Isolated and modified VP1 capsid protein of AAV5

Abstract

This application relates to the fields of gene therapy and molecular biology. More specifically, the present invention relates to an isolated altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid, which contains one or more amino acid substitutions that enhance transduction efficiency as compared to the VP1 protein of the wild-type AAV5 capsid, and to capsids and vectors based thereon.

Description

Field of the Invention

[0001] The present application relates to the fields of gene therapy and molecular biology. More specifically, the present invention relates to an isolated, altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid, which contains one or more amino acid substitutions that improve transduction efficiency as compared to the VP1 protein of the wild-type AAV5 capsid, and to capsids and vectors based thereon. Background of the Invention

[0003] Adeno-associated virus (AAV) is a small (20 nm), autonomously replication-defective, non-enveloped virus. Many different AAV serotypes have been described in humans and primates. The adeno-associated virus genome consists of a single-stranded DNA (ssDNA) that is approximately 4,700 nucleotides long (+ or -). The genomic DNA has inverted terminal repeats (ITRs) at the ends. The genome contains two open reading frames (ORFs), Rep and Cap, which contain several alternative reading frames encoding various protein products. The rep products are essential for AAV replication, while the three capsid proteins (VP1, VP2, and VP3) and other alternative products are encoded by the Cap gene. The VP1, VP2, and VP3 proteins form an icosahedral capsid in a 1:1:10 ratio (Xie Q. et al., The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA, 2002; 99:10405-10410). During the production of recombinant AAV (rAAV) vectors, an expression cassette flanked by ITRs is packaged into the AAV capsid. The genes required for AAV replication are not included in the cassette. Recombinant AAV is considered one of the safest and most widely used viral vectors for in vivo gene transfer. The vectors can infect cells of a variety of tissue types to provide robust and persistent transgene expression. They are also non-pathogenic and have low immunogenicity characteristics (High KA et al., "rAAV human trial experience" Methods Mol Biol. 2011; 807:429-57).

[0004] One of the fundamental goals of trials in the field of effective gene therapy development is to optimize vectors to maximize tissue transduction while minimizing the vector dose.

[0005] The various AAV serotypes are characterized by their affinities for different host cell surface receptors to which they are tropic. Thus, the major known receptor for AAV2 is heparan sulfate proteoglycan, and the co-receptors are integrin heterodimer αVβ5, fibroblast growth factor receptor type 1, and hepatocyte growth factor receptor c-Met. AAV12 binds to heparan sulfate proteoglycan and sialic acid. AAV4 and AAV5 bind to N- and O-linked sialic acid, respectively. AAV5 activates the platelet-derived growth factor receptor. At the same time, a correlation has been established between the amino acid sequence of the AAV capsid protein and its assembly process, encapsidation of the genome, and affinity for different types of receptors presented on the surface of host cells (Govindasamy L. et al., Structural insights into adeno-associated virus serotype 5. J Virol. 2013 Oct;87(20): 11187-99).

[0006] International Application WO 2012145601 discloses adeno-associated virus (AAV) virions having variant capsid proteins, which exhibit greater infectivity for retinal cells when administered by intravitreal injection as compared to wild-type AAV.

[0007] International Application WO 2013158879 discloses an adeno-associated virus (AAV) vector for delivering a heterologous nucleic acid sequence to a subject, which comprises a VP1 capsid protein comprising one or more lysine substitutions, wherein one of the lysine substitutions is K137R, and wherein said lysine substitution effectively inhibits ubiquitination of the capsid protein, thereby increasing transduction of the AAV vector in target cells.

[0008] There is a current need for AAVs with improved transduction capabilities that include various transgenes, including clinically important transgenes, in their structure for patients in need thereof. Improved tissue transduction enables minimization of the vector dose administered to a subject.

[0009] The inventors have unexpectedly found that there are one or more amino acid substitutions in the VP1 protein of the wild-type AAV5 capsid selected from:

[0010] S651A,

[0011] S2A and T711S or

[0012] S2A, S651A and T711S,

[0013] This results in an increased efficiency of transducing target cells using an AAV serotype 5 vector with such modification(s) and a significantly increased efficiency of transgene delivery by the rAAV vector with the above mutations.

[0014] BRIEF DESCRIPTION OF THE INVENTION

[0015] In one aspect, the present invention relates to an isolated altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid for highly efficient transduction of target cells, which comprises the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid encoded by the Cap gene, and one or more substitutions selected from the following:

[0016] S651A,

[0017] S2A and T711S,

[0018] S2A, S651A and T711S.

[0019] In some embodiments, the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid has the amino acid sequence represented by SEQ ID NO:1.

[0020] In some embodiments, the isolated altered VP1 protein of the AAV5 capsid comprises a substitution at the S651A position.

[0021] In some embodiments, the isolated altered VP1 protein of the AAV5 capsid has the amino acid sequence represented by SEQ ID NO: 2.

[0022] In some embodiments, the isolated altered VP1 protein of the AAV5 capsid comprises S2A and T711S substitutions.

[0023] In some embodiments, the isolated altered VP1 protein of the AAV5 capsid has the amino acid sequence represented by SEQ ID NO: 3.

[0024] In some embodiments, the isolated altered VP1 protein of the AAV5 capsid comprises S2A, S651A and T711S substitutions.

[0025] In some embodiments, the isolated altered VP1 protein of the AAV5 capsid has the amino acid sequence represented by SEQ ID NO: 4.

[0026] In one aspect, the present invention relates to an isolated nucleic acid encoding the altered VP1 protein of the above adeno-associated virus serotype 5 (AAV5) capsid, which is used for highly efficient transduction of target cells.

[0027] In some embodiments, an isolated nucleic acid encoding an altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid having an amino acid S651A substitution is represented by a nucleic acid sequence having SEQ ID NO: 5 or any other sequence encoding the corresponding amino acid sequence of an altered protein of adeno-associated virus serotype 5 (AAV5) capsid having an amino acid S651A substitution.

[0028] In some embodiments, an isolated nucleic acid encoding an altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid having amino acid S2A and T711S substitutions is represented by a nucleic acid sequence having SEQ ID NO: 6 or any other sequence encoding the corresponding amino acid sequence of an altered protein of adeno-associated virus serotype 5 (AAV5) capsid having amino acid S2A and T711S substitutions.

[0029] In some embodiments, an isolated nucleic acid encoding an altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid having amino acid S2A, S651A and T711S substitutions is represented by a nucleic acid sequence having SEQ ID NO: 7 or any other sequence encoding the corresponding amino acid sequence of an altered protein of adeno-associated virus serotype 5 (AAV5) capsid having amino acid S2A, S651A and T711S substitutions.

[0030] In one aspect, the present invention relates to an isolated capsid for highly efficient transduction of target cells, which comprises the altered VP1 protein of the above adeno-associated virus serotype 5 (AAV5) capsid.

[0031] In some embodiments, the isolated capsid comprises the altered VP1 protein of the above adeno-associated virus serotype 5 (AAV5) capsid, the VP2 protein of the AAV5 capsid or an altered variant thereof, and the VP3 protein of the AAV5 capsid or an altered variant thereof.

[0032] In some embodiments, the isolated capsid comprises the VP2 protein of the wild-type AAV5 capsid.

[0033] In some embodiments, the isolated capsid comprises the VP1 protein of the wild-type AAV5 capsid, which has an amino acid sequence represented by SEQ ID NO: 8.

[0034] In some embodiments, the isolated capsid comprises an altered VP2 protein of adeno-associated virus serotype 5 (AAV5) capsid.

[0035] In some embodiments, the isolated capsid comprises an altered VP2 protein of the AAV5 capsid, which comprises a T575S substitution.

[0036] In some embodiments, the isolated capsid comprises an altered VP2 protein of the AAV5 capsid, which comprises a T575S substitution and has an amino acid sequence represented by SEQ ID NO: 9.

[0037] In some embodiments, the isolated capsid comprises an altered VP2 protein of the AAV5 capsid, which comprises S515A and T575S substitutions.

[0038] In some embodiments, the isolated capsid comprises an altered VP2 protein of the AAV5 capsid, which comprises S515A and T575S substitutions and has an amino acid sequence represented by SEQ ID NO: 10.

[0039] In some embodiments, the isolated capsid comprises the VP3 protein of the wild-type AAV5 capsid.

[0040] In some embodiments, the isolated capsid comprises the VP3 protein of the wild-type AAV5 capsid, which has an amino acid sequence represented by SEQ ID NO: 11.

[0041] In some embodiments, the isolated capsid comprises an altered VP3 protein of the adeno-associated virus serotype 5 (AAV5) capsid.

