Isolation modification VP1 capsid protein of AAV5

Abstract

To provide AAV having modified transduction ability, the AAV including in its structure, various kinds of transducing genes including a clinically important transducing gene for a patient who requires a transducing gene.SOLUTION: There are provided: an isolation modification VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid, including one or more amino acid replacements for improving transduction efficiency, relative to VP1 protein of a wild type AAV5 capsid; and capsid and vector based on the isolation modification VP1 protein.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Adeno-associated virus (AAV) is a small (20 nm), independent, replication-incompetent, non-enveloped virus. Many different AAV serotypes have been described in humans and primates. The adeno-associated virus genome is composed of single-stranded DNA (ssDNA) approximately 4,700 nucleotides in length (+ or -). The genomic DNA has inverted terminal repeats (ITRs) at its termini. The genome contains two open reading frames (ORFs), Rep and Cap, and several alternative reading frames encoding diverse protein products. The Rep product is essential for AAV replication, while the three capsid proteins (VP1, VP2, and VP3) as well as other alternative products are encoded by the Cap gene. The VP1, VP2, and VP3 proteins are in a 1:1:10 ratio and form an icosahedral capsid (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 recombinant AAV (rAAV) vector production, an expression cassette flanked by ITRs is packaged into the AAV capsid. 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 multiple tissue types, providing strong and sustained transgene expression. They are also nonpathogenic and have a low immunogenicity profile (High KA et al., "rAAV human trial experience" Methods Mol Biol. 2011;807:429-57).

[0003] One of the essential goals of trials in the field of developing effective gene therapy is to optimize the vector to maximize tissue transduction while minimizing vector dosage.

[0004] It is known that various AAV serotypes are characterized by their affinity for distinct host cell surface receptors, which are their tropisms. Thus, the primary known receptor for AAV2 is heparan sulfate proteoglycan, and the coreceptors are the integrin heterodimer aVβ5, type 1 fibroblast growth factor receptor, and hepatocyte growth factor receptor, c-Met. AAV12 binds to heparan sulfate proteoglycans and sialic acid. AAV4 and AAV5 bind to N- and O-linked sialic acid, respectively. AAV5 activates the platelet-derived growth factor receptor. Concurrently, the link between the amino acid sequence of the AAV capsid protein and its assembly process, genome encapsidation, and affinity for different types of receptors displayed on the host cell surface has been established (Govindasamy L. et al. Structural insights into adeno-associated virus serotype 5. J Virol. 2013 Oct;87(20):11187-99).

[0005] International application WO 2012145601 discloses adeno-associated virus (AAV) virions comprising mutant capsid proteins, which exhibit higher infectivity for retinal cells when administered via intravitreal injection compared to wild-type AAV.

[0006] International application WO 2013158879 discloses an adeno-associated virus (AAV) vector for delivering a heterologous nucleic acid sequence to a subject, the vector comprising a VP1 capsid protein containing one or more lysine substitutions, one lysine substitution being K137R, which is effective to inhibit ubiquitination of the capsid protein, thereby increasing transduction of the AAV vector in target cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International application WO 2012145601 [Patent Document 2] International application WO 2013158879 [Non-patent literature]

[0008] [Non-Patent Document 1] Xie Q. et al. Proc Natl Acad Sci USA, 2002;99:10405-10410 [Non-patent document 2] High KA et al., Methods Mol Biol. 2011;807:429-57 [Non-patent document 3] Govindasamy L. et al. J Virol. 2013 Oct;87(20):11187-99 Summary of the Invention [Problem to be solved by the invention]

[0009] Currently, there is a need for AAVs with improved transduction capabilities that contain within their structure a variety of transgenes, including clinically important transgenes, for patients in need thereof. Improved tissue transduction would allow for minimizing the dose of vector administered to a subject. [Means for solving the problem]

[0010] The inventors surprisingly found that in the VP1 protein of the wild-type AAV5 capsid: S651A, S2A and T711S or S2A, S651A, and T711S We have found that the presence of one or more amino acid substitutions selected from the group comprising: results in increased transduction efficiency of target cells using AAV serotype 5 vectors bearing this / these modification(s), and a significant increase in transgene delivery efficiency by rAAV vectors bearing said mutations.

[0011] In one aspect, the invention provides an isolated modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid for highly efficient transduction of target cells, comprising: S651A, S2A and T711S, S2A, S651A and T711S The present invention relates to the isolated modified VP1 protein, which comprises the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid encoded by the Cap gene, containing one or more substitutions selected from the group comprising:

[0012] In some embodiments, the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid has the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the isolated modified VP1 protein of the AAV5 capsid includes a substitution at position S651A.

[0013] In some embodiments, the isolated modified VP1 protein of the AAV5 capsid has the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the isolated modified VP1 protein of the AAV5 capsid includes an S2A and a T711S substitution.

[0014] In some embodiments, the isolated modified VP1 protein of the AAV5 capsid has the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the isolated modified VP1 protein of the AAV5 capsid includes S2A, S651A, and T711S substitutions.

[0015] In some embodiments, the isolated modified VP1 protein of the AAV5 capsid has the amino acid sequence set forth in SEQ ID NO:4. In one aspect, the present invention relates to an isolated nucleic acid encoding the above-described modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid for use in highly efficient transduction of target cells.

[0016] In some embodiments, the isolated nucleic acid encoding the modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising an amino acid S651A substitution is represented by the nucleic acid sequence of SEQ ID NO: 5 or any other sequence encoding the corresponding amino acid sequence of a modified protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising an amino acid S651A substitution.

[0017] In some embodiments, the isolated nucleic acid encoding the modified VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S2A and T711S substitutions is represented by the nucleic acid sequence of SEQ ID NO: 6 or any other sequence encoding the corresponding amino acid sequence of the modified protein of adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S2A and T711S substitutions.

[0018] In some embodiments, the isolated nucleic acid encoding the modified VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S2A, S651A and T711S substitutions is represented by the nucleic acid sequence of SEQ ID NO: 7 or any other sequence encoding the corresponding amino acid sequence of the modified protein of adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S2A, S651A and T711S substitutions.

[0019] In one aspect, the present invention relates to an isolated capsid for highly efficient transduction of target cells, comprising the above-described modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid.

[0020] In some embodiments, the isolated capsid includes the above-described modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid, a VP2 protein of an AAV5 capsid or a modified variant thereof, and a VP3 protein of an AAV5 capsid or a modified variant thereof.

[0021] In some embodiments, the isolated capsid includes the VP2 protein of a wild-type AAV5 capsid. In some embodiments, the isolated capsid comprises the amino acid sequence set forth by SEQ ID NO:8. The VP1 protein of the wild-type AAV5 capsid is included, which has the sequence:

[0022] In some embodiments, the isolated capsid includes a modified VP2 protein of an adeno-associated virus serotype 5 (AAV5) capsid. In some embodiments, the isolated capsid includes a modified VP2 protein of an AAV5 capsid that includes a T575S substitution.

[0023] In some embodiments, the isolated capsid includes a modified VP2 protein of an AAV5 capsid that includes a T575S substitution and has the amino acid sequence set forth in SEQ ID NO:9.

[0024] In some embodiments, the isolated capsid includes a modified VP2 protein of an AAV5 capsid that includes a S515A and a T575S substitution. In some embodiments, the isolated capsid includes a modified VP2 protein of an AAV5 capsid that includes S515A and T575S substitutions and has the amino acid sequence set forth in SEQ ID NO:10.

[0025] In some embodiments, the isolated capsid includes the VP3 protein of a wild-type AAV5 capsid. In some embodiments, the isolated capsid includes the VP3 protein of a wild-type AAV5 capsid having the amino acid sequence set forth in SEQ ID NO:11.

[0026] In some embodiments, the isolated capsid includes a modified VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid. In some embodiments, the isolated capsid includes a modified VP3 protein of an AAV5 capsid that includes a T519S substitution.

[0027] In some embodiments, the isolated capsid includes a modified VP3 protein of an AAV5 capsid that includes a T519S substitution and has the amino acid sequence set forth in SEQ ID NO:12.

[0028] In some embodiments, the isolated capsid includes a modified VP3 protein of an AAV5 capsid that includes S459A and T519S substitutions. In some embodiments, the isolated capsid includes a modified VP3 protein of an AAV5 capsid that includes S459A and T519S substitutions and has the amino acid sequence set forth in SEQ ID NO:13.

