AAV capsid proteins for nucleic acid transfer

Abstract

The present invention provides recombinant adeno-associated virus (rAAV) vectors that express recombinant capsid proteins having enhanced transduction properties in humans and mice. The invention also provides methods for producing rAAV capsid proteins and assays for assessing transduction efficiency. The invention also describes a method for determining the efficacy of a treatment in two mammalian species with respect to a gene of interest by delivering to them an rAAV carrying the gene of interest encased in a recombinant capsid protein.

Description

[0001] Statement Regarding Federally Sponsored Research

[0002] This invention was made with government support under Contract AI116698 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Reference to a sequence listing, table or computer program

[0004] This application contains a sequence listing, which is submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on April 19, 2023 is named "091511-0648_SEQ" and is 7,144 bytes in size. Technical Field

[0005] The subject matter described herein relates to nucleotide sequences encoding recombinant adeno-associated virus (rAAV) capsid proteins with enhanced cross-species transduction properties. The subject matter also relates to plasmids and viruses comprising the identified sequences to provide high transduction efficiency and low levels of neutralization by the human immune system. The subject matter also relates to methods for transferring target nucleic acid sequences by using rAAV vectors encoding recombinant capsid proteins. The subject matter also relates to methods for determining the therapeutic efficacy of target genes in rodents and humans. Background of the Invention

[0007] In recent years, a variety of recombinant gene transfer vectors based on different types of viruses have been developed and tested in clinical trials. Gene transfer vectors based on adeno-associated viruses (AAV), i.e., AAV vectors, have become the preferred vectors because they have features such as the ability to transduce different types of dividing and non-dividing cells of different tissues, and the ability to establish stable, long-term transgene expression. Although vectors based on other viruses (e.g., adenoviruses and retroviruses) may have certain desirable features, the use of other vectors has been associated with toxicity or some human diseases. These side effects have not been detected with AAV-based gene transfer vectors (Manno et al., Nature Medicine, 12(3): 342(2006)). In addition, techniques have been developed that can produce and purify AAV vectors without excessive effort.

[0008] At least 11 AAV serotypes have been identified, cloned, sequenced, and converted into vectors, and at least 100 new AAV variants have been isolated from non-primates, primates, and humans. However, to date, most preclinical data involving AAV vectors have been generated with vectors based on the human AAV-2 serotype, which is considered the prototype of AAV.

[0009] There are several disadvantages to the AAV-2 vectors currently used. For example, many clinically relevant cell types and tissues cannot be effectively transduced with these vectors. In addition, a large percentage of the population is immune to AAV-2 due to previous exposure to wild-type AAV-2 virus. It has been estimated that up to 96% of people are seropositive for AAV-2, and up to 67% of seropositive individuals carry neutralizing anti-AAV-2 antibodies, which can eliminate or greatly reduce transduction by AAV-2 vectors. In addition, it has been reported that AAV-2 induces a cell-mediated immune response in patients when administered systemically (Manno et al., Nature Medicine, 12(3):342(2006)).

[0010] Methods to overcome the limitations of AAV-2 vectors have been proposed. For example, random mutagenesis of the nucleotide sequence encoding the AAV-2 capsid by error-prone PCR has been proposed as a method to generate AAV-2 mutants that can escape neutralizing antibodies that affect wild-type AAV-2. However, it is expected that the generation of significantly improved AAV-2 variants with a single random point mutation will be difficult because naturally occurring serotypes have only about 85% homology in the capsid nucleotide sequence at best.

[0011] Methods have also been developed to use mixtures of AAV serotype constructs for AAV vectors. The resulting chimeric vectors have capsid proteins from different serotypes, and ideally have the properties of the different serotypes used. However, the ratio of different capsid proteins varies between vectors and cannot be consistently controlled and reproduced (due to lack of genetic templates), which is unacceptable for clinical use and unsatisfactory for experimental use.

[0012] AAV-LK03, a recombinant AAV vector that is the subject of U.S. Patent 9,169,299, exhibits 30 times better transduction than AAV-DJ (U.S. Patent 8,067,014). AAV-LK03 is primate-specific, and therefore preclinical studies (typically conducted in non-primates, particularly rodents) require the use of alternative serotypes, resulting in higher costs and time for preclinical development. AAV-based vectors that can be used effectively across species do not exist in the art. Therefore, there is an urgent need for AAV vectors with cross-species compatibility, which can bypass the need for alternative serotypes. The present invention addresses this long-standing need in the art.

[0013] The above examples of the related art and limitations associated therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become clear to those skilled in the art after reading the specification and studying the drawings. SUMMARY OF THE INVENTION

[0015] The following aspects and embodiments thereof described and illustrated below are intended to be exemplary and illustrative, not limiting in scope.

[0016] In one aspect, a recombinant capsid protein and a method for producing the recombinant capsid protein are provided. The capsid protein includes regions or domains derived from different serotypes of AAV. The AAV serotype can be human or non-human. A recombinant AAV comprising the capsid protein and a plasmid encoding the capsid protein are also provided.

[0017] In one aspect, the capsid protein comprises the first amino acid sequence indicated in SEQ ID NO: 1 (AAV-AM).

[0018] In one embodiment, the capsid protein comprises an amino acid residue sequence that is substantially similar or substantially identical to AAV-LK03, except for an additional glycine residue inserted into the amino acid sequence of AAV-LK03.

[0019] In some aspects, the capsid protein is encoded by the nucleotide sequence indicated in SEQ ID NO:2, or a sequence having at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity thereto.

[0020] In one embodiment, the capsid protein comprises the amino acid sequence shown in SEQ ID NO:1, which is encoded by the nucleotide sequence indicated in SEQ ID NO:2.

[0021] In one embodiment, the capsid protein has a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, with the proviso that the protein is not identical to SEQ ID NO: 3. In one embodiment, the capsid protein is not SEQ ID NO: 3.