[0042] In some embodiments, the isolated capsid comprises an altered VP3 protein of the AAV5 capsid, which comprises a T519S substitution.

[0043] In some embodiments, the isolated capsid comprises an altered VP3 protein of the AAV5 capsid, which comprises a T519S substitution and has an amino acid sequence represented by SEQ ID NO: 12.

[0044] In some embodiments, the isolated capsid comprises an altered VP3 protein of the AAV5 capsid, which comprises S459A and T519S substitutions.

[0045] In some embodiments, the isolated capsid comprises an altered VP3 protein of the AAV5 capsid, which comprises S459A and T519S substitutions and has an amino acid sequence represented by SEQ ID NO: 13.

[0046] In one aspect, the present invention relates to an isolated nucleic acid encoding the above capsid, which is used for highly efficient transduction of target cells.

[0047] In one aspect, the present invention relates to a recombinant adeno-associated virus serotype 5 (rAAV5)-based vector for delivering a heterologous nucleic acid sequence to a subject, which comprises:

[0048] 1) the above capsid, and

[0049] 2) A heterologous nucleic acid sequence comprising regulatory sequences that promote the expression of a product encoded by the heterologous nucleic acid sequence in a target cell.

[0050] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding a therapeutic polypeptide or a reporter polypeptide as the product.

[0051] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding a therapeutic polypeptide as the product, wherein the therapeutic polypeptide is a coagulation factor selected from Factor VIII, Factor IX, or a functional variant thereof.

[0052] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding Factor VIII or a functional variant thereof as the product.

[0053] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding Factor IX or a functional variant thereof as the product.

[0054] In one aspect, the present invention relates to a pharmaceutical composition for delivering a gene product to a subject in need thereof, comprising:

[0055] a) The above rAAV5-based vector; and

[0056] b) A pharmaceutically acceptable excipient.

[0057] In some embodiments, the pharmaceutical composition is used for delivering a gene product to a human in need thereof.

[0058] In one aspect, the present invention relates to a method for delivering a gene product to a subject in need thereof, comprising administering to the subject the above rAAV5-based vector or the above pharmaceutical composition.

[0059] In some embodiments, the method for delivering a gene product is used for delivering a gene product to a human in need thereof.

[0060] In one aspect, the present invention relates to the use of the above rAAV5-based vector or the above pharmaceutical composition for treating a disease in a subject in need thereof.

[0061] In some embodiments, the use is for treating a disease in a human in need thereof.

[0062] In some embodiments of the use, the disease is selected from blood diseases, central nervous system diseases, metabolic diseases, muscle diseases, genetic diseases.

[0063] In some embodiments of the use, the disease is a blood disease.

[0064] In some embodiments of the use, the expression product of the heterologous nucleic acid sequence is Factor IX or a functional variant thereof.

[0065] In some embodiments of the use, the expression product of the heterologous nucleic acid sequence is Factor VIII or a functional variant thereof.

[0066] In some embodiments of the use, the disease is a muscle disease.

[0067] In some embodiments of the use, the disease is a genetic disease.

[0068] In one aspect, the present invention relates to a method for producing the above rAAV5-based vector, which comprises transfecting a production cell with the above nucleic acid encoding the above capsid. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 . Circular scheme of plasmid pAAV-Linker intended for cloning a random variant library of AAV serotype 5 capsid genes.

[0071] AmpR is the β-lactamase gene providing resistance to ampicillin,

[0072] pUC origin is the pUC replication origin in bacteria,

[0073] ITR is the inverted terminal repeat,

[0074] CMV-promoter is the promoter of the cytomegalovirus early gene,

[0075] PolyA is the polyadenylation signal sequence for increasing mRNA stability,

[0076] HBG intron is the human β-globin intron,

[0077] GFP is the green fluorescent protein gene,

[0078] T2A is the 2A self-cleaving peptide allowing co-expression of the target protein and the reporter protein.

[0079] Figure 2 . Circular scheme of plasmid pAAV-Rep intended for generating recombinant viral products of wild-type AAV serotype 5 from a random variant library.

[0080] AmpR is the β-lactamase gene providing resistance to ampicillin,

[0081] pUC origin is the pUC replication origin in bacteria,

[0082] Rep gene is the Rep gene sequence encoding the AAV replication protein.

[0083] Figure 3 . Circular scheme of plasmid pHelper for recombinant viral products expected to generate wild-type AAV serotype 5 from a random variant library.

[0084] AmpR is the β-lactamase gene providing resistance to ampicillin,

[0085] Ori is the origin of replication in bacteria,

[0086] Adeno E2A is the helper adenovirus gene sequence involved in viral DNA replication,

[0087] Adeno E4 is the helper adenovirus gene sequence involved in viral DNA replication,

[0088] Adeno VARNA is the helper adenovirus gene sequence responsible for translating both early and late viral genes.

[0089] Figure 4 . Efficiency analysis of CHO-K1-S cell transduction with AAV5-GFP-based viral products, where the VP1 protein of the wild-type AAV5 capsid includes one or more amino acid substitutions.

[0090] Figure 5 . Analysis of the hFIX protein concentration in the medium harvested from CHO-K1-S cells 7 days after transduction with AAV5-hFIX-based viral products, where the VP1 protein of the wild-type AAV5 capsid includes one or more amino acid substitutions.

[0091] Figure 6 . Location of AAV5 capsid proteins in the genome.

[0092] 2087 - 4258 bp - VP1

[0093] 2495 - 4258 bp - VP2

[0094] 2663 - 4258 bp - VP3

[0095] Description of the Invention

[0096] Definitions and General Methods

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0098] Further, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. In general, the cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicine and pharmaceutical chemistry, and the classification and methods of hybridization and chemistry of proteins and nucleic acids described herein are well known and widely used by those skilled in the art. Enzyme reactions and purification methods are carried out according to the manufacturer's guidelines, as commonly practiced in the art or as described herein.

[0099] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in an animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment (such as a genetically modified cell).

[0100] The terms "naturally occurring", "natural" or "wild-type" are used to describe an object that can be found in nature and is different from an artificially produced one. For example, a protein or nucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been deliberately modified by a laboratory worker is naturally occurring.

[0101] The term "genome" refers to the complete genetic material of an organism.

[0102] As used in this specification and the subsequent claims, unless the context otherwise dictates, the words "include", "comprise" or variations thereof such as "have", "includes", "including", "comprises" or "comprising" will be understood to mean including the stated integer or group of integers, but not excluding any other integer or group of integers.

[0103] Protein (peptide)

[0104] As used in this specification, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up the protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used in this specification, the term refers to both short chains that are commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and longer chains that are commonly referred to in the art as proteins (of which there are many types). "Polypeptide" includes, in particular, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides or combinations thereof.

[0105] The terms "transformation", "transfection" and "transduction" refer to any method or means by which nucleic acid is introduced into a cell or host organism and can be used interchangeably to express the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, infection, PEG-fusion, etc.

[0106] Nucleic acid molecule

[0107] The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably in this specification, mean the precise sequence of nucleotides, whether modified or not, determining a fragment or region of a nucleic acid, whether containing unnatural nucleotides or not, and being double-stranded DNA or RNA, single-stranded DNA or RNA or the transcript of said DNA.

[0108] Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used in this specification, polynucleotides include (as non-limiting examples) all nucleic acid sequences obtained by any means available in the art, said means including (as non-limiting examples) recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques such as PCR, and synthetic means.

[0109] It should also be noted here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. The sequences of the present invention have been isolated and / or purified, i.e., they have been sampled directly or indirectly, for example by copying, and their environment has been at least partially altered. Thus, reference should also be made here to isolated nucleic acids obtained by recombinant genetics (e.g., through host cells) or by chemical synthesis.

[0110] A "separated" nucleic acid molecule is a nucleic acid molecule identified and separated from at least one nucleic acid molecule-impurity, which is generally bound to the nucleic acid molecule-impurity in the natural source of the nuclease nucleic acid. The separated nucleic acid molecule is different from the form or collection in which it is found under natural conditions. Thus, the separated nucleic acid molecule is different from the nucleic acid molecule present in a cell under natural conditions. However, the separated nucleic acid molecule includes the nucleic acid molecule located in the cell in which the nuclease is normally expressed, for example, if the nucleic acid molecule has a chromosomal location different from its location in the cell under natural conditions.

[0111] Unless otherwise specified, the term nucleotide sequence includes its complementary sequence. Thus, a nucleic acid having a specific sequence should be understood to include the nucleic acid of its complementary strand containing its complementary sequence.