[0029] In one aspect, the invention relates to isolated nucleic acids encoding the capsids described above for use in highly efficient transduction of target cells. In one aspect, the invention provides a recombinant adeno-associated virus serotype 5 (rAAV5)-based vector for delivery of a heterologous nucleic acid sequence to a subject, comprising: 1) the capsid described above, and 2) a heterologous nucleic acid sequence comprising a control sequence that promotes expression of the product encoded by the heterologous nucleic acid sequence in the target cell; The present invention relates to the vector comprising:

[0030] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence that encodes a product that is a therapeutic polypeptide or a reporter polypeptide. In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding a product that is a therapeutic polypeptide, the therapeutic polypeptide being Factor VIII, Factor IX, or or functional variants thereof.

[0031] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding a product that is Factor VIII or a functional variant thereof. 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.

[0032] In one aspect, the present invention provides a pharmaceutical composition for delivering a gene product to a subject in need thereof, comprising: a) the above vector based on rAAV5; and b) Pharmaceutically acceptable excipients The present invention relates to the pharmaceutical composition comprising:

[0033] In some embodiments, the pharmaceutical composition is used to deliver the gene product to a human in need thereof. In one aspect, the present invention relates to a method for delivering a gene product to a subject in need thereof, the method comprising administering to the subject the above-described rAAV5-based vector or the above-described pharmaceutical composition.

[0034] In some embodiments, the methods for delivery of gene products are used to deliver the gene product to a human in need thereof. In one aspect, the present invention relates to the use of the rAAV5-based vector or the pharmaceutical composition for the treatment of a disease in a subject in need thereof.

[0035] In some embodiments, the use is for the treatment of a disease in a human in need thereof. In some embodiments of the use, the disease is selected from the group comprising: a blood disease; a central nervous system disease; a metabolic disease; a muscular disease; a genetic disease.

[0036] In some embodiments of the use, the disease is a blood disease. In some embodiments of the use, the expression product of the heterologous nucleic acid sequence is Factor IX or a functional variant thereof.

[0037] In some embodiments of the use, the expression product of the heterologous nucleic acid sequence is Factor VIII or a functional variant thereof. In some embodiments of the use, the disease is a muscle disease.

[0038] In some embodiments of the use, the disease is a genetic disease. In one aspect, the invention relates to a method for producing the rAAV5-based vector, comprising transfecting a producer cell with the nucleic acid encoding the capsid. [Brief explanation of the drawings]

[0039] [Figure 1] Circular diagram of the plasmid pAAV-linker, intended for cloning a random mutant library of the capsid gene of AAV serotype 5. AmpR is the beta-lactamase gene that confers resistance to ampicillin, pUC origin is the pUC origin of replication in bacteria, ITR is an inverted terminal repeat, CMV promoter is the promoter of the cytomegalovirus early gene, polyA is a polyadenylation signal sequence to increase mRNA stability, HBG intron is the human beta-globin intron, GFP is the green fluorescent protein gene, and T2A is a self-cleaving peptide that allows for simultaneous expression of target and reporter proteins. [Figure 2] Circular representation of the plasmid pAAV-Rep, which is intended to produce recombinant viral products of wild-type AAV serotype 5 from a library of random mutants.

[0040] AmpR is a beta-lactamase gene that confers resistance to ampicillin; The pUC origin is the bacterial origin of replication of pUC. The Rep gene is a Rep gene sequence that encodes an AAV replication protein. [Figure 3] Circular representation of the plasmid pHelper, which is intended to produce recombinant viral products of wild-type AAV serotype 5 from a library of random mutants.

[0041] AmpR is a beta-lactamase gene that confers resistance to ampicillin; Ori is the origin of replication in bacteria, AdenoE2A is a helper adenovirus gene sequence involved in viral DNA replication. AdenoE4 is a helper adenovirus gene sequence involved in viral DNA replication. Adeno-VARNA is a helper adenovirus gene sequence involved in the translation of both early and late viral genes. [Figure 4] Analysis of the efficiency of CHO-K1-S cell transduction with AAV5-GFP-based viral products containing one or more amino acid substitutions in the VP1 protein of the wild-type AAV5 capsid. [Figure 5] Analysis of hFIX protein concentration in medium collected from CHO-K1-S cells 7 days after transduction with an AAV5-hFIX-based viral product containing one or more amino acid substitutions in the VP1 protein of the wild-type AAV5 capsid. [Figure 6] Position of AAV5 capsid proteins in the genome.

[0042] 2087~4258bp-VP1 2495~4258bp-VP2 2663~4258bp-VP3 DETAILED DESCRIPTION OF THE INVENTION

[0043] Definitions and general methods Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0044] Further, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular. Typically, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known and widely used by those of skill in the art. Enzymatic reactions and purification methods are performed according to manufacturer's instructions, as common in the art or as described herein.

[0045] "Isolated" means altered or removed from the natural state. For example, a 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 materials with which it coexists in the natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a genetically modified cell.

[0046] The terms "naturally occurring," "natural," or "wild-type" are used to describe an entity that can be found in nature, as opposed to one that is artificially produced. 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 intentionally modified by man in the laboratory is naturally occurring.

[0047] The term "genome" refers to the complete genetic material of an organism. As used in this specification and the claims that follow, unless the context otherwise dictates, the words "include" and "comprise" or variations thereof, such as "having," "includes," "including," "comprises," or "comprising," will be understood to refer to the inclusion of a reference integer or group of integers, but not to the exclusion of any other integer or group of integers.

[0048] Proteins (peptides) As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting 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 on the maximum number of amino acids a protein or peptide sequence may contain. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, as well as longer chains of which there are many types, commonly referred to in the art as proteins. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0049] 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 may be used interchangeably to carry the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, infection, PEG fusion, etc.

[0050] nucleic acid molecule The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably in this description and refer to an exact sequence of nucleotides, modified or not, determining a fragment or region of a nucleic acid, containing or not containing non-naturally occurring nucleotides, and being either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0051] Those skilled in the art have the general knowledge that nucleic acids are polynucleotides that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, nucleic acid sequences isolated from recombinant libraries or cellular genomes using recombinant means available in the art, including but not limited to, conventional cloning techniques and PCR. The term "nucleic acid sequence" includes all nucleic acid sequences obtained by any means, including cloning, and by synthetic means.

[0052] It should also be noted herein 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 are 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 modified. Thus, isolated nucleic acids obtained by recombinant genetics, for example, in a host cell, or obtained by chemical synthesis, should also be mentioned herein.

[0053] An "isolated" nucleic acid molecule is one that has been identified and separated from at least one nucleic acid molecule impurity with which the molecule is associated in the natural source of the nuclease nucleic acid. An isolated nucleic acid molecule is different from the type or set found under natural conditions. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule present in a cell under natural conditions. However, an isolated nucleic acid molecule includes a nucleic acid molecule that is located in a cell in which the nuclease is normally expressed, for example, if the nucleic acid molecule has a chromosomal location that is different from its location in the cell under natural conditions.

[0054] Unless otherwise indicated, the term nucleotide sequence includes its complement. Thus, a nucleic acid having a particular sequence should be understood to include its complementary sequence, along with its complementary strand.

[0055] Adeno-associated virus (AAV) Parvoviridae viruses are small, DNA-containing animal viruses. The Parvoviridae family 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 viruses have been described (Mori, S. et al., 2004, "Two novel adeno-associated viruses from cynomolgus monkeys: pseudotyping characterization of capsid protein", Virology, T. 330(2):375-83). All known serotypes can infect cells from multiple tissue types. Tissue specificity is determined by the capsid protein serotype; therefore, adeno-associated virus-based vectors are constructed by assigning the desired serotype. Further information on parvoviruses and other members of the Parvoviridae family can be found in the literature (Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," in Fields Virology, Chapter 69 (3rd ed. 1996)).

[0056] The genomic organization of all known AAV serotypes is very similar. The AAV genome is a linear, single-stranded DNA molecule less than approximately 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the nonstructural replication (Rep) and structural (Cap) proteins. The Cap gene encodes the capsid-forming VP proteins (VP1, VP2, and VP3). The terminal 145 nucleotides are self-complementary and organized to form an energetically stable intramolecular duplex, forming a T-shaped hairpin. This hairpin structure serves as an origin of viral DNA replication and as a primer for the cellular DNA polymerase complex. After wild-type AAV (wtAAV) infection in mammalian cells, Rep genes (e.g., Rep78 and Rep52) are expressed using the P5 and P19 promoters, respectively, and both Rep proteins have specific functions in viral genome replication. Splicing events in the Rep open reading frame (Rep ORF) actually result in four Rep proteins (e.g., Rep78, Rep68, Rep52, and Rep68). However, it has been shown that unspliced ​​mRNAs encoding the Rep78 and Rep52 proteins are sufficient for AAV vector production in mammalian cells.

[0057] Recombinant adeno-associated virus (rAAV)-based vectors The term "vector," as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0058] The term "recombinant AAV vector" (or "rAAV vector"), as used herein, refers to a vector containing one or more polynucleotide sequences of interest, a parvovirus, or a gene of interest or "transgene" flanked by inverted terminal repeats (ITRs).

