Synthetic hepatophilic adeno-associated virus capsid and uses thereof

Abstract

The invention relates to a synthetic adeno-associated virus capsid targeting liver and a virus vector containing the capsid. The invention further relates to methods of targeting the liver and providing liver-specific expression and transducing human primary hepatocytes and cell lines using the vectors.

Description

Priority declaration

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 683,868, filed on June 12, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to a synthetic adeno-associated virus capsid targeted to the liver and a viral vector comprising the capsid. The present invention further relates to a method for using the vector to target the liver and provide liver-specific expression and transduce human primary hepatocytes and cell lines. Background Art

[0003] Adeno-associated virus (AAV) is a promising in vivo gene therapy vector due to its efficient gene transfer and durable transgene expression in various tissues. AAV vectors have shown good safety and therapeutic effects in gene therapy clinical trials. Some new AAV serotypes are characterized by greatly improved transduction efficiency of the heart, muscle, and central nervous system (CNS) through systemic gene delivery. Others are effective in liver gene transfer in small animals. These findings provide inspiration for gene therapy of many genetic and metabolic diseases. In addition to identifying new serotypes, gene modification can further improve the clinical application of AAV vectors. One problem with the use of AAV vectors is that their broad tissue tropism often leads to gene transfer in unwanted tissues and may raise safety issues of ectopic gene expression. In order to improve the tissue specificity and tissue targeting ability of AAV virus particles, enhance capsid diversity and change the target tissue tropism of capsids, in addition to rational engineering design, random mutagenesis of capsid genes is one of the most effective methods. Directed evolution (such as DNA shuffling) is a recently developed method for genetic engineering of AAV capsid genes.

[0004] DNA shuffling is a random fragmentation method followed by PCR reorganization to introduce or exchange new genetic mutations into genes through patch homologous recombination. AAV naturally evolves into different serotypes with different tissue tropisms or immunogenicity. The AAV cap (capsid) genes of these serotypes are also ideal templates for generating new recombinants by shuffling their DNA and selecting functionally viable mutants and variants. The modified AAV cap genes are cloned into appropriate plasmid vectors to generate chimeric virus libraries with highly diverse biological properties and further characterize their tissue tropism.

[0005] After DNA shuffling, many in vitro systems based on cultured cell lines are used to select viral vectors. However, in gene therapy applications, AAV vectors must face a more complex physiological environment in humans or animals, such as a large number of serum proteins, potential neutralizing and non-neutralizing antibodies, endothelial barriers, direct tropism and infectivity for target cells, etc. Often, modified vectors that show hepatotropism in in vitro testing do not adequately target the liver when used in vivo.

[0006] Therefore, there is a need in the art for AAV vectors with improved hepatotropism that provide benefits for in vivo use. Summary of the invention

[0007] The present invention is based in part on the use of the following scheme: the cap genes of different natural AAV serotypes are significantly diversified through in vitro recombination using DNA shuffling. The resulting AAV capsid gene library containing a wide range of mutants and variants is first screened directly in mice. Given the obstacles to identifying in vivo vector effectiveness, AAV library screening is performed directly in the liver of C57BL / 6J mice to simulate the real in vivo environment in preclinical or clinical situations. Capsid genes that are initially enriched on cultured cells are then selected to further select novel AAV capsids with cell type and tissue tropism, including hepatotropic capsids. We found that a capsid gene is highly enriched in mouse liver and is also highly infective to human hepatoma cell lines and primary human hepatocytes.

[0008] Therefore, one aspect of the present invention relates to a nucleic acid encoding an AAV capsid, which comprises an AAV capsid coding sequence that is at least 90% identical to the following sequences: (a) a nucleotide sequence of any one of SEQ ID NOS: 1-3; or (b) a nucleotide sequence encoding any one of SEQ ID NOS: 4-6, as well as cells and viral particles comprising the nucleic acid.

[0009] Another aspect of the invention relates to an AAV capsid comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 4-6, and an AAV particle comprising an AAV vector genome and an AAV capsid of the invention.

[0010] Another aspect of the present invention relates to a method for producing recombinant AAV particles comprising an AAV capsid, the method comprising: providing cells in vitro with a nucleic acid of the present invention, an AAV rep coding sequence, an AAV vector genome comprising a heterologous nucleic acid, and an auxiliary function body for producing productive AAV infection; assembling recombinant AAV particles comprising the AAV capsid and encapsidating the AAV vector genome.

[0011] Another aspect of the invention relates to a pharmaceutical formulation comprising a nucleic acid, viral particle, AAV capsid or AAV particle of the invention in a pharmaceutically acceptable carrier.

[0012] Another aspect of the invention relates to a method of delivering a target nucleic acid to a hepatocyte, the method comprising contacting the cell with an AAV particle of the invention.

[0013] Another aspect of the invention relates to a method of delivering a target nucleic acid to a hepatocyte of a mammalian subject, the method comprising administering to the mammalian subject an effective amount of an AAV particle or a pharmaceutical formulation of the invention.

[0014] Another aspect of the invention relates to a method of treating a disease in a mammalian subject in need thereof, wherein the disease can be treated by expressing a product in the liver of the subject, the method comprising administering to the mammalian subject a therapeutically effective amount of an AAV particle or pharmaceutical formulation of the invention.

[0015] These and other aspects of the present invention are described in more detail below in the detailed description of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A-1B Figure 2 shows an in vivo comparison of the hepatic gene transfer efficiency of the hepatotropic AAV capsids XL12 and XL32 with that of AAV9. LacZ and GFP reporter vectors were packaged in AAV9, AAVXL12, and AAVXL32. These vectors were purified, titrated, and injected into adult C57B / 6 mice via the tail vein at a dose of 5x10 12 vg (vector genome) / kg body weight. Mice were killed 3 weeks later, and liver thin sections were stained with X-gal to detect LacZ expression ( Figure 1A ); or directly install and take fluorescent photos to detect GFP expression ( Figure 1B ).

[0017] Figure 2 Figure 2 shows the in vitro comparison of gene transfer efficiency of hepatotropic AAV capsids XL12 and XL32 and AAV9 on the human hepatoma Huh7 cell line. The LacZ reporter vectors packaged in AAV9, XL12, and XL32 were infected at a multiplicity of infection of 1x10 5 vg / cell and 1x10 4 Huh7 cells were transduced with 5% vg / cell (moi). Three days later, X-gal staining was performed to detect LacZ expression.

[0018] Figures 3A-3DThe results show a comparison of the gene transfer efficiency of hepatotropic AAV capsids XL12 and XL32 with other serotypes of AAV in human primary hepatocyte cultures in vitro. The GFP reporter vector was packaged in AAVXL12, AAVXL32, and multiple other commonly used serotype capsids. The AAV-GFP vector was transfected at a multiplicity of infection (MOI) of 1x10 5 vg / cells infected with human primary hepatocyte cultures. Green fluorescence photos were taken at 24 hours and 48 hours or every day (up to 7 days). (A) GFP expression after 24 hours of infection of human primary hepatocytes from three donors with AAV-GFP vectors. (B) GFP expression after 48 hours of infection. (C) AAVXL12 and AAVXL32 GFP gene expression after 1 to 7 days of infection of hepatocytes from donor patient #4021. (D) AAVXL12 and AAVXL32 GFP gene expression after 1 to 7 days of infection of hepatocytes from donor patient #4073. Efficient and increased expression of the GFP gene was observed.

[0019] Figures 4A-4C The results show a comparison of the gene transfer efficiency of hepatotropic AAV capsids XL12 and XL32 with other AAV serotypes in canine primary hepatocyte cultures in vitro. The GFP reporter vector was packaged in AAVXL12, AAVXL32, and multiple other commonly used serotype capsids. The AAV-GFP vector was transfected at a multiplicity of infection (MOI) of 1x10 5 vg / cells infected with canine primary hepatocyte cultures. Green fluorescence images were taken at 24 and 48 hours. (A) GFP expression in cryopreserved primary hepatocytes (DogDBCP01) at 24 and 48 hours. (B) GFP expression in freshly isolated primary hepatocytes (Dog DBF24) at 24 hours. (C) GFP expression in freshly isolated primary hepatocytes (Dog DBF24) at 48 hours. Efficient and increased expression of the GFP gene was observed in cells infected with AAV6, AAVXL12, and AAVXL32.

[0020] Figure 5 A comparison of the transduction efficiency of AAVXL32 and AAV8 in the liver of adult macaques via intravenous vector delivery is shown. AAV vectors were injected intravenously into adult male rhesus macaques aged 3-5 years with serum neutralizing antibody titers less than 1:8. Two macaques were injected with AAVXL32-CB-GFP and the other two macaques were injected with AAV-CB-GFP at a dose of 1x10 12 vg / kg body weight. The AAV vector expression cassette is in a double-stranded (also called self-complementary) form, which allows for faster and more efficient gene expression. The macaques were euthanized two weeks later, and liver tissue was extracted for frozen sections and green fluorescence photography. Some GFP-positive hepatocytes are highlighted with arrows.

[0021] Figure 6 Protein gel analysis of AAVXL32 and AAVXL32.1 capsids is shown. Highly purified AAVXL32 and AAVXL32.1 were analyzed at a concentration of 1x10 11 The particles were loaded, analyzed on SDS-PAGE gels, and visualized by silver staining. Arrows highlight the typical AAV capsid proteins VP1, VP2, and VP3. Arrows highlight the additional capsid protein VPX in AAVXL32, which is likely due to the weaker start codon generated by the CTC to CTG mutation. Reverse mutation of CTG to CTC eliminated this additional protein in AAVXL32.1. DETAILED DESCRIPTION

[0022] The present invention is based in part on the development of a synthetic AAV capsid sequence that is capable of transducing hepatocytes in vivo and in vitro. This synthetic capsid can be used to generate AAV vectors for research or therapeutic applications that require liver-specific gene transfer without the need for broad biodistribution of the vector to other organs.

[0023] The present invention is described in more detail below. These descriptions are not intended to be an exhaustive list of all the different ways in which the present invention may be implemented or all the features that may be incorporated into the present invention. For example, features discussed in one embodiment may be adopted in other embodiments, and features illustrated in a particular embodiment may also be omitted from that embodiment. In addition, many variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art, in accordance with the present invention, and they do not depart from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively specify all permutations, combinations, and variations thereof.

[0024] Unless the context otherwise states, it should be specifically noted that the various features of the invention described herein may be used in any combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. For example, if the specification states that a composite comprises components A, B, and C, it should be specifically noted that any of the components A, B, or C, or a combination thereof, may be omitted and abandoned individually or in any combination.

[0025] Unless otherwise specified, the meanings of all terms (including technical terms and scientific terms) used in the present invention are the same as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the present invention description are only for describing specific embodiments and are not intended to limit the present invention.

[0026] Unless otherwise specified, nucleotide sequences herein are shown as single strands from left to right in the 5' to 3' direction. Nucleotides and amino acids are indicated herein in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission, or (amino acids) in accordance with 37 CFR §1.822 and customary usage using single-letter or three-letter codes.

[0027] Unless otherwise indicated, standard methods familiar to those skilled in the art can be used to prepare recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, manipulate nucleic acid sequences, generate transformed cells, construct rAAV constructs, modified capsid proteins, packaging vectors expressing AAV rep and / or cap sequences, and transient and stable transfected packaging cells. These techniques are familiar to those skilled in the art. See, for example, SAMBROOK et al., MOLECULAR CLONING: ALABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); FMAUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0028] All publications, patent applications, nucleotide sequences, amino acid sequences, and other references mentioned herein are incorporated by reference in their entirety. I. definition

[0029] Throughout the present specification and appended claims, all amino acid positions in AAV capsid subunits are designated relative to the VP1 capsid subunit numbering.

[0030] Throughout the description of the invention and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0031] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as combinations set forth in the absence of alternatives ("or").

[0032] Furthermore, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted.

[0033] In addition, the word "about" used in reference to a measurable value, such as an amount of a compound or agent of the invention, dosage, time, temperature, etc., is meant to include a range of variation of ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the stated value.

[0034] As used herein, the phrase "consisting essentially of" in connection with a nucleic acid, protein, or capsid structure means that the nucleic acid, protein, or capsid structure does not contain any elements other than the listed elements that significantly alter (e.g., greater than about 1%, 5%, or 10%) the function of the target nucleic acid, protein, or capsid structure (e.g., the tropism of the protein or capsid or the nucleic acid encoding the protein or capsid).

[0035] As described herein, in the context of the present invention, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY (Vol. 2, Chapter 69) (4th Edition, Lippincott-Raven Publishers). A variety of other AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virol. 78:6381-6388 and Table 1), which are also included in the term "AAV".

[0036] The genomic sequences of various AAVs and autonomous parvoviruses, as well as the sequences of ITRs, Rep proteins and capsid subunits are familiar in the art. Such sequences can be found in the literature or in public databases such as See, for example, Accession No. NC 002077, NC 001401, NC 001729, NC 001863, NC 001829, NC001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC001358, NC 001540, AF513851, AF513852, AY530579, AY631965, AY631966; the disclosures of which are incorporated herein in their entirety. See also, e.g., Srivistava et al., (1983) J. Virol. 45:555; Chiorini et al., (1998) J. Virol. 71:6823; Chiorini et al., (1999) J. Virol. 73:1309; Bantel-Schaal et al., (1999) J. Virol. 73:939; Xiao et al., (1999) J. Virol. 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; International Patent Publication Nos. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; U.S. Patent No. 6,156,303; the disclosures of which are incorporated herein in their entirety. See also Table 1. An early description of the terminal repeat sequences of AAV1, AAV2, and AAV3 is found in Xiao, X., (1996) "Characterization of Adeno-associated virus (AAV) DNA replication and integration", PhD thesis, University of Pittsburgh, Pittsburgh, Pennsylvania (incorporated herein in its entirety).

[0037] A "chimeric" AAV nucleic acid capsid coding sequence or AAV capsid protein is a sequence or protein that combines portions of two or more capsid sequences. A "chimeric" AAV virion or particle comprises a chimeric AAV capsid protein.