[0112] Adeno-associated virus (AAV)

[0113] Parvoviridae viruses are small DNA-containing animal viruses. Parvoviridae can be divided into two subfamilies: the Parvovirinae whose members infect vertebrates and the Densovirinae whose members infect insects. As of 2006, 11 serotypes of adeno-associated virus have been described (Mori, S. et al., 2004, "Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein", Virology, T. 330 (2): 375-83). All known serotypes can infect cells from a variety of tissue types. Tissue specificity is determined by the capsid protein serotype; thus, vectors based on adeno-associated virus are constructed by specifying the desired serotype. Further information on parvoviruses and other members of Parvoviridae is described in the literature (Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication", Chapter 69 in Fields Virology (3rd Edition, 1996)).

[0114] The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear single-stranded DNA molecule less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for replication of the non-structural protein (Rep) and the structural protein (Cap). The Cap gene encodes the VP proteins (VP1, VP2, and VP3) that form the capsid. The terminal 145 nucleotides are self-complementary and are organized such that an energetically stable intramolecular duplex that forms a T-shaped hairpin can form. Such hairpin structures serve as the origin of viral DNA replication and function as primers for the cellular DNA polymerase complex. After infection with wild-type AAV (wtAAV) in mammalian cells, the Rep genes (such as Rep78 and Rep52) are expressed using the P5 promoter and the P19 promoter, respectively, and both Rep proteins have a function in the replication of the viral genome. Splicing events in the Rep open reading frame (Rep ORF) result in the expression of actually four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep40). However, it has been shown that unspliced mRNAs encoding the Rep78 and Rep52 proteins are sufficient to produce AAV vectors in mammalian cells.

[0115] Vectors based on recombinant adeno-associated virus (rAAV)

[0116] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0117] As used in this specification, the term "recombinant AAV vector" (or "rAAV vector") refers to a vector that contains one or more polynucleotide sequences of interest, genes of interest, or "transgenes" flanked by parvovirus or inverted terminal repeat sequences (ITRs).

[0118] As used with respect to virus titer, the terms "infectious unit (iu)", "infectious particle", or "replication unit" refer to the number of infectious recombinant AAV vector particles as measured by the infectious center assay (also known as the replication center assay, as described, for example, in McLaughlin et al., J. Virol. (1988) 62:1963-1973).

[0119] When the term "heterologous" refers to nucleic acid sequences such as coding sequences and regulatory sequences, it means sequences that are not normally joined together and / or are not normally associated with a particular cell. Thus, a "heterologous" region of a nucleic acid construct or vector is a nucleic acid fragment within or ligated to another nucleic acid molecule that is not found in nature to be associated with the other molecule. For example, a heterologous region of a nucleic acid construct can include a coding sequence flanked by sequences not found in nature to be associated with the coding sequence. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., a synthetic sequence with codons different from a native gene).

[0120] As used herein, the term "operably linked" refers to the joining of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if a transcriptional regulatory sequence affects the transcription of a coding sequence, it is operably linked to the coding sequence. The term "operably linked" means that the DNA sequences being linked are generally contiguous and, where necessary to join two protein-coding regions, contiguous and in the reading frame.

[0121] As used in this specification, the term "promoter" or "transcriptional regulatory sequence" or "regulatory sequence" refers to a nucleic acid fragment that controls the transcription of one or more coding sequences and is located upstream in the reading direction (relative to the direction of transcription from the transcription start site of the coding sequence) and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to those of skill in the art to directly or indirectly regulate the level of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under typical physiological and developmental conditions. An "inducible" promoter is a promoter that is regulated physiologically or developmentally, e.g., in response to the influence of a chemical inducer. A "tissue-specific" promoter is active only in a particular type of tissue or cell.

[0122] As used herein, the term "enhancers" or "enhancer" may refer to a DNA sequence located adjacent to a DNA sequence encoding a recombinant product. Enhancer elements are generally located in the 5' direction of a promoter element or may be located downstream or within a coding DNA sequence (e.g., a DNA sequence that is transcribed or translated into one or more recombinant products). Thus, an enhancer element may be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a DNA sequence encoding a recombinant product. Enhancer elements can increase the amount of recombinant product expressed from a DNA sequence to levels higher than the expression level associated with a single promoter element. A variety of enhancer elements are readily available to those of ordinary skill in the art.

[0123] The term "selectable marker gene" refers to a gene that confers a selectable phenotype (e.g., antibiotic resistance) on a transformed cell when expressed.

[0124] As used in this specification, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0125] Therapeutic use

[0126] "Gene therapy" is the insertion of a gene into a subject's cells and / or tissues to treat a disease, generally a genetic disease, in which a defective mutant allele is replaced by a functional allele.

[0127] "Treat", "treatment", and "therapy" refer to a method of alleviating or eliminating a biological disorder and / or at least one of its attendant symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Further, "treatment" as referred to herein includes curative, palliative, and prophylactic treatment.

[0128] In one aspect, the subject or patient being treated is a mammal, preferably a human subject. The subject can be male or female of any age.

[0129] The term "disorder" means any condition that would benefit from treatment according to the present invention. This includes chronic and acute disorders or diseases, including those pathological conditions that render a mammal susceptible to the disorder under discussion.

[0130] "Disease" is the state of health of an animal in which the animal is unable to maintain homeostasis and in which the animal's health condition will continue to deteriorate if the disease is not improved.

[0131] The terms "subject", "patient", "individual", etc. are used interchangeably in this specification and refer to any animal to which the methods described in this specification are applicable. In certain non-limiting embodiments, the subject, patient, or individual is a human.

[0132] "Therapeutically effective amount" refers to the amount of a therapeutic agent that, when administered during treatment, will relieve one or more symptoms of the disease being treated to some extent.

[0133] The term "chronic" is used to refer to continuous (uninterrupted) use of a drug, rather than an acute (short-term) administration route, in order to maintain the initial therapeutic effect (activity) over a long period of time.

[0134] "Intermittent" use refers to treatment that is not continuous without interruption, but rather is quite periodic in nature. DETAILED DESCRIPTION OF THE INVENTION

[0136] Isolated altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid

[0137] In one aspect, the present invention relates to an isolated altered VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid for highly efficient transduction of target cells, which comprises the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid encoded by the Cap gene, and one or more substitutions selected from the following:

[0138] S651A,

[0139] S2A and T711S,

[0140] S2A, S651A and T711S.

[0141] The amino acid S2A substitution is understood to mean that the serine (Ser, S) at position 2 of the VP1 protein of the wild-type adeno-associated virus serotype 5 capsid is replaced by alanine (Ala, A).

[0142] The amino acid S651A substitution is understood to mean that the serine (Ser, S) at position 651 of the VP1 protein of the wild-type adeno-associated virus serotype 5 capsid is replaced by alanine (Ala, A).

[0143] The amino acid T711S substitution is understood to mean that the threonine (Thr, T) at position 711 of the VP1 protein of the wild-type adeno-associated virus serotype 5 capsid is replaced by serine (Ser, S).

[0144] In some embodiments, the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid has the amino acid sequence represented by: MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL (SEQ ID NO: 1).

[0145] In some embodiments, the isolated and altered VP1 protein of the AAV5 capsid comprises a substitution at the S651A position.

[0146] In some embodiments, the isolated and altered VP1 protein of the AAV5 capsid has the amino acid sequence represented by:

[0147] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFADVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL (SEQ ID NO: 2).

[0148] In some embodiments, the isolated, altered VP1 protein of the AAV5 capsid comprises the S2A and T711S substitutions.

[0149] In some embodiments, the isolated, altered VP1 protein of the AAV5 capsid has an amino acid sequence represented by: MAFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRSTRPIGTRYLTRPL (SEQ ID NO: 3).

[0150] In some embodiments, the isolated, altered VP1 protein of the AAV5 capsid comprises S2A, S651A, and T711S substitutions.

[0151] In some embodiments, the isolated, altered VP1 protein of the AAV5 capsid has the amino acid sequence represented by: MAFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFADVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRSTRPIGTRYLTRPL (SEQ ID NO: 4).

[0152] Isolated, altered VP2 and VP3 proteins of adeno-associated virus serotype 5 (AAV5) capsid

[0153] The "right side" of the genomic DNA (+)-strand of adeno-associated virus contains overlapping sequences encoding three capsid proteins (VP1, VP2, and VP3). Transcription of these genes starts from a promoter p40. The molecular weights of the corresponding proteins are 87, 72, and 62 kDa respectively. All three of these proteins are translated from a single mRNA. After transcription, the mRNA precursor can be spliced in two different ways, where the longer or shorter intron is excised to form an mRNA 2300 or 2600 nucleotides in length.