[0059] The terms "infectious unit (iu)," "infectious particle," or "replication unit," when used in reference to viral titer, refer to the number of infectious recombinant AAV vector particles as measured by the infectious center assay, also known as the replication center assay, e.g., as described in McLaughlin et al., J. Virol. (1988) 62:1963-1973.

[0060] The term "heterologous," when referring to nucleic acid sequences, such as coding sequences and control sequences, refers to sequences that are not normally linked 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 attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct can include a coding sequence that is flanked by sequences not found in association with the coding sequence in nature. 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 the native gene).

[0061] As used herein, the term "operably linked" refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is present in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. The term "operably linked" means that the DNA sequences being linked are typically contiguous, and, where necessary to join two protein-coding regions, also contiguous and in reading frame.

[0062] As used herein, the term "promoter" or "transcriptional control sequence" or "control sequence" refers to a sequence that controls the transcription of one or more coding sequences and is located upstream of the transcriptional start site of the coding sequence in the reading direction of transcription, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription initiation site, and any other DNA sequences, including but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to those skilled in the art that directly or indirectly control the transcription level at 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 physiologically or developmentally regulated, for example, under the influence of a chemical inducer. A "tissue-specific" promoter is only active in specific types of tissues or cells.

[0063] The term "enhancer" or "enhancers" as used herein refers to an enhancer or enhancers that enhance the coding sequence of a recombinant product. The term "enhancer" may refer to a DNA sequence located adjacent to a DNA sequence encoding it. Enhancer elements are typically located 5' to a promoter element, or may be located downstream of 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, 200, or 300 base pairs or more upstream of a DNA sequence encoding a recombinant product or downstream of said sequence. Enhancer elements may increase the amount of recombinant product expressed from a DNA sequence above the expression level associated with a single promoter element. Numerous enhancer elements are readily available to those of ordinary skill in the art.

[0064] The term "selectable marker gene" refers to a gene that, when expressed, confers a selectable phenotype, such as antibiotic resistance, on transformed cells. As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0065] Therapeutic Use "Gene therapy" is the insertion of a gene into a subject's cells and / or tissues to treat a disease, typically a genetic disease, where a defective mutant allele is replaced with a functional one.

[0066] "Treating," "treatment," and "therapy" refer to a method of alleviating or inhibiting a biological disorder and / or at least one of its associated symptoms. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder, or condition. Furthermore, references herein to "treatment" include curative, symptomatic, and prophylactic treatment.

[0067] In one aspect, the subject or patient of treatment is a mammal, preferably a human subject, who may be male or female of any age. The term "disorder" means any condition that would benefit from treatment in accordance with the present invention, including chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question.

[0068] A "disease" is a state of health in an animal in which the animal is no longer able to maintain homeostasis and, if the disease is not alleviated, the animal's health continues to deteriorate. The terms "subject," "patient," "individual," and the like are used interchangeably herein and refer to any animal amenable to the methods described herein. In certain non-limiting embodiments, the subject, patient, or individual is a human.

[0069] A "therapeutically effective amount" refers to that amount of a therapeutic agent administered during treatment that will relieve to some extent one or more of the symptoms of the disease being treated. The term "chronic" use refers to continuous (uninterrupted) use of an agent(s), as opposed to acute (transient) routes of administration, so as to sustain the initial therapeutic effect (activity) for an extended period of time.

[0070] "Intermittent" use refers to treatment that is cyclic in nature, rather than continuously without interruption. Detailed Description of the Invention Isolated modified VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid In one aspect, the invention provides an isolated modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid for highly efficient transduction of target cells, comprising: S651A, S2A and T711S, S2A, S651A, and T711S The present invention relates to the isolated modified VP1 protein, which comprises the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid encoded by the Cap gene, containing one or more substitutions selected from the group comprising:

[0071] Amino acid S2A substitution is understood to mean the substitution of serine (Ser, S) at position 2 of the VP1 protein of wild-type adeno-associated virus serotype 5 capsid with alanine (Ala, A).

[0072] The amino acid S651A substitution is understood to mean the substitution of serine (Ser, S) at position 651 of the VP1 protein of wild-type adeno-associated virus serotype 5 capsid with alanine (Ala, A).

[0073] The amino acid T711S substitution is understood to mean the substitution of threonine (Thr, T) at position 711 of the VP1 protein of wild-type adeno-associated virus serotype 5 capsid with serine (Ser, S).

[0074] In some embodiments, the VP1 protein amino acid sequence of the wild-type AAV5 capsid is

[0075] [ka]

[0076] The amino acid sequence is represented by the formula: In some embodiments, the isolated modified VP1 protein of the AAV5 capsid includes a substitution at position S651A.

[0077] In some embodiments, the isolated modified VP1 protein of the AAV5 capsid

[0078] [ka]

[0079] The amino acid sequence is represented by the formula: In some embodiments, the isolated modified VP1 protein of the AAV5 capsid includes an S2A and a T711S substitution.

[0080] In some embodiments, the isolated modified VP1 protein of the AAV5 capsid

[0081] [ka]

[0082] The amino acid sequence is represented by the formula: In some embodiments, the isolated modified VP1 protein of the AAV5 capsid includes S2A, S651A, and T711S substitutions.

[0083] In some embodiments, the isolated modified VP1 protein of the AAV5 capsid

[0084] [ka]

[0085] The amino acid sequence is represented by the formula: Isolated modified VP2 and VP3 proteins of adeno-associated virus serotype 5 (AAV5) capsid The "right" (+) strand of the adeno-associated virus genomic DNA contains overlapping sequences encoding three capsid proteins, VP1, VP2, and VP3. Transcription of these genes is initiated by a single promoter, p40. The molecular weights of the corresponding proteins are 87, 72, and 62 kDa, respectively. All three proteins are translated from a single mRNA. After transcription, the pre-mRNA can be spliced ​​in two different ways, where longer or shorter introns are excised to form mRNAs of 2300 or 2600 nucleotides in length.

[0086] Therefore, the introduction of mutations into the Cas gene affects not only the VP1 protein of the AAV5 capsid, but also the VP2 and VP3 proteins of the AAV5 capsid. Figure 6 is a schematic representation of the location of the AAV5 capsid proteins in the AAV genome: 2087~4258bp-VP1 2495~4258bp-VP2 2663~4258bp-VP3.

[0087] From the above, it follows that mutations similar to mutation S2A in VP1 are not present in VP2 and VP3, while mutations similar to mutations S651A and / or T711S in VP1 are present in both VP2 and VP3.

[0088] Considering the overlapping sequences encoding the three capsid proteins VP1, VP2 and VP3, the amino acid substitution S651A in VP1 is: an amino acid substitution at position S515A in VP2; Amino acid substitution at position S459A in VP3 would correspond to

[0089] Considering the overlapping sequences encoding the three capsid proteins VP1, VP2 and VP3, the amino acid substitution T711S is: an amino acid substitution at position T575S in VP2; Amino acid substitution at position T519S in VP3 would correspond to

[0090] Furthermore, the applicants consider it appropriate to identify the circumstances of the mutations found by showing short amino acid sequences containing said mutations in VP1 / VP2 / VP3:

[0091] [ka]

[0092] In some embodiments, the amino acid sequence of the VP2 protein of the wild-type AAV5 capsid is

[0093] [ka]

[0094] The amino acid sequence is represented by the formula: In some embodiments, the isolated modified VP2 protein of the AAV5 capsid includes a T575S substitution.

[0095] In some embodiments, the isolated modified VP2 protein of the AAV5 capsid

[0096] [ka]

[0097] The amino acid sequence is represented by the formula: In some embodiments, the isolated modified VP2 protein of the AAV5 capsid includes a S515A and a T575S substitution.

[0098] In some embodiments, the isolated modified VP2 protein of the AAV5 capsid

[0099] [ka]

[0100] The amino acid sequence is represented by the formula: In some embodiments, the amino acid sequence of the VP3 protein of the wild-type AAV5 capsid is

[0101] [ka]

[0102] The amino acid sequence is represented by the formula: In some embodiments, the isolated modified VP3 protein of the AAV5 capsid includes a T519S substitution.

[0103] In some embodiments, the isolated modified VP3 protein of the AAV5 capsid

[0104] [ka]

[0105] The amino acid sequence is represented by the formula: In some embodiments, the isolated modified VP3 protein of the AAV5 capsid includes a S459A and a T519S substitution.

[0106] In some embodiments, the isolated modified VP3 protein of the AAV5 capsid

[0107] [ka]

[0108] The amino acid sequence is represented by the formula: Capsid In one aspect, the present invention relates to an isolated capsid for highly efficient transduction of target cells, the isolated capsid comprising the above-described modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid.