[0038] As used herein, the term "tropism" refers to the preferential entry of a virus into certain types of cells or tissues and / or preferential interaction with the cell surface, promoting entry into certain types of cells or tissues, and optionally and preferably subsequently expressing (e.g., transcribing and (optionally) translating) sequences carried by the viral genome in the cell, e.g., expression of heterologous nucleotide sequences for recombinant viruses). It will be appreciated by those skilled in the art that in the absence of trans-acting factors, for example, for inducible promoters or other regulatory nucleic acid sequences, transcription of heterologous nucleic acid sequences from the viral genome may not be initiated. In the case of rAAV genomes, gene expression of the viral genome can be from stably integrated proviruses and / or from non-integrated episomes, as well as any other form that the viral nucleic acid can take within the cell. Table 1

[0039] The term "tropism pattern" refers to the pattern of transduction of one or more target cells, tissues and / or organs. Representative examples of synthetic AAV capsids have a tropism pattern characterized by efficient hepatocyte transduction and low transduction of other organs.

[0040] As used herein, the phrase "hepatocyte specific" refers to a viral vector that, when administered in vivo, preferentially transduces hepatocytes, while transduction of extrahepatic cells is very minimal. In some embodiments, at least about 80% of the transduced cells are hepatocytes, for example, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the transduced cells are hepatocytes.

[0041] As used herein, the phrase "a disease treatable by expression of a product in the liver" refers to a disease, disorder or injury in which expression of a product (eg, a protein or polynucleotide) in the liver provides effective treatment or prevention.

[0042] As used herein, "transduction" of cells by the viral vector (e.g., AAV vector) refers to entry of the vector into the cell and transfer of genetic material into the cell by incorporating nucleic acid into the viral vector, which is then transferred into the cell by the viral vector.

[0043] Unless otherwise indicated, "efficient transduction" or "efficient tropism" or similar terms can be determined by reference to an appropriate positive or negative control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism of a positive control, respectively, or at least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of the transduction or tropism of a negative control, respectively).

[0044] Similarly, whether a virus "does not efficiently transduce" or "does not have effective tropism" for a target tissue, or similar terms, can be determined by reference to an appropriate control. In certain embodiments, the viral vector does not efficiently transduce (i.e., does not have effective tropism for) extrahepatic tissues, e.g., muscle, kidney, gonads, and / or germ cells. In certain embodiments, transduction of an undesirable tissue (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of transduction of a desired target tissue (e.g., hepatocytes).

[0045] Unless otherwise indicated, as used herein, the term "polypeptide" includes peptides and proteins.

[0046] A "nucleic acid" or "nucleotide sequence" is a sequence of nucleotide bases, which may be RNA, DNA, or a DNA-RNA hybrid sequence (including natural and non-natural nucleotides), but is preferably a single-stranded or double-stranded DNA sequence.

[0047] As used herein, the "isolated" nucleic acid or nucleotide sequence (e.g., "isolated DNA" or "isolated RNA") refers to a nucleic acid or nucleotide that is separated from at least some other components of a naturally occurring organism or virus or is substantially free of at least some other components of a naturally occurring organism or virus, such as cellular or viral structural components or other polypeptides or nucleic acids that are normally found in association with the nucleic acid or nucleotide sequence.

[0048] Likewise, an "isolated" polypeptide refers to a polypeptide that is separated from or substantially free of at least some other components of the native organism or virus (e.g., cellular or viral structural components or other polypeptides or nucleic acids with which the polypeptide is normally found).

[0049] The term "treating" (or its grammatical equivalent) refers to reducing or at least partially improving or ameliorating the severity of a subject's disease and / or alleviating, alleviating or reducing at least one clinical symptom and / or delaying the progression of the disease and / or preventing or delaying the onset of a disease or condition.

[0050] As used herein, the term "prevention" (and its grammatical equivalents) refers to delaying the onset of a disease or condition or alleviating symptoms after the onset of a disease or condition. The term does not refer to complete elimination of the disease, and includes any type of preventive treatment that reduces the incidence of the disease or delays the onset and / or development of the disease.

[0051] As used herein, an "effective" or "therapeutically effective" dose is a dose sufficient to provide some improvement or benefit to a subject. Alternatively, an "effective" or "therapeutically effective" dose is a dose that alleviates, relieves, or reduces at least one clinical symptom of a subject. It will be appreciated by those skilled in the art that the therapeutic effect need not be comprehensive or curative, as long as some benefit is provided to the subject.

[0052] A "heterologous nucleotide sequence" or "heterologous nucleic acid" is a sequence that does not naturally occur in a virus. Typically, a heterologous nucleic acid or nucleotide sequence comprises an open reading frame encoding a polypeptide and / or a non-translated RNA.

[0053] A "therapeutic polypeptide" is a polypeptide that can alleviate or reduce symptoms caused by protein deficiency or defect in cells or subjects. In addition, a "therapeutic polypeptide" is a polypeptide that brings benefits to subjects (e.g., anticancer effects or improved transplant survival).

[0054] As used herein, the words "vector", "viral vector", "delivery vector" (and similar words) generally refer to viral particles that act as nucleic acid delivery vectors and contain viral nucleic acids (i.e., vector genomes) packaged in virions. The viral vectors of the invention comprise synthetic AAV capsids of the invention and can package AAV or rAAV genomes or any other nucleic acids (including viral nucleic acids). Alternatively, in some cases, the words "vector", "viral vector", "delivery vector" (and similar words) can be used to refer to vector genomes (e.g., vDNA) in the absence of virions and / or viral capsids that act as transporters for delivering molecules bound or packaged in capsids.

[0055] A "recombinant AAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) comprising at least one inverted terminal repeat (e.g., one, two, or three inverted terminal repeats) and one or more heterologous nucleotide sequences. rAAV vectors typically retain 145 cis-base terminal repeats (TRs) to produce viruses; however, modified AAV TRs and non-AAV TRs including partial or complete synthetic sequences can also achieve this purpose. All other viral sequences are optional and may be provided in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158: 97). rAAV vectors optionally include two TRs (e.g., AAV TRs), which will typically be at the 5' and 3' ends of the heterologous nucleotide sequence, but not necessarily adjacent to it. These TRs may be identical or different from each other. The vector genome may also include an ITR at its 3' or 5' end.

[0056] The term "terminal repeat" or "TR" includes any viral terminal repeat sequence or synthetic sequence that forms a hairpin structure and acts as an inverted terminal repeat sequence (i.e., mediates the desired function, such as replication, viral packaging, integration and / or provirus rescue, etc.). TR can be AAV TR or non-AAV TR. For example, non-AAV TR sequences, such as sequences of other parvoviruses (e.g., canine parvovirus (CPV), murine parvovirus (MVM), human parvovirus B-19), or SV40 hairpins that serve as SV40 replication sources can be used as TRs, which can be further modified by truncation, substitution, deletion, insertion and / or addition. In addition, TRs can be partially or fully synthetic, for example, the "double-D sequence" described in U.S. Pat. No. 5,478,745 to Samulski et al.

[0057] "AAV terminal repeats" or "AAV TRs" can be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 11, or any other AAV now known or later discovered (see, e.g., Table 1). The AAV terminal repeat sequences need not possess native terminal repeat sequences (e.g., native AAV TR sequences may be altered by insertions, deletions, truncations, and / or missense mutations), as long as the terminal repeat sequences mediate the desired function, e.g., replication, viral packaging, integration, and / or proviral rescue, etc.

[0058] The terms "rAAV particle" and "rAAV virion" are used interchangeably herein. An "rAAV particle" or "rAAV virion" comprises an rAAV vector genome packaged within an AAV capsid.

[0059] AAV capsid structure is described in more detail in BERNARD N. FIELDS et al., VIROLOGY, (Vol. 2, Chapters 69 & 70) (4th ed., Lippincott-Raven Publishers).

[0060] "Substantially retaining" a property means retaining at least about 75%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of the property (eg, activity or other measurable characteristic). Synthetic AAV capsids targeted to the liver

[0061] The inventors have identified synthetic AAV capsid structures that are capable of transducing human and other mammalian hepatocytes in vivo and in vitro but have very low tropism for other organs. Thus, one aspect of the present invention relates to synthetic AAV capsid structures that are capable of providing liver gene transfer in a subject (e.g., a wild-type subject). In some embodiments, the present invention relates to a nucleic acid encoding an AAV capsid, the nucleic acid comprising, consisting essentially of, or consisting of an AAV capsid coding sequence that is at least 90% identical to the following sequence: (a) a nucleotide sequence of any one of SEQ ID NOS: 1-3; or (b) a nucleotide sequence encoding any one of SEQ ID NOS: 4-6; and a virus comprising a chimeric AAV capsid. In some embodiments, the AAV capsid coding sequence is at least 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%, or 99.9% identical to the nucleotide sequence of (a) or (b). In another embodiment, the AAV capsid coding sequence comprises, consists essentially of, or consists of the nucleotide sequence of (a) or (b).

[0062] SEQ ID NOS:4-6 show the VP1 capsid protein sequence. In the present specification and the appended claims, all amino acid position designations are relative to the VP1 numbering. It will be appreciated by those skilled in the art that the AAV capsid generally also contains smaller VP2 and VP3 capsid proteins. Due to the overlap in the coding sequences of the AAV capsid proteins, the nucleic acid coding sequences and amino acid sequences of the VP2 and VP3 capsid proteins will be apparent from the VP1 sequences shown in the public sequences. In particular, VP2 begins at nucleotide 412 (acg) of SEQ ID NO:1 and threonine 138 of SEQ ID NO:4. VP3 begins at nucleotide 610 (atg) of SEQ ID NO:1 and methionine 204 of SEQ ID NO:4. In some embodiments, isolated VP2 and VP3 capsid proteins comprising sequences from SEQ ID NOS:4-6 and isolated nucleic acids encoding VP2 or VP3 proteins or both are contemplated.

[0063] The present invention also provides synthetic AAV capsid proteins and synthetic capsids, the capsids comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 4-6. In some embodiments, the AAV capsid sequence is at least 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% or 99.9% identical to any one of SEQ ID NOS: 4-6. In some embodiments, the capsid protein comprises, consists essentially of, or consists of an amino acid sequence as shown in SEQ ID NOS: 4-6, wherein SEQ ID 1, 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 12 or less, 15 or less, 20 or less, 25 or less, 30 or less, 40 or less or 50 or less amino acids in the capsid protein coding sequence of NOS: 4-6 are substituted by another (natural, modified and / or synthetic) amino acid (optionally conservative amino acid substitutions), and / or are deleted and / or 1, 2 or less, 3 or less amino acids are present In some embodiments, the capsid protein coding sequence of SEQ ID NOS: 4-6 is substituted with another amino acid at position 220 and / or 643 (numbered relative to SEQ ID NO: 4). For example, in representative embodiments of the present invention, the AAV virion comprising the synthetic capsid protein substantially retains at least one characteristic of the synthetic virion comprising the synthetic capsid protein set forth in SEQ ID NOS: 4-6. For example, virions comprising synthetic capsid proteins can substantially retain the hepatotropism of virions comprising synthetic AAV capsid proteins as set forth in SEQ ID NOS: 4-6. Methods for assessing biological properties such as viral transduction are well known in the art (see, e.g., Examples).

[0064] Conservative amino acid substitutions are familiar in the art. In a particular embodiment, conservative amino acid substitutions include substitutions within one or more of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and / or phenylalanine, tyrosine.

[0065] It will be apparent to those skilled in the art that the amino acid sequences of the chimeric AAV capsid proteins of SEQ ID NOS: 4-6 can be further modified to incorporate other modifications familiar in the art to impart desired properties. As a non-limiting possibility, the capsid protein can be modified to incorporate a targeting sequence (e.g., RGD) or a sequence that facilitates purification and / or detection. For example, the capsid protein can be fused to all or part of a glutathione-S-transferase, a maltose binding protein, a heparin / heparan sulfate binding domain, a polyhistidine, a ligand and / or a reporter protein (e.g., green fluorescent protein, β-glucuronidase, β-galactosidase, luciferase, etc.), an immunoglobulin Fc fragment, a single-chain antibody, a hemagglutinin, a c-myc, a FLAG epitope, etc., to form a fusion protein. Methods for inserting targeting peptides into AAV capsids are familiar in the art (see, e.g., International Patent Publication No. WO 00 / 28004; Nicklin et al., (2001) Mol. Ther. 474-181; White et al., (2004) Circulation 109:513-319; Muller et al., (2003) Nature Biotech. 21:1040-1046).

[0066] The viruses of the present invention may further comprise a double-stranded viral genome as described in International Patent Publication No. WO 01 / 92551 and US Pat. No. 7,465,583.

[0067] The present invention also provides an AAV capsid comprising a synthetic AAV capsid protein of the present invention and a viral particle (i.e., a virion) comprising the AAV capsid, wherein the viral particle packages (i.e., encapsidates) a vector genome, optionally an AAV vector genome. In a specific embodiment, the present invention provides an AAV particle comprising an AAV capsid, wherein the AAV capsid comprises an AAV capsid protein of the present invention, wherein the AAV capsid packages an AAV vector genome. The present invention also provides an AAV particle comprising an AAV capsid or an AAV capsid protein encoded by a synthetic nucleic acid capsid coding sequence of the present invention.

[0068] In a particular embodiment, the virion is a recombinant vector comprising a target heterologous nucleic acid, for example for delivery to a cell. Thus, the present invention can be used for delivery of nucleic acids to cells in vitro, in vitro, and in vivo. In a representative embodiment, the recombinant vector of the present invention can be advantageously used for delivery or transfer of nucleic acids to animal (e.g., mammalian) cells.

[0069] Any heterologous nucleotide sequence can be delivered using the viral vectors of the invention. Target nucleic acids include nucleic acids encoding polypeptides, optionally therapeutic (eg, for medical or veterinary use) and / or immunogenic (eg, for vaccines) polypeptides.