[0154] Therefore, introducing mutations into the Cas gene will affect not only the VP1 protein of the AAV5 capsid, but also the VP2 and VP3 proteins of the AAV5 capsid.

[0155] Figure 6 Schematic diagram of the positions of AAV5 capsid proteins in the AAV genome:

[0156] 2087 - 4258 bp - VP1

[0157] 2495 - 4258 bp - VP2

[0158] 2663 - 4258 bp - VP3.

[0159] In summary, there will be no mutations in VP2 and VP3 similar to the mutation S2A in VP1, while mutations similar to the mutations S651A and / or T711S in VP1 will be present in both VP2 and VP3.

[0160] Considering the overlapping sequences encoding the three capsid proteins (VP1, VP2, and VP3), the amino acid substitution S651A in VP1 will correspond to:

[0161] An amino acid substitution at position S515A in VP2;

[0162] An amino acid substitution at position S459A in VP3.

[0163] Considering the overlapping sequences encoding the three capsid proteins (VP1, VP2, and VP3), the amino acid substitution T711S will correspond to:

[0164] An amino acid substitution at position T575S in VP2;

[0165] An amino acid substitution at position T519S in VP3.

[0166] Furthermore, the applicant believes it is appropriate to specify the environment in which the mutations have been found by indicating the short amino acid sequences including the mutations in VP1 / VP2 / VP3:

[0167] For S2A in VP1 (absent in VP2 and VP3) - M S FVDHP;

[0168] For S651A in VP1 (S515A in VP2 / S459A in VP3) - TSF S DVP;

[0169] For T711S in VP1 (T575S in VP2 / T519S in VP3) - EYR T TRP.

[0170] In some embodiments, the amino acid sequence of the VP2 protein of the wild - type AAV5 capsid has the amino acid sequence represented by: TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL (SEQ ID NO: 8).

[0171] In some embodiments, the isolated, altered VP2 protein of the AAV5 capsid includes the T575S substitution.

[0172] In some embodiments, the isolated, altered VP2 protein of the AAV5 capsid has an amino acid sequence represented by: TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRSTRPIGTRYLTRPL (SEQ ID NO: 9).

[0173] In some embodiments, the isolated, altered VP2 protein of the AAV5 capsid comprises S515A and T575S substitutions.

[0174] In some embodiments, the isolated, altered VP2 protein of the AAV5 capsid has an amino acid sequence represented by: TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFADVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRSTRPIGTRYLTRPL (SEQ ID NO:10).

[0175] In some embodiments, the amino acid sequence of the VP3 protein of the wild-type AAV5 capsid has the amino acid sequence represented by: MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL (SEQ ID NO: 11).

[0176] In some embodiments, the isolated, altered VP3 protein of the AAV5 capsid comprises the T519S substitution.

[0177] In some embodiments, the isolated, altered VP3 protein of the AAV5 capsid has an amino acid sequence represented by: MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRSTRPIGTRYLTRPL (SEQ ID NO: 12).

[0178] In some embodiments, the isolated, altered VP3 protein of the AAV5 capsid comprises S459A and T519S substitutions.

[0179] In some embodiments, the isolated, altered VP3 protein of the AAV5 capsid has an amino acid sequence represented by: MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFADVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRSTRPIGTRYLTRPL (SEQ ID NO: 13).

[0180] capsid

[0181] In one aspect, the present invention relates to an isolated capsid for highly efficient transduction of target cells, which comprises an altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid.

[0182] In one embodiment, the isolated capsid comprises an altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid, the VP2 protein of the AAV5 capsid or an altered variant thereof, and the VP3 protein of the AAV5 capsid or an altered variant thereof.

[0183] Particularly preferred embodiments include substitutions that are substantially conservative, i.e., substitutions that occur within amino acid families added by their side chains. Specifically, amino acids are generally divided into 4 families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that substituting leucine alone with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or amino acids similarly conservatively substituted with structurally related amino acids will not have a significant impact on biological activity. For example, the polypeptide of interest may include up to about 5 - 10 conservative or non-conservative amino acid substitutions, or even up to about 15 - 25 or 50 conservative or non-conservative amino acid substitutions, or any integer between 5 - 50, as long as the desired molecular function remains intact.

[0184] In one embodiment, the isolated capsid comprises the VP2 protein of the wild-type AAV5 capsid.

[0185] In one embodiment, the isolated capsid comprises the VP2 protein of the wild-type AAV5 capsid, which has the amino acid sequence represented by SEQ ID NO: 8.

[0186] In one embodiment, the isolated capsid comprises an altered VP2 protein of the adeno-associated virus serotype 5 (AAV5) capsid.

[0187] In one embodiment, the isolated capsid comprises an altered VP2 protein of the AAV5 capsid, which comprises the T575S substitution.

[0188] In one embodiment, the isolated capsid comprises an altered VP2 protein of the AAV5 capsid, which comprises the T575S substitution and has the amino acid sequence represented by SEQ ID NO: 9.

[0189] In one embodiment, the isolated capsid comprises an altered VP2 protein of the AAV5 capsid, which comprises the S515A and T575S substitutions.

[0190] In one embodiment, the isolated capsid comprises an altered VP2 protein of the AAV5 capsid, which comprises the S515A and T575S substitutions and has the amino acid sequence represented by SEQ ID NO: 10.

[0191] In one embodiment, the isolated capsid comprises the VP3 protein of the wild-type AAV5 capsid.

[0192] In one embodiment, the isolated capsid comprises the VP3 protein of the wild-type AAV5 capsid, which has the amino acid sequence represented by SEQ ID NO: 11.

[0193] In one embodiment, the isolated capsid comprises an altered VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid.

[0194] In one embodiment, the isolated capsid comprises an altered VP3 protein of an AAV5 capsid, which comprises a T519S substitution.

[0195] In one embodiment, the isolated capsid comprises an altered VP3 protein of an AAV5 capsid, which comprises a T519S substitution and has the amino acid sequence represented by SEQ ID NO: 12.

[0196] In one embodiment, the isolated capsid comprises an altered VP3 protein of an AAV5 capsid, which comprises S459A and T519S substitutions.

[0197] In one embodiment, the isolated capsid comprises an altered VP3 protein of an AAV5 capsid, which comprises S459A and T519S substitutions and has the amino acid sequence represented by SEQ ID NO: 13.

[0198] Isolated nucleic acid

[0199] In one aspect, the invention relates to an isolated nucleic acid encoding an altered VP1 protein of the above adeno-associated virus serotype 5 (AAV5) capsid, which is used for highly efficient transduction of target cells.

[0200]

[0201] "Other sequences encoding the corresponding amino acid sequence of an altered VP1 protein encoding an adeno-associated virus serotype 5 (AAV5) capsid having an amino acid substitution of S651A" means a nucleic acid sequence that can replace the nucleic acid sequence having SEQ ID NO: 5, because due to the degeneracy of the genetic code, a wide range of different DNA sequences can encode the amino acid sequence disclosed herein as SEQ ID NO: 2. Generating these alternative DNA sequences encoding the same amino acid sequence is entirely within the skill of those in the art. Such variant DNA sequences are within the scope of the present invention.

[0202]

[0203] "Other sequences that encode the corresponding amino acid sequences of an altered VP1 protein that encodes an adeno-associated virus serotype 5 (AAV5) capsid having amino acid S2A and T711S substitutions" means nucleic acid sequences that can replace the nucleic acid sequence having SEQ ID NO: 6, because due to the degeneracy of the genetic code, a wide range of different DNA sequences can encode the amino acid sequences disclosed herein such as SEQ ID NO: 3. Generating these alternative DNA sequences that encode the same amino acid sequence is entirely within the skill of those in the art. Such variant DNA sequences are within the scope of the present invention.

[0204]

[0205] "Other sequences corresponding to the amino acid sequence of an altered VP1 protein encoding an adeno-associated virus serotype 5 (AAV5) capsid having amino acid substitutions of S2A, S651A, and T711S" means a nucleic acid sequence that can replace the nucleic acid sequence having SEQ ID NO: 7, because due to the degeneracy of the genetic code, a wide range of different DNA sequences can encode the amino acid sequence disclosed herein as SEQ ID NO: 4. Generating these alternative DNA sequences that encode the same amino acid sequence is entirely within the skill of those in the art. Such variant DNA sequences are within the scope of the present invention.

