Vector system for recombinant AAV production, comprising stuffer

Abstract

Provided are a vector system for producing recombinant adeno-associated virus (AAV), the vector system comprising a stuffer, and a method for producing recombinant AAV using same. The vector system for producing recombinant AAV comprises (a) a helper virus gene, (b) an AAV Rep gene, (c) an AAV Cap gene, (d) a transgene arranged between ITRs, and at least one stuffer, and by comprising at least one vector, is able to reduce impurities during recombinant AAV production.

Description

Vector system for recombinant AAV production containing stuffers

[0001] The present invention relates to a vector for producing a recombinant adeno-associated virus (AAV) including a stuffer and a method for producing a recombinant AAV using the same.

[0002] Recombinant adeno-associated virus (rAAV) vectors are safe and effective vectors that are attracting attention as gene therapy. rAAV vectors can be produced using a triple transfection method in HEK293 cells without the aid of a helper virus. To produce normal AAV with transduction function, rAAV produced using this method requires the simultaneous delivery of three plasmids to a single cell. Inevitably, injecting only one or two plasmids into a single cell can result in recombinant AAV with poor quality or no therapeutic effect. Therefore, large quantities of all three plasmids must be simultaneously injected into the production cells. Furthermore, administering high doses of low-quality AAV to achieve therapeutic effects poses the safety risk of inducing an excessive immune response. Currently, the production of AAV viruses for therapeutic purposes has problems such as the use of large quantities of three types of plasmids, high cost of raw materials due to high-purity purification processes, and increased price of therapeutics, which are issues that cannot be overlooked in the production process.

[0003] To develop high-quality, high-productivity recombinant AAV production methods and processes, as well as to improve the current triple transfection process, a single-vector AAV production system that integrates Rep, Cap, Helper, and GOI (gene-of-interest) into a single DNA molecule or vector has been proposed. However, even single-vector systems still require improvement to produce high-quality viruses.

[0004] AAV is considered the vector of choice for in vivo gene transfer, boasting the highest safety and efficacy. Among the developed AAV vector systems, the single-vector system boasts improved AAV production quality and efficiency compared to the traditional triple-transfection system. However, the single-vector system raises safety concerns due to the potential introduction of genes that could potentially produce new AAVs, or the potential introduction of certain Rep, Cap, and Helper genes, during the AAV production process, which involves transfecting HEK293 cells with a single gene.

[0005] The present disclosure relates to a vector system for producing recombinant AAV comprising one or more stuffers, and relates to an AAV vector system that enables recombinant AAV production efficiency and production process robustness at the same or improved levels as compared to existing vector systems, improved %Full / Empty capsid ratio, and reduced production costs, while reducing side effects by reducing impurities due to reverse packaging, etc.

[0006] One aspect provides a vector system for producing recombinant adeno-associated virus (AAV), comprising a stuffer.

[0007] Another aspect provides a method for producing recombinant AAV using the vector system for producing recombinant AAV.

[0008] Another aspect is to provide a recombinant AAV produced by the above method.

[0009] Another aspect provides a composition comprising a recombinant AAV produced by the above method.

[0010] Another aspect provides a gene therapy method using a recombinant AAV produced by the above method.

[0011] Another aspect provides a use of the vector system for producing the recombinant AAV or the recombinant AAV produced by the vector system for the manufacture of a medicament for gene therapy.

[0012] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. Furthermore, while preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. Furthermore, numerical values ​​described herein are considered to include the meaning of "about," even if not explicitly stated. The contents of all publications cited as references herein are incorporated herein by reference in their entirety.

[0013] The terms "about" or "approximately" in this specification can generally be interpreted to mean a value or range that is within 10%, 5%, 4%, 3%, 2%, or 1% above or below a given value or range.

[0014]

[0015] One aspect provides a vector system for producing a recombinant adeno-associated virus (AAV) comprising a stuffer. Specifically, the vector system comprises a nucleotide sequence of the following genes and one or more stuffers, and comprises one or more vectors or nucleic acid molecules:

[0016] (a) Helper virus genes required for AAV production;

[0017] (b) Rep gene of AAV;

[0018] (c) Cap gene of AAV; and

[0019] (d) Transgene arranged between ITRs (Inverted Terminal Repeats).

[0020] Additionally, in the above vector system, the nucleotide sequence of the gene is included in the vector or nucleic acid molecule, and specifically, one or more of the nucleotide sequences of the gene may be arranged or included in one or more vectors or nucleic acid molecules, respectively.

[0021] In one specific example, the vector system for AAV production can produce recombinant AAV using one (single) vector, and provides an all-in-one vector system including a stuffer.