[0070] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator (CFTR), dystrophin (including protein products of dystrophin minigenes or minigenes, see, e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Publication No. 2003017131; Wang et al., (2000) Proc. Natl. Acad. Sci. USA 97:13714-9, [mini-dystrophin]; Harper et al., (2002) Nature Genetics 5:13714-9, [mini-dystrophin]; Harper et al., (2002) Nature Genetics 5:13714-9, [mini-dystrophin]; Med.8:253-61 [micro-dystrophin]); mini-aggregin, laminin-α2, sarcoglycan (α, β, γ or δ), fukutin-related protein, myostatin propeptide, follistatin, dominant negative myostatin, angiogenic factors (e.g., VEGF, angiopoietin-1 or 2), anti-apoptotic factors (e.g., heme oxygenase-1, TGF-β, inhibitors of pro-apoptotic signals (e.g., caspases), proteases, kinases, death receptors [e.g., CD-095], regulators of cytochrome c release, inhibitors of mitochondrial pore opening and swelling); activin type II soluble receptor, anti-inflammatory peptides (e.g., Ikappa B dominant mutant), sarcospan, utrophin, mini-utrophin, antibodies or antibody fragments against myostatin or myostatin propeptide, cell cycle regulators, Rho kinase regulators (such as Cethrin, which is a modified bacterial C3 exoenzyme [provided by BioAxone Therapeutics, Inc., Saint-Laurent, Quebec, Canada], BCL-xL, BCL2, XIAP, FLICEc-s, dominant negative caspase-8, dominant negative caspase-9, SPI-6 (see, e.g., U.S. Patent Application No.20070026076), transcription factor PGC-α1, Pinch gene, ILK gene and thymosin β4 gene), coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, intracellular and / or extracellular superoxide dismutase, leptin, LDL receptor, enkephalinase, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α1-antitrypsin, methylcytosine binding protein 2, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, Lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., interferon-α, interferon-β, interferon-γ, interleukin-1 to -14, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors and hormones (e.g., growth hormone, insulin, insulin-like growth factor (including IGF-1 and IGF-2), GLP-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factors-3 and -4, brain-derived neurotrophic factor, glial-derived growth factor, transforming growth factor-α and -β, etc.), Bone morphogenetic proteins (including RANKL and VEGF), lysosomal proteins, glutamate receptors, lymphokines, soluble CD4, Fc receptors, T cell receptors, ApoE, ApoC, inhibitor of protein phosphatase inhibitor 1 (I-1), phospholamban, serca2a, lysosomal acid α-glucosidase, α-galactosidase A, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), calsarcin, receptors (e.g., soluble tumor necrosis growth factor-α receptor), anti-inflammatory factors (e.g., IRAP, Pim-1, PG C-1α, SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-β4), hypoxia-inducible transcription factor [HIF], angiogenic factor, S100A1, parvalbumin, adenylate cyclase type 6, molecules that affect knockdown of G-protein coupled receptor type 2 kinase, such as truncated constitutively active bARKct; phospholamban inhibitory or dominant negative molecules, such as phospholamban S16E, monoclonal antibodies (including single chain monoclonal antibodies) or suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin and tumor necrosis factor such as TNF-α), and any other polypeptides that have a therapeutic effect on a subject in need thereof. .

[0071] The heterologous nucleotide sequence of the coded polypeptide includes those sequences encoding reporter polypeptide (for example, enzyme).Reporter polypeptide is familiar in the art, including but not limited to fluorescent protein (for example, EGFP, GFP, RFP, BFP, YFP or dsRED2), enzymes producing detectable products, such as luciferase (for example, Gaussia, Renilla or Photinus supply), β-galactosidase, β-glucuronidase, alkaline phosphatase and chloramphenicol acetyltransferase gene, or proteins that can be directly detected.In fact, any protein can be directly detected by using specific antibodies such as the protein.Other markers suitable for positive or negative eukaryotic cell selection (and related antibiotics) are disclosed in Sambrook and Russell (2001), Molecular Cloning, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al. (1992), Current Protocols in Molecular Biology, John Wiley & Sons, including regular updates.

[0072] Alternatively, the heterologous nucleic acid may encode a functional RNA, such as an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that affects spliceosome-mediated trans-splicing (see Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), an interfering RNA (RNAi), including a small interfering RNA (siRNA) that mediates gene silencing (see Sharp et al., (2000) Science 287:2431), a microRNA, or other non-translated "functional" RNA, such as a "guide" RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; US Patent No. 5,869,248 to Yuan et al.), etc. Exemplary untranslated RNAs include RNAi or antisense RNAs to multidrug resistance (MDR) gene products (e.g., for treating tumors and / or for administration to the heart to prevent damage caused by chemotherapy), RNAi or antisense RNAs to myostatin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNAs to VEGF or tumor immunogens, including but not limited to those tumor immunogens specifically described herein (for treating tumors), RNAi or antisense oligonucleotides targeting mutant dystrophin (Duchenne or Becker muscular dystrophy), RNAi or antisense RNAs to hepatitis B surface antigen genes (preventing and / or treating hepatitis B infection), RNAi or antisense RNAs to HIV tat and / or rev genes (preventing and / or treating HIV), and / or RNAi or antisense RNAs to any other immunogens of pathogens (to protect subjects from pathogen infection) or defective gene products (to prevent or treat disease). RNAi or antisense RNAs to the above targets or any other targets can also be used as research reagents.

[0073] As is familiar in the art, antisense nucleic acid (e.g., DNA or RNA) and inhibitory RNA (e.g., microRNA and RNAi, such as siRNA or shRNA) sequences can be used to induce "exon skipping" in patients with muscular dystrophy caused by defects in the dystrophin gene. Thus, the heterologous nucleic acid can encode an antisense nucleic acid or inhibitory RNA that induces appropriate exon skipping. It will be appreciated by those skilled in the art that the specific method of exon skipping depends on the nature of the potential defect in the dystrophin gene, and many such schemes are known in the art. Exemplary antisense nucleic acid and inhibitory RNA sequences target upstream branch points and / or downstream donor splice sites and / or internal splicing enhancer sequences of one or more dystrophin exons (e.g., exon 19 or 23). For example, in a particular embodiment, the heterologous nucleic acid encodes an antisense nucleic acid or inhibitory RNA directed to the upstream branch point and downstream splice donor site of exon 19 or 23 of the dystrophin gene. These sequences can be incorporated into AAV vectors that deliver modified U7 snRNA and antisense or inhibitory RNA (see, e.g., Goyenvalle et al., (2004) Science 306: 1796-1799). As another strategy, modified U1 snRNA and siRNA, microRNA or antisense RNA complementary to upstream and downstream splice sites of dystrophin exons (e.g., exon 19 or 23) can be incorporated into AAV vectors (see, e.g., Denti et al., (2006) Proc. Nat. Acad. Sci. USA 103: 3758-3763). In addition, antisense and inhibitory RNAs can target splicing enhancer sequences within exons 19, 43, 45 or 53 (see, e.g., U.S. Pat. No. 6,653,467; U.S. Pat. No. 6,727,355; and U.S. Pat. No. 6,653,466).

[0074] Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim et al., (1987) Proc. Natl. Acad. Sci. USA 84:8788; Gerlach et al., (1987) Nature 328:802; Forster and Symons, (1987) Cell 49:211). For example, a large number of ribozymes promote phosphate transfer reactions with high specificity, typically cleaving only one of several phosphates in an oligonucleotide substrate (Michel and Westhof, (1990) J. Mol. Biol. 216:585; Reinhold-Hurek and Shub, (1992) Nature 357:173). This specificity is attributed to the requirement that the substrate bind to the internal guide sequence ("IGS") of the ribozyme through specific base pairing interactions prior to the chemical reaction.

[0075] It has been observed that ribozyme catalysis is primarily a part of sequence-specific cleavage / ligation reactions involving nucleic acids (Joyce, (1989) Nature 338:217). For example, according to U.S. Pat. No. 5,354,855, certain ribozymes can act as endonucleases with sequence specificity greater than that of known ribonucleases and approaching that of DNA restriction enzymes. Therefore, sequence-specific ribozyme-mediated inhibition of nucleic acid expression may be particularly suitable for therapeutic applications (Scanlon et al., (1991) Proc. Natl. Acad. Sci. USA 88:10591; Sarver et al., (1990) Science 247:1222; Sioud et al., (1992) J. Mol. Biol. 223:831).

[0076] MicroRNA (mir) is a natural cell RNA molecule that can regulate the expression of multiple genes by controlling the stability of mRNA. Overexpression or reduction of specific microRNA can be used to treat dysfunction and has been shown to be effective in many diseases and disease animal models (see, for example, Couzin, (2008) Science 319: 1782-4). Chimeric AAV can be used to deliver microRNA to cells, tissues and subjects to treat genetic diseases and acquired diseases, or to enhance function and promote the growth of certain tissues. For example, mir-1, mir-133, mir-206 and / or mir-208 can be used to treat heart and skeletal muscle diseases (see, for example, Chen et al., (2006) Genet.38: 228-33; van Rooij et al., (2008) Trends Genet.24: 159-66). MicroRNA can also be used to regulate the immune system after gene delivery (Brown et al., (2007) Blood 110: 4144-52).

[0077] The term "antisense oligonucleotide" (including "antisense RNA") as used herein refers to a nucleic acid that is complementary to and specifically hybridizes to a specified DNA or RNA sequence. Antisense oligonucleotides and nucleic acids encoding antisense oligonucleotides can be prepared according to conventional methods. See, for example, U.S. Patent No. 5,023,243 to Tullis; U.S. Patent No. 5,149,797 to Pederson et al.

[0078] Those skilled in the art will appreciate that an antisense oligonucleotide need not be completely complementary to a target sequence, as long as the sequence similarity is sufficient to enable the antisense nucleotide sequence to specifically hybridize to its target (as defined above) and reduce the production of a protein product (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more).

[0079] To determine the specificity of hybridization, hybridization of such oligonucleotides to target sequences can be performed under low stringency, medium stringency or even high stringency conditions. Suitable conditions for achieving low stringency, medium stringency and high stringency hybridization conditions are as described herein.

[0080] In other words, in certain embodiments, the antisense oligonucleotides of the invention have at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or more sequence identity to the complementary sequence of the target sequence and reduce the production of protein products (as defined above). In some embodiments, the antisense sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches compared to the target sequence.

[0081] Methods for determining percent nucleic acid sequence identity are described in more detail elsewhere in this application.

[0082] The length of the antisense oligonucleotide is not critical, as long as it specifically hybridizes to the intended target and reduces the amount of protein product produced (as defined above), and the length can be determined according to routine procedures. Typically, the antisense oligonucleotide is at least about eight, ten, twelve, or fifteen nucleotides in length and / or less than about 20, 30, 40, 50, 60, 70, 80, 100, or 150 nucleotides in length.

[0083] RNA interference (RNAi) is another useful method for reducing the amount of protein products (e.g., shRNA or siRNA) produced. RNAi is a post-transcriptional gene silencing mechanism in which a double-stranded RNA (dsRNA) corresponding to a target sequence is introduced into a cell or organism, resulting in degradation of the corresponding mRNA. The mechanism by which RNAi achieves gene silencing is reviewed in Sharp et al., (2001) Genes Dev 15:485-490; and Hammond et al., (2001) Nature Rev. Gen. 2:110-119). The RNAi effect lasts for multiple cell divisions before gene expression is restored. Therefore, RNAi is a powerful method for targeted knockout or "knockdown" at the RNA level. RNAi has been shown to be successful in human cells (including human embryonic kidney and HeLa cells) (see, e.g., Elbashir et al., Nature (2001) 411:494-8).

[0084] Initial attempts to use RNAi in mammalian cells resulted in an antiviral defense mechanism involving PKR in response to dsRNA molecules (see, e.g., Gil et al., (2000) Apoptosis 5:107). Since then, it has been demonstrated that short synthetic dsRNAs of approximately 21 nucleotides, termed "short interfering RNAs" (siRNAs), can mediate silencing in mammalian cells without triggering an antiviral response (see, e.g., Elbashir et al., Nature (2001) 411:494-8; Caplen et al., (2001) Proc. Nat. Acad. Sci. USA 98:9742).

[0085] RNAi molecules (including siRNA molecules) can be short hairpin RNA (shRNA; see Paddison et al., (2002), Proc. Nat. Acad. Sci. USA 99: 1443-1448), which is believed to be processed into 20-25mer siRNA molecules in cells by the action of RNase III, which is similar to the Dicer enzyme. shRNAs generally have a stem-loop structure in which two inverted repeat sequences are separated by a short spacer sequence that loops out. shRNAs with loop lengths of 3 to 23 nucleotides have been reported. The loop sequence is generally not important. Exemplary loop sequences include the following motifs: AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA.

[0086] RNAi can further include circular molecules, which include sense and antisense regions, with two loop regions on either side to form a "dumbbell" shaped structure when dsRNA is formed between the sense and antisense regions. Such molecules can be processed in vitro or in vivo to release a dsRNA portion, such as siRNA.

[0087] International Patent Publication WO 01 / 77350 describes vectors for bidirectional transcription to produce sense and antisense transcripts of heterologous sequences in eukaryotic cells. This approach can be used to generate RNAi for use in the present invention.

[0088] Shinagawa et al., (2003) Genes Dev. 17: 1340 reported a method for expressing long dsRNA from a CMV promoter (pol II promoter), which is also applicable to tissue-specific pol II promoters. Similarly, the method of Xia et al., (2002) Nature Biotech. 20: 1006 avoids poly (A) tailing and can be used in combination with tissue-specific promoters.

[0089] Methods for generating RNAi include chemical synthesis, in vitro transcription, digestion of long dsRNA using Dicer (in vitro or in vivo), in vivo expression from a delivery vector, and in vivo expression from a PCR-derived RNAi expression cassette (see, e.g., Tech Notes 10(3) "Five Ways to Produce siRNAs," Ambion, Austin, Texas).

[0090] A guide to designing siRNA molecules can be provided (see, e.g., literature from Ambion, Austin, Texas). In a particular embodiment, the siRNA sequence has a G / C content of about 30-50%. In addition, if RNA polymerase III is used to transcribe the RNA, long stretches longer than four T or A residues are generally avoided. An online siRNA target search tool can be provided, e.g., provided by Ambion, Whitehead Institute of Biomedical Research, or Dharmacon Research, Inc.

[0091] The antisense region of the RNAi molecule can be completely complementary to the target sequence, but it does not need to be so, as long as it specifically hybridizes to the target sequence (as defined above) and reduces the amount of protein product produced (e.g., by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, hybridization of such oligonucleotides to the target sequence can be carried out under conditions of low stringency, medium stringency, or even high stringency, as defined above.

[0092] In other embodiments, the antisense region of the RNAi has at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher sequence identity with the complementary sequence of the target sequence, and reduces the amount of protein product produced (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more). In some embodiments, the antisense region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mismatches compared to the target sequence. Generally, mismatches at the ends of the dsRNA are better tolerated than those in the central portion.