[0022] In some embodiments, viral particles comprising the capsid protein sequence described above are contemplated.Disclosed herein are viral particles comprising a capsid protein encoded by a nucleotide sequence as indicated in SEQ ID NO: 2 of the sequence listing or a nucleotide sequence having at least 95% sequence identity thereto.

[0023] Also disclosed is a plasmid comprising the nucleotide sequence indicated in SEQ ID NO: 2, or a sequence having at least 95% sequence identity thereto.

[0024] Also disclosed is a recombinant AAV vector (rAAV) comprising a capsid protein having an amino acid sequence encoded by the nucleotide sequence indicated in SEQ ID NO: 2 or a nucleotide sequence having at least 95% sequence identity thereto.

[0025] The present disclosure also provides a method for transferring a target nucleic acid sequence into a cell or a mammal, which comprises introducing a recombinant AAV (rAAV) vector into a cell or a mammal, wherein the recombinant AAV vector encodes a target gene, and the target gene is encapsidated in a recombinant capsid protein indicated by the amino acid sequence of SEQ ID NO: 1 (AAV-AM).

[0026] The present disclosure further provides a method for transferring a target nucleic acid into a cell or a mammal, comprising introducing a recombinant AAV (rAAV) vector into a cell or a mammal, wherein the recombinant AAV vector encodes a recombinant capsid nucleotide sequence and a target gene, wherein the target gene is encapsidated in a recombinant capsid protein having an amino acid sequence encoded by the following sequence: the nucleotide sequence indicated in SEQ ID NO:2, or a sequence having at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity or at least about 99% sequence identity thereto. In this embodiment, the rAAV vector is produced by the following steps: site-directed mutagenesis (SDM) of the AAV-LK03 plasmid (U.S. Pat. No. 9,169,299) using overlapping primers containing a foreign triplet nucleotide sequence; exponential amplification by PCR to generate amplicons, the PCR using the AAV-LK03 plasmid as a template and a primer pair containing the foreign triplet nucleotide sequence; incubating the amplicon with a kinase, a ligase and DpnII; ​​performing high-efficiency transformation in bacteria; culturing the bacteria in the presence of a selective antibiotic to isolate bacterial clones containing the target nucleic acid sequence; confirming the presence of the recombinant capsid nucleotide sequence by sequencing; and purifying the plasmid containing the target nucleic acid sequence from the bacterial clone to produce the rAAV vector.

[0027] In another embodiment, a method for determining the therapeutic efficacy of a target gene is provided, comprising: delivering a recombinant AAV (rAAV) vector to a first mammal, the rAAV vector comprising a nucleotide sequence encoding a recombinant capsid protein having the amino acid sequence shown in SEQ ID NO: 1 (AAV-AM), and a target gene, wherein the target gene is encapsidated in the recombinant capsid protein; evaluating the transduction efficiency in the first mammal; when the transduction efficiency in the first mammal is higher than the transduction efficiency observed after infecting the first mammal with a control rAAV vector, delivering the rAAV vector to a second mammal; evaluating the transduction efficiency in the second mammal, whereby a higher transduction efficiency of the rAAV vector in the first mammal and the second mammal relative to the control rAAV vector indicates a higher therapeutic efficacy of the target gene.

[0028] Adeno-associated virus has a broad host range, i.e., it can infect many mammalian cell lines or primary cells, including replicating as well as non-replicating cells, and including cells resident in mammalian tissues. Some lymphoid cell lines may be more resistant to adeno-associated virus infection, and therefore high viral loads may be required to achieve adequate infection levels. Cell types that can be used in the methods disclosed herein include, but are not limited to, CHO cells, monocytes, dendritic cells (DCs), freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, Langerhans cells and dermal DCs, human T-cell leukemia DND-41 cells, p53-deficient cancer cells, tumor cells retaining wild-type p53, tumor cells with unknown p53 status, adenocarcinoma human alveolar basal epithelial cells (also referred to as "A549 cells"), human KB cells, Madin Darby bovine kidney (MDBK) cells, mouse Hepa1-6 cells, mouse embryonic fibroblasts (MEF cells), human HuH-7 cells, pulmonary artery endothelial cells (hPAEC), NIH-3T3 cells, Hep G2 cells, HEp-2 cells, HeLa cells, Dempsey cells, human embryonic kidney 293 cells (also referred to as "HEK 293" or "293 cells"), fetal rhesus monkey kidney (FRhK-4) cells, rat hepatoma H4TG cells, LMH chicken hepatoma epithelial cells, primary human hepatocytes, and primary human keratinocytes. In some aspects, the rAAV is used to infect 293 cells. In some aspects, the rAAV is used to infect Hepa1-6 cells. In some aspects, the rAAV is used to infect HuH-7 cells. In some embodiments, a helper adenovirus is used.

[0029] In some embodiments, humanized FRG mice are transfected with an AAV in vitro selection library. In some embodiments, non-humanized FRG mice are transfected with an AAV in vitro selection library.

[0030] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figures 1A-1F Shown are the transduction efficiencies of AAV (1e4 vg / cell) carrying AAV-LK03 vector (also referred to as LK03) or AAV-AM vector in human (HuH-7) and mouse (Hepa1-6) cells. Figure 1A Luciferase activity at 16 hours after transduction with LK03 or AAV-AM is shown. Figure 1B Luciferase activity at 48 hours after transduction with LK03 or AAV-AM is shown. Figure 1C Shown is the assessment of luciferase mRNA expression by qPCR 16 hours after transduction with LK03 or AAV-AM. Figure 1D Shown is the assessment of luciferase mRNA expression by qPCR 48 hours after transduction with LK03 or AAV-AM. Figure 1E Shown are assessments of nuclear vector copy number by qPCR 16 hours after transduction with LK03 or AAV-AM. Figure 1F Shown is the assessment of nuclear vector copy number by qPCR 48 hours after transduction with LK03 or AAV-AM.