[0109] In one embodiment, the isolated capsid includes the above-described modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid, a VP2 protein of an AAV5 capsid or a modified variant thereof, and a VP3 protein of an AAV5 capsid or a modified variant thereof.

[0110] Particularly preferred embodiments include substitutions that are conservative in nature, i.e., substitutions made within a family of amino acids that are related in their side chains. In particular, amino acids are typically divided into four families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, and threonine with serine, or similar conservative substitutions of structurally related amino acids, will not significantly affect biological activity. For example, a 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 value between 5 and 50, as long as the desired function of the molecule remains intact.

[0111] In one embodiment, the isolated capsid includes the VP2 protein of a wild-type AAV5 capsid. In one embodiment, the isolated capsid includes the VP2 protein of a wild-type AAV5 capsid having the amino acid sequence set forth by SEQ ID NO:8.

[0112] In one embodiment, the isolated capsid includes a modified VP2 protein of an adeno-associated virus serotype 5 (AAV5) capsid. In one embodiment, the isolated capsid comprises a T575S substitution in the AAV5 capsid. A modified VP2 protein is included.

[0113] In one embodiment, the isolated capsid includes a modified VP2 protein of an AAV5 capsid that includes a T575S substitution and has the amino acid sequence set forth by SEQ ID NO:9.

[0114] In one embodiment, the isolated capsid includes a modified VP2 protein of an AAV5 capsid that includes S515A and T575S substitutions. In one embodiment, the isolated capsid includes a modified VP2 protein of an AAV5 capsid that includes S515A and T575S substitutions and has the amino acid sequence set forth in SEQ ID NO:10.

[0115] In one embodiment, the isolated capsid includes the VP3 protein of a wild-type AAV5 capsid. In one embodiment, the isolated capsid includes the VP3 protein of a wild-type AAV5 capsid having the amino acid sequence set forth by SEQ ID NO:11.

[0116] In one embodiment, the isolated capsid includes a modified VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid. In one embodiment, the isolated capsid includes a modified VP3 protein of an AAV5 capsid that includes a T519S substitution.

[0117] In one embodiment, the isolated capsid includes a modified VP3 protein of an AAV5 capsid that includes a T519S substitution and has the amino acid sequence set forth in SEQ ID NO:12.

[0118] In one embodiment, the isolated capsid includes a modified VP3 protein of an AAV5 capsid that includes S459A and T519S substitutions. In one embodiment, the isolated capsid includes a modified VP3 protein of an AAV5 capsid that includes S459A and T519S substitutions and has the amino acid sequence set forth in SEQ ID NO:13.

[0119] Isolated nucleic acids In one aspect, the present invention relates to an isolated nucleic acid encoding the above-described modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid for use in highly efficient transduction of target cells.

[0120] In some embodiments, the isolated nucleic acid encoding the modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S651A substitution is

[0121] [ka]

[0122] [ka]

[0123] or by any other sequence encoding the corresponding amino acid sequence of a modified protein of adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid S651A substitution.

[0124] "Another sequence encoding the corresponding amino acid sequence of the modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid S651A substitution" refers to an alternative nucleic acid sequence to the nucleic acid sequence having SEQ ID NO:5, since 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. A person trained in the art is fully capable of generating these alternative DNA sequences that encode the same amino acid sequence. Such variant DNA sequences are within the scope of the present invention.

[0125] In some embodiments, the isolated nucleic acid encoding the modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S2A and T711S substitutions is

[0126] [ka]

[0127] [ka]

[0128] or by any other sequence encoding the corresponding amino acid sequence of the modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid S2A and T711S substitutions.

[0129] "Other sequences encoding the corresponding amino acid sequence of the modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid S2A and T711S substitutions" refers to alternative nucleic acid sequences to the nucleic acid sequence having SEQ ID NO:6, since 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:3. One trained in the art is fully capable of generating these alternative DNA sequences that encode the same amino acid sequence. Such variant DNA sequences are within the scope of the present invention.

[0130] In some embodiments, the isolated nucleic acid encoding the modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid S2A, S651A, and T711S substitutions is

[0131] [ka]

[0132] [ka]

[0133] or by any other sequence encoding the corresponding amino acid sequence of the modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid substitutions S2A, S651A and T711S.

[0134] "Other sequences encoding the corresponding amino acid sequence of the modified VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid S2A, S651A, and T711S substitutions" refers to alternative nucleic acid sequences to the nucleic acid sequence having SEQ ID NO:7, since 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. One trained in the art is fully capable of generating these alternative DNA sequences that encode the same amino acid sequence. Such variant DNA sequences are within the scope of the present invention.

[0135] The isolated nucleic acid encoding the VP1 of the wild-type adeno-associated virus serotype 5 (AAV5) capsid is represented by the nucleic acid sequence

[0136] [ka]

[0137] [ka]

[0138] or by any other sequence encoding the corresponding amino acid sequence of the VP1 protein of a wild-type adeno-associated virus serotype 5 (AAV5) capsid. "Another sequence encoding the corresponding amino acid sequence of the VP1 protein of wild-type adeno-associated virus serotype 5 (AAV5) capsid" refers to a nucleic acid sequence that is an alternative to the nucleic acid sequence having SEQ ID NO: 14, since 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. A person skilled in the art is fully capable of generating these alternative DNA sequences which encode the same amino acid sequence, and such variant DNA sequences are within the scope of the present invention.

[0139] In one aspect, the invention relates to an isolated nucleic acid encoding the above-described modified VP2 protein of the adeno-associated virus serotype 5 (AAV5) capsid. In some embodiments, the isolated nucleic acid encoding the modified VP2 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid T575S substitution is

[0140] [ka]

[0141] or by any other sequence encoding the corresponding amino acid sequence of the modified VP2 protein of an adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid T575S substitution.

[0142] "Other sequences encoding the corresponding amino acid sequence of the modified VP2 protein of an adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid T575S substitution" refers to alternative nucleic acid sequences to the nucleic acid sequence having SEQ ID NO: 15, since 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. One trained in the art is fully capable of generating these alternative DNA sequences that encode the same amino acid sequence. Such variant DNA sequences are within the scope of the present invention.

[0143] In some embodiments, the isolated nucleic acid encoding the modified VP2 protein of an adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid S515A and T575S substitutions has any nucleic acid sequence that encodes the amino acid sequence disclosed herein as SEQ ID NO: 10. A person skilled in the art is fully capable of generating these alternative DNA sequences that encode the same amino acid sequence. Such mutant DNA sequences are within the scope of the present invention.

[0144] The isolated nucleic acid encoding the VP2 of the wild-type adeno-associated virus serotype 5 (AAV5) capsid is represented by the nucleic acid sequence

[0145] [ka]

[0146] or by any other sequence encoding the corresponding amino acid sequence of the VP2 protein of a wild-type adeno-associated virus serotype 5 (AAV5) capsid. "Other sequences encoding the corresponding amino acid sequence of the VP2 protein of wild-type adeno-associated virus serotype 5 (AAV5) capsid" refers to nucleic acid sequences that are alternatives to the nucleic acid sequence having SEQ ID NO: 16, since 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. Those trained in the art will be able to generate these alternative DNA sequences that encode the same amino acid sequence. Such mutant DNA sequences are within the scope of the present invention.

[0147] In one aspect, the invention relates to an isolated nucleic acid encoding the above-described modified VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid. In some embodiments, the isolated nucleic acid encoding the modified VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising the amino acid T519S substitution is

[0148] [ka]

[0149] or by any other sequence encoding the corresponding amino acid sequence of the modified VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid T519S substitution.

[0150] "Other sequences encoding the corresponding amino acid sequence of the modified VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid T519S substitution" refers to alternative nucleic acid sequences to the nucleic acid sequence having SEQ ID NO: 17, since 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: 12. One trained in the art is fully capable of generating these alternative DNA sequences that encode the same amino acid sequence. Such variant DNA sequences are within the scope of the present invention.

[0151] In some embodiments, the isolated nucleic acid encoding the modified VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid containing the amino acid S459A and T519S substitutions has any nucleic acid sequence encoding the amino acid sequence disclosed herein as SEQ ID NO: 13. A person skilled in the art would be fully capable of generating these alternative DNA sequences that encode the same amino acid sequence. Such mutant DNA sequences are within the scope of the present invention.

[0152] The isolated nucleic acid encoding the VP3 of the wild-type adeno-associated virus serotype 5 (AAV5) capsid is represented by the nucleic acid sequence

[0153] [ka]

[0154] or by any other sequence encoding the corresponding amino acid sequence of the VP3 protein of a wild-type adeno-associated virus serotype 5 (AAV5) capsid. "Other sequences encoding the corresponding amino acid sequence of the VP3 protein of a wild-type adeno-associated virus serotype 5 (AAV5) capsid" refers to nucleic acid sequences that are alternatives to the nucleic acid sequence having SEQ ID NO: 18, since 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. A person trained in the art is fully capable of generating these alternative DNA sequences that encode the same amino acid sequence. Such variant DNA sequences are within the scope of the present invention.