[0093] In a particular embodiment, RNAi is formed by intermolecular complexation between two separated sense and antisense molecules. RNAi comprises a ds region formed by intermolecular base pairing between two separated chains. In other embodiments, RNAi comprises a ds region formed by intramolecular base pairing within a single nucleic acid molecule, which includes sense and antisense regions, typically as inverted repeats (e.g., shRNA or other stem-loop structures, or circular RNAi molecules). RNAi may further comprise a spacer between the sense and antisense regions.

[0094] Typically, RNAi molecules are highly selective. If desired, one skilled in the art can easily eliminate candidate RNAi that may interfere with expression of nucleic acids beyond the target by searching relevant databases to identify RNAi sequences that have no substantial sequence homology to other known sequences, e.g., using BLAST (available from www.ncbi.nlm.nih.gov / BLAST).

[0095] Kits for generating RNAi are available from, for example, New England Biolabs and Ambion.

[0096] The recombinant viral vector may also contain heterologous nucleotide sequences that have homology with and recombinate with loci on the host chromosome. This approach can be used to correct genetic defects in host cells.

[0097] The present invention also provides recombinant viral vectors expressing immunogenic polypeptides, for example, for vaccination. The heterologous nucleic acid may encode any immunogen of interest known in the art, including but not limited to immunogens of human immunodeficiency virus, influenza virus, gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc. Alternatively, the immunogen may be present in (e.g., incorporated into) or bound to (e.g., by covalent modification) a viral capsid.

[0098] The use of parvovirus as a vaccine is familiar in the art (see, e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci. USA 91:8507; U.S. Pat. No. 5,916,56 to Young et al., U.S. Pat. No. 5,905,040 to Mazzara et al., U.S. Pat. No. 5,882,652, No. 5,863,541 to Samulski et al.; the contents of which are incorporated herein by reference in their entirety). The antigen may be present in the viral capsid. Alternatively, the antigen may be expressed from a heterologous nucleic acid introduced into the genome of a recombinant vector.

[0099] The immunogenic polypeptide or immunogen can be any polypeptide suitable for protecting a subject from disease, including but not limited to microbial, bacterial, protozoan, parasitic, fungal and viral diseases. For example, the immunogen can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein or an influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus (SIV) immunogen, or a human immunodeficiency virus (HIV) immunogen, such as HIV or SIV envelope GP160 protein, HIV or SIV matrix / capsid protein, and HIV or SIV gag, pol and env gene products). The immunogen can also be an arenavirus immunogen (e.g., a Lassa fever virus immunogen, such as a Lassa fever virus nucleocapsid protein gene and a Lassa fever envelope glycoprotein gene), a poxvirus immunogen (e.g., vaccinia, such as a vaccinia L1 or L8 gene), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen or a Marburg virus immunogen, such as the NP and GP genes), a bunyavirus immunogen (e.g., RVFV, CCHF, and SFS viruses), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein gene, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen, or a severe acute respiratory syndrome (SARS) immunogen, such as the S [S1 or S2], M, E, or N protein or an immunogenic fragment thereof). The immunogen can also be a polio immunogen, a herpes immunogen (e.g., a CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, or hepatitis C) immunogen, or any other vaccine immunogen known in the art.

[0100] Alternatively, the immunogen can be any tumor or cancer cell antigen. Optionally, the tumor or cancer cell antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are described in A. Rosenberg, (1999) Immunity 10:281).Exemplary cancer and tumor antigens include, but are not limited to, BRCA1 gene products, BRCA2 gene products, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124), including MART-1 (Coulie et al., (1991) J. Exp. Med. 180:35), gp100 (Wick ... al., (1988) J. Cutan. Pathol. 4:201) and MAGE antigens (MAGE-1, MAGE-2 and MAGE-3) (Van der Bruggen et al., (1991) Science, 254:1643), CEA, TRP-1; TRP-2; P-15 and tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2 / neurogenic gene product (U.S. Pat. No. 4,968,603); CA 125; HE4; LK26; FB5 (endosialin); TAG 72; AFP; CA19-9; NSE; DU-PAN-2; CA50; Span-1; CA72-4; HCG; STN (sialyl Tn antigen); c-erbB-2 protein; PSA; L-CanAg; estrogen receptor; milk fat globule protein; p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62: 623); mucin antigen (International Patent Publication WO 90 / 05142); telomerase; nuclear matrix protein; prostatic acid phosphatase; papillomavirus antigens; and antigens associated with the following cancers: melanoma, adenocarcinoma, thymoma, sarcoma, lung cancer, liver cancer, colorectal cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, etc. (see, e.g., Rosenberg, (1996) Annu. Rev. Med. 47:481-91).

[0101] Alternatively, the heterologous nucleotide sequence may encode any polypeptide that is desired to be produced in cells in vitro, ex vivo or in vivo. For example, a viral vector may be introduced into cultured cells and the expressed protein product isolated therefrom.

[0102] It will be appreciated by those skilled in the art that the heterologous nucleic acid of interest can be operably linked to appropriate control sequences. For example, the heterologous nucleic acid can be operably linked to expression control elements, such as transcription / translation control signals, replication origins, polyadenylation signals, internal ribosome entry sites (IRES), promoters, enhancers, and the like.

[0103] Those skilled in the art will further appreciate that a variety of promoter / enhancer elements may be used, depending on the desired level and tissue-specific expression. The promoter / enhancer may be constitutive or inducible, depending on the desired expression pattern. The promoter / enhancer may be native or foreign, and may be a natural or synthetic sequence. Foreign means that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0104] The promoter / enhancer element can be a target cell or a subject to be treated natural and / or a natural element of a heterologous nucleic acid sequence. Usually the promoter / enhancer element is selected so that it plays a role in the target target cell. In addition, in a representative embodiment, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element can be a promoter based on RNA polymerase II or a promoter based on RNA polymerase III. The promoter / enhancer element can be a constitutive or inducible.

[0105] Inducible expression control elements are generally used in applications where it is desired to provide regulated overexpression of heterologous nucleic acid sequences. Inducible promoter / enhancer elements for gene delivery can be tissue-specific or tissue-preferred promoter / enhancer elements, including muscle-specific or preferred (including cardiac muscle, skeletal muscle and / or smooth muscle), neural tissue-specific or preferred (including brain-specific), eye-specific or preferred (including retinal specific and corneal specific), liver-specific or preferred, bone marrow-specific or preferred, pancreas-specific or preferred, spleen-specific or preferred, and lung-specific or preferred promoter / enhancer elements. In one embodiment, hepatocyte-specific or hepatocyte-preferred promoters are used. Examples of hepatocyte-specific or preferred promoters include, but are not limited to, apolipoprotein AII, albumin, α1-antitrypsin, thyroxine-binding globulin, cytochrome P450 CYP3A4 or microRNA122 or synthetic liver-specific regulatory sequences. The use of hepatocyte-specific or preferred promoters can increase the specificity achieved by the synthetic AAV vector by further limiting the expression of heterologous nucleic acids to the liver. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, Tet on / off elements, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters.

[0106] In embodiments where heterologous nucleic acid sequences are transcribed and then translated in target cells, specific initiation signals are generally employed to efficiently translate the inserted protein coding sequence. These exogenous translation control sequences, which may include the ATG initiation codon and adjacent sequences, can be derived from a variety of natural and synthetic sources.

[0107] The invention also provides a synthetic AAV particle comprising an AAV capsid and an AAV genome, wherein the AAV genome "corresponds to" (i.e., encodes) the AAV capsid. Also provided is a collection or library of such chimeric AAV particles, wherein the collection or library comprises 2 or more, 10 or more, 50 or more, 10 ... 4 or more, 10 5 or more, or 10 6 or more different sequences.

[0108] The present invention further encompasses "empty" capsid particles (i.e., without a vector genome) comprising, consisting of, or substantially consisting of synthetic AAV capsid proteins of the present invention. As described in U.S. Pat. No. 5,863,541, the synthetic AAV capsids of the present invention can be used as "capsid carriers". Molecules that can be covalently linked, bound or packaged to the viral capsid and transferred to cells include DNA, RNA, lipids, carbohydrates, polypeptides, small organic molecules, or combinations thereof. In addition, the molecule can be associated with the outside of the viral capsid (e.g., "bound to") to transfer the molecule to the host target cell. In one embodiment of the present invention, the molecule is covalently linked to the capsid protein (i.e., bound or chemically coupled). Methods of covalently linking molecules are familiar to those skilled in the art.

[0109] Virus capsids of the present invention can also be used to generate antibodies against novel capsid structures. As another option, exogenous amino acid sequences can be inserted into virus capsids to present antigens to cells, for example, for use in subjects to generate immune responses to exogenous amino acid sequences.

[0110] The present invention also provides nucleic acids (e.g., isolated nucleic acids) encoding synthetic viral capsids and synthetic capsid proteins of the present invention. Vectors comprising the nucleic acids, and cells (in vivo or in culture) comprising nucleic acids and / or vectors of the present invention are also provided. The nucleic acids, vectors, and cells can be used, for example, as reagents (e.g., auxiliary constructs or packaging cells) for producing viral vectors described herein.

[0111] In an exemplary embodiment, the present invention provides a nucleic acid sequence encoding an AAV capsid of SEQ ID NOS: 4-6 or a nucleic acid sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NOS: 1-3. The present invention also provides nucleic acids encoding AAV capsid variants, capsid protein variants, and fusion proteins as described above. In a specific embodiment, the nucleic acid hybridizes with the complementary sequence of the nucleic acid sequence specifically disclosed herein under standard conditions familiar to those skilled in the art, and encodes a variant capsid and / or capsid protein. Optionally, the variant capsid or capsid protein substantially retains at least one property of the capsid and / or capsid protein encoded by the nucleic acid sequence of SEQ ID NOS: 1-3. For example, a virus particle comprising the variant capsid or variant capsid protein may substantially retain the hepatotropism of a virus particle comprising a capsid or capsid protein encoded by a nucleic acid coding sequence of SEQ ID NOS: 1-3.

[0112] For example, hybridization of the sequences can be carried out under low stringency, medium stringency or even high stringency conditions. Exemplary conditions for low stringency hybridization, medium stringency hybridization and high stringency hybridization are as follows: (e.g., conditions represented by washing stringency of 35-40% formamide, 5x Denhardt's solution, 0.5% SDS and 1x SSPE at 37°C; conditions represented by washing stringency of 40-45% formamide, 5x Denhardt's solution, 0.5% SDS and 1x SSPE at 42°C; and conditions represented by washing stringency of 50% formamide, 5x Denhardt's solution, 0.5% SDS and 1x SSPE at 42°C). See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory).

[0113] In other embodiments, the nucleic acid sequence encoding a variant capsid or capsid protein of the invention has at least about 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% or 99.9% or more sequence identity to the nucleic acid sequence of SEQ ID NOs: 1-3, and optionally the encoded variant capsid or capsid protein substantially retains at least one property of the capsid or capsid protein encoded by the nucleic acids of SEQ ID NOs: 1-3.

[0114] As is familiar in the art, a variety of different programs can be used to identify whether a nucleic acid or polypeptide has sequence identity with a known sequence. The percent identity described herein refers to the percentage of identity that a nucleic acid or fragment thereof has with another nucleic acid when optimally aligned (with appropriate nucleotide insertions or deletions) using BLASTN with another nucleic acid (or its complementary strand). In order to determine the percent identity between two different nucleic acids, the BLASTN program "BLAST2sequences" is used to determine the percent identity. This program is publicly available from the National Center for Biotechnology Information (NCBI) via the Internet (Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402). The parameters used were any combination of the following that produced the highest calculated percent identity (calculated as follows), with default parameters shown in parentheses: Program--blastn Matrix--0BLOSUM62 Reward for a match--0or 1(1)Penalty for a mismatch--0,-1,-2or-3(-2)Open gap penalty--0,1,2,3,4or 5(5)Extension gap penalty--0or 1(1)Gap x_dropoff--0or 50(50)Expect--10.

[0115] When referring to polypeptides, the percentage of identity or similarity refers to the percentage of identity or similarity of the polypeptide in question compared to another protein or a portion thereof over a common length, as determined using BLASTP. This program is also publicly available from the National Center for Biotechnology Information (NCBI) via the Internet (Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402). The percentage of identity or similarity of polypeptides is usually determined using sequence analysis software. See, for example, the sequence analysis software package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (910 University Avenue, Madison, Wisconsin, 53705). Protein analysis software uses homology metrics assigned to various substitutions, deletions, and other modifications to match similar sequences. Conservative substitutions generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0116] In certain embodiments, the nucleic acid may comprise, consist essentially of, or consist essentially of a vector including, but not limited to, a plasmid, a phage, a viral vector (e.g., an AAV vector, an adenoviral vector, a herpes virus vector, or a baculovirus vector), a bacterial artificial chromosome (BAC), or a yeast artificial chromosome (YAC). For example, the nucleic acid may comprise, consist essentially of, or consist essentially of an AAV vector comprising a 5' and / or 3' terminal repeat sequence (e.g., a 5' and / or 3' AAV terminal repeat sequence).

[0117] In some embodiments, the nucleic acid encoding the synthetic AAV capsid protein further comprises an AAV rep coding sequence. For example, the nucleic acid can be a helper construct for generating a viral stock.

[0118] The invention also provides packaging cells that stably contain the nucleic acid of the invention.For example, the nucleic acid can be stably incorporated into the genome of the cell or can be stably maintained in episomal form (eg, "EBV-based nuclear episome").

[0119] The nucleic acid can be incorporated into a delivery vector, such as a viral delivery vector. For example, the nucleic acid of the invention can be packaged in an AAV particle, an adenovirus particle, a herpes virus particle, a baculovirus particle, or any other suitable virus particle.

[0120] In addition, the nucleic acid can be operably linked to a promoter element. Promoter elements are described in more detail herein.

[0121] The present invention further provides a method for producing a viral vector of the present invention. In a representative embodiment, the present invention provides a method for producing a recombinant viral vector, the method comprising providing (a) a template comprising (i) a heterologous nucleic acid and (ii) a packaging signal sequence to a cell in vitro, the packaging signal sequence being sufficient to encapsidate the AAV template into a viral particle (e.g., one or more (e.g., two) terminal repeats, such as AAV terminal repeats), and (b) an AAV sequence sufficient to replicate and encapsidate the template into a viral particle (e.g., an AAV rep encoding the AAV capsid of the present invention and an AAV cap sequence). The template and the AAV replication sequence and the capsid sequence are provided under certain conditions so that recombinant viral particles containing the template packaged in the capsid are produced in the cell. The method may further include the step of collecting viral particles from the cell. The viral particles can be collected from the culture medium and / or collected by lysing the cells.