[0033] Figure 2A-2B Shown are the results of an in vivo luciferase reporter assay showing transduction efficiency in mice following intravenous injection. Figure 2A Shown are luciferase activities in animals at day 3 after injection of AAV-LK03 (3e11 viral genomes), exposure time 120 seconds. Figure 2B Shown are luciferase activities in animals at day 3 after injection of AAV AAV-AM (3e11 viral genomes), exposure time 1 sec.

[0034] Figures 3A-3F Shown are the evaluations of mouse tissues for transduction efficiency of AAV carrying LK03 or AAV-AM vectors following intravenous injection. Figure 3A Shown are luciferase activities 3 days after animals were transduced with LK03 or AAV-AM (3e11 viral genomes). Figure 3BShown are luciferase activities at 15 days after animals were transduced with LK03 or AAV-AM. Figure 3C Shown is the assessment of luciferase mRNA expression by qPCR 3 days after animals were transduced with LK03 or AAV-AM. Figure 3D Shown is the assessment of luciferase mRNA expression by qPCR 15 days after animals were transduced with LK03 or AAV-AM. Figure 3E Shown is the assessment of nuclear vector copy number by qPCR 3 days after transduction of animals with LK03 or AAV-AM. Figure 3F Shown is the assessment of nuclear vector copy number by qPCR 15 days after transduction of animals with LK03 or AAV-AM.

[0035] Figure 4 Prediction model for AAV-AM compared to AAV-LK03.

[0036] Sequence Description

[0037] SEQ ID NO: 1 is the amino acid sequence encoded by SEQ ID NO: 2 and is referred to herein as AAV-AM. See Table 1.

[0038] It will be appreciated that each of the nucleotide sequences disclosed herein can be translated to predict an amino acid sequence representing a rAAV capsid protein. DETAILED DESCRIPTION OF THE INVENTION

[0040] Several embodiments of the present disclosure are described in detail below. These embodiments may take many different forms and should not be construed as being limited to those embodiments explicitly set forth herein. On the contrary, these embodiments are provided so that the disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art.

[0041] I. definition

[0042] When providing a range of values, it should be understood that each intermediate value between the upper and lower limits of that range is also specifically disclosed, to one tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in that stated range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the range or excluded, and each range in which any, none or both limits are included in the smaller range is also encompassed within the present invention, subject to any specifically excluded limits in the stated range. When the stated range includes one or both of the limits, the range excluding any or both of those included limits is also included in the present invention.

[0043] Unless otherwise defined, all technical and scientific terms used in this article have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described.

[0044] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "protein" includes more than one protein, and reference to a "compound" includes more than one compound. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set forth below.

[0045] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (when the polynucleotide is RNA, uracil (U) replaces thymine (T)). Unless otherwise stated, the abbreviated nucleotides can be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides can be designated individually or collectively as ribonucleosides or 2'-deoxyribonucleosides. When designated individually, the single-letter abbreviation is preceded by "d" or "r", where "d" indicates that the nucleoside is a 2'-deoxyribonucleoside, and "r" indicates that the nucleoside is a ribonucleoside. For example, "dA" indicates 2'-deoxyriboadenosine, and "rA" indicates riboadenosine. When designated collectively, a specific nucleic acid or polynucleotide is designated as an RNA molecule or a DNA molecule. Nucleotides are abbreviated by adding a "p" to represent each phosphate and whether the phosphate is attached to the 3'-position or the 5'-position of the sugar. Thus, 5'-nucleotides are abbreviated as "pN", and 3'-nucleotides are abbreviated as "Np", where "N" represents A, G, C, T or U. When a nucleic acid sequence is presented as a string of single-letter abbreviations, by convention, the sequence is presented in the 5'→3' direction and the phosphate is not specified. Thus, the term polynucleotide sequence is an alphabetic representation of a polynucleotide molecule. This alphabetic representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searches.

[0046] An "isolated polynucleotide" molecule is a nucleic acid molecule that is separated and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule that lacks, in whole or in part, the sequence with which it is normally associated in nature; or a sequence as it occurs in nature but with heterologous sequences associated therewith.

[0047] The technology for measuring nucleic acid and amino acid "sequence identity" is also known in the art. Typically, such technology includes determining the nucleotide sequence of the mRNA about the gene and / or determining the amino acid sequence encoded by it, and comparing these sequences with a second nucleotide or amino acid sequence. In general, "identity" refers to the precise nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences. Two or more sequences (polynucleotides or amino acids) can be compared by measuring their "identity percentage". The identity percentage of two sequences (whether nucleic acid or amino acid sequence) is the number of exact matches between the two compared sequences divided by the length of the shorter sequence and multiplied by 100. The identity percentage can also be determined, for example, by using the advanced BLAST computer program (including version 2.2.9) comparative sequence information available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul. Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990), and as discussed in Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). In short, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. The program can be used to determine the percent identity over the entire length of the compared protein. Default parameters are provided to optimize searches with short query sequences, such as in blastp using the program. The program also allows the use of SEG filters to mask segments of query sequences determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The desired degree of sequence identity ranges from about 80% to 100% and integer values ​​therebetween. Typically, the percent identity between the disclosed sequence and the claimed sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%.

[0048] Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of the polynucleotides under conditions where stable duplexes are formed between homologous regions, followed by digestion with a single-stranded specific nuclease, and size determination of the digested fragments. Two DNA or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80-85%, preferably 85-90%, more preferably 90-95%, and most preferably 98-100% sequence identity to a reference sequence over a specified length of the molecule, as determined using the above methods. As used herein, "substantially homologous" also refers to sequences that show complete identity to a specified DNA or polypeptide sequence. Substantially homologous DNA sequences can be identified in a Southern hybridization experiment, for example, under stringent conditions (as defined for that particular system). Defining appropriate hybridization conditions is within the skill of the art.