[0206]

[0207] "Other sequences encoding the corresponding amino acid sequence of the VP1 protein of the wild-type adeno-associated virus serotype 5 (AAV5) capsid" means nucleic acid sequences that can replace the nucleic acid sequence having SEQ ID NO: 14, because due to the degeneracy of the genetic code, a wide range of different DNA sequences can encode the amino acid sequence disclosed herein as SEQ ID NO: 1. Generating these alternative DNA sequences that encode the same amino acid sequence is entirely within the skill of those in the art. Such variant DNA sequences are within the scope of the present invention.

[0208] In one aspect, the present invention relates to an isolated nucleic acid encoding an altered VP2 protein of the above adeno-associated virus serotype 5 (AAV5) capsid.

[0209]

[0210] "Other sequences encoding the corresponding amino acid sequences of an altered VP2 protein encoding an adeno-associated virus serotype 5 (AAV5) capsid with an amino acid T575S substitution" means nucleic acid sequences that can replace the nucleic acid sequence having SEQ ID NO: 15, because due to the degeneracy of the genetic code, a wide range of different DNA sequences can encode the amino acid sequence disclosed herein as SEQ ID NO: 9. Generating these alternative DNA sequences that encode the same amino acid sequence is entirely within the skill of those skilled in the art. Such variant DNA sequences are within the scope of the present invention.

[0211] In some embodiments, the isolated nucleic acid encoding an altered VP2 protein encoding an adeno-associated virus serotype 5 (AAV5) capsid with amino acid S515A and T575S substitutions has any nucleic acid sequence encoding the amino acid sequence disclosed herein as SEQ ID NO: 10. Generating these alternative DNA sequences that encode the same amino acid sequence is entirely within the skill of those skilled in the art. Such variant DNA sequences are within the scope of the present invention.

[0212]

[0213] "Other sequences encoding the corresponding amino acid sequence of the VP2 protein of the wild-type adeno-associated virus serotype 5 (AAV5) capsid" means nucleic acid sequences that can replace the nucleic acid sequence having SEQ ID NO: 16, because due to the degeneracy of the genetic code, a wide range of different DNA sequences can encode the amino acid sequence disclosed herein as SEQ ID NO: 8. Generating these alternative DNA sequences that encode the same amino acid sequence is entirely within the skill of those in the art. Such variant DNA sequences are within the scope of the present invention.

[0214] In one aspect, the present invention relates to an isolated nucleic acid encoding an altered VP3 protein of the above adeno-associated virus serotype 5 (AAV5) capsid.

[0215]

[0216] "Other sequences encoding the corresponding amino acid sequences of the altered VP3 protein that encodes the adeno-associated virus serotype 5 (AAV5) capsid with an amino acid T519S substitution" means nucleic acid sequences that can replace the nucleic acid sequence having SEQ ID NO: 17, because due to the degeneracy of the genetic code, a wide range of different DNA sequences can encode the amino acid sequences disclosed herein as SEQ ID NO: 12. Generating these alternative DNA sequences that encode the same amino acid sequence is entirely within the skill of those skilled in the art. Such variant DNA sequences are within the scope of the present invention.

[0217] In some embodiments, the isolated nucleic acid encoding the altered VP3 protein of the adeno-associated virus serotype 5 (AAV5) capsid having amino acid S459A and T519S substitutions has any nucleic acid sequence encoding the amino acid sequence disclosed herein as SEQ ID NO: 13. Generating these alternative DNA sequences that encode the same amino acid sequence is entirely within the skill of those skilled in the art. Such variant DNA sequences are within the scope of the present invention.

[0218]

[0219] "Other sequences encoding the corresponding amino acid sequence of the VP3 protein of the wild-type adeno-associated virus serotype 5 (AAV5) capsid" means nucleic acid sequences that can replace the nucleic acid sequence having SEQ ID NO: 18, because due to the degeneracy of the genetic code, a wide range of different DNA sequences can encode the amino acid sequence disclosed herein as SEQ ID NO: 11. Generating these alternative DNA sequences that encode the same amino acid sequence is well within the skill of those in the art. Such variant DNA sequences are within the scope of the present invention.

[0220] In one aspect, the present invention relates to an isolated nucleic acid encoding the above capsid, which is used for highly efficient transduction of target cells.

[0221] In some embodiments, the isolated nucleic acid encoding the above capsid comprises any of the above nucleic acid sequences.

[0222] Recombinant adeno-associated virus serotype 5 (rAAV5)-based vector

[0223] In one aspect, the present invention relates to a recombinant adeno-associated virus serotype 5 (rAAV5)-based vector for delivering a heterologous nucleic acid sequence to a subject, which comprises:

[0224] 1) The above capsid, and

[0225] 2) A heterologous nucleic acid sequence, which comprises regulatory sequences that promote the expression of the target product encoded by the heterologous nucleic acid sequence in target cells.

[0226] The rAAV vector of the present invention does not contain the nucleotide sequences of the genes encoding the non-structural protein (Rep) and the structural protein (Cap).

[0227] The capsid is characterized in detail in the above part of this specification.

[0228] In some embodiments, the rAAV5-based vector has an expression product of the heterologous nucleic acid sequence, which is a therapeutic polypeptide or a reporter polypeptide.

[0229] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence, and the product encoded by the sequence is a therapeutic polypeptide, wherein the therapeutic polypeptide is a coagulation factor selected from factor VIII, factor IX or a functional variant thereof.

[0230] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence, and the product encoded by the sequence is factor VIII or a functional variant thereof.

[0231] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding a product that is Factor IX or a functional variant thereof.

[0232] Pharmaceutical composition

[0233] In one aspect, the present invention relates to a pharmaceutical composition for delivering a gene product to a subject in need thereof, comprising:

[0234] a) the above rAAV5-based vector; and

[0235] b) a pharmaceutically acceptable excipient.

[0236] In some embodiments, the pharmaceutical composition is used to deliver a gene product to a human in need thereof.

[0237] In certain embodiments, the present invention relates to a pharmaceutical composition comprising the rAAV5 viral particles of the present invention in a pharmaceutically acceptable carrier or other medical substance, pharmaceutical substance, carrier, adjuvant, diluent, etc. For injection, the carrier is generally a liquid carrier. For other administration methods, the carrier can be solid or liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate buffered saline solution. For inhaled administration, the carrier is inhalable and preferably in the form of solid or liquid particles. As an injection medium, it is preferred to use water containing additives commonly used in injection solutions (such as stabilizers, salts or saline and / or buffers).

[0238] In other embodiments, the present invention relates to a pharmaceutical composition comprising cells in a pharmaceutically acceptable carrier or other medical substance, pharmaceutical substance, carrier, adjuvant, diluent, etc., wherein the rAAV5-based vector is integrated into the genome of the cells.

[0239] "Pharmaceutical composition" means a composition comprising the above rAAV5-based vector of the present invention and at least one component selected from pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, aids, distributors, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, extended delivery control agents, the selection and proportion of which depend on the type and route of administration and the dose. The pharmaceutical compositions of the present invention and methods for their preparation will undoubtedly be obvious to those skilled in the art. The pharmaceutical compositions should preferably be manufactured in accordance with GMP (Good Manufacturing Practice) requirements. The compositions may comprise buffer compositions, tonicity agents, stabilizers and solubilizers.

[0240] "Pharmaceutically acceptable" means a material that does not have biological or other adverse side effects, e.g., the material can be administered to a subject without causing any undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, to transfect cells ex vivo or to administer viral particles or cells directly to a subject in vivo.

[0241] The term "excipient" is used herein to describe any ingredient other than the ingredients of the present invention above. These are substances of inorganic or organic nature, which are used in pharmacy to give the pharmaceutical product the necessary physicochemical properties.

[0242] A "stabilizer" refers to an excipient or a mixture of two or more excipients that provides physical and / or chemical stability to the active agent.

[0243] The terms "buffer", "buffer composition", "buffering agent" refer to a solution that can resist changes in pH through the action of its acid-base conjugate components, which allows the rAAV5-based product carrier to resist changes in pH. Generally, the pharmaceutical composition preferably has a pH in the range of 4.0 - 8.0. Examples of buffers that can be used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate and other buffer solutions.

[0244] A pharmaceutical composition is "stable" if the active agent retains its physical stability and / or chemical stability and / or biological activity during the specified shelf life at a storage temperature, for example, of 2 - 8°C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The storage period is adjusted based on the results of stability tests under accelerated or natural aging conditions.

[0245] The pharmaceutical compositions of the present invention can be presented in dosage form and manufactured, packaged or sold in single unit doses or in multiple single unit doses. As used herein, the term "single unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient generally equals the dose of the active ingredient to be administered to a subject in vivo, or a convenient fraction of this dose, e.g., half or one third of this dose.