[0022] Therefore, specifically, the vector system for AAV production provides a nucleic acid molecule comprising (a) a helper virus gene necessary for AAV production; (b) a Rep gene of AAV; (c) a Cap gene of AAV; and (d) a transgene arranged between the ITRs, and comprising one or more stuffer sequences. More specifically, the vector system provides a nucleic acid molecule comprising (a) a nucleotide sequence of a helper virus gene necessary for AAV production; (b) a nucleotide sequence of a Rep gene of AAV; (c) a nucleotide sequence of a Cap gene of AAV; and (d) a nucleotide sequence of a transgene arranged between the ITRs, and comprising one or more stuffer sequences. In the vector or nucleic acid molecule, the (a), (b), (c), (d), and one or more stuffer sequences are all integrated together in a single nucleic acid molecule.

[0023] In one specific example, the vector system for AAV production can produce recombinant AAV using two vectors, and provides a dual vector system including a stuffer.

[0024] Therefore, specifically, the vector system for AAV production provides a vector system comprising two vectors, wherein the vector system comprises (a) a helper virus gene required for AAV production; (b) a Rep gene of AAV; (c) a Cap gene of AAV; and (d) a transgene arranged between ITRs, and wherein the vector system comprises at least one stuffer. More specifically, the first vector comprises at least one of (a) a nucleotide sequence of a helper virus gene required for AAV production; (b) a nucleotide sequence of a Rep gene of AAV; (c) a nucleotide sequence of a Cap gene of AAV; and (d) a nucleotide sequence of a transgene arranged between ITRs, and the second vector comprises a nucleotide sequence that is not included in any of (a), (b), (c), and (d), and wherein the at least one stuffer may be included in any one of the first vector and the second vector.

[0025] In one specific example, the vector system for AAV production can produce recombinant AAV using three vectors, and provides a triple vector system including a stuffer.

[0026] Therefore, specifically, the vector system for AAV production provides a vector system comprising three vectors, wherein the vector system comprises (a) a helper virus gene required for AAV production; (b) a Rep gene of AAV; (c) a Cap gene of AAV; and (d) a transgene arranged between ITRs, and wherein the vector system comprises at least one stuffer. More specifically, the first vector comprises (a) a nucleotide sequence of a helper virus gene required for AAV production, the second vector comprises (b) a nucleotide sequence of a Rep gene of AAV; and (c) a nucleotide sequence of a Cap gene of AAV, and the third vector comprises at least one of (d) a nucleotide sequence of a transgene arranged between ITRs, wherein the at least one stuffer may be included in any one of the first vector, the second vector, and the third vector.

[0027] The following description applies commonly to vectors according to the above aspects or nucleic acid molecules according to the above aspects.

[0028] The above "adeno-associated virus (AAV)" is a virus belonging to the genus Dependovirus in the family Parvoviridae. It does not have the ability to replicate on its own and therefore requires the coexistence of a helper virus for its replication. The AAV has a single-stranded DNA (ssDNA) of approximately 4.7 kb. More than 100 serotypes of AAV are known, including naturally occurring serotypes and variants.

[0029] The above "recombinant AAV (rAAV)" may be used interchangeably with "AAV vector", "AAV particle", "AAV vector particle", "rAAV particle", and "rAAV vector particle", and may refer to an AAV vector capable of expressing a target protein in a host cell. The term "recombinant" means that the AAV or sequence has been manipulated in a way that does not generally occur in nature. For example, a recombinant vector, such as an AAV vector, may refer to a case where a polynucleotide that does not generally exist in the wild-type AAV genome has been inserted into the viral genome. Therefore, the recombinant AAV produced by the vector or vector system for producing the recombinant AAV may be applied as a gene therapy agent.

[0030] As used herein, the term "vector system for recombinant AAV production" may refer to a vector or vector system capable of producing recombinant AAV in a host cell into which the vector or a vector system comprising the same has been introduced. The vector may refer to a plasmid vector.

[0031] In one specific example, the vector system for recombinant AAV production may relate to vector technology that integrates all genes necessary for AAV production into a single vector. Accordingly, in the present specification, the term "vector or vector system for recombinant AAV production" may include the term "all-in-one vector" or "AAV single vector system." The vector for recombinant AAV production may be a single plasmid.

[0032] The term "vector" may refer to a vehicle capable of artificially transporting heterologous genetic material into another cell. Examples of such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. The plasmids may include plasmids derived from Escherichia coli (pBR322, pBR325, pUC118, pUC119, pET21a(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5), plasmids derived from yeast (YEp13, YEp24, and YCp50), etc. The viruses may include animal viruses such as retroviruses, adenoviruses, or vaccinia viruses; insect viruses such as baculoviruses, etc.