[0049] II. Chimeric AAV capsid

[0050] Capsid protein can be derived from two or more different serotypes of AAV. For example, the capsid protein can have a first region derived from a first AAV serotype or a known recombinant AAV capsid protein (e.g., AAV-DJ) or having a high level of sequence similarity or identity thereto, a second region derived from a second AAV serotype or a known recombinant AAV capsid protein or having a high level of sequence similarity or identity thereto, and a third, fourth, fifth, sixth, seventh and eighth regions derived from another AAV serotype or a known recombinant AAV capsid protein or having a high level of sequence similarity or identity thereto, and the like. The AAV serotype can be a human AAV serotype or a non-human AAV serotype, such as a murine, cattle, bird and goat AAV serotype. In particular, non-primate mammalian AAV serotypes can be used, such as AAV sequences from rodents (e.g., mice, rats, rabbits and hamsters) and carnivores (e.g., dogs, cats and raccoons). By including individual amino acids or regions from multiple AAV serotypes in one capsid protein, capsid proteins with multiple desirable properties derived separately from multiple AAV serotypes can be obtained.

[0051] The capsid protein referred to herein as "AAV-LK03" is primarily derived from AAV3B, except for a hypervariable region in the 5" of the gene and a single point mutation at the 3' end of the gene. It was obtained by screening an AAV-shuffled library selective for primate cells and was found to transduce human cells to a degree up to 30 times better than AAV-DJ. The selectivity was independent of the transgene (gene of interest) or cell type. The AAV-LK03 capsid protein is described in U.S. Patent 9,169,299 and is incorporated herein by reference in its entirety.

[0052] Although the embodiments described above are primarily about rAAV capsid proteins, it should be recognized that capsids having amino acid and / or nucleotide sequences that are similar in sequence and have the same function can be used and considered. In one embodiment, a recombinant capsid protein having at least about 60% sequence identity, further at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity is considered to be with the amino acid sequence indicated in the sequence listing.

[0053] It will be appreciated that conservative amino acid substitutions can be made in a polypeptide sequence to obtain a protein having, for example, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a polypeptide encoded by a nucleotide sequence disclosed herein, and preferably retaining the activity of the native sequence. As known in the art and as referred to herein, conservative amino acid substitutions involve replacing an amino acid in a protein with an amino acid having a side chain that is similar in, for example, structure, size and / or chemical properties. For example, the amino acids within each of the following groups can be interchanged with other amino acids in the same group: amino acids with aliphatic side chains, including glycine, alanine, valine, leucine and isoleucine; amino acids with non-aromatic, hydroxyl-containing side chains, such as serine and threonine; amino acids with acidic side chains, such as aspartic acid and glutamic acid; amino acids with amide side chains, including glutamine and asparagine; basic amino acids, including lysine, arginine and histidine; amino acids with aromatic ring side chains, including phenylalanine, tyrosine and tryptophan; and amino acids with sulfur-containing side chains, including cysteine ​​and methionine. In addition, amino acids with acidic side chains (e.g., aspartic acid and glutamic acid) are considered herein to be interchangeable with amino acids with amide side chains (e.g., asparagine and glutamine).

[0054] A mouse model system of severe immunodeficiency has been developed. These fumarylacetoacetate hydrolase (Fah)-deficient mice can be pretreated with adenovirus expressing urokinase and then highly engrafted (up to 90%) with human hepatocytes from multiple sources (including liver biopsies). Further, human cells can be continuously transplanted from the original donor and repopulated in the liver for at least four consecutive rounds. The expanded cells exhibit typical human drug metabolism. The system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It can also be useful for testing the toxicity of drug metabolites and evaluating pathogens that rely on human hepatocytes for replication (Azuma et al., (2007) Nature Biotech.25:903-910).

[0055] A humanized mouse model called the FRG mouse (Yecuris Corporation, Portland, OR) has been designed to allow researchers to grow and expand human hepatocyte populations in vivo for research and drug testing. The FRG model has knockout genes for Fah, Rag, and Ilrg. Knockout of Fah produces liver damage in mice; deficiency of Rag removes that part of the innate immune system that rejects other mouse cells; and knockout of Ilrg inactivates that part of the immune system that would prevent the engraftment of cells from other species, including humans. Thus, FRG mice can be repopulated with selected human donor cells or repopulated from a pool of prequalified donors. Animals can be provided with human hepatocytes ranging from 5-95% of total liver mass. FRG mice that have not been repopulated can also be used as research controls. In one embodiment, the recombinant AAV capsid protein comprises an amino acid residue sequence derived from an AAV serotype. In one aspect, the first recombinant AAV capsid protein (first capsid protein) has a first amino acid sequence that is closely homologous to an amino acid sequence in a second capsid protein sequence in the AAV-LK03 serotype. In one embodiment, close homology means at least about 80% sequence identity. In one embodiment, close homology means at least about 90% sequence homology. The length of the contiguous amino acid sequence in such a conservative set can be about 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, or 2 to 50 amino acid residues. In one aspect, the first recombinant AAV capsid protein has a first amino acid sequence shown in SEQ ID NO: 1, which is identical to the second capsid protein sequence (SEQ ID NO: 3) in the AAV-LK03 serotype, except for a single foreign amino acid residue inserted into the second capsid sequence. In one aspect, the single foreign amino acid residue is inserted at position 266 of the second capsid protein sequence. In one aspect of this embodiment, the foreign amino acid residue is glycine.

[0056] In this embodiment, a virus particle is also provided, which comprises an amino acid sequence for a first recombinant capsid protein. In one aspect, the first recombinant capsid protein has an amino acid sequence as shown in SEQ ID NO:1.

[0057] Further, in this embodiment, a plasmid is provided, which comprises an amino acid sequence for a recombinant capsid protein. In one aspect, the recombinant capsid protein has an amino acid sequence as shown in SEQ ID NO:1.