[0246] Methods for gene product delivery

[0247] In one aspect, the present invention relates to a method for delivering a gene product to a subject in need thereof, which comprises administering to the subject the above rAAV5-based vector or the above pharmaceutical composition.

[0248] In some embodiments, the method for delivering a gene product is used to deliver a gene product to a human in need thereof.

[0249] Any method recognized in the art for delivering rAAV5-based vectors may be suitable for the rAAV5-based vectors of the present invention described above.

[0250] The rAAV5-based recombinant viral vector is preferably delivered to cells in a biologically effective amount. A "biologically effective" amount of the viral vector is an amount sufficient to effect infection (or transduction) and expression of a heterologous nucleic acid sequence in the cells. If the virus is delivered to cells in vivo (e.g., to a subject as described below), the "biologically effective" amount of the viral vector is an amount sufficient to effect transduction and expression of a heterologous nucleic acid sequence in the target cells.

[0251] The cells for delivering the rAAV5 viral vector of the present invention can be any type of cell, including but not limited to nerve cells (including cells of the peripheral and central nervous systems, particularly brain cells), lung cells, epithelial cells (e.g., intestinal and respiratory epithelial cells), muscle cells, pancreatic cells (including islet cells), liver cells, cardiomyocytes, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. Alternatively, the cells for delivering the rAAV5 viral vector can be any progenitor cell. As a further alternative, the cell can be a stem cell (e.g., neural stem cell, liver stem cell). In addition, as described above, the cell can be from a species of any origin.

[0252] Use

[0253] In one aspect, the present invention relates to the use of the above rAAV5-based vector or the above pharmaceutical composition for treating a disease in a subject in need thereof.

[0254] In some embodiments, the use is for treating a disease in a human in need thereof.

[0255] Delivery of the rAAV5-based vector of the present invention to a human subject or an animal in need thereof can be effected by any means known in the art for delivering viral vectors.

[0256] Exemplary modes of delivery include topical, oral, rectal, transmucosal, transdermal, inhalation, parenteral delivery (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intra-articular delivery), etc., as well as direct tissue or organ injection, and alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. Injectables can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for preparation of solutions or suspensions in a liquid prior to injection, or as emulsions. Alternatively, the rAAV5-based vector can be delivered in a local rather than a systemic manner, e.g., in a depot or sustained release formulation.

[0257] In some embodiments of the use, the disease is selected from blood diseases, central nervous system diseases, metabolic diseases, muscle diseases, genetic diseases.

[0258] In some embodiments of the use, the disease is a blood disease.

[0259] In some embodiments of the use, the disease is a muscle disease.

[0260] In some embodiments of the use, the disease is a genetic disease.

[0261] In certain embodiments of the invention, the nucleotide sequence of interest is delivered to the liver of a subject via an rAAV5-based vector. Liver administration can be performed by any method known in the art, including but not limited to intravenous administration, intraportal administration, intra-biliary administration, intra-arterial administration, and direct injection into the liver parenchyma.

[0262] Preferably, cells (such as hepatocytes) are infected with an rAAV5-based vector encoding a peptide or protein, and the cells express the encoded peptide or protein and secrete it into the blood circulatory system in a therapeutically effective amount (as described below). Alternatively, the vector is delivered to another cell or tissue and expressed by it, and the other cell or tissue includes but is not limited to the brain, pancreas, spleen, or muscle.

[0263] "Therapeutically effective amount" is understood to mean an amount sufficient to alleviate (e.g., reduce, lower, decrease) at least one symptom associated with a disease state. Alternatively, a "therapeutically effective" amount is an amount sufficient to provide some improvement in the condition of a subject.

[0264] In some embodiments of the use, the expression product of the heterologous nucleic acid sequence is factor IX or a functional variant thereof.

[0265] In some embodiments of the use, the expression product of the heterologous nucleic acid sequence is factor VIII or a functional variant thereof.

[0266] In other preferred embodiments, the rAAV5-based vector of the invention is administered intramuscularly, more preferably by intramuscular injection or by topical administration (as described above). In other preferred embodiments, the parvovirus particles of the invention are administered to the lungs.

[0267] The rAAV5-based vectors disclosed in the present invention can be administered to the lungs of a subject by any suitable means, but are preferably administered in the form of an aerosol suspension of inhalable particles containing the rAAV5-based vector of the present invention that the subject inhales. The inhalable particles can be liquid or solid. An aerosol of liquid particles containing the parvovirus rAAV5 vector of the present invention can be generated by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer known to those skilled in the art. An aerosol of solid particles containing the viral rAAV5 vector of the present invention can also be generated with any solid particle drug aerosol generator by techniques known in the pharmaceutical art.

[0268] The dose of the parvovirus rAAV5 particles of the present invention will depend on the mode of administration, the disease or disorder to be treated, the condition of the subject, the particular viral vector, and the gene to be delivered, and can be determined in a conventional manner. Exemplary doses for achieving a therapeutic effect are 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 or more transduction units, preferably about 10 8 -10 13 transduction units, still more preferably a viral titer of 10 12 transduction units.

[0269] Thus, the rAAV5-based parvovirus vectors, reagents, and methods of the present invention can be used to direct nucleic acids to dividing or non-dividing cells and stably express heterologous nucleic acids therein. Using this vector system, genes encoding proteins that affect cell physiology can now be introduced into cells under in vivo conditions. Accordingly, the vectors of the present invention can be used for gene therapy of disease states.

[0270] Generally, the present invention can be used to deliver any exogenous nucleic acid having a biological effect to treat or ameliorate symptoms associated with any disorder related to gene expression. Exemplary disease states include, but are not limited to, cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood clotting disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, diabetes, muscular dystrophy (e.g., Duchenne, Becker), Gaucher's disease, Hurler's disease, adenosine deaminase deficiency, glycogen storage diseases and other metabolic defects, diseases of solid organs (e.g., brain, liver, kidney, heart), etc.

[0271] Gene transfer has great potential use in understanding and providing therapies for disease states. Defective genes for many genetic diseases are known and have been cloned. In some cases, the functions of these cloned genes are known. Generally, the above disease states are divided into two categories: deficiency states, generally enzyme deficiencies, which are usually inherited in a recessive manner; and imbalance states, sometimes involving at least regulatory or structural proteins, which are inherited in a dominant manner. For deficiency state diseases, gene transfer can be used to bring normal genes into affected tissues for replacement therapy. For imbalance disease states, gene transfer can be used to create disease states in model systems, and then efforts can be made to counteract the disease states with them. Thus, the methods of the present invention allow the treatment of genetic diseases. According to the present invention, disease states are treated by partially or completely remedying the deficiencies or imbalances that cause the disease or make it worse. It is also possible to use site-specific integration of nucleic acid sequences to induce mutations or correct deficiencies.

[0272] Methods for producing rAAV5-based vectors

[0273] In one aspect, the present invention relates to a method for producing the above rAAV5-based vector, which comprises transfecting a production cell with the above nucleic acid comprising a sequence encoding a capsid, said capsid comprising an altered VP1 capsid protein of adeno-associated virus serotype 5 (AAV5).

[0274] In some embodiments of the method for producing an rAAV5-based vector, the above nucleic acid is used, which comprises sequences encoding an altered VP1 protein of the above adeno-associated virus serotype 5 (AAV5) capsid, a VP2 protein of the AAV5 capsid or an altered variant thereof, and a VP3 protein of the AAV5 capsid or an altered variant thereof.

[0275] Altered variants of the VP2 protein of the wild-type AAV5 capsid and the VP3 protein of the AAV5 capsid protein are understood to mean variants of the VP2 protein of the wild-type AAV5 capsid and the VP3 protein of the wild-type AAV5 capsid comprising one or more amino acid substitutions.

[0276] Particularly preferred embodiments include substitutions that are substantially conservative, i.e., substitutions that occur within families of amino acids added by their side chains. Specifically, amino acids are generally divided into 4 families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that substituting leucine alone with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or amino acids similarly conservatively substituted with structurally related amino acids will not have a significant impact on biological activity. For example, the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 or 50 conservative or non-conservative amino acid substitutions, or any integer between 5-50, as long as the desired molecular function remains intact.

[0277] In some embodiments of the method for generating an rAAV5-based vector, the above nucleic acid is used, which contains sequences encoding an altered VP1 protein of the above adeno-associated virus serotype 5 (AAV5) capsid, the VP2 protein of the wild-type AAV5 capsid, and the VP3 protein of the wild-type AAV5 capsid.