[0033] The nucleic acid molecule may be a DNA molecule. The nucleic acid molecule may be single-stranded (ss) or double-stranded (ds). The nucleic acid molecule may be a linear DNA molecule or a circular DNA molecule. The nucleic acid molecule may comprise the gene, specifically the sequence of the gene, more specifically the nucleotide sequence of the gene, and even more specifically the nucleotide sequence encoding the gene. The term "nucleotide sequence" may be used interchangeably with the terms "nucleic acid sequence" and "DNA sequence."

[0034] The nucleic acid molecule may exist in various forms. For example, the nucleic acid molecule may be linear or circular. Accordingly, the vector may be a linear vector or a circular vector. In certain embodiments, the vector may be a circular vector.

[0035] The recombinant AAV produced by the vector system for producing the above recombinant AAV may be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV).

[0036] The above vector system or a vector included in the above system comprises (a) a helper virus gene necessary for AAV production.

[0037] The above helper virus can refer to a virus that aids the replication of a virus that cannot replicate on its own through simultaneous infection. Because AAV lacks the ability to replicate on its own, it requires the genes of a helper virus for AAV replication.

[0038] The above helper virus gene may be a gene of a helper virus required for AAV production. In this specification, the term "helper virus gene" may be used interchangeably with the term "helper gene."

[0039] The nucleotide sequence of the above helper virus gene may be derived from one or more selected from among adenovirus, herpes simplex virus (HSV), baculovirus, papillomavirus, and bocavirus, but is not limited thereto.

[0040] The adenovirus is known to have more than 50 serotypes. In one specific example, the nucleotide sequence of the helper virus gene may be derived from an adenovirus. The adenovirus may be selected from adenovirus 2 and adenovirus 5. The adenovirus may be adenovirus 2.

[0041] The above herpes simplex virus may be type 1 or type 2.

[0042] The above papillomavirus may be a human papillomavirus (HPV). More than 150 types of HPV are known. For example, the HPV may be, but is not limited to, HPV-16.

[0043] The helper virus gene may include at least one selected from E1, E2, E2a, E4, E4orf1, E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA (also referred to as “VA RNA gene”), DBP (DNA-binding protein), and variants thereof. The helper virus gene is a gene encoding the helper protein.

[0044] The above variant may be an engineered helper virus gene.

[0045] As used herein, the term "engineered" may mean that a gene has been intentionally modified and manipulated using genetic engineering techniques. Genetic engineering techniques for manipulating genes are well known. As used herein, "engineered A" may include a variant comprising one or more mutations in the wild-type sequence of A, a variant in which a portion of the sequence of A is truncated, etc. The mutations may be insertions, substitutions, deletions, or a combination thereof.

[0046] The nucleotide sequence of the helper virus gene may be derived from adenovirus 2. In one specific example, the helper virus gene may include E2a, E4, and VA. The helper virus gene may include E2a and E4.

[0047] The above vector system or a vector included in the above system comprises (b) a Rep gene of AAV.

[0048] The Rep (Replication) gene may be a gene required for AAV replication. The Rep gene may be derived from any AAV serotype. The nucleotide sequence of the Rep gene may be derived from one or more selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh39, AAVrh64R1, and AAVrh74, but is not limited thereto. The AAV serotype may also include other AAV serotypes currently known or to be discovered in the future. The AAV serotype may also include an artificial AAV serotype. When there are two or more Rep genes, each gene may be derived from the same or different AAV serotypes.

[0049] In one specific example, the nucleotide sequence of the Rep gene may be derived from AAV2.

[0050] The above Rep gene may include a gene encoding a known Rep protein or a variant thereof.

[0051] The Rep gene may include at least one selected from Rep78, Rep68, Rep52, Rep40, and variants thereof. The Rep gene may include Rep68. The Rep gene may include Rep78 or Rep68. The Rep gene may include at least one selected from (i) Rep78, and (ii) Rep68, Rep52, and Rep40. The Rep gene may include at least one selected from (i) Rep68, and (ii) Rep78, Rep52, and Rep40. The Rep gene may include at least one selected from (i) Rep68, and (ii) Rep52 and Rep40. The Rep gene may include all of Rep78, Rep68, Rep52, and Rep40.

[0052] In one specific example, the Rep gene may include one or more (one, two, or three) selected from Rep68, Rep52, and Rep40. Rep78, when overexpressed, may increase cytotoxicity and decrease AAV productivity. Therefore, the Rep gene may include a Rep gene other than Rep78.

[0053] The above variant may be an engineered Rep (ERep) protein. The variant may be a variant having one or more mutations from the wild-type sequence, or a truncated variant.

[0054] In one specific embodiment, the Rep gene may comprise a Rep gene of AAV2. In a specific specific embodiment, the Rep gene may comprise Rep78, Rep68, Rep52, and Rep40.

[0055] The above vector system or a vector included in the above system comprises (c) the Cap gene of AAV.