[0058] Also provided is a recombinant AAV vector comprising a capsid protein having the amino acid sequence shown in SEQ ID NO:1.

[0059] In another embodiment of the present invention, a first nucleotide sequence encoding a first recombinant capsid protein is provided. In one aspect, the first nucleotide sequence is closely homologous to the nucleotide sequence encoding the capsid protein in the AAV-LK03 serotype. In one embodiment, close homology means at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity. In one aspect, the first nucleotide sequence is shown in SEQ ID NO: 2, which is identical to the nucleotide sequence in AAV-LK03, except for the foreign triplet nucleotide sequence (triplet codon sequence) inserted into the nucleotide sequence in AAV-LK03. In one aspect, the triplet codon sequence encodes a single foreign amino acid residue, which is inserted at position 266 in the first recombinant AAV capsid protein sequence. In one aspect of this embodiment, the foreign amino acid residue is glycine.

[0060] In this embodiment, a virus particle is also provided, which comprises an amino acid sequence of a first recombinant capsid protein encoded by the first nucleotide sequence. In one aspect, the first nucleotide sequence is shown in SEQ ID NO:2.

[0061] Further, in this embodiment, a plasmid is provided, which comprises an amino acid sequence of a first recombinant capsid protein encoded by the first nucleotide sequence. In one aspect, the first nucleotide sequence is shown in SEQ ID NO:2.

[0062] Also provided is a recombinant AAV vector comprising a capsid protein encoded by the first nucleotide sequence shown in SEQ ID NO:2.

[0063] In another embodiment of the present invention, a method for transferring a nucleic acid sequence of interest into a cell or a mammal is provided, comprising introducing a recombinant AAV (rAAV) vector into a cell or a mammal, wherein the rAAV vector encodes a recombinant capsid nucleotide sequence and a gene of interest, wherein the gene of interest is encapsidated in the recombinant capsid protein. In one aspect, the recombinant AAV capsid protein has a first amino acid sequence that is closely homologous to an amino acid sequence in a second capsid protein sequence in the AAV-LK03 serotype. In one embodiment, close homology means at least about 80% sequence identity. In one embodiment, close homology means at least about 90% sequence homology. The length of the continuous amino acid sequence in such a conservative set can be approximately 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, or 2 to 50 amino acid residues. In one aspect, the first recombinant AAV capsid protein has a first amino acid sequence shown in SEQ ID NO: 1, which is identical to the second capsid protein sequence (SEQ ID NO: 3) in the AAV-LK03 serotype, except for a single additional or foreign amino acid residue inserted into the second capsid sequence. In one aspect, the single additional or foreign amino acid residue is inserted at position 266 of the second capsid protein sequence. In one aspect of this embodiment, the foreign or additional amino acid residue is glycine.

[0064] In another embodiment, the method of transferring a nucleic acid sequence of interest into a cell or a mammal comprises introducing a recombinant AAV (rAAV) vector into a cell or a mammal, wherein the recombinant AAV vector encodes a recombinant capsid nucleotide sequence and a gene of interest, wherein the gene of interest is encapsidated in a recombinant capsid protein having an amino acid sequence encoded by a nucleotide sequence encoding a first recombinant capsid protein. In one aspect, the first nucleotide sequence is closely homologous to the nucleotide sequence of the capsid protein encoded in the AAV-LK03 serotype. In one embodiment, close homology means at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity. In one aspect, the first nucleotide sequence is that shown in SEQ ID NO: 2, which is identical to the nucleotide sequence in AAV-LK03, except for the foreign triplet nucleotide sequence (triplet codon sequence) inserted into the nucleotide sequence in AAV-LK03. In one aspect, the triplet codon sequence encodes a single foreign amino acid residue that is inserted at position 266 in the first recombinant AAV capsid protein shown in SEQ ID NO:1.

[0065] In this embodiment, the cell types into which the rAAV can be introduced include, but are not limited to, cell lines and primary cells. Examples of such cells include: CHO cells, monocytes, dendritic cells (DC), myeloid DC, plasmacytoid DC and monocyte-derived DC, Langerhans cells and dermal DC, T cell leukemia cells, tumor cells, alveolar basal epithelial cells, Madin Darby bovine kidney (MDBK) cells, mouse Hepa1-6 cells, mouse embryonic fibroblasts (MEF), human HuH-7 cells, pulmonary artery endothelial cells (hPAEC), NIH-3T3 cells, Hep G2 cells, HEp-2 cells, HeLa cells, Dempsey cells, human embryonic kidney 293 cells (HEK 293), fetal rhesus monkey kidney (FRhK-4) cells, rat hepatoma H4TG cells, LMH chicken hepatoma epithelial cells, primary human hepatocytes, and primary human keratinocytes.

[0066] In this embodiment, the rAAV can also be introduced into mammals, including, but not limited to, primates, non-human primates and rodents (e.g., mice, rats, rabbits, hamsters) and carnivores (e.g., dogs, cats, raccoons). In one aspect, the mammal is a rodent (e.g., mouse), a human, or both a rodent and a human, which is relevant to preclinical testing of the rAAV in rodents before entering clinical studies.

[0067] In this embodiment, it may also be desirable to introduce the rAAV into specific mammalian tissues for therapeutic purposes. Mammalian tissues include, but are not limited to, liver, pancreas, blood, bone, brain, prostate, ovary, breast, bladder, and muscle. For example, in one aspect, the liver is a desirable target for rAAV transduction for gene therapy.