[0155] In one aspect, the invention relates to isolated nucleic acids encoding the capsids described above, for use in highly efficient transduction of target cells. In some embodiments, the isolated nucleic acid encoding the capsid includes any of the above nucleic acid sequences.

[0156] Recombinant adeno-associated virus serotype 5 (rAAV5)-based vectors In one aspect, the invention provides a recombinant adeno-associated virus serotype 5 (rAAV5)-based vector for delivery of a heterologous nucleic acid sequence to a subject, comprising: 1) the capsid described above, and 2) a heterologous nucleic acid sequence comprising a control sequence that promotes expression of a target product encoded by the heterologous nucleic acid sequence in a target cell; The present invention relates to the vector comprising:

[0157] The rAAV vectors of the present invention do not contain the nucleotide sequences of the genes encoding the nonstructural proteins (Rep) and the structural proteins (Cap). The capsid is characterized in detail in the above section of this specification.

[0158] In some embodiments, the rAAV5-based vector has an expression product of a heterologous nucleic acid sequence that is a therapeutic polypeptide or a reporter polypeptide. In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding a product that is a therapeutic polypeptide, wherein the therapeutic polypeptide is a clotting factor selected from the group consisting of factor VIII, factor IX, or a functional variant thereof.

[0159] In some embodiments, the rAAV5-based vector comprises a heterologous nucleic acid sequence encoding a product that is Factor VIII or a functional variant thereof. 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.

[0160] Pharmaceutical Compositions In one aspect, the present invention provides a pharmaceutical composition for delivering a gene product to a subject in need thereof, comprising: a) the above vector based on rAAV5; and b) Pharmaceutically acceptable excipients The present invention relates to the pharmaceutical composition comprising:

[0161] In some embodiments, the pharmaceutical composition is used to deliver the gene product to a human in need thereof. In certain embodiments, the present invention relates to pharmaceutical compositions comprising the rAAV5 viral particles of the present invention in a pharmaceutically acceptable carrier or other medicinal agent, pharmaceutical agent, carrier, adjuvant, diluent, etc. For injection, the carrier will typically be a liquid carrier. For other modes of administration, the carrier may be solid or liquid, such as sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline solution. For inhalation administration, the carrier is inhalable and is preferably in solid or liquid particulate form. As the injection medium, it is preferred to use water containing additives common to injection solutions, such as stabilizers, salts or saline, and / or buffers.

[0162] In other aspects, the invention relates to pharmaceutical compositions comprising cells having an rAAV5-based vector integrated into their genome in a pharmaceutically acceptable carrier or other medicinal agent, pharmaceutical agent, carrier, adjuvant, diluent, etc.

[0163] A "pharmaceutical composition" refers to a pharmaceutical composition containing the rAAV5-based vector of the present invention and pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, etc. The term "pharmaceutical composition" refers to a composition comprising at least one component selected from the group consisting of an adjuvant, a dispersing agent, a delivery agent, a preservative, a stabilizer, an emulsifier, a suspending agent, a thickener, and a sustained-delivery modulating agent, the selection and proportion of which depend on the type and route of administration and the dosage. The pharmaceutical composition of the present invention and its preparation method will be clearly apparent to those skilled in the art. The pharmaceutical composition should preferably be manufactured in accordance with Good Manufacturing Practice (GMP) requirements. The composition may also include a buffer composition, an isotonicity agent, a stabilizer, and a solubilizer.

[0164] "Pharmaceutically acceptable" means a substance that has no biological or other negative side effects, e.g., the substance can be administered to a subject without causing any undesirable biological effects. Thus, such pharmaceutical compositions may be used, for example, in transfection of cells ex vivo or in administration of viral particles or cells directly to a subject in vivo.

[0165] The term "excipient" is used herein to describe any ingredient other than those mentioned above of the present invention. These are inorganic or organic substances used in pharmaceutical manufacturing to impart necessary physicochemical properties to the drug product.

[0166] "Stabilizer" refers to an excipient, or a mixture of two or more excipients, that provides physical and / or chemical stability to an active agent. The terms "buffer," "buffering composition," and "buffering agent" refer to a solution that can resist changes in pH due to the action of its acid-base conjugate components, allowing vectors based on rAAV5 products to resist pH changes. Generally, pharmaceutical compositions preferably have a pH in the range of 4.0 to 8.0. Examples of buffering agents that may be used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, and the like buffer solutions.

[0167] A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical stability and / or biological activity during a specified shelf life, e.g., at a storage temperature of 2-8°C. Preferably, the active agent retains both physical and chemical stability and biological activity. The shelf life is adjusted based on the results of stability testing under accelerated or natural aging conditions.

[0168] The pharmaceutical compositions of the present invention may be manufactured, packaged, or widely sold in the form of a ready-to-use formulation, a single unit dose, or multiple single unit doses. The term "single unit dose," as used herein, refers to a discrete amount of pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is typically equal to the dose of the active ingredient to be administered to a subject, or a suitable fraction of such a dose, for example, one-half or one-third of such a dose.

[0169] Methods for delivery of gene products In one aspect, the present invention relates to a method for delivering a gene product to a subject in need thereof, the method comprising administering to the subject the above-described rAAV5-based vector or the above-described pharmaceutical composition.

[0170] In some embodiments, the method for delivering a gene product is used to deliver the gene product to a human in need thereof. Any art-recognized method for administering a rAAV5-based vector may be suitably used for the rAAV5-based vectors of the present invention.

[0171] The rAAV5-based recombinant viral vector is preferably administered to cells in a biologically effective amount. A "biologically effective" amount of a viral vector is one that is sufficient to induce infection (or When a virus is administered to a cell in vivo (e.g., when the virus is administered to a subject, as described below), a "biologically effective" amount of a viral vector is an amount sufficient to cause transduction and expression of a heterologous nucleic acid sequence in the target cell.

[0172] Cells for administering the rAAV5 viral vectors of the present invention may be any type of cell, including, but not limited to, neural 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), hepatocytes, cardiac myocytes, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. Alternatively, cells for administering the rAAV5 viral vectors may be any progenitor cells. As a further alternative, cells may be stem cells (e.g., neural stem cells, hepatic stem cells). Furthermore, cells may be derived from any species of origin, as specified above.

[0173] use In one aspect, the present invention relates to the use of the rAAV5-based vector or the pharmaceutical composition for treating a disease in a subject in need thereof.

[0174] In some embodiments, the use is for treating a disease in a human in need thereof. Administration of the rAAV5-based vectors of the invention to a human subject or animal in need thereof may be by any means known in the art for administering viral vectors.

[0175] Exemplary modes of administration include topical, oral, rectal, transmucosal, transdermal, inhalation, parenteral administration (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular administration), and direct tissue or organ injection, and alternatively, intrathecal, direct intramuscular, intracerebroventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. Injections may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for preparing solutions or suspensions in liquids prior to injection, or as emulsions. Alternatively, rAAV5-based vectors may be administered in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0176] In some embodiments of the use, the disease is selected from the group comprising: a blood disease; a central nervous system disease; a metabolic disease; a muscular disease; a genetic disease. In some embodiments of the use, the disease is a blood disease.

[0177] In some embodiments of the use, the disease is a muscle disease. In some embodiments of the use, the disease is a genetic disease. In a specific embodiment of the present invention, the nucleotide sequence of interest is delivered to the liver of subject by the vector based on rAAV5.Administered to the liver can be carried out by any method known in the art, including but not limited to intravenous administration, intraportal administration, intrabiliary administration, intraarterial administration and direct injection into the liver parenchyma.

[0178] Preferably, cells (e.g., liver cells) are infected with an rAAV5-based vector encoding a peptide or protein, and the cells express and secrete the encoded peptide or protein into the blood circulation in therapeutically effective amounts (as described below). Alternatively, the vector is delivered to and expressed by another cell or tissue, including, but not limited to, the brain, pancreas, spleen, or muscle.

[0179] A "therapeutically effective amount" refers to an amount that relieves (e.g., alleviates, reduces, or diminishes) at least one symptom associated with a disease state. In other words, a "therapeutically effective" amount is an amount sufficient to provide some improvement in the subject's condition.

[0180] In some embodiments of the use, the expression product of the heterologous nucleic acid sequence is Factor IX or a functional variant thereof. In some embodiments of the use, the expression product of the heterologous nucleic acid sequence is Factor VIII or a functional variant thereof.

