 40-41, wherein the enhancer sequence is located upstream of the promoter sequence.

43. The rAAV of any one of claims 26-30, wherein the packaged vector genome further comprises one or more intron sequences.

44. The rAAV according to claim 43, wherein the intron sequence is selected from the group consisting of SV40 small T intron sequence, rabbit hemoglobin subunit β (rHBB) intron sequence, human β-globin IVS2 intron sequence, Promega chimeric intron sequence and hFIX intron sequence.

45. The rAAV of claim 44, wherein the intron sequence is the SV40 small T intron sequence.

46. ​​The rAAV of claim 45, wherein the intron sequence comprises SEQ ID NO:

4.

47. The rAAV of claim 44, wherein the intron sequence is a rHBB intron sequence.

48. The rAAV of claim 47, wherein the intron sequence comprises SEQ ID NO:

5.

49. The rAAV of any one of claims 26-30, wherein the packaged vector genome further comprises a polyadenylation signal sequence.

50. The rAAV of claim 49, wherein the polyadenylation signal sequence is selected from the group consisting of an SV40 polyadenylation signal sequence, a bovine growth hormone (BGH) polyadenylation signal sequence, and a rabbit β-globin polyadenylation signal sequence.

51. The rAAV of claim 50, wherein the polyadenylation signal sequence is a bovine growth hormone (BGH) polyadenylation signal sequence.

52. The rAAV of claim 51, wherein the polyadenylation signal sequence comprises SEQ ID NO:

6.

53. The rAAV of claim 50, wherein the polyadenylation signal sequence is the SV40 polyadenylation signal sequence.

54. The rAAV of claim 53, wherein the polyadenylation signal sequence comprises SEQ ID NO:

7.

55. An rAAV comprising the recombinant nucleic acid construct of any one of claims 1-25.

56. A recombinant nucleic acid construct comprising: a. AAV 5'-inverted terminal repeat (ITR) sequence of SEQ ID NO: 2; b. the enhancer sequence of SEQ ID NO: 3; c. the promoter sequence of SEQ ID NO: 12; d. a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15 encoding a truncated human copper-transporting ATPase 2 (ATP7B) having the amino acid sequence shown in SEQ ID NO: 8; and e. AAV 3'-ITR sequence of SEQ ID NO:

2.

57. The recombinant nucleic acid construct of claim 56, wherein the nucleic acid sequence encoding truncated human copper-transporting ATPase 2 (ATP7B) is SEQ ID NO:

1.

58. The recombinant nucleic acid construct of claim 56, wherein the nucleic acid sequence encoding truncated human copper-transporting ATPase 2 (ATP7B) is SEQ ID NO:

15.

59. A rAAV comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising: a. AAV 5'-inverted terminal repeat (ITR) sequence of SEQ ID NO: 2; b. the enhancer sequence of SEQ ID NO: 3; c. the promoter sequence of SEQ ID NO: 12; d. a nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15 encoding a truncated human copper-transporting ATPase 2 (ATP7B) having the amino acid sequence shown in SEQ ID NO: 8; and e. AAV 3'-ITR sequence of SEQ ID NO:

2.

60. The rAAV of claim 59, wherein the packaged vector genome comprises a nucleic acid sequence encoding a truncated human copper-transporting ATPase 2 (ATP7B) of SEQ ID NO:

1.

61. The rAAV of claim 59, wherein the packaged vector genome comprises a nucleic acid sequence encoding a truncated human copper-transporting ATPase 2 (ATP7B) of SEQ ID NO:

15.

62. A host cell comprising a recombinant nucleic acid construct according to any one of claims 1-25 or 56-58 or a rAAV according to any one of claims 26-55 or 59-61.

63. A composition comprising the rAAV of any one of claims 26-55 or 59-61 and a pharmaceutically acceptable carrier.

64. A recombinant vector comprising a recombinant nucleic acid construct according to any one of claims 1-25 or 56-58.

65. The recombinant vector of claim 64, wherein the vector is an adeno-associated virus (AAV) vector.

66. The recombinant vector of claim 65, wherein the AAV vector is an AAV serotype 9 (AAV9) vector.

67. Use of a method for increasing rAAV production in the preparation of a medicament for treating Wilson's disease, comprising delivering a recombinant vector according to any one of claims 64 to 66 to a eukaryotic host cell culture, and harvesting the rAAV from the eukaryotic cell culture.

68. Use of the rAAV according to any one of claims 26-55 or 59-61 or the composition according to claim 63 in the preparation of a medicament for treating Wilson's disease in a human subject, the treatment comprising administering to the human subject a therapeutically effective amount of the rAAV or the composition thereof.

69. The use according to claim 68, wherein the medicament is for subcutaneous, intramuscular, intradermal, intraperitoneal or intravenous administration.

70. The use according to claim 69, wherein the medicament is for intravenous administration.

71. The recombinant nucleic acid construct of claim 1, the rAAV of claim 26, the composition of claim 63, the host cell of claim 62, the recombinant vector of claim 64, or the use of claim 67 or 68, wherein the nucleic acid sequence encoding the truncated human copper-transporting ATPase 2 (ATP7B) comprises SEQ ID NO: 1 or SEQ ID NO:

15.

72. A recombinant nucleic acid encoding a truncated human copper-transporting ATPase 2 (ATP7B) having an amino acid sequence as shown in SEQ ID NO:8, the recombinant nucleic acid comprising a nucleic acid sequence at least 80% identical to SEQ ID NO:

14.

73. The recombinant nucleic acid of claim 72, wherein the nucleic acid sequence comprises SEQ ID NO:14.

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