[0068] Also provided is a method for generating a recombinant AAV vector with enhanced transduction properties across animal species. The method includes using overlapping primers containing an external triplet nucleotide sequence to perform site-directed mutagenesis (SDM) of an AAV-LK03 plasmid. The SDM product is subjected to a PCR amplification reaction to generate an amplicon, and the PCR amplification reaction uses an AAV-LK03 plasmid as a template and a primer pair containing the external triplet nucleotide sequence. The amplicon is incubated with a kinase, a ligase, and DpnII, and then efficiently transformed in bacteria. Using a suitable antibiotic for selection pressure, the transformed bacteria are cultured to obtain a clone containing a recombinant nucleic acid sequence of interest. The nucleic acid sequence is verified by sequencing. The plasmid containing the recombinant capsid nucleic acid sequence is separated, thereby generating the recombinant AAV vector. In one aspect, the recombinant capsid nucleic acid sequence is shown in SEQ ID NO:2, which encodes the recombinant capsid protein sequence shown in SEQ ID NO:1.

[0069] In another embodiment, a method for determining therapeutic efficacy regarding a gene of interest is provided, comprising: delivering a recombinant AAV (rAAV) vector to a first mammal, the rAAV vector comprising a nucleotide sequence encoding a recombinant capsid protein having the amino acid sequence shown in SEQ ID NO: 1 (AAV-AM), and a gene of interest, wherein the gene of interest is encapsidated in the recombinant capsid protein; evaluating the transduction efficiency in the first mammal; when the transduction efficiency in the first mammal is higher than the transduction efficiency observed after infection of the first mammal with a control rAAV vector, delivering the rAAV vector to a second mammal; evaluating the transduction efficiency in the second mammal, whereby a higher transduction efficiency of the rAAV vector in the first mammal and the second mammal relative to the control rAAV vector indicates a higher therapeutic efficacy regarding the gene of interest.

[0070] In this embodiment, in one aspect, the first mammalian species is a rodent (eg, mouse, rat, rabbit, hamster), and the second mammalian species is a primate (eg, human).

[0071] In this embodiment, the transduction efficiency is determined by measuring at least one of the following: nuclear vector copy number, expression of the gene of interest, and activity of a protein encoded by the gene of interest.

[0072] It will also be appreciated that the recombinant vectors described herein are contemplated for use in methods for expressing a target gene in a variety of cells and mammals. Transduction into cell lines other than the cell lines described herein is exemplary, and other cell lines, particularly stem cells, are contemplated. In vivo use, the method preferably includes introducing a recombinant AAV (rAAV) into a mammal, the recombinant AAV vector encoding the target gene and comprising a first capsid protein having an amino acid sequence with a single foreign amino acid residue inserted into a second capsid sequence. The vector expressing the target gene is introduced into a mammal, typically by injection, intravenously, subcutaneously, intraperitoneally, etc. The target gene can be any gene, and many suitable genes for expression for therapeutic or non-therapeutic purposes are easily identified by a technician. The nucleotide sequence of the target gene is typically "operably linked" to one or more other nucleotide sequences (including, but not limited to, genes, promoters, and enhancers for selected capsid proteins, etc.).

[0073] In this embodiment, the rAAV can be introduced into any mammal, including but not limited to primates, non-human primates, rodents (eg, mice, rats, rabbits, hamsters), and carnivores (eg, dogs, cats, raccoons).

[0074] When a gene is placed in a functional relationship with another nucleotide sequence, the gene is "operably connected" to another nucleotide sequence. For example, if a coding sequence is operably connected to a promoter sequence, this generally means that the promoter can promote the transcription of the coding sequence. Operably connected means that the DNA sequence connected is typically continuous and is continuous and in frame when two protein coding regions need to be connected. However, since enhancers can work when separated from promoters by several thousand bases and intron sequences can have variable lengths, some nucleotide sequences can be operably connected but not continuous. In addition, as defined herein, nucleotide sequences are intended to refer to natural or synthetic linear and sequential queues of nucleotides and / or nucleosides and derivatives thereof. The term "coding" refers to such a process by which a nucleotide sequence provides information to a cell through the mechanism of transcription and translation, from which a series of amino acids can be assembled into a specific amino acid sequence to produce a polypeptide.

[0075] III. Embodiment

[0076] The following examples are illustrative and are not intended to be limiting. For technical procedures, reference may be made to U.S. Pat. No. 8,067,014 (which is incorporated herein in its entirety by reference), and Grimm, D. et al. (Blood, 102: 2412-2419 (2003)). For in vivo procedures and methods for the generation of AAV capsid libraries (including the generation of rAAV such as AAV-LK03), reference may be made to U.S. Pat. No. 9,169,299 (which is incorporated herein in its entirety by reference).

[0077] Example 1

[0078] Methods used to assess transduction efficiency

[0079] Luciferase assay

[0080] Cells were plated in tissue culture dishes. After 12-24 hours, cells were transduced with rAAV and incubated for another 16 hours or 48 hours to allow expression of luciferase. TM Luciferase assay reagent (Promega) is used to quantify luciferase activity. Growth medium is removed, and a 1:4 dilution of the assay reagent in PBS is added to the cells. The cells are incubated for 10 minutes under shaking, and luminescence is subsequently measured using a luminometer. The plotting of relative light units (RLU) is used for quantification.

[0081] Luciferase mRNA expression by qPCR

[0082] The cells were plated in tissue culture dishes. After 12-24 hours, the cells were transduced with rAAV and incubated for another 16 hours or 48 hours. The cells were plated in tissue culture dishes. After 12-24 hours, the cells were transduced with rAAV and incubated for another 16 hours or 48 hours. The growth medium was removed and TRIZOL was added to the cells. TM Reagents were added and then incubated for 10 minutes. Samples were then collected and RNA was isolated using the Direct-zol RNA miniprep kit (Zymo Research) according to the manufacturer's protocol.

[0083] Use High-Capacity RNA-TO-cDNA according to the company's experimental protocol TM Kit (Applied Biosciences) was used to reverse transcribe 50 ng of the isolated RNA.

[0084] SYBER Green qPCR using primers for the gene of interest (eg, luciferase) and a normalization gene (actin) was employed in this assay. Plots of relative enrichment to actin were used for quantification.