[0122] In another exemplary embodiment, the present invention provides a method for producing rAAV particles containing AAV capsids, the method comprising: providing cells in vitro with a nucleic acid encoding a synthetic AAV capsid of the present invention, an AAV rep coding sequence, an AAV vector genome comprising a heterologous nucleic acid, and an auxiliary function body for producing productive AAV infection; assembling AAV particles containing AAV capsids and encapsidated AAV vector genomes.

[0123] The cells are usually cells that allow AAV virus replication. Any suitable cells familiar in the art can be used, such as mammalian cells. In addition, suitable cells include trans-complementing packaging cell lines that provide replication-defective helper virus deletion functions, for example, 293 cells or other E1a trans-complementing cells.

[0124] The AAV replication sequence and capsid sequence can be provided by any method familiar in the art. Current protocols typically express the AAV rep / cap gene on a single plasmid. The AAV replication sequence and packaging sequence do not need to be provided together, although it may be more convenient to provide them together. The AAV rep and / or cap sequences can be provided by any viral or non-viral vector. For example, the rep / cap sequence can be provided by a hybrid adenovirus or herpes virus vector (for example, inserted into the E1a or E3 region of a deletion adenovirus vector). EBV vectors can also be used to express the AAV cap and rep genes. One advantage of this method is that the EBV vector is an episomal vector, but will maintain a high copy number throughout continuous cell division (i.e., stably integrated into the cell as an extrachromosomal element, referred to as an "EBV-based nuclear episome").

[0125] As another alternative, the rep / cap sequences may be stably carried within the cell (either episomal or integrated).

[0126] Typically, the AAV rep / cap sequences will not be flanked by AAV packaging sequences (e.g., AAV ITRs) to prevent rescue and / or packaging of these sequences.

[0127] The template (e.g., rAAV vector genome) can be provided to the cell using any method known in the art. For example, the template can be provided by a non-viral (e.g., plasmid) or viral vector. In a specific embodiment, the template is provided by a herpes virus or adenovirus vector (e.g., inserted into the E1a or E3 region of a deleted adenovirus). For another example, Palombo et al., (1998) J. Virol. 72: 5025, describes a baculovirus vector carrying a reporter gene flanking AAV ITR. As described above when talking about the rep / cap gene, the template can also be delivered using an EBV vector.

[0128] In another representative embodiment, the template is provided by replicating rAAV virus. In other embodiments, the AAV provirus is stably integrated into the chromosome of the cell.

[0129] In order to increase the viral titer, the cells are usually provided with helper virus functions (e.g., adenovirus or herpes virus) that are essential for productive AAV infection. The helper virus sequences necessary for AAV replication are known in the art. Typically, these sequences are provided by helper adenovirus or herpes virus vectors. Alternatively, adenovirus sequences or herpes virus sequences can be provided by another non-viral or viral vector, for example, as a non-infectious adenovirus miniplasmid, carrying all the helper genes that effectively produce AAV, as described in Ferrari et al., (1997) Nature Med. 3: 1295 and U.S. Patent Nos. 6,040,183 and 6,093,570.

[0130] In addition, helper virus functions can be provided by packaging cells that have helper genes integrated into the chromosome or maintained as stable extrachromosomal elements. In representative embodiments, the helper virus sequences cannot be packaged into the AAV virion, for example, are not flanked by the AAV ITRs.

[0131] Those skilled in the art will appreciate that it is advantageous to provide the AAV replication and capsid sequences and the helper virus sequences (e.g., adenovirus sequences) on a single helper construct. Such a helper construct may be a non-viral or viral construct, but is optionally a hybrid adenovirus or hybrid herpes virus comprising the AAV rep / cap genes.

[0132] In a specific embodiment, the AAV rep / cap sequence and the adenoviral helper sequence are provided by a single adenoviral helper vector. Such a vector further comprises a rAAV template. The AAV rep / cap sequence and / or the rAAV template can be inserted into a deleted region of an adenovirus (e.g., within the E1a or E3 region).

[0133] In another embodiment, the AAV rep / cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector. The rAAV template is provided as a plasmid template.

[0134] In another exemplary embodiment, the AAV rep / cap sequences and adenovirus helper sequences are provided by a single adenovirus helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector maintained as an extrachromosomal element in the cell (e.g., as an EBV-based nuclear episome, see Margolski, (1992) Curr. Top. Microbiol. Immun. 158: 67).

[0135] In another exemplary embodiment, the AAV rep / cap sequences and adenovirus helper sequences are provided by a single helper adenovirus. The rAAV template can be provided as an independent replicating viral vector. For example, the rAAV template can be provided by rAAV particles or a second recombinant adenovirus particle.

[0136] According to the foregoing methods, hybrid adenoviral vectors typically include adenoviral 5' and 3' cis sequences (i.e., adenoviral terminal repeats and PAC sequences) sufficient for adenoviral replication and packaging. AAV rep / cap sequences, and rAAV template (if present) are embedded in the adenoviral backbone, flanked by 5' and 3' cis sequences, so that these sequences can be packaged into the adenoviral capsid. As described above, in representative embodiments, adenoviral helper sequences and AAV rep / cap sequences are typically not flanked by AAV packaging sequences (e.g., AAV ITRs), so that these sequences are not packaged into the AAV virus body.

[0137] Herpes virus can also be used as a helper virus in the AAV packaging method. Hybrid herpes virus encoding AAV rep protein can advantageously promote scalable AAV vector production scheme. It has been described (Conway et al., (1999) Gene Ther. 6:986 and WO 00 / 17377, these disclosures are incorporated herein in their entirety) a hybrid herpes simplex virus type I (HSV-1) expressing AAV-2 rep and cap genes.

[0138] As another alternative, the viral vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the rep / cap genes and the rAAV template as described by Urabe et al., (2002) Human Gene Ther. 13:1935-43.

[0139] Other methods of producing AAV use stably transformed packaging cells (see, e.g., U.S. Pat. No. 5,658,785).

[0140] AAV vector stocks free of contaminating helper viruses can be obtained by any method known in the art. For example, it is easy to distinguish AAV from helper viruses based on size. AAV can also be separated from helper viruses based on affinity for heparin substrates (Zolotukhin et al., (1999) Gene Therapy 6:973). In a representative embodiment, a deletion-defective helper virus can be used, so that any contaminating helper virus does not have replication ability. As another alternative, a helper adenovirus lacking late gene expression can be used, because only early adenovirus gene expression is required to mediate AAV virus packaging. Adenovirus mutants with defective late gene expression are familiar in the art (e.g., ts100K and ts149 adenovirus mutants).

[0141] The packaging methods of the present invention can be used to produce high titer virus particle stocks. In certain embodiments, the titer of the virus stock is at least about 10 5 transduction units (tu) / ml, at least about 10 6 tu / ml, at least about 10 7 tu / ml, at least about 10 8 tu / ml, at least about 10 9 tu / ml, or at least about 10 10 tu / ml.

[0142] This novel capsid protein and capsid structure can be used to generate antibodies, for example for diagnostic or therapeutic purposes or as research reagents. Therefore, the present invention also provides antibodies against the novel capsid protein and capsid of the present invention.

[0143] As used herein, the term "antibody" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD and IgE. The antibody may be a monoclonal antibody or a polyclonal antibody, may be derived from any species, including, for example, mice, rats, rabbits, horses, goats, sheep or humans, or may be a chimeric antibody. See, for example, Walker et al., Mol. Immunol. 26, 403-11 (1989). The antibody may be a recombinant monoclonal antibody, for example, an antibody produced according to the method disclosed in U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. The antibody may also be chemically constructed, for example, according to the method disclosed in U.S. Pat. No. 4,676,980.

[0144] Antibody fragments included within the scope of the present invention include, for example, Fab, F(ab')2 and Fc fragments, and corresponding fragments obtained from antibodies other than IgG. The fragments can be prepared using methods familiar to people. For example, F(ab')2 fragments can be prepared by pepsin digestion of antibody molecules, and Fab fragments can be produced by reducing the disulfide bonds of F(ab')2 fragments. Alternatively, a Fab expression library can be constructed to quickly and easily identify monoclonal Fab fragments with the desired specificity (Huse et al., (1989) Science 254, 1275-1281).

[0145] Polyclonal antibodies can be produced by immunizing a suitable animal (eg, rabbit, goat, etc.) with an antigen that binds to a target monoclonal antibody, collecting immune serum from the animal, and isolating polyclonal antibodies from the immune serum according to known procedures.

[0146] Monoclonal antibodies can be produced in hybridoma cell lines according to the method described in Kohler and Milstein, (1975) Nature 265, 495-97. For example, a solution containing an appropriate antigen can be injected into mice, and the mice can be killed after a sufficient period of time to obtain spleen cells. Then, the spleen cells are fused with myeloma cells or lymphoma cells, usually in the presence of polyethylene glycol, to immortalize them, thereby producing hybridoma cells. The hybridoma cells are then grown in a suitable culture medium, and the supernatant is screened to obtain monoclonal antibodies with the desired specificity. Monoclonal Fab fragments can be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, for example, W.Huse, (1989) Science 246, 1275-81.

[0147] Antibodies specific for the target polypeptide can also be obtained by phage display methods familiar in the art.

[0148] Various immunoassays can be used to screen to identify antibodies with the desired specificity. Many protocols for competitive binding or immunoradiometric assays using polyclonal or monoclonal antibodies with defined specificity are familiar in the art. Such immunoassays generally involve the formation of a complex between an antigen and its specific antibody (e.g., antigen / antibody complex formation). Monoclonal two-point immunoassays using monoclonal antibodies reactive to two non-interfering epitopes as well as competitive binding assays can be employed.

[0149] The antibodies can be bound to a solid support (e.g., microspheres, plates, slides, or wells formed from materials such as latex or polystyrene) according to known techniques. Likewise, the antibodies can be coupled to a detectable group, such as a radiolabel (e.g., 35 S. 125I. 131 I), enzyme labeling (such as horseradish peroxidase, alkaline phosphatase) is directly or indirectly connected with fluorescent labeling (such as, fluorescein). As is familiar in the art, the formation of antibody / antigen complex in the method of the present invention can be measured by detecting, for example, precipitation, agglutination, flocculation, radioactivity, color development or change, fluorescence, luminescence, etc. III. Methods using synthetic AAV capsids

[0150] The present invention also relates to methods for delivering heterologous nucleotide sequences to the liver while minimizing delivery to other organs. The viral vectors of the present invention can be used to deliver target nucleotide sequences to hepatocytes in vitro, for example, to produce polypeptides or nucleic acids in vitro or for ex vivo gene therapy. In addition, the vectors are used in methods for delivering nucleotide sequences to subjects in need, for example, for expressing therapeutic or immunogenic polypeptides or nucleic acids. In this way, polypeptides or nucleic acids can be produced in the subject. The subject may need the polypeptide or nucleic acid because the subject lacks the polypeptide, or because the production of the polypeptide or nucleic acid in the subject can confer a certain therapeutic effect, as a treatment method or other methods, which will be further explained below.

[0151] In certain embodiments, the vector can be used to express a polypeptide or nucleic acid that generally provides a beneficial effect to a subject. In other embodiments, the vector can be used to express a polypeptide or nucleic acid that provides a beneficial effect to cells in the liver (e.g., hepatocytes).

[0152] Thus, another aspect of the invention relates to a method of delivering a target nucleic acid to a hepatocyte, the method comprising contacting the hepatocyte with an AAV particle of the invention.

[0153] In another aspect, the present invention relates to a method for delivering a target nucleic acid into hepatocytes of a mammalian subject, the method comprising administering to the mammalian subject an effective dose of the AAV particles or pharmaceutical preparations of the present invention, thereby delivering the target nucleic acid into the hepatocytes of the mammalian subject.

[0154] Another aspect of the invention relates to a method of treating a disease in a mammalian subject in need thereof, wherein the disease is treatable by expressing a product in the liver of the subject, the method comprising administering to the mammalian subject a therapeutically effective dose of an AAV particle of the invention, wherein the product is expressed, thereby treating the disease.