[0181] In other preferred embodiments, the rAAV5-based vectors of the invention are administered intramuscularly, more preferably by intramuscular injection, or by local administration (as described above). In other preferred embodiments, the parvovirus particles of the invention are administered to the lungs.

[0182] The rAAV5-based vectors disclosed herein may be administered to a subject's lungs by any suitable means, preferably in the form of an aerosol suspension of inhalable particles comprised of the rAAV5-based vectors of the invention, which are inhaled by the subject. The inhalable particles may be liquid or solid. Aerosols of liquid particles comprising the parvoviral rAAV5 vectors of the invention may be produced by any suitable means, for example, with a pressure-driven aerosol nebulizer or ultrasonic nebulizer, as known to those skilled in the art. Aerosols of solid particles comprising the viral rAAV5 vectors of the invention may be produced with any solid particulate drug aerosol generator, using techniques known in the pharmaceutical industry.

[0183] The dosage of the parvovirus rAAV5 particles of the present invention depends on the mode of administration, the disease or condition to be treated, the condition of the subject, the particular viral vector, and the gene to be delivered, and can be determined routinely. Exemplary doses to achieve 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 transducing units or more, preferably about 10 8 ~10 13 transducing units, even more preferably 10 12 Viral titer in transducing units.

[0184] Therefore, the parvovirus vectors, reagents, and methods based on the rAAV5 of the present invention can be used to deliver nucleic acids to either dividing or non-dividing cells and stably express heterologous nucleic acids therein. Using this vector system, it is now possible to introduce genes encoding proteins that affect cell physiology into cells under in vivo conditions. Therefore, the vectors of the present invention can be useful in gene therapy for disease conditions.

[0185] In general, the present invention may be used to deliver any exogenous nucleic acid gene having a biological effect of treating or alleviating symptoms associated with any disorder associated with 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 clotting disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, diabetes, muscular dystrophies (e.g., Duchenne, Becker), Gaucher disease, Hurler disease, adenosine deaminase deficiency, glycogen storage diseases, and other metabolic defects, diseases of solid organs (e.g., brain, liver, kidney, heart), and the like.

[0186] Gene transfer has substantial potential use in understanding disease states and providing therapies therefor. There are many inherited diseases for which the defective genes are known and have been cloned. In some cases, the function of these cloned genes is known. Generally, the disease states fall into two classes: deficiency conditions, which are generally inherited in a recessive manner. , a dominantly inherited imbalance condition, typically involving an enzyme deficiency, and sometimes at least a regulatory or structural protein. For deficiency state diseases, gene transfer may be used to deliver a normal gene into affected tissue for replacement therapy. For imbalance disease states, gene transfer may be used to generate a disease state in a model system, which may then be used to attempt to combat the disease state. Thus, the method of the present invention makes it possible to treat genetic diseases. In accordance with the present invention, a disease state is treated, partially or completely, by remedying the deficiency or imbalance that causes or makes the disease more severe. Site-specific integration of nucleic acid sequences to induce mutations or correct the deficiency is also possible.

[0187] Methods for producing rAAV5-based vectors In one aspect, the invention relates to a method for producing an rAAV5-based vector, comprising transfecting a producer cell with a nucleic acid comprising a sequence encoding a capsid comprising a modified VP1 capsid protein of adeno-associated virus serotype 5 (AAV5).

[0188] In some embodiments of the methods for producing rAAV5-based vectors, the above-described nucleic acids are used, which include sequences encoding the above-described modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid, the VP2 protein of an AAV5 capsid or a modified variant thereof, and the VP3 protein of an AAV5 capsid or a modified variant thereof.

[0189] Modified 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 that contain one or more amino acid substitutions.

[0190] Particularly preferred embodiments include substitutions that are conservative in nature, i.e., substitutions made within a family of amino acids that are related in their side chains. In particular, amino acids are typically divided into four families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, and threonine with serine, or similar conservative substitutions of structurally related amino acids, will not significantly affect biological activity. For example, a 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 value between 5 and 50, as long as the desired function of the molecule remains intact.

[0191] In some embodiments of the methods for producing rAAV5-based vectors, the above-described nucleic acid is used, which includes a sequence encoding the above-described modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid, the VP2 protein of a wild-type AAV5 capsid, and the VP3 protein of a wild-type AAV5 capsid.

[0192] Engineered variants of the VP2 and VP3 proteins of the AAV5 capsid, as well as the nucleic acids encoding them, are disclosed in detail in the corresponding sections of this specification. [Example]

[0193] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

[0194] All publications, patents, and patent applications cited herein are hereby incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art, upon consideration of the description of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the accompanying embodiments.

[0195] Materials and general methods recombinant DNA technology DNA manipulations were 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 manufacturers' instructions.