[0278] Altered variants of the VP2 and VP3 proteins of the AAV5 capsid and nucleic acids encoding them are disclosed in detail in the corresponding parts of this specification. Examples

[0279] The following examples are provided to better understand the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0280] All publications, patents and patent applications cited in this specification are incorporated herein by reference. Although the foregoing invention has been described in considerable detail for purposes of clear understanding, it will be apparent to those of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments, according to the teachings of the present invention.

[0281] Materials and General Methods

[0282] Recombinant DNA Technology

[0283] DNA manipulation was performed by standard techniques as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions.

[0284] Gene synthesis

[0285] The desired gene segment was prepared from oligonucleotides made by chemical synthesis. Gene segments 300 - 4000 bp in length flanked by single restriction sites on both sides were assembled by annealing and ligation of oligonucleotides (including PCR amplification), and then cloned through the designated restriction sites. The DNA sequence of the subcloned gene fragment was confirmed by DNA sequencing.

[0286] DNA sequence determination

[0287] The DNA sequence was determined by Sanger sequencing.

[0288] DNA and protein sequence analysis and sequence data management

[0289] The Infomax Vector NTI Advance Suite version 8.0 and SnapGene Viewer were used for sequence creation, mapping, analysis, annotation, and illustration.

[0290] Example 1. Generation of an AAV5 capsid variant library

[0291] The AAV5 capsid variant library was generated by random mutagenesis of the Cap gene sequence (Davidsson M. et al., 2016). Briefly, the wild - type sequence of the serotype 5 Cap gene (GenBank ID AF085716.1) was assembled de novo, after which the synthesized wild - type AAV5 capsid gene was fragmented using uracil - DNA glycosylase, and the resulting fragments were assembled into the full - length Cap gene using a DNA polymerase without proofreading activity (as a result, random mutations occurred in the sequence). The full - length mutant variants were cloned into the vector plasmid pAAV - linker at the AscI / EcoRI restriction sites in the co - reading frame with green fluorescent protein (GFP) Figure 1 ) to generate a diverse AAV5 capsid random library, and then this capsid library was used to select capsid variants with increased transduction activity.

[0292] Positive selection of viral particles with increased transduction activity was performed on CHO-K1-S cells in vitro. Thus, for transduction, we used particles purified by ultracentrifugation in an iodixanol gradient. After 48 hours, the cells were harvested and genomic DNA was isolated for subsequent amplification of the viral genomic sequences capable of efficient transduction. Subsequently, the resulting sequences were recloned and reproduced for subsequent selection iterations to enrich the library containing variants with the highest transduction efficiency. After 5 rounds of selection, the capsid genes of 30 clones were sequenced to determine the most successful mutations and their combinations. The sequencing results showed that the major mutation combinations were S2A, T711S in AAV5 VP1 and capsid variants containing S2A, T711S, S651A in AAV5 VP1, accounting for approximately 20% of the clones. Capsid variants containing the mutation S651A in AAV5 VP1 were also selected. These capsid variants were cloned into vectors for the production of viral particles and further used to visualize and compare the transduction profiles relative to wild-type AAV5.

[0293] Example 2. Generation and subsequent selection of recombinant viral particles from the resulting sequence library

[0294] To generate recombinant viral particles and subsequently select recombinant viral particles from the resulting sequence library, a series of plasmids were developed as follows: a carrier plasmid, a plasmid containing the Rep gene sequence, and a construct containing adenoviral genes required for viral particle replication.

[0295] Using the restriction enzyme-ligase method cloned at the HindIII / EcoRI sites, by replacing the sequence of the modified green fluorescent protein in the initial construct pAAV-GFP control plasmid (VPK-402) from CellBiolab (USA) with the de novo synthesized sequence T2A-GFP, a carrier plasmid pAAV-linker ( Figure 1 ) was generated for cloning a library of random variants of the capsid gene of AAV serotype 5 into a reading frame with a reporter protein. The sequence T2A-GFP contains the addition of an EcoRI restriction site from the 5'-end and a HindIII restriction site from the 3'-end.

[0296] The plasmid pAAV-Rep containing the Rep gene sequence ( Figure 2) was generated by de novo cloning of the synthetic sequence of the AAV serotype 2 Rep gene (GenBank ID: AYF043303.1) at the PciI / PsiI restriction sites (New England Biolabs, USA), followed by treatment with T4 DNA polymerase (New England Biolabs, USA) to generate blunt ends within the plasmid pGem-T Easy (Promega, USA), which was also treated with the PciI / PsiI restriction enzyme (New England Biolabs, USA).

[0297] The adenovirus genes for generating recombinant viral particles were derived from the construct pHelper of the AAV-2 Packaging System (VPK-402) commercial kit from CellBiolab (USA) ( Figure 3 ), which contains: AmpR, the β-lactamase gene providing resistance to ampicillin; Ori, the origin of replication in bacteria; Adeno E2A, the helper adenovirus gene sequence involved in viral DNA replication; Adeno E4, the helper adenovirus gene sequence involved in viral DNA replication; Adeno VARNA, the helper adenovirus gene sequence responsible for translating both early and late viral genes.

[0298] Example 3. Method for generating a vector based on altered adeno-associated virus serotype 5 (rAAV)

[0299] To generate rAAV particles with an altered serotype 5 capsid, producer cells were transfected simultaneously with 3 plasmids as follows:

[0300] 1) Transfected with a plasmid (helper plasmid) containing the adenovirus nucleotide sequence encoding the proteins and RNAs required for assembling rAAV particles;

[0301] 2) Transfected with a plasmid containing the native nucleotide sequence of the Rep gene of adeno-associated virus serotype 2 and the sequence of the altered Cap gene, the sequence of the altered Cap gene being selected from the nucleotide sequences of SEQ ID NO: 5, 6 or 7 or any other nucleotide sequence encoding a VP1 protein having the amino acid sequence of SEQ ID No: 2, 3 or 4 and VP2 and VP3 proteins from the alternative reading frames of the nucleotide sequences used, wherein

[0302] VP2 can have any amino acid sequence of SEQ ID No: 8, 9 or 10;

[0303] VP3 may have any amino acid sequence of SEQ ID No: 11, 12 or 13;

[0304] 3) Transfect with a plasmid containing a heterologous genome of rAAV particles, the heterologous genome encoding a target gene intended for delivery into patient cells.

[0305] This set of genes provides for the assembly of rAAV viral particles and the encapsidation of the target genome therein within 72 hours. At 72 hours post-transfection, the production cells are lysed to release rAAV particles for purification by subsequent filtration and chromatography steps. The titer of the purified rAAV particles is verified by enzyme-linked immunosorbent assay and quantitative PCR.

[0306] Example 4. Increasing the efficiency of cell transduction with an rAAV5-based product in the presence of mutations S2A, S651A, T711S in the VP1 protein of the wild-type AAV5 capsid.

[0307] Experimental design:

[0308] Plate CHO-K1-S cells into the wells of a 12-well plate. Seed into the following growth medium: DMEM / F12 supplemented with glutamine, glucose content of 4.5 g / l, 5% bovine serum. The cell seeding density is 10,000 cells / cm 2 . During the transduction run, pre-prepared cells are transduced at an MOI of 100,000 vg / cell. All samples are run in triplicate. Use intact cells as a negative control.

[0309] Use a Guava EasyCyte flow cytometer and GuavaSoft software for transduction efficiency analysis.

[0310] The inventors have unexpectedly found that the presence of one or more mutations selected from S2A, S651A or T711S in the VP1 protein of the wild-type AAV5 capsid results in a significant increase in the efficiency of transgene delivery by rAAV vectors having the above mutations. For example, flow cytometry methods show that the amount of GFP-positive cells changes 48 hours after transduction of the CHO-K1-S line with an rAAV-based product containing the VP1 protein of the wild-type AAV5 capsid or the VP1 protein of the wild-type AAV5 capsid with one or more mutations selected from S2A, S651A, T711S ( Figure 4 ).

[0311] When there is a mutation S651A (AAV5-01Mut-GFP), compared with the control AAV5 with wild-type VP1 capsid protein (AAV5-NullMut-GFP), the amount of cells expressing GFP increased to 2.2-fold (from 22.54% to 49.45%).

[0312] When there are both mutations S2A and T711S (AAV5-02Mut-GFP), compared with the control AAV5 with wild-type VP1 capsid protein (AAV5-NullMut-GFP), the amount of cells expressing GFP increased to 2.6-fold (from 22.54% to 58.51%).