[0155] In general, the viral vectors of the present invention can be used to deliver any foreign nucleic acid with a biological effect to treat or alleviate any disease symptoms associated with gene expression. In addition, the present invention can be used to treat any disease for which delivery of a therapeutic polypeptide is beneficial. Exemplary diseases include, but are not limited to, cystic fibrosis (cystic fibrosis transmembrane regulator) and other lung diseases, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globulin), anemia (erythropoietin) and other blood diseases, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (inhibitory RNA, including but not limited to RNAi, such as siRNA or shRNA, antisense RNA or microRNA to remove repetitive sequences), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors) and other nervous system diseases, cancer (endostatin, angiostatin, TRAIL, FAS ligands, cytokines including interferon;Inhibitory RNAs include, but are not limited to, RNAi (e.g., siRNA or shRNA), antisense RNA and microRNA, including inhibitory RNA to VEGF, a multidrug resistance gene product or a cancer immunogen), diabetes (insulin, PGC-α1, GLP-1, myostatin propeptide, glucose transporter 4), muscular dystrophy, including Duchenne and Becker muscular dystrophy (e.g., dystrophin, mini-dystrophin, micro-dystrophin, insulin-like growth factor I, myoglycoprotein [e.g., α, β, γ], inhibitory RNA [e.g., RNAi, antisense RNA or microRNA] against myostatin or myostatin propeptide), laminin-α2, Fukutin-related protein, dominant negative myostatin, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides, such as Ikappa B dominant mutant, sarcospan (muscle length protein), utrophin (dystrophin-related protein), mini-utrophin, splice junctions in the dystrophin gene to induce exon skipping inhibitory RNA [[e.g., RNAi, antisense RNA or microRNA] [see, e.g., WO / 2003 / 095647], anti-U7 snRNA to induce exon skipping inhibitory RNA (e.g., RNAi, antisense RNA or microRNA) RNA) [see, e.g., WO / 2006 / 021724], and antibodies or antibody fragments against myostatin or myostatin propeptide), Gaucher disease (glucocerebrosidase), Hurler's disease (α-L-iduronidase), adenylate deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) and other metabolic defects, including other lysosomal storage diseases and glycogen storage diseases, congenital emphysema (α1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), Niemann-Pick disease (sphingomyelinase), maple syrup urine disease (branched-chain ketoacid dehydrogenase), retinal degenerative diseases (other diseases of the eye and retina);For example, PDGF, endostatin and / or angiostatin (macular degeneration), solid organ diseases such as the brain (including Parkinson's disease [GDNF], astrocytoma [endostatin, angiostatin and / or RNAi to VEGF], glioblastoma [endostatin, angiostatin] and / or RNAi to VEGF]), liver disease (RNAi, such as siRNA or shRNA, microRNA or antisense RNA to hepatitis B and / or hepatitis C genes), kidney disease, heart disease including congestive heart failure or peripheral arterial disease (PAD) (e.g., by delivery of Protein phosphatase inhibitor I [I-1], phospholamban, sarcosporic Ca2+-ATPase [serca2a], zinc finger protein regulating phospholamban gene, Pim-1, PGC-1α, SOD-1, SOD-2, ECF-SOD, kallikrein, thymosin-β4, hypoxia-inducible transcription factor [HIF], βarkct, β2-adrenergic receptor, β2-adrenergic receptor kinase [βARK], phosphoinositide-3 kinase [PI3 kinase], calretinin, angiogenic factor, S100A1, parvalbumin, adenylate cyclase type 6, G-affecting Protein-coupled receptor kinase type 2 knockdown molecules, such as truncated constitutively active bARKct, inhibitory RNA against phospholamban [e.g., RNAi, antisense RNA or microRNA]; phospholamban inhibitory or dominant negative molecules, such as phospholamban S16E, etc.), arthritis (insulin-like growth factor), joint disease (insulin-like growth factor), intimal hyperplasia (e.g., by delivery of enos (endothelial nitric oxide synthase), inos (inducible nitric oxide synthase)), improved cardiac transplant survival (superoxide dismutase), AIDS (soluble CD4), muscle atrophy Disease (insulin-like growth factor I, myostatin peptide, anti-apoptotic factors, follistatin), limb ischemia (VEGF, FGF, PGC-1α, EC-SOD, HIF), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors, such as IRAP and TNFα soluble receptors), hepatitis (α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), spinal cord ataxia including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc. The present invention is further used after the following organ transplantation to improve the transplantation success rate and / or reduce the adverse effects of organ transplantation or adjuvant therapy (for example, administration of immunosuppressants or inhibitory nucleic acids, blocking cytokine production). As another example, bone morphogenetic proteins (including RANKL and / or VEGF) can be administered concurrently with a bone allograft, e.g., following surgical resection of a bone fracture or cancer patient.

[0156] Exemplary lysosomal storage diseases that may be treated in accordance with the present invention include, but are not limited to, Hurler syndrome (MPS IH), Scheie syndrome (MPS IS), and Hurler-Scheie syndrome (MPS IH / S) (α-L-iduronidase); Hunter syndrome (MPS II) (iduronic acid sulfate sulfatase); Sanfilippo syndrome A (MPS IIIA) (heparan-S-sulfaminase), Sanfilippo syndrome B (MPS IIIB) (N-acetyl-D-glucosaminidase), Sanfilippo syndrome C (MPS IIIC) (acetyl-CoA-glucoside N-acetyltransferase), Sanfilippo syndrome D (MPS IIID) (N-acetyl-glucosamine-6-sulfate sulfatase); Morquio disease A (MPS IVA) (galactosamine-6-sulfate sulfatase), Morquio disease B (MPS IV B) (β-galactosidase); Maroteaux-Lmay disease (MPS VI) (arylsulfatase B); Thrush syndrome (MPS VII) (β-glucuronidase); hyaluronidase deficiency (MPS IX) (hyaluronidase); sialidosis (mucopolysaccharidosis I), mucopolysaccharidosis II (I-cell disease) (N-acetylglucosamine-1-phosphotransferase catalytic subunit); mucopolysaccharidosis III (pseudo-Heller's dystrophy) (N-acetylglucosamine-1-phosphotransferase; type IIIA [catalytic subunit] and type IIIC [substrate recognition subunit]); GM1 gangliosidosis (ganglioside β-galactosidase), GM2 gangliosidosis type I (Tay-Sachs disease) (β-hexosidase A), GM2 gangliosidosis type II (Sandhoff disease) (β-hexosidase B); Niemann-Pick disease (types A and B) (sphingomyelinase); Gaucher disease (glucocerebrosidase); Farber disease (ceramidase); Fabry disease (alpha-galactosidase A); Krabbe's disease (galactosylceramide beta-galactosidase); metachromatic leukodystrophy (arylsulfatase A); lysosomal acid lipase deficiency, including Wolman disease (lysosomal acid lipase); Batten disease (juvenile neuronal ceroid lipofuscinosis) (lysosomal transmembrane CLN3 protein); sialidase (neuraminidase 1); galactosialidase (Goldberg syndrome) (protectin / cathepsin A); alpha-mannosidosis (alpha-D-mannosidase); beta-mannosidosis (beta-D-mannosidase); fucosidosis (alpha-D-fucosidase); aspartylglucosaminuria (N-aspartylglucosaminidase); and sialuria (Na-phosphate cotransporter).

[0157] Exemplary glycogen storage diseases that may be treated according to the present invention include, but are not limited to, type Ia GSD (von Gierke's disease) (glucose-6-phosphatase), type Ib GSD (glucose-6-phosphate translocase), type Ic GSD (microsomal phosphate or pyrophosphate transporter), type Id GSD (microsomal glucose transporter), type II GSD, including Pompe disease or infantile GSD type IIa (lysosomal acid) α-glucosidase) and type IIb (Danon) (lysosomal membrane protein-2), type IIIa and type IIIb GSD (debranching enzymes; amyloglucosidase and oligoglucosyltransferase), type IV GSD (Anderson's disease) (branching enzymes), type V GSD (McArdle's disease) (muscle phosphorylase), GSD type VI (Hulse's disease) (liver phosphorylase), GSD type VII (Tarry's disease) (phosphofructokinase), GSD type VIII / IXa (X-linked phosphorylase kinase), GSD type IXb (liver and muscle phosphorylase kinase), GSD type IXc (liver phosphorylase kinase), GSD type IXd (muscle phosphorylase kinase), GSD type O (glycogen synthase), Fanconi-Bickel syndrome (glucose transporter-2), phosphoglucose isomerase deficiency, muscle phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, fructose 1,6-bisphosphatase deficiency, phosphoenolpyruvate carboxykinase deficiency, and lactate dehydrogenase deficiency.

[0158] Gene transfer has great potential applications in understanding and providing disease treatment. The defective genes of many genetic diseases are already well known and have been cloned. Generally, the above diseases can be divided into two categories: defective diseases, usually enzyme deficiencies, and usually inherited in a recessive manner, and unbalanced diseases, which may involve regulatory proteins or structural proteins, and are usually inherited in a dominant manner. For defective diseases, gene transfer can be used to introduce normal genes into affected tissues for replacement therapy, and inhibitory RNA (such as RNAi (such as siRNA or shRNA), microRNA or antisense RNA) can be used to establish animal models of the disease. For unbalanced diseases, gene transfer can be used to establish the disease in a model system, and then, it can be used to try to eliminate the disease. Therefore, the viral vectors of the present invention can treat genetic diseases. As described herein, the disease is treated by partially or completely rescuing the defects or imbalances that cause the disease or make the disease more severe. Site-specific recombination of nucleic acid sequences can also be used to induce mutations or correct defects.

[0159] The viral vector of the present invention can also be used to provide antisense nucleic acids or inhibitory RNA (e.g., microRNA or RNAi, such as siRNA or shRNA) to cells in vitro or in vivo. The expression of inhibitory RNA in target cells reduces the expression of specific proteins in the cells. Therefore, inhibitory RNA can be used to reduce the expression of specific proteins in subjects in need. Inhibitory RNA can also be administered to cells in vitro for regulating cell physiology, for example, to optimize cell or tissue culture systems.

[0160] On the other hand, the viral vector of the present invention can be used to generate an immune response in a subject. According to this embodiment, a viral vector comprising a nucleic acid encoding an immunogen can be administered to a subject, and the subject generates an active immune response (optionally a protective immune response) against the immunogen. The immunogen is as described above.

[0161] Alternatively, the viral vector can be administered to the cell ex vivo, and the altered cell is then administered to a subject. A heterologous nucleic acid is introduced into a cell, and the cell is administered to a subject, wherein the heterologous nucleic acid encoding the immunogen is optionally expressed, and an immune response to the immunogen is induced in the subject. In a specific embodiment, the cell is an antigen-presenting cell (e.g., a dendritic cell).

[0162] An "active immune response" or "active immunity" is characterized by "the involvement of host tissues and cells following encounter with an immunogen. It involves differentiation and proliferation of immunocompetent cells in the lymphoreticular tissue, leading to the synthesis of antibodies or the development of cell-mediated reactivity, or both." Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). In other words, an active immune response is elicited following exposure of the host to an immunogen, either by infection or vaccination. Active immunity is in contrast to passive immunity, which is obtained by "transfer of preformed substances (antibodies, transfer factors, thymic grafts, interleukin-2) from an actively immune host to a non-immune host." Id.

[0163] As used herein, a "protective" immune response or "protective" immunity refers to an immune response that brings certain benefits to a subject by preventing or reducing the incidence of a disease. Alternatively, a protective immune response or protective immunity can be used to treat a disease, particularly a cancer or tumor (e.g., regression of a cancer or tumor and / or prevention of metastasis and / or prevention of metastatic nodule growth). The protective effect can be comprehensive or partial, as long as the benefits of the treatment outweigh its adverse effects.

[0164] The viral vectors of the present invention can also be used to carry out cancer immunotherapy by administering a viral vector expressing a cancer cell antigen (or an immunologically similar molecule) or any other immunogen that produces an immune response against a cancer cell. For example, an immune response against a cancer cell antigen can be produced in a subject by administering a viral vector comprising a heterologous nucleotide sequence encoding a cancer cell antigen, such as to treat a cancer patient. As described herein, the viral vector can be administered in vivo or in vitro.

[0165] As used herein, the term "cancer" includes neoplastic cancers. Likewise, the term "cancer tissue" includes tumors. "Cancer cell antigens" include tumor antigens.

[0166] The term "cancer" has the meaning understood in the art, e.g., uncontrolled growth of tissue that may spread to distant parts of the body (i.e., metastasis). Exemplary cancers include, but are not limited to, leukemia, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), colorectal cancer, kidney cancer, liver cancer, breast cancer, lung cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, cervical cancer, brain cancer (e.g., glioma and glioblastoma), bone cancer, sarcoma, melanoma, head and neck cancer, esophageal cancer, thyroid cancer, etc. In embodiments of the present invention, the present invention is implemented to treat and / or prevent neoplastic cancer.

[0167] In the art, the term "tumor" is also understood to be an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In representative embodiments, the methods disclosed herein are used to prevent and treat malignant tumors.

[0168] Cancer cell antigens have been described above. The words "treating cancer" or "treatment of cancer" refer to reducing the severity of cancer or preventing or at least partially eliminating cancer. For example, in certain cases, these words refer to preventing or reducing or at least partially eliminating cancer metastasis. In other representative embodiments, these words refer to preventing or reducing or at least partially eliminating the growth of metastatic nodules (e.g., after surgical resection of the primary tumor). The words "cancer prevention" or "preventing cancer" refer to the method at least partially eliminating or reducing the occurrence or onset of cancer. In other words, the onset or progression of cancer in a subject can be slowed, controlled, and the likelihood or probability reduced or delayed.

[0169] In certain embodiments, cells can be removed from a cancer subject and contacted with the viral vector of the present invention. The modified cells are then administered to the subject, thereby inducing an immune response against the cancer cell antigen. This method is advantageous for subjects with immunosuppression who are unable to induce a sufficient immune response in the body (i.e., unable to produce a sufficient number of enhanced antibodies).

[0170] It is well known in the art that immune response can be enhanced by immunomodulatory cytokines (e.g., α-interferon, β-interferon, γ-interferon, ω-interferon, τ-interferon, interleukin-1α, interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-18, B cell growth factor, CD40 ligand, tumor necrosis factor-α, tumor necrosis factor-β, monocyte chemoattractant protein-1, granulocyte macrophage colony stimulating factor and lymphocyte toxin). Therefore, immunomodulatory cytokines (e.g., CTL inducing cytokines) can be administered to a subject together with a viral vector.

[0171] Cytokines can be administered by any method known in the art. Exogenous cytokines can be administered to a subject, or a nucleic acid sequence encoding a cytokine can be delivered to a subject using a suitable vector and the cytokine can be produced in vivo.

[0172] Viral vectors can further target hepatocytes for research purposes, such as for in vitro or animal liver function studies, or for establishing and / or studying animal models of disease. For example, in animal models of liver damage (e.g., fibrosis or cirrhosis) or animal models of liver disease (e.g., viral infection (e.g., hepatitis virus)), vectors can be used to deliver heterologous nucleic acids to hepatocytes.

[0173] In addition, the viral vectors of the present invention are further used in diagnostic and screening methods, whereby target genes are transiently or stably expressed in cell culture systems or transgenic animal models. The present invention can also be practiced to deliver nucleic acids for protein production purposes, such as for laboratory, industrial or commercial purposes.

[0174] The recombinant viral vectors of the present invention are used for veterinary and medical applications. Suitable subjects include birds and mammals. The term "birds" described herein includes but is not limited to chickens, ducks, geese, quails, turkeys, pheasants, parrots, parakeets, etc. The term "mammals" described herein includes but is not limited to humans, primates, non-human primates (e.g., monkeys and baboons), cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats, mice, hamsters, etc.). Human subjects include neonates, infants, teenagers and adults. The subject "needs" the method of the present invention, for example, because the subject suffers from or is believed to have a risk of suffering from diseases including those described herein, or will benefit from delivering nucleic acids including those described herein. As another option, the subject can be an experimental animal and / or an animal model of the disease.

[0175] In a specific embodiment, the present invention provides a pharmaceutical composition comprising a viral vector of the present invention in a pharmaceutically acceptable carrier, and optionally other drugs, pharmaceutical preparations, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection administration, the carrier is generally a liquid. For other methods of administration, the carrier can be solid or liquid. For inhalation administration, the carrier is inhalable and is preferably in the form of solid or liquid particles.