[0196] Gene synthesis The desired gene segments were prepared from chemically synthesized oligonucleotides. Gene segments of 300-4000 bp in length, flanked by single restriction sites, were assembled by annealing and ligation of PCR-amplified oligonucleotides and subsequently cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0197] DNA sequencing The DNA sequence was determined by Sanger sequencing. DNA and protein sequence analysis and sequence data management Infomax's Vector NTI Advance suite of software, version 8.0, and SnapGene Viewer were used for sequence generation, mapping, analysis, annotation, and illustration. The present invention includes, but is not limited to, the following aspects. [Aspect 1] 1. An isolated modified VP1 protein of adeno-associated virus serotype 5 (AAV5) capsid for highly efficient transduction of target cells, comprising: S651A, S2A and T711S, S2A, S651A and T711S The isolated modified VP1 protein comprises the amino acid sequence of the VP1 protein of a wild-type AAV5 capsid encoded by the Cap gene, wherein the VP1 protein comprises one or more substitutions selected from the group comprising: [Aspect 2] 2. The isolated modified VP1 protein of the AAV5 capsid of embodiment 1, wherein the amino acid sequence of the VP1 protein of the wild-type AAV5 capsid has the amino acid sequence set forth in SEQ ID NO:1. [Aspect 3] 2. An isolated modified VP1 protein of the AAV5 capsid of embodiment 1, comprising a single substitution at position S651A. [Aspect 4] 4. The isolated modified VP1 protein of the AAV5 capsid of embodiment 3, having the amino acid sequence set forth in SEQ ID NO:2. [Aspect 5] 2. An isolated modified VP1 protein of the AAV5 capsid of embodiment 1, comprising S2A and T711S substitutions. [Aspect 6] 6. The isolated modified VP1 protein of the AAV5 capsid of embodiment 5, having the amino acid sequence set forth in SEQ ID NO:3. [Aspect 7] 2. An isolated modified VP1 protein of the AAV5 capsid of embodiment 1, comprising the substitutions S2A, S651A and T711S. [Aspect 8] 8. The isolated modified AAV5 VP1 capsid protein of embodiment 7, having the amino acid sequence set forth in SEQ ID NO:4. [Aspect 9] 9. An isolated nucleic acid encoding the modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid of any one of embodiments 1 to 8, for use in highly efficient transduction of target cells. [Aspect 10] 10. The isolated nucleic acid of embodiment 9, encoding a modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising an amino acid S651A substitution, wherein the isolated 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. [Aspect 11] 10. The isolated nucleic acid of embodiment 9, encoding a modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising amino acid S2A and T711S substitutions, wherein the isolated 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. [Aspect 12] 10. The isolated nucleic acid of embodiment 9, encoding a modified VP1 protein of an adeno-associated virus serotype 5 (AAV5) capsid comprising amino acid S2A, S651A, and T711S substitutions, wherein the isolated 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. [Aspect 13] 9. An isolated capsid for highly efficient transduction of target cells, comprising a modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid of any one of embodiments 1 to 8. [Aspect 14] 14. The isolated capsid of embodiment 13, comprising a modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid of any one of embodiments 1 to 8, a VP2 protein of the AAV5 capsid or a modified variant thereof, and a VP3 protein of the AAV5 capsid or a modified variant thereof. [Aspect 15] 15. The isolated capsid of embodiment 14, comprising the VP2 protein of a wild-type AAV5 capsid. [Aspect 16] 16. The isolated capsid of embodiment 15, comprising the VP2 protein of the wild-type AAV5 capsid protein, having the amino acid sequence set forth in SEQ ID NO:8. [Aspect 17] 15. The isolated capsid of embodiment 14, comprising a modified VP2 protein of an adeno-associated virus serotype 5 (AAV5) capsid. [Aspect 18] 20. The isolated capsid of embodiment 17, comprising a modified VP2 protein of an AAV5 capsid comprising a T575S substitution. [Aspect 19] 20. The isolated capsid of embodiment 18, comprising a modified VP2 protein of an AAV5 capsid comprising a T575S substitution and having the amino acid sequence set forth in SEQ ID NO:9. [Aspect 20] 20. The isolated capsid of embodiment 18, comprising a modified VP2 protein of an AAV5 capsid comprising a S515A and a T575S substitution. [Aspect 21] 21. The isolated capsid of embodiment 20, comprising a modified VP2 protein of an AAV5 capsid comprising S515A and T575S substitutions and having the amino acid sequence set forth in SEQ ID NO:10. [Aspect 22] 15. The isolated capsid of embodiment 14, comprising the VP3 protein of a wild-type AAV5 capsid. [Aspect 23] 23. The isolated capsid of embodiment 22, comprising a VP3 protein of a wild-type AAV5 capsid having the amino acid sequence set forth in SEQ ID NO:11. [Aspect 24] 15. The isolated capsid of embodiment 14, comprising a modified VP3 protein of an adeno-associated virus serotype 5 (AAV5) capsid. [Aspect 25] 25. The isolated capsid of embodiment 24, comprising a modified VP3 protein of an AAV5 capsid comprising a T519S substitution. [Aspect 26] 26. The isolated capsid of embodiment 25, comprising a modified VP3 protein of an AAV5 capsid comprising a T519S substitution and having the amino acid sequence set forth in SEQ ID NO: 12. [Aspect 27] 25. The isolated capsid of embodiment 24, comprising a modified VP3 protein of an AAV5 capsid comprising S459A and T519S substitutions. [Aspect 28] 28. The isolated capsid of embodiment 27, comprising a modified VP3 protein of an AAV5 capsid comprising S459A and T519S substitutions and having the amino acid sequence set forth in SEQ ID NO: 13. [Aspect 29] 29. An isolated nucleic acid encoding the capsid of any one of embodiments 13 to 28 for use in highly efficient transduction of target cells. [Aspect 30] 1. A recombinant adeno-associated virus serotype 5 (rAAV5)-based vector for delivery of a heterologous nucleic acid sequence to a subject, comprising: 1) a capsid according to any one of embodiments 13 to 28, and 2) a heterologous nucleic acid sequence comprising a control sequence that promotes expression of the product encoded by the heterologous nucleic acid sequence in the target cell; The vector comprising: [Aspect 31] The rAAV5-based vector of embodiment 30, wherein the expression product of the heterologous nucleic acid sequence is a therapeutic polypeptide or a reporter polypeptide. [Aspect 32] 32. The rAAV5-based vector of embodiment 31, wherein the therapeutic polypeptide is a clotting factor selected from the group consisting of factor VIII, factor IX, or a functional variant thereof. [Aspect 33] 33. The rAAV5-based vector of embodiment 32, wherein the therapeutic peptide is Factor VIII or a functional variant thereof. [Aspect 34] 33. The rAAV5-based vector of embodiment 32, wherein the therapeutic peptide is Factor IX or a functional variant thereof. [Aspect 35] 1. A pharmaceutical composition for delivering a gene product to a subject in need thereof, comprising: a) the rAAV5-based vector of any one of embodiments 30 to 34; and b) Pharmaceutically acceptable excipients The pharmaceutical composition comprising: [Aspect 36] The pharmaceutical composition of embodiment 35, wherein the subject is a human subject. [Aspect 37] 36. A method for delivering a gene product to a subject in need thereof, the method comprising administering to the subject an AAV5-based vector of any one of aspects 30 to 34 or a pharmaceutical composition of aspect 35. [Aspect 38] The method for delivery of a gene product of embodiment 37, wherein the subject is a human subject. [Aspect 39] Use of the rAAV5-based vector of any one of embodiments 30 to 34 or the pharmaceutical composition of embodiment 35 for the treatment of a disease in a subject in need thereof. [Aspect 40] The use of embodiment 39, wherein the subject is a human subject. [Aspect 41] The use of embodiment 39, wherein the disease is selected from the group comprising: a blood disease; a central nervous system disease; a metabolic disease; a muscular disease; a genetic disease. [Aspect 42] The use of embodiment 41, wherein the disease is a blood disease. [Aspect 43] 43. The use of embodiment 42, wherein the expression product of the heterologous nucleic acid sequence is Factor IX or a functional variant thereof. [Aspect 44] 43. The use of embodiment 42, wherein the expression product of the heterologous nucleic acid sequence is Factor VIII or a functional variant thereof. [Aspect 45] The use of embodiment 41, wherein the disease is a muscle disease. [Aspect 46] The use of embodiment 41, wherein the disease is a genetic disease. [Aspect 47] 35. A method for obtaining the rAAV5-based vector of any one of aspects 30 to 34, comprising transfecting a producer cell with a nucleic acid of aspect 29.

[0198] Example 1. Generation of an AAV5 capsid mutant library A library of AAV5 capsid mutants 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. The synthesized wild-type AAV5 capsid gene was then fragmented using uracil-DNA glycosylase. The resulting fragments were then assembled into a full-length Cap gene using a DNA polymerase lacking proofreading activity (resulting in random mutations in the sequence). The full-length mutants were cloned into the carrier plasmid pAAV-linker (Figure 1) at the AscI / EcoRI restriction sites in a common reading frame containing green fluorescent protein (GFP), thereby generating a diverse random library of AAV5 capsids. This library was then used to select capsid mutants with increased transduction activity.

[0199] Positive selection of viral particles with increased transduction activity was performed in vitro on CHO-K1-S cells. For transduction, we used particles purified by ultracentrifugation in an iodixanol gradient. After 48 hours, cells were harvested and genomic DNA was isolated for subsequent amplification of viral genome sequences capable of efficient transduction. Subsequently, to enrich the library for mutants with the highest transduction efficiency, The resulting sequences were recloned and reproduced for subsequent rounds of selection. After five rounds of selection, the capsid genes of 30 clones were sequenced to determine the most successful mutations and their combinations. Based on the sequencing results, the predominant mutation combinations were S2A, T711S in AAV5 VP1, and a capsid mutant containing S2A, T711S, and S651A in AAV5 VP1, accounting for approximately 20% of the clones. A capsid mutant containing the S651A mutation in AAV5 VP1 was also selected. These capsid mutants were cloned into vectors to produce viral particles and further used to visualize and compare their transduction profiles compared to wild-type AAV5.

[0200] Example 2. Production and subsequent selection of recombinant viral particles from the resulting sequence library To produce 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 the adenoviral genes necessary for viral particle replication.

[0201] The carrier plasmid pAAV-linker (Fig. 1), intended for cloning a random mutant library of the AAV serotype 5 capsid gene in one reading frame with the reporter protein, was generated by substituting the sequence of a modified green fluorescent protein in the original construct pAAV-GFP control plasmid (VPK-402) from CellBiolab (USA) using the restrictase-ligase method to clone the de novo synthesized sequence T2A-GFP, which added an EcoRI restriction site at the 5' end and a HindIII restriction site at the 3' end, at the HindIII / EcoRI site.

[0202] The plasmid pAAV-Rep (Figure 2) containing the Rep gene sequence was generated by de novo cloning a synthetic sequence of the AAV serotype 2 Rep gene (GenBank ID AF043303.1) at PciI / PsiI restriction sites (New England Biolabs, USA) followed by treatment with T4 DNA polymerase (New England Biolabs, USA) to generate blunt ends into the plasmid pGem-T Easy (Promega, USA) also treated with PciI / PsiI restriction enzymes (New England Biolabs, USA).

[0203] The adenovirus genes for producing recombinant viral particles were sourced from the construct pHelper (Figure 3) derived from the commercial kit AAV-2 Packaging System (VPK-402) from CellBiolab (USA), including AmpR, a beta-lactamase gene that provides resistance to ampicillin; Ori, an origin of replication in bacteria; adenoE2A, a helper adenovirus gene sequence involved in viral DNA replication; adenoE4, a helper adenovirus gene sequence involved in viral DNA replication; and adenoVARNA, a helper adenovirus gene sequence involved in the translation of both early and late viral genes.

[0204] Example 3. Methods for producing modified adeno-associated virus serotype 5 (rAAV)-based vectors To produce rAAV particles containing modified serotype 5 capsids, producer cells were co-transfected with three plasmids as follows: 1) a plasmid containing adenoviral nucleotide sequences encoding proteins and RNAs required for rAAV particle assembly (helper plasmid); 2) A plasmid containing the native nucleotide sequence of the Rep gene of adeno-associated virus serotype 2 together with the sequence of a modified Cap gene, wherein the modified Cap gene is: VP1 comprising the nucleotide sequence of SEQ ID NO: 5, 6 or 7 or the amino acid sequence of SEQ ID NO: 2, 3 or 4. any other nucleotide sequence encoding a protein, as well as VP2 and VP3 proteins derived from alternative reading frames of the nucleotide sequence used. VP2 may have the amino acid sequence of any of SEQ ID NOs: 8, 9, or 10; and VP3 may have any of the amino acid sequences of SEQ ID NOs: 11, 12, or 13. the plasmid; 3) A plasmid containing the heterologous genome of the rAAV particle, encoding the target gene intended for delivery into the patient's cells.