[0313] When there are mutations S2A, S651A and T711S simultaneously (AAV5-03Mut-GFP), compared with the control AAV5 with wild-type VP1 capsid protein (AAV5-NullMut-GFP), the amount of cells expressing GFP increased to 1.7-fold (from 22.54% to 38.27%).

[0314] Example 5. Increase the production of the target protein encoded by the transgene after cell transduction with an rAAV5-based product in the presence of mutations S2A, S651A, T711S in the VP1 protein of the wild-type AAV5 capsid.

[0315] Experimental design:

[0316] Plate CHO-K1-S cells into the wells of a 12-well plate. Seed into the following growth medium: DMEM / F12 supplemented with glutamine, glucose content of 4.5 g / l, 5% bovine serum. The cell seeding density is 10,000 cells / cm 2 . During the transduction run, transduce the pre-prepared cells at an MOI of 100,000 vg / cell. All samples are run in triplicate. Use intact cells as the negative control.

[0317] Evaluate the amount of FIX protein in the culture medium 7 days after transduction using a human factor IX ELISA kit. We use samples diluted 1:25. The procedure is carried out according to the manufacturer's instructions.

[0318] The inventors have unexpectedly found that the presence of one or more mutations selected from S2A, S651A, and T711S in the VP1 protein of the wild-type AAV5 capsid results in a significant increase in the production of hFIX protein after transduction of CHO-K1-S cells with a vector based on rAAV having the above mutations. For example, enzyme-linked immunosorbent assay (ELISA) methods showed that 7 days after transduction of CHO-K1-S cells with an rAAV product containing the wild-type AAV5 VP1 capsid protein or the VP1 protein of the wild-type AAV5 capsid with one or more mutations selected from S2A, S651A, and T711S, the amount of hFIX protein in the culture medium increased ( Figure 5 ).

[0319] When the mutation S651A (AAV5-01Mut-FIX) is present, the amount of protein produced increased to 4.6-fold (from 0.17 ng / ml to 0.74 ng / ml) compared to the control AAV5 with wild-type VP1 capsid protein (AAV5-NullMut-GFP).

[0320] When both mutations S2A and T711S are present simultaneously (AAV5-02Mut-GFP), the amount of protein produced increased to 7.1-fold (from 0.17 ng / ml to 1.24 ng / ml) compared to the control AAV5 with wild-type VP1 capsid protein (AAV5-NullMut-GFP).

[0321] When mutations S2A, S651A, and T711S are present simultaneously (AAV5-03Mut-GFP), the amount of protein produced increased to 3.3-fold (from 0.17 ng / ml to 0.57 ng / ml) compared to the control AAV5 with wild-type VP1 capsid protein (AAV5-NullMut-GFP).

Claims

1. An isolated altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid for highly efficient transduction of target cells, which comprises the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid encoded by the Cap gene, and the amino acid sequence comprises one or more substitutions selected from the following: S2A and T711S, S2A, S651A and T711S.

2. The isolated altered VP1 protein of the AAV5 capsid of claim 1, which comprises S2A and T711S substitutions.

3. The isolated altered VP1 protein of the AAV5 capsid of claim 2, which has the amino acid sequence represented by SEQ ID NO:

3.

4. The isolated altered VP1 protein of the AAV5 capsid of claim 1, which comprises the substitutions S2A, S651A and T711S.

5. The isolated altered AAV5 VP1 capsid protein of claim 4, which has the amino acid sequence represented by SEQ ID NO:

4.

6. An isolated nucleic acid encoding the altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid of any one of claims 1-5, which is for highly efficient transduction of target cells.

7. The isolated nucleic acid of claim 6, which encodes an altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S651A substitution, and the nucleic acid is represented by the nucleic acid sequence of SEQ ID NO:5 or by any other sequence encoding the corresponding amino acid sequence.

8. The isolated nucleic acid of claim 6, which encodes an altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S2A and T711S substitutions, and the nucleic acid is represented by the nucleic acid sequence of SEQ ID NO:6 or by any other sequence encoding the corresponding amino acid sequence.

9. The isolated nucleic acid of claim 6, which encodes an altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S2A, S651A and T711S substitutions, and the nucleic acid is represented by the nucleic acid sequence of SEQ ID NO:7 or by any other sequence encoding the corresponding amino acid sequence.

10. An isolated capsid for highly efficient transduction of target cells, which comprises the altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid of any one of claims 1-5.

11. The isolated capsid of claim 10, which comprises the altered VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid of any one of claims 1-5, the VP2 protein of the AAV5 capsid or its altered variant, and the VP3 protein of the AAV5 capsid or its altered variant.

12. The isolated capsid of claim 11, which comprises the VP2 protein of the wild-type AAV5 capsid.

13. The isolated capsid of claim 12, which comprises the VP2 protein of the wild-type AAV5 capsid protein having the amino acid sequence represented by SEQ ID NO:

8.

14. The isolated capsid of claim 11, which comprises the altered VP2 protein of an adeno-associated virus serotype 5 (AAV5) capsid.

15. The isolated capsid of claim 14, which comprises an altered VP2 protein of an AAV5 capsid containing a T575S substitution.

16. The isolated capsid of claim 15, which comprises an altered VP2 protein of an AAV5 capsid containing a T575S substitution and having the amino acid sequence represented by SEQ ID NO:

9.

17. The isolated capsid of claim 15, which comprises an altered VP2 protein of an AAV5 capsid containing S515A and T575S substitutions.

18. The isolated capsid of claim 17, which comprises an altered VP2 protein of an AAV5 capsid containing S515A and T575S substitutions and having the amino acid sequence represented by SEQ ID NO:

10.

19. The isolated capsid of claim 11, which comprises a VP3 protein of a wild-type AAV5 capsid.

20. The isolated capsid of claim 19, which comprises a VP3 protein of a wild-type AAV5 capsid having the amino acid sequence represented by SEQ ID NO:

11.

21. The isolated capsid of claim 11, which comprises an altered VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid.

22. The isolated capsid of claim 21, which comprises an altered VP3 protein of an AAV5 capsid containing a T519S substitution.

23. The isolated capsid of claim 22, which comprises an altered VP3 protein of an AAV5 capsid containing a T519S substitution and having the amino acid sequence represented by SEQ ID NO:

12.

24. The isolated capsid of claim 21, which comprises an altered VP3 protein of an AAV5 capsid containing S459A and T519S substitutions.

25. The isolated capsid of claim 24, which comprises an altered VP3 protein of an AAV5 capsid containing S459A and T519S substitutions and having the amino acid sequence represented by SEQ ID NO:

13.

26. An isolated nucleic acid encoding the capsid of any one of claims 10-25, which is used for highly efficient transduction of target cells.

27. A recombinant adeno-associated virus serotype 5 (rAAV5)-based vector for delivering a heterologous nucleic acid sequence to a subject, which comprises: 1) The capsid of any one of claims 10-25, and 2) A heterologous nucleic acid sequence, which comprises regulatory sequences that promote the expression of the product encoded by the heterologous nucleic acid sequence in target cells.

28. The rAAV5-based vector of claim 27, wherein the expression product of the heterologous nucleic acid sequence is a therapeutic polypeptide or a reporter polypeptide.

29. The rAAV5-based vector of claim 28, wherein the therapeutic polypeptide is a coagulation factor selected from factor VIII, factor IX, or a functional variant thereof.

30. The rAAV5-based vector of claim 29, wherein the therapeutic peptide is factor VIII or a functional variant thereof.

31. The rAAV5-based vector of claim 29, wherein the therapeutic peptide is factor IX or a functional variant thereof.

32. A pharmaceutical composition for delivering a gene product to a subject in need thereof, which comprises: a) An rAAV5-based vector according to any one of claims 27-31; and b) A pharmaceutically acceptable excipient.

33. The pharmaceutical composition of claim 32, wherein the subject is a human subject.

34. Use of an rAAV5-based vector according to any one of claims 27-31 or the pharmaceutical composition of claim 32 in the manufacture of a medicament for the treatment of a disease selected from the group consisting of blood diseases, central nervous system diseases, metabolic diseases, muscle diseases, and genetic diseases.

35. The use of claim 34, wherein the disease is a blood disease.

36. The use of claim 35, wherein the expression product of the heterologous nucleic acid sequence is factor IX or a functional variant thereof.

37. The use of claim 35, wherein the expression product of the heterologous nucleic acid sequence is factor VIII or a functional variant thereof.

38. The use of claim 34, wherein the disease is a muscle disease.

39. The use of claim 34, wherein the disease is a genetic disease.

40. A method for obtaining an rAAV5-based vector according to any one of claims 27-31, which comprises transfecting a production cell with the nucleic acid of claim 26.

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