[0176] "Pharmaceutically acceptable" refers to a material that is non-toxic or otherwise acceptable, ie, the material can be administered to a subject without causing any undesirable biological effects.

[0177] One aspect of the invention is a method for transferring a nucleotide sequence to a cell in vitro. The viral vector may be introduced into the cell at an appropriate multiplicity of infection according to standard transduction methods appropriate for the specific target cell. The titer of the viral vector or capsid administered may vary depending on the type and number of target cells and the specific viral vector or capsid, and may be determined by one skilled in the art without undue experimentation. In a specific embodiment, at least about 10 3 infectious units, more preferably at least about 10 5 Infectious units are introduced into cells.

[0178] The cell introduced into the viral vector can be any type of cell, including but not limited to neural cells (including peripheral and central nervous system cells, in particular, brain cells, such as neurons, oligodendrocytes, glial cells, astrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelial cells and corneal cells), epithelial cells (for example, intestinal and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes and myofibers), diaphragmatic cells, dendritic cells, pancreatic cells (including islet cells), hepatocytes, gastrointestinal cells (including smooth muscle cells, epithelial cells), heart cells (including cardiomyocytes), bone cells (such as bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells (including, for example, cartilage, meniscus, synovium and bone marrow), germ cells, etc. Alternatively, the cell can be any progenitor cell. As another alternative, the cell can be a stem cell (for example, neural stem cells, liver stem cells). As another alternative, the cell can be a cancer cell or a tumor cell (cancer and tumor are described above). Furthermore, the cells may be derived from any of the species described above.

[0179] The viral vector can be introduced into the cell in vitro for administering the modified cell to the subject. In a specific embodiment, the cell is removed from the subject, the viral vector is introduced into the cell, and then the cell is administered back into the subject. The method of removing cells from the subject for ex vivo treatment and then introducing the cells back into the subject is familiar in the art (see, e.g., U.S. Patent No. 5,399,346). Alternatively, the recombinant viral vector can be introduced into the cells of another subject, into cultured cells, or into cells of any other suitable source, and the cell is administered to a subject in need.

[0180] Cells suitable for ex vivo gene therapy are described above. The dosage of cells administered to a subject will vary depending on the age, condition and species of the subject, the cell type, the nucleic acid expressed by the cell, and the mode of administration. Typically, the number of cells administered per dose in a pharmaceutically acceptable carrier is at least about 10 2 Up to about 10 8 cells or at least about 10 3 cells to approximately 10 6 In certain embodiments, the viral vector-transduced cells are administered to a subject together with a pharmaceutical carrier at an effective dose.

[0181] In some embodiments, cells that have been transduced with a viral vector can be administered to elicit an immunogenic response to the delivered polypeptide (e.g., expressed in a transgenic or capsid form). Typically, cells expressing an effective amount of polypeptide are administered together with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to produce a protective immune response (as defined above). The degree of protection elicited is not required to be comprehensive or permanent, as long as the benefits of administering the immune polypeptide outweigh any adverse effects.

[0182] Another aspect of the invention is a method of administering a viral vector or capsid of the invention to a subject. In a specific embodiment, the method includes a method of delivering a target nucleic acid to an animal subject, the method comprising: administering an effective amount of a viral vector of the invention to an animal subject. The viral vector of the invention can be administered to a human subject or animal in need in any manner known in the art. The viral vector is optionally delivered in an effective dose in a pharmaceutically acceptable carrier.

[0183] The viral vector of the present invention can be further administered to a subject to induce an immunogenic response (e.g., as a vaccine). Typically, the vaccine of the present invention comprises an effective dose of a virus and a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to produce a protective immune response (as defined above). The degree of protection elicited is not required to be comprehensive or permanent, as long as the benefits of administering the immune polypeptide outweigh any adverse effects. The subject and the immunogen are as described above.

[0184] The dosage of the viral vector administered to a subject depends on the mode of administration, the disease or condition being treated, the condition of the subject, the specific viral vector, and the nucleic acid being delivered, etc., and can be determined in a routine manner. An exemplary dosage for achieving a therapeutic effect is a viral titer of at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 Transduction units or higher, preferably about 10 7 or 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15transduction units, more preferably about 10 12 Up to 10 14 Transduction unit.

[0185] In certain embodiments, to achieve the desired gene expression level, multiple administrations (e.g., two, three, four or more administrations) may be performed at various intervals, e.g., daily, weekly, monthly, yearly, etc.

[0186] Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., by aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including skeletal muscle, diaphragmatic and / or myocardial administration], intradermal, intrapleural, intracerebral and intraarticular), topical (e.g., skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g., injection into the liver, skeletal muscle, myocardial muscle, diaphragm or brain). Administration to a tumor (e.g., within or near a tumor or lymph node) is also possible. In any case, the most appropriate route will depend on the nature and severity of the disease being treated and the nature of the specific carrier used.

[0187] In some embodiments, the viral vector is administered directly to the liver. Direct administration results in high specificity of hepatocyte transduction, e.g., wherein at least 80%, 85%, 90%, 95% or more of the transduced cells are hepatocytes. The vector may be administered directly to the liver using any method known in the art. The vector may be introduced by direct injection into the liver or into an artery or vein of the liver (e.g., intraportal delivery).

[0188] Typically, the viral vector will be administered in the form of a liquid formulation by systemic delivery or direct injection into the desired area or compartment of the liver. In some embodiments, the vector can be delivered by a reservoir and / or a pump. In other embodiments, the vector can be provided by topical application to the desired area or by intranasal administration of an aerosol formulation. Administration in the eyes or ears can be topical application of liquid drops. As another alternative, the vector can be administered in the form of a solid sustained-release formulation. The controlled release of parvovirus and AAV vectors is described in International Patent Publication WO 01 / 91803.

[0189] Delivery to any of these tissues can also be accomplished by delivering a reservoir containing the viral vector, which can be implanted in the tissue, or the tissue can be contacted with a membrane or other matrix containing the viral vector. Suitable such implantable matrices or substrates are described in U.S. Pat. No. 7,201,898.

[0190] Injections can be prepared in traditional forms, in the form of liquid solutions or suspensions, in solid forms suitable for preparation as liquid solutions or suspensions before injection, or in the form of emulsions. Alternatively, the viral vectors of the present invention can be administered locally rather than systemically, for example, in the form of reservoirs or sustained-release preparations. In addition, the viral vectors can be delivered dry to surgically implantable matrices, such as bone graft substitutes, sutures, stents, etc. (e.g., as described in U.S. Patent No. 7,201,898).

[0191] Pharmaceutical compositions suitable for oral administration may be present in discrete units, such as capsules, cachets, lozenges or tablets, each of which contains a predetermined amount of the composition of the present invention; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Oral administration may be performed by compounding the viral vector of the present invention with a carrier that can withstand degradation by digestive enzymes in the animal intestine. Examples of carriers that withstand degradation by digestive enzymes in the animal intestine include plastic capsules or tablets known in the art. Such preparations are prepared by any suitable pharmaceutical method, including the step of mixing the composition with a suitable carrier (which may contain one or more auxiliary ingredients as described above). Typically, the pharmaceutical composition of the embodiments of the present invention is prepared by uniformly and intimately mixing the composition with a liquid or finely divided solid carrier or both, and then, if necessary, shaping the resulting mixture. For example, tablets may be prepared by compressing or molding a powder or granules containing the composition, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing a composition in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent and / or surfactant / dispersant, in a suitable machine. Molded tablets can be prepared by wetting the powdered compound with an inert liquid binder and molding in a suitable machine.

[0192] Pharmaceutical compositions suitable for buccal (sublingual) administration include lozenges comprising the composition of the invention in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia.

[0193] Pharmaceutical compositions suitable for parenteral administration may include sterile aqueous and non-aqueous injection solutions of the compositions of the present invention, the preparations being optionally isotonic with the blood of the intended recipient. These preparations may include antioxidants, buffers, antibacterial agents and solutes to make the compositions isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions may include suspending agents and thickening agents. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous carriers include liquid and nutrient supplements, electrolyte supplements (e.g., supplements based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, etc.

[0194] These compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, which may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, saline or water for injection, prior to use.

[0195] The aforementioned sterile powders, granules and tablets can be used to prepare instant injections and suspensions. For example, injectable, stable sterile compositions of the present invention in unit dosage form in a sealed container can be provided. The composition can be provided in the form of a lyophilized product, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form can contain about 1 μg to about 10g of the composition of the present invention. When the composition is substantially water-insoluble, a sufficient amount of a physiologically acceptable emulsifier can be included to emulsify the composition in an aqueous carrier. A useful emulsifier is phosphatidylcholine.

[0196] Pharmaceutical compositions suitable for rectal administration may be presented as unit-dose suppositories. These may be prepared by mixing the composition with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[0197] The pharmaceutical composition of the present invention suitable for topical application to the skin can take the form of an ointment, cream, lotion, paste, gel, spray, aerosol or oil. Useful carriers include, but are not limited to, vaseline, lanolin, polyethylene glycol, alcohol, transdermal enhancers and combinations of two or more thereof. In some embodiments, for example, topical delivery can be carried out by mixing the pharmaceutical composition of the present invention with a lipophilic agent (e.g., DMSO) that can pass through the skin.

[0198] The pharmaceutical composition suitable for transdermal administration can be in the form of discrete patches, which are suitable for keeping close contact with the epidermis of the experimenter for a long time. Compositions suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharm.Res.3:318 (1986)), and the form of optional buffered aqueous solution of the present composition is generally adopted. Suitable preparations can include citrate or bis\tris buffer (pH 6) or ethanol / water, and can include 0.1 to 0.2M active ingredient.

[0199] The viral vector disclosed herein can be administered to the lungs of a subject in any suitable manner, for example, by administering an aerosol suspension of inhalable particles composed of the viral vector that is inhaled by the subject. The inhalable particles can be liquid or solid. Liquid particle aerosols comprising the viral vector can be prepared by any suitable means, for example, using a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is familiar to those skilled in the art. See, for example, U.S. Patent No. 4,501,729. Similarly, solid particle aerosols comprising the viral vector can be prepared using any solid particle pharmaceutical aerosol generator using methods familiar to the pharmaceutical field.

[0200] The present invention has been described above, and these aspects will be explained in more detail in the following examples, which are only used for illustrative purposes and are not used to limit the present invention. Example 1 Construction of AAV capsid gene shuffling library

[0201] The AAV capsid gene shuffling library was constructed as described previously (Yang et al., Proc. Natl. Acad. Sci. USA 106 (10): 3946 (2009); Yang et al., Meth. Mol. Biol. 709: 127 (2011)). The capsid genes of AAV serotypes 1, 2, 3B, 4, 6, 7, 8 and 9 were cloned into the same vector pXX-UF1-AAV containing the AAV2 inverted terminal repeats (ITRs) and the AAV2 Rep gene. These plasmids containing ITRs, AAV2 Rep and each of the eight different Cap genes were named pXX-UF1-AAVn in turn. These capsid genes were amplified by primers CAP-5'5'-CCC-AAGCTTCGATCAACTACGCAGACAGGTACCAA-3' (SEQ ID NO: 7) and CAP-3'5'-ATAAGAAT-GCGGCCGC-AGAGACCAAAGTTCAACTGAAACGA-3' (SEQ ID NO: 8) and mixed in the same ratio for DNA shuffling (Stemmer, Proc. Natl. Acad. Sci. USA 91: 10747 (1994)). Briefly, 4 μg of DNA template was briefly treated with 0.04 U DNase I at 15°C, and 300-500 and 500-1000 bp fragments were purified from agarose gel using pfu DNA polymerase for recombinant capsid genes. The recombinant capsid gene was re-amplified by pfu DNA polymerase using CAP5' / CAP3' primers, digested with HindIII / NotI, and ligated into HindIII / NotI-digested pXX-UF1-AAV vector containing the AAV2 ITR flanked by the AAV2 Rep and Cap genes. HindIII / Not I digestion effectively removed the AAV2 capsid gene to replace the PCR-amplified fragment of the liver-enriched Cap gene. The PCR program was 94°C for 5 min; 94°C for 1 min, 75°C for 5 min for hot start, 60°C for 1 min, 72°C for 4.5 min; 29 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 4.5 min, and 72°C for 10 min. The reconstructed plasmid was transformed into E. coli DH10B cells, and multiple clones were randomly selected for restriction enzyme analysis to determine the recombination frequency and packaging viability. The resulting library of infectious AAV plasmids containing ITRs was used to generate a library of self-packaging AAV viral particles. Example 2 In vivo enrichment in mouse liver

[0202] For in vivo screening of liver-targeted AAV variants, 1x1011 vg (vector genome) / mouse The AAV chimeric library was administered to adult C57BL / 6J mice via the tail vein. Two weeks later, mouse tissues, including liver and other tissues, were collected and genomic DNA was isolated using phenol / chloroform extraction. After PCR amplification of total liver DNA using universal primers for the capsid gene, the PCR product was recloned into the AAV plasmid backbone containing the AAV2 ITR and Rep genes. Therefore, both the capsid gene PCR product and the pXX-UF1-AAV vector were digested with HindIII / NotI and ligated for cloning. A second round of infectious AAV particles was generated from the first round of in vivo enrichment and then injected into mice again. This in vivo enrichment process was repeated three times in mice.