[0085] Nuclear vector copy number, by qPCR

[0086] Cells are seeded in tissue culture dishes. After 12-24 hours, cells are transduced with rAAV and incubated for another 16 hours or 48 hours. Cells are subjected to trypsin digestion, and the cell nucleus is separated with NE-PER nucleus and cytoplasm extraction reagent (ThermoFisher Scientific) according to the manufacturer's experimental scheme. After washing the nucleus granular precipitation, DNA is extracted using QIAamp DNA extraction kit (Qiagen). SYBER Green qPCR using primers for target gene (e.g., luciferase) and standardization gene (PTGER2) is adopted in this assay. Standard curve is used for quantitative, and the ratio of viral genome (vg) / cell is measured and it is plotted.

[0087] Example 2

[0088] In vitro studies

[0089] Human cells (HuH-7) and mouse cells (Hepa1-6) were transduced with control AAV-LK03 or AAV-AM (MOI1e4) carrying a luciferase reporter gene (surrogate for the "target gene"). The activity of the luciferase protein was quantified using a luminometer as described above. Surprisingly, transduction with AAV-AM for 16 hours resulted in 4.7-fold higher luciferase expression in human cells and 45.2-fold higher luciferase expression in mouse cells when compared to AAV-LK03 ( Figure 1A After 48 hours, luciferase expression in AAV-AM-transduced mouse cells further increased to 88.6-fold ( Figure 1B A similar trend was observed in luciferase mRNA levels ( Figure 1C-1D ).

[0090] Next, the copy number of the vector in the nucleus was determined by qPCR, which was performed after isolating the nuclear fraction from these cells using the method described above. Surprisingly, less than a 2-fold difference in nuclear vector copy number was observed between species for AAV-LK03 and AAV-AM ( Figure 1E and 1F ), which cannot explain the huge differences in mRNA and protein expression ( Figures 1A-1D ).

[0091] To further understand this apparent anomaly, the histone enrichment and histone modifications associated with the viral genome were investigated. Three core histones (H3, H2A, and H4) and four post-translational modifications (PTMs) of H3 were analyzed. Among these, H3K4me2 and H3K27ac are typically associated with actively transcribed chromatin, while H3K9me3 and H3K27me3 are repressive histone marks. It was determined that histone H3 and its active PTMs (H3K7me3 and H3K27ac) were depleted in mouse cells transduced with AAV-LK03. In contrast, H3 and its active PTMs (H3K7me3 and H3K27ac) were enriched in mouse cells transduced with AAV-AM.

[0092] These results led to the hypothesis that capsid proteins that package or expose the viral genome in the nucleus interact with host proteins to help establish the epigenetic state of the viral episome; and that these interactions differ between mouse and human species.

[0093] In vivo studies

[0094] To determine whether the enhanced transduction efficiency observed in vitro with AAV-AM could be reproduced in vivo, BALB / c scid mice injected with control AAV-LK03 or AAV-AM (3e11 viral genomes) were imaged for luciferase activity 3 days later. Figure 2A and 2B As seen in Figure 3, mice injected with AAV-AM showed significantly higher luciferase signals than those injected with AAV-LK03.

[0095] On days 3 and 15, animals in each group were sacrificed and liver tissues were harvested for gene expression, protein activity, and nuclear copy number analyses as described above. Figure 3A , 3C Figure 4 shows significantly higher luciferase expression in the AAV-AM group relative to the AAV-LK03 group at day 3 post-injection. Similar to the in vitro data, nuclear vector copy number was only modestly higher for the AAV-AM group ( Figure 3E ). Importantly, copy number levels at day 15 were different between AAV-LK03 and AAV-AAV-AM, indicating better preservation of the “gene of interest” transduced with the AAV-AM capsid.

[0096] Although many exemplary aspects and embodiments have been illustrated and described, those skilled in the art will recognize that certain modifications, permutations, additions, and sub-combinations thereof may be made without departing from the spirit and scope of the present invention. It is therefore intended that the claims appended hereto and claims introduced thereafter be interpreted as including all such modifications, permutations, additions, and sub-combinations, as within their true spirit and scope, and it should be understood that the present disclosure is not limited to the specific embodiments disclosed herein, as they are presented by way of example. Although specific terms are employed herein, they are used in a general and descriptive sense only and not for limiting purposes.

[0097] All documents and similar materials cited in this application (including, but not limited to, patents, patent applications, articles, books, papers, internet web pages, and other publications) are expressly incorporated by reference in their entirety for any purpose, to the same extent as if each were individually indicated to be incorporated by reference, regardless of the format of such documents and similar materials. In the event that one or more of the incorporated documents and similar materials differs from or contradicts the present disclosure (including, but not limited to, defined terms, term usage, described techniques, etc.), the present disclosure controls.

[0098] Example 3

[0099] Figure 4 Comparative prediction models for AAV-AM and AAV-LK03 are shown. Amino acid residues are marked with asterisks. Non-covalent bonds are shown with light grey and dark grey dashed lines for AAV-AM and AAV-LK03, respectively.

[0100] Table 1. Amino acid and nucleic acid sequences of rAAV capsid proteins

[0101]

[0102]

[0103]

Claims

1. A capsid protein sequence comprising the amino acid sequence of SEQ ID NO: 1 (AAV-AM).

2. A nucleotide sequence encoding a recombinant capsid protein, wherein the nucleotide sequence is SEQ ID NO: 2, or a sequence having at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity thereto.

3. An amino acid sequence encoded by the nucleotide sequence according to claim 2.

4. A virus particle comprising the amino acid sequence of SEQ ID NO: 1 (AAV-AM).

5. A virus particle comprising the amino acid sequence of the recombinant capsid protein encoded by the nucleotide sequence according to claim 2.