[0205] This set of genes provides for the assembly of rAAV viral particles and the encapsidation of the target genome within the particles within 72 hours. 72 hours after transfection, the producer cells are lysed to release the rAAV particles for subsequent purification by filtration and chromatography steps. The titer of the purified rAAV particles is verified by enzyme-linked immunosorbent assay and quantitative PCR.

[0206] Example 4. Increased efficiency of cell transduction with rAAV5-based products in the presence of mutations S2A, S651A, T711S in the VP1 protein of wild-type AAV5 capsid Experimental design: CHO-K1-S cells were plated in wells of a 12-well plate. They were seeded in the following growth medium: DMEM / F12 supplemented with glutamine, glucose content 4.5 g / L, and 5% bovine serum. The cell seeding density was 10,000 cells / cm2. During the transduction run, previously prepared cells were transduced at an MOI of 100,000 vg / cell. Three independent experiments were performed for all samples. Intact cells were used as a negative control.

[0207] Analysis of transduction efficiency was performed using a Guava EasyCyteflow cytometer and GuavaSoft software. The present inventors surprisingly found that the presence of one or more mutations selected from the group consisting of S2A, S651A, or T711S in the VP1 protein of wild-type AAV5 capsid significantly increased the efficiency of transgene delivery by rAAV vectors containing the above mutations. For example, flow cytometry showed changes in the amount of GFP-positive cells 48 hours after transduction of the CHO-K1-S strain with rAAV-based products containing the VP1 protein of wild-type AAV5 capsid or the VP1 protein of wild-type AAV5 capsid carrying one or more mutations selected from the group consisting of S2A, S651A, and T711S (Figure 4).

[0208] In the presence of the mutation S651A (AAV5-01Mut-GFP), the amount of GFP-expressing cells increased 2.2-fold, from 22.54% to 49.45%, when compared with control AAV5 containing the wild-type VP1 capsid protein (AAV5-NullMut-GFP).

[0209] When both mutations S2A and T711S were present simultaneously (AAV5-02Mut-GFP), the amount of GFP-expressing cells increased 2.6-fold, from 22.54% to 58.51%, when compared with control AAV5 containing the wild-type VP1 capsid protein (AAV5-NullMut-GFP).

[0210] When mutations S2A, S651A, and T711S were simultaneously present (AAV5-03Mut-GFP), the amount of GFP-expressing cells increased 1.7-fold, from 22.54% to 38.27%, when compared with control AAV5 containing the wild-type VP1 capsid protein (AAV5-NullMut-GFP).

[0211] Example 5. Increased production of transgene-encoded target protein after cell transduction with rAAV5-based products in the presence of mutations S2A, S651A, T711S in the VP1 protein of wild-type AAV5 capsid Experimental design: CHO-K1-S cells were plated in wells of a 12-well plate. They were seeded in the following growth medium: DMEM / F12 supplemented with glutamine, with a glucose content of 4.5 g / L and 5% bovine serum. The cell seeding density was 10,000 cells / cm2. During the transduction run, previously prepared cells were transduced at an MOI of 100,000 vg / cell. All experiments were performed three times independently. Intact cells were used as a negative control.

[0212] A human Factor IX ELISA kit was used to assess the amount of FIX protein in the culture medium 7 days after transduction. We used a 1:25 dilution of the sample. The method was performed according to the manufacturer's instructions.

[0213] The present inventors surprisingly found that the presence of one or more mutations selected from the group consisting of S2A, S651A, or T711S in the VP1 protein of wild-type AAV5 capsid resulted in a significant increase in hFIX protein production after transduction of CHO-K1-S cells with rAAV-based vectors containing the above mutations. For example, enzyme-linked immunosorbent assay (ELISA) showed an increase in the amount of hFIX protein in the culture medium 7 days after transduction of CHO-K1-S cells with rAAV products containing the wild-type AAV5 VP1 capsid protein or the VP1 protein of wild-type AAV5 capsid carrying one or more mutations selected from the group consisting of S2A, S651A, or T711S (Figure 5).

[0214] When the mutation S651A was present (AAV5-01Mut-FIX), the amount of protein produced increased 4.6-fold, from 0.17 ng / ml to 0.74 ng / ml, when compared with control AAV5 containing the wild-type VP1 capsid protein (AAV5-NullMut-GFP).

[0215] When both mutations S2A and T711S were present simultaneously (AAV5-02Mut-GFP), the amount of protein produced increased 7.1-fold, from 0.17 ng / ml to 1.24 ng / ml, when compared to control AAV5 containing the wild-type VP1 capsid protein (AAV5-NullMut-GFP).

[0216] When the mutations S2A, S651A, and T711S were present simultaneously (AAV5-03Mut-GFP), the amount of protein produced increased 3.3-fold, from 0.17 ng / ml to 0.57 ng / ml, when compared with control AAV5 containing the wild-type VP1 capsid protein (AAV5-NullMut-GFP).

Claims

1. The isolated modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid was hand: The amino acid sequence of SEQ ID NO:3, which contains the substitutions S2A and T711S. have columns, or S2A, S651A and T711S substitutions and represented by SEQ ID NO:

4. having an amino acid sequence The isolated modified VP1 protein.

2. The modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid of claim 1. An isolated nucleic acid encoding

3. Adeno-associated virus serotype 5 (AAV5) containing amino acid S2A and T711S substitutions 3. The isolated nucleic acid of claim 2, encoding a modified VP1 protein of the capsid, comprising SEQ ID NO: 6 or by a nucleic acid sequence other than SEQ ID NO: 6 that encodes the amino acid sequence of SEQ ID NO:

3. The isolated nucleic acid is represented by any other nucleic acid sequence of

4. Adeno-associated virus serotype 5 containing amino acid substitutions S2A, S651A and T711S 3. The isolated nucleic acid of claim 2, encoding a modified VP1 protein of the (AAV5) capsid. , by the nucleic acid sequence of SEQ ID NO: 7 or by a sequence encoding the amino acid sequence of SEQ ID NO:

4. The isolated nucleic acid represented by any other nucleic acid sequence other than sequence number 7.

5. The modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid of claim 1. An isolated capsid comprising:

6. The modified VP1 protein of the adeno-associated virus serotype 5 (AAV5) capsid of claim 1. , an AAV5 capsule having the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10 VP2 protein of the present invention, and the amino acid sequence represented by SEQ ID NO: 12 or SEQ ID NO:

13.

6. The isolated capsid of claim 5, comprising the VP3 protein of an AAV5 capsid having the sequence

7. An isolated nucleic acid encoding the capsid of any one of claims 5 to 6.

8. Recombinant adeno-associated virus serotype 5 (rAA) for delivery of heterologous nucleic acid sequences to a subject V5), a vector based on: 1) a capsid according to any one of claims 5 to 6, and 2) Regulation that promotes expression of the product encoded by the heterologous nucleic acid sequence in the target cell. Heterologous nucleic acid sequences, including sequences The vector comprising:

9. the expression product of the heterologous nucleic acid sequence is a therapeutic polypeptide or a reporter polypeptide; The therapeutic polypeptide is a coagulation factor selected from the group consisting of factor VIII and factor IX.

9. The rAAV5-based vector of claim 8, wherein

10. 10. The rAAV5-based vector of claim 9, wherein the therapeutic polypeptide is Factor VIII. -.

11. 10. The rAAV5-based vector of claim 9, wherein the therapeutic polypeptide is Factor IX.

12. Pharmaceutical compositions for delivering gene products to a subject in need thereof Where: a) the rAAV5-based vector of any one of claims 8 to 11; and b) Pharmaceutically acceptable excipients The pharmaceutical composition comprising:

13. 13. The pharmaceutical composition of claim 12, wherein the subject is a human subject.

14. 1. A pharmaceutical composition for use in the treatment of a disease, comprising: The disease is selected from the group consisting of: blood disease; central nervous system disease; metabolic disease; muscular disease; genetic disease The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is selected from the group consisting of:

15. 15. The pharmaceutical composition of claim 14, wherein the disease is a blood disease.

16. 16. The pharmaceutical composition of claim 15, wherein the expression product of the heterologous nucleic acid sequence is Factor IX.

17. 16. The pharmaceutical composition of claim 15, wherein the expression product of the heterologous nucleic acid sequence is Factor VIII.

18. 15. The pharmaceutical composition of claim 14, wherein the disease is a muscular disease or a genetic disease.

19. 12. An in vitro method for obtaining the rAAV5-based vector of any one of claims 8 to 11, comprising transfecting a producer cell with the nucleic acid of claim 7.

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