[0203] The final PCR product, which was highly enriched for capsid genes, was cloned into a universal precursor of an AAV packaging plasmid that contains the AAV2 Rep gene without the AAV inverted terminal repeats (ITRs). The capsid gene was ligated downstream of the Rep gene and upstream of the AAV polyadenylation signal sequence to reconstruct the Rep and Cap gene expression cassette. The ligation product was electroporated into the E. coli DH10B strain and grown on Amp-resistant plates. Single colonies were picked and microprepared DNA was isolated for DNA sequencing of the capsid genes. Many novel capsid genes were discovered, two of which were highly enriched. The two capsid genes were named AAVXL32 and AAVXL12, respectively. Their DNA sequences are shown in SEQ ID NOS: 1 and 2, and the amino acid sequences are shown in SEQ ID NOS: 4 and 5. Although the AAVXL12 and AAVXL32 DNA sequences differ by 23 nucleotides (23 of 2214 nucleotides), their amino acid sequences differ by only 2 residues (2 of 737 amino acids). Amino acid 220 is S in XL12 and D in XL32; amino acid 643 is N in XL12 and H in XL32. Example 3 Generation of AAV vectors containing reporter genes

[0204] Capsid-specific packaging plasmids were prepared by conventional large-scale preparation methods and purified twice by CsCl density ultracentrifugation. These plasmids are used to package AAV vectors containing reporter genes (e.g., green fluorescent protein GFP and β-galactosidase Lac-Z gene) under the transcriptional control of the ubiquitous promoter CMV or CB (CMV enhancer plus chicken β-actin basic promoter). The AAV vector was packaged in each new capsid and purified by conventional double CsCl density ultracentrifugation. The titer of the reporter vector was determined by DNA dot blotting relative to the known copy number of the corresponding reporter vector plasmid DNA. All vector yields were within the normal range. Example 4 In vivo investigation of AAV capsid enrichment in the liver of mice

[0205] To investigate whether the novel capsids enriched and selected in vivo are effective in transducing the liver, two reporter genes, GFP and LacZ, were packaged using AAVXL12 and AAVXL32, respectively. These vectors were purified, titrated, and injected into C57B / 6 mice via the tail vein at a dose of 5x10 12 vg / kg body weight. AAV9 vectors packaging the same GFP and LacZ genes were used as positive controls because AAV9 gene transfer in mouse liver is very robust. Figure 1A-1B As shown, the results show that for both GFP and LacZ reporters, AAVXL12 and AAVXL32 achieved essentially the same levels of hepatic gene expression as control AAV9, thus validating the in vivo selection strategy for enrichment of mouse hepatotropic capsid genes. Example 5 In vitro investigation of liver-enriched AAV capsids in human hepatoma cell lines

[0206] In order to observe whether the hepatotropic capsid can transduce human hepatocytes, the vector was first investigated in vitro on the human hepatoma cell line Huh7. Huh7 is widely used in in vitro determination of gene expression of AAV vectors with liver-specific promoters. This cell line retains certain characteristics of human hepatocytes and is positively correlated with cultured primary human hepatocytes. Therefore, this cell line can be used for preliminary testing of the hepatotropic properties of AAV capsids in human cells. Again, AAV9 was used as a positive control. Figure 2 As shown, both AAVXL12 and AAVXL32 can be used at a multiplicity of infection (MOI) of 1x10 5 Efficient gene expression was obtained under the conditions of 10 vg / cell and 1x104 vg / cell. On the other hand, AAV9 was not efficient in transducing Huh7 cells. At higher moi, only a small number of lacZ-positive cells were detected. These results indicate that AAVXL12 and AAVXL32 have high affinity for human hepatocytes in addition to mouse hepatocytes. Example 6 In vitro investigation of liver-enriched AAV capsids in human and dog primary hepatocytes

[0207] Because some AAV serotypes can transduce mouse liver very well but not human or non-human (monkey) liver well (Hurlbut et al., Mol. Ther. 18:1983 (2010)), while other serotypes have poor transduction ability in mouse liver but perform well in mouse models of humanized liver (Lisowski et al., Nature 506(7488):382 (2014)) and monkeys (Li et al., Mol. Ther. 12:1867 (2015)), this difference prompted people to examine whether the new hepatotropic AAV capsids can also effectively transduce human and canine primary hepatocyte cultures.

[0208] The vectors were first investigated in vitro on primary human hepatocytes isolated from three human donors. Hepatocytes were purchased from TRL (Triangle Research Laboratories, now a subsidiary of Invitrogen) and cultured in medium provided by the supplier according to the protocol provided by the supplier. GFP under the control of the ubiquitous CMV promoter was used as a reporter gene and packaged in AAVXL12, AAVXL32 and AAV2, 6, 7, 8, 9, etc. These vectors were used to infect primary hepatocyte cultures with a cell confluency of approximately 80% and a vector dose of 1x10 at a multiplicity of infection (moi). 5 vg / cell. Take cell fluorescence photos at different time points to understand the expression of GFP in green. Figures 3A-3D The results show that, although widely reported serotype capsids (e.g., AAV8 and AAV9) can efficiently transduce mouse liver, these vectors are very poorly infective for primary human hepatocytes. In contrast, the novel hepatotropic AAV capsid enriched in mouse liver also transduced primary human hepatocytes with the highest efficiency among all other naturally occurring vectors tested in this study. Figure 3A ), other AAV serotypes had almost no GFP expression, while AAVXL12 and AAVXL32 had obvious GFP expression. Figure 3B ), AAVXL12 and AAVXL32 again showed the highest level of GFP expression. Hepatocytes infected with the above two vectors were cultured for 7 days, and GFP images were taken every day. The GFP fluorescence intensity increased over time and reached the highest level on the 7th day ( Figure 3C and 3D). Therefore, the new capsid is in sharp contrast to other AAV capsids (such as AAV3 and AAVLK03) (Lisowski et al., Nature 506 (7488): 382 (2014)) and monkeys that have poor ability to transduce mouse hepatocytes in humanized mouse liver and non-human primate liver but can robustly transduce human hepatocytes (Li et al., Mol. Ther. 12: 1867 (2015)). Other AAV serotypes (such as AAV2 and AAV5) have been reported to not transduce mouse and non-human primate livers well. The above findings strongly suggest that both AAVXL12 and AAVXL32 have unique properties not seen in other known AAV capsids.

[0209] Subsequently, the infectivity of hepatotropic AAV was also investigated using primary dog ​​hepatocytes and compared with a number of commonly used AAV capsids containing the GFP reporter gene. The dose of AAV vector genome particles used was an infection multiplicity of 1x105 vg / cell. Ultra-cryopreserved and fresh hepatocytes from two dogs were used. Green fluorescence images of AAV GFP vector-infected cells were taken 24 hours and 48 hours after infection. Figures 4A-4C As shown, the gene transfer efficiency of AAVXL12 and AAVXL32 was higher than that of other serotypes except AAV6, which was similar or slightly higher than that of AAVXL12 and AAVXL32. It is worth noting that although AAV6 showed high levels of gene transfer in canine hepatocytes, this was not observed in human primary hepatocyte cultures. These results indicate that both AAVXL12 and AAVXL32 have high infectivity on canine hepatocytes. Example 7 In vivo investigation of AAVXL32 in non-human primate liver

[0210] Next, we investigated whether the hepatotropic capsids were also able to achieve efficient gene transfer in the liver of non-human primates. AAVXL32 was chosen for comparison with AAV8. The latter has been previously reported to transduce monkey liver and has also been used for liver-directed gene therapy in human hemophilia B clinical trials. Based on previously published papers, adult male rhesus macaques aged 3-5 years were screened for serum neutralizing antibodies against AAVXL32 and AAV8. Four monkeys with neutralizing antibody titers below 1:8 were selected and injected intravenously with AAV vectors. Two macaques were injected with AAVXL32-CB-GFP and the other two with AAV-CB-GFP at a dose of 1x10 12 vg / kg body weight. The AAV vector expression cassette adopts a double-stranded (also called self-complementary) form, which can achieve faster and more efficient gene expression. The monkeys were euthanized two weeks later, and liver tissue was extracted for frozen sectioning and green fluorescence photography. Figure 5As shown, in monkey liver, the GFP expression level of AAVXL32 was significantly higher than that of AAV8. Due to the higher fluorescence intensity and more positive cells (as shown by arrows), GFP-positive cells were easily detected in monkeys treated with AAVXL32. On the other hand, monkeys treated with AAV8 had far fewer positive cells and weaker fluorescence intensity. These results strongly suggest that AAVXL32 is significantly more hepatotropic than AAV8 in non-human primates. Example 8 Characterization of AAVXL32 capsid proteins and removal of the weak start codon of the third capsid protein

[0211] To characterize the capsid protein, AAVXL32 capsids purified by double CsCl ultracentrifugation and titrated by DNA dot blot were analyzed. The purified vector was loaded on an SDS PAGE gel to determine the molecular weight of the VP protein after silver staining. Figure 6 As shown, AAVXL32 shows 4 bands. In addition to the typical VP1, VP2 and VP3 proteins of all natural AAVs, there is an additional band (referred to herein as VPX) between VP1 and VP2. This additional band is the result of a single nucleotide site-directed mutagenesis of the AAV7 and AAV8 capsid genes at the XhoI restriction site in the unique region of the large protein VP1. The original purpose of this mutation was to facilitate DNA cloning in the AAV capsid gene DNA shuffling library, which includes capsid genes of AAV serotypes 1, 2, 3, 4, 6, 7, 8 and 9 (Yang et al., Proc. Natl. Acad. Sci. USA 106 (10): 3946 (2009)). Counting from the VP1 start codon, a single C to G mutation (CTCGAG to CTGGAG) at the XhoI site at position 219 produced an amino acid leucine sense mutation in the AAV7 and AAV8 capsid genes. This mutation did not change the amino acid sequence of the VP1 protein. However, it was suspected that this sense mutation produced a weaker non-consensus start codon, CTG (relative to the classical ATG start codon). This may have led to the generation of an additional capsid protein, VPX, between VP1 and VP2. Apparently, this additional band did not impair the packaging and infectivity of the vector DNA in an obvious way. To confirm that the sense mutation produced a weak start codon for an additional capsid protein, site-directed mutagenesis was performed to reverse the C to G mutation back to the original C of the wild-type AAV7 and AAV8 sequences. This new capsid gene with the reverse mutation, called AAVXL32.1, indeed inactivated the weak start codon and removed the additional capsid band between VP1 and VP2 ( Figure 6LacZ reporter vectors packaged in either XL32 or XL32.1 showed no obvious differences in vector yield or infectivity, suggesting that the additional capsid band plays an insignificant or unknown role in AAV capsid function. Example 9 Prevalence of neutralizing antibodies in human serum samples

[0212] Pre-existing neutralizing antibodies (Nabs) against AAV capsids in the population are an important problem encountered in AAV-mediated in vivo gene therapy. Certain serotypes of AAV, such as AAV2 and AAV3, have a high incidence of Nabs (Ling, J. Integr. Med. 5:341 (2015)). The incidence of Nabs against the hepatotropic AAVXL32 capsid was preliminarily evaluated. The incidence of Nabs in a group of Chinese patient serum samples was investigated, and the patient identities were kept completely confidential. Specifically, pre-existing NAbs against AAVXL32 capsids in the sera of 100 different populations were measured. The Nab assay was essentially based on the method previously published by Ling et al. and the references therein (Ling, J. Integr. Med. 5:341 (2015)). It was modified by using secreted luciferase (Gaussia luciferase) as a reporter gene and the human liver cell line Huh7 as a test cell. Briefly, Huh7 cells were cultured at 5×10 4 Cells / well were seeded in round-bottom 96-well plates. On the second day, individual serum samples were serially diluted in 2-fold increments. The positive control for Nab was from the serum of mice immunized with AAVXL32, and the negative control was from the serum of young mice purchased from Sigma. The diluted serum samples and AAV were incubated at 37°C for 1 hour and added to the plates seeded with Huh7 cells one day earlier. 48 hours after AAVXL32 infection, a small aliquot of the cell culture medium was taken from each well for luciferase activity assay. Unlike firefly luciferase, Gaussia luciferase is a secreted enzyme, and enzymatic activity can be measured without cell lysis. The results showed that more than 50% of the serum samples had Nab titers below a 1:8 dilution, and approximately 70% were below a 1:16 dilution. These data suggest that the incidence of Nab for AAVXL32 in the Chinese population is much lower than previously published data for AAV2, AAV3, AAVKL3, AAV5, and AAV8. In those studies, more than 90% of the subjects were found to be neutralizing antibody positive for AAV2, AAV3, and AAVK03, with a serum dilution cutoff of 1:20, while at the same dilution cutoff of 1:20, the Nab positivity rate for AAV8 was higher than 80%, and the positivity rate for AAV5 was higher than 70%.

[0213] The above description is intended to illustrate the present invention and should not be considered as limiting the present invention. The present invention is defined by the following claims, and the contents equivalent to the claims are also included in the present invention. AAV capsid sequences SEQ ID NO: 1AAVXL12 capsid gene DNA sequence SEQ ID NO:2AAVXL32 capsid gene DNA sequence SEQ ID NO:3AAVXL32.1 capsid gene DNA sequence SEQ ID NO:4 AAVXL12 capsid amino acid sequence SEQ ID NO:5 AAVXL32 capsid amino acid sequence SEQ ID NO:6 AAVXL32.1 capsid amino acid sequence SEQ ID NO:7 DNA primer CAP-5' SEQ ID NO:8 DNA primer CAP-3'

Claims

1. A nucleic acid encoding an AAV capsid, the nucleic acid comprising an AAV capsid coding sequence that is at least 90% identical to: (a) a nucleotide sequence of any one of SEQ ID NOS: 1-3; or (b) A nucleotide sequence encoding any one of SEQ ID NOS: 4-6.

2. An in vitro cell comprising the nucleic acid of claim 1 stably integrated into its genome.

3. A viral particle comprising the nucleic acid according to claim 1.

4. An AAV capsid comprising an amino acid sequence that is at least 90% identical to any one of SEQ ID NOS: 4-6.

5. An AAV particle comprising: AAV vector genome; and The AAV capsid of claim 4, wherein the AAV capsid encapsidates the AAV vector genome.

6. A method for preparing a recombinant AAV particle comprising an AAV capsid, the method comprising: providing cells in vitro with the nucleic acid of claim 1, an AAV rep coding sequence, an AAV vector genome comprising a heterologous nucleic acid, and helper functions for producing a productive AAV infection; and Assembling recombinant AAV particles comprising the AAV capsid and encapsidating the AAV vector genome.

7. A pharmaceutical formulation comprising the nucleic acid of claim 1, the viral particle of claim 3, the AAV capsid of claim 4, or the AAV particle of claim 5 in a pharmaceutically acceptable carrier.

8. A method for delivering a target nucleic acid to a hepatocyte, the method comprising contacting the hepatocyte with the AAV particle of claim 5.

9. A method for delivering a target nucleic acid into a hepatocyte of a mammalian subject, the method comprising: An effective amount of the AAV particle of claim 5 or the pharmaceutical formulation of claim 7 is administered to a mammalian subject, thereby delivering the target nucleic acid to hepatocytes of the mammalian subject.

10. A method for treating a disease in a mammalian subject in need thereof, wherein the disease is treatable by expressing a product in the subject's liver, the method comprising administering to the mammalian subject a therapeutically effective dose of the AAV particles of claim 5 or the pharmaceutical formulation of claim 7, wherein the product is expressed, thereby treating the disease.

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