6. A plasmid comprising nucleotides encoding an amino acid sequence for a recombinant capsid protein, wherein the amino acid sequence is shown in SEQ ID NO: 1 (AAV-AM).

7. A plasmid comprising a nucleotide sequence for a recombinant capsid protein encoded by the nucleotide sequence according to claim 2.

8. A recombinant AAV vector comprising an amino acid sequence for a capsid protein, wherein the amino acid sequence is as shown in SEQ ID NO: 1 (AAV-AM).

9. A recombinant AAV vector comprising an amino acid sequence for a capsid protein, wherein the amino acid sequence is encoded by the nucleotide sequence according to claim 3.

10. A method for transferring a target nucleic acid sequence into a cell or a mammal, comprising: A recombinant AAV (rAAV) vector is introduced into a cell or a mammal, wherein the rAAV vector encodes a recombinant capsid nucleotide sequence and a target gene, wherein the target gene is encapsidated in a recombinant capsid protein having an amino acid sequence of SEQ ID NO: 1 (AAV-AM).

11. A method for transferring a target nucleic acid sequence into a cell or a mammal, comprising: A recombinant AAV (rAAV) vector is introduced into a cell or a mammal, wherein the recombinant AAV vector encodes a recombinant capsid nucleotide sequence and a target gene, wherein the target gene is encapsidated in a recombinant capsid protein having amino acids encoded by the nucleotide sequence according to claim 2.

12. The method according to any one of claims 10-11, wherein the recombinant AAV vector is generated by steps comprising: (a) Site-directed mutagenesis of AAV-LK03 plasmid using overlapping primers containing foreign triplet nucleotide sequences; (b) performing exponential amplification by PCR to generate amplicons, the PCR using the AAV-LK03 plasmid as a template and a primer pair comprising the foreign triplet nucleotide sequence; (c) incubating the amplicon with a kinase, a ligase and DpnII; (d) Efficient transformation in bacteria; (e) culturing the bacteria in the presence of a selective antibiotic to isolate positive bacterial clones comprising the recombinant capsid nucleic acid sequence; (f) confirming the presence of the recombinant capsid nucleotide sequence by sequencing; and (g) purifying a plasmid comprising the recombinant capsid nucleic acid sequence from the positive bacterial clone; The recombinant AAV vector is thereby generated.

13. The method according to any one of claims 10 to 12, wherein the capsid nucleotide sequence encodes a capsid protein sequence comprising foreign amino acids.

14. The method according to any one of claims 12-13, wherein the foreign amino acid is inserted at position 266 in the capsid protein sequence.

15. The method according to any one of claims 13-14, wherein the foreign amino acid is glycine.

16. The method according to any one of claims 11 to 15, wherein the cell is a HuH-7 cell, a Hepa1-6 cell, a HEK293 cell or a hPAEC cell.

17. The method of any one of claims 11-16, wherein the mammal is a rodent or a human.

18. The method of claim 17, wherein the rodent is a mouse.

19. The method of any one of claims 11-18, wherein the rAAV is introduced into liver tissue or skin of the mammal.

20. A method for determining the therapeutic efficacy of a target gene, comprising: delivering a recombinant AAV (rAAV) vector to a first mammalian species, the rAAV vector comprising a nucleotide sequence encoding a recombinant capsid protein having an amino acid sequence as shown in SEQ ID NO: 1 (AAV-AM), and a gene of interest, wherein the gene of interest is encapsidated in the recombinant capsid protein; assessing transduction efficiency in the first mammal; delivering the rAAV vector to a second mammalian species when the transduction efficiency in the first mammalian species is greater than the transduction efficiency observed after infection of the first mammalian species with a control rAAV vector; The transduction efficiency in the second mammalian species is assessed, whereby a higher transduction efficiency of the rAAV vector in the first mammalian species and the second mammalian species relative to the control rAAV vector indicates a higher therapeutic efficacy with respect to the gene of interest.

21. The method of claim 20, wherein the first mammalian species is a rodent.

22. The method of any one of claims 20-21, wherein the second mammalian species is human.

23. The method of claim 20, wherein the first mammalian species is a mouse and the second mammalian species is a human.

24. The method according to any one of claims 20-23, wherein the rAAV vector comprising the gene of interest is transduced into the liver.

25. The method of any one of claims 20-24, wherein the control rAAV vector comprises an AAV-LK03 plasmid.

26. The method according to any one of claims 20-25, wherein the transduction efficiency is determined by measuring at least one of the following: nuclear vector copy number, expression of the gene of interest, and activity of a protein encoded by the gene of interest.

27. A nucleotide sequence encoding a recombinant capsid protein, wherein the nucleotide sequence has at least about 96% or at least about 97% sequence identity with SEQ ID NO:

2.

28. A nucleotide sequence encoding a recombinant capsid protein, wherein the nucleotide sequence has at least about 98% or at least about 99% sequence identity with SEQ ID NO:

2.

29. The nucleotide sequence as in any one of claims 27 to 30, wherein the nucleotide sequence encodes an amino acid sequence that is not identical to SEQ ID NO:

3.

30. A nucleotide sequence encoding a recombinant capsid protein, wherein the nucleotide sequence comprises SEQ ID NO:

2.

31. A nucleotide sequence encoding a recombinant capsid protein, wherein the nucleotide sequence consists of SEQ ID NO:

2.

32. A plasmid comprising a nucleotide sequence as in any one of claims 27-31.

33. A plasmid comprising a nucleotide sequence as in any one of claims 27 to 31, wherein the nucleotide sequence encodes an amino acid sequence that is not identical to SEQ ID NO:

3.

34. A recombinant AAV vector comprising a nucleotide sequence as in any one of claims 27-33.

35. A recombinant AAV vector comprising a nucleotide sequence as in any one of claims 27-33, wherein the nucleotide sequence encodes an amino acid sequence that is not identical to SEQ ID NO:3.

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