[0056] The above Cap (Capsid) gene is a gene that encodes the viral capsid protein.

[0057] The Cap gene may be derived from any AAV serotype. The nucleotide sequence of the Cap gene may be derived from one or more selected from, but not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh39, AAVrh64R1, and AAVrh74. The AAV serotype may also include other AAV serotypes currently known or to be discovered in the future. The AAV serotype may also include an artificial AAV serotype. When there are two or more Cap genes, each gene may be derived from the same or different AAV serotypes.

[0058] In one specific example, the nucleotide sequence of the Cap gene may be derived from AAV8.

[0059] The above Cap gene may include a gene encoding a known Cap protein or a variant thereof.

[0060] The Cap gene may encode one or more selected from, but not limited to, a capsid protein, a VP1 protein, a VP2 protein, a VP3 protein, and variants thereof. The Cap gene may encode all of the VP1, VP2, and VP3 proteins.

[0061] In one specific example, the Cap gene may be a wild-type Cap gene of AAV8.

[0062] The variant may be an engineered Cap (ECap) protein. The variant may be an engineered capsid protein. The variant may be a variant having one or more mutations from the wild-type sequence, or a truncated variant.

[0063] The nucleotide sequences of the Rep gene and Cap gene may be derived from the same or different AAV serotypes. In one specific example, the Rep gene may be derived from AAV2, and the Cap gene may be derived from AAV8.

[0064] The above vector system or a vector included in the above system comprises (d) a transgene arranged between ITRs (Inverted Terminal Repeats).

[0065] The transgene described above is a gene transferred from one organism to another. The transgene may be a heterologous polynucleotide. The transgene may be a gene of interest (GOI) to be packaged into a recombinant AAV capsid. The transgene is not limited to a specific type, as long as it is a gene intended for introduction into a host cell.

[0066] The term "polynucleotide" can refer to any form of nucleic acid, including DNA and RNA, or oligonucleotides. Polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. The sequence or structure of a particular polynucleotide can be described according to the convention of providing the sequence in the 5' to 3' direction.

[0067] The "polypeptide," "protein," and "peptide" encoded by a polynucleotide sequence may include functional subsequences, modified forms, or sequence variants, as long as they retain the functionality of the native protein. The term "modified" or "variant" means that the sequence of the polynucleotide or polypeptide deviates from the reference sequence. Thus, the modified or variant sequence may have substantially the same activity or function as the reference sequence, or a greater or lesser activity or function, but retain at least a partial activity or function of the reference sequence.

[0068] Non-limiting examples of modifications include substitutions, insertions, and / or deletions of one or more nucleotides or amino acids. An example of an amino acid substitution is a conservative amino acid substitution. Examples of conservative amino acid substitutions are well known. A "conservative substitution" is the replacement of an amino acid with a biologically, chemically, or structurally similar residue. Biological similarity means that the substitution does not destroy biological activity. Structural similarity means that the amino acids have similar lengths (e.g., alanine, glycine, and serine) or similar sizes. Chemical similarity means that the residues have the same charge, or have the same hydrophilic or hydrophobic properties, or both. For example, conservative amino acid substitutions include substitutions within the following groups: glycine / alanine, valine / isoleucine / leucine, aspartic acid / glutamic acid, asparagine / glutamine, serine / threonine, lysine / arginine, and phenylalanine / tyrosine.

[0069] At the nucleotide sequence level, naturally occurring and non-naturally occurring variant genes can have at least 50%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with a reference gene.

[0070] At the amino acid sequence level, the naturally occurring and non-naturally occurring variant proteins can have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with the reference protein.

[0071] The term "sequence identity" refers to the degree of similarity in amino acid residues or bases between two sequences after aligning them to achieve maximum agreement over a specific comparison region. Sequence identity can be determined using methods known in the art. The percentage of sequence identity can be determined using known sequence comparison programs, such as NCBI's BLAST.

[0072] The transgene may be a therapeutic gene. For example, the transgene may encode antisense RNA, a ribozyme, a protein, a toxin, an antigen, or an antibody. Therefore, the vector for producing the recombinant AAV can be used to produce a gene therapy clinical agent for treating a patient.

[0073] The above transgene may be one or two or more.

[0074] The transgene may be arranged between Inverted Terminal Repeats (ITRs). The transgene may have ITRs arranged on both sides. The transgene may have two ITRs arranged on either side. The transgene may be arranged between L-ITRs and R-ITRs. For example, the L-ITR (first ITR), the transgene, and the R-ITR (second ITR) may be arranged sequentially in the 5' to 3' direction or in the 3' to 5' direction.

[0075] The above Inverted Terminal Repeat (ITR) is involved in the replication of the AAV genome and the packaging of AAV particles. The ITR includes the Rep binding element (RBE), RBE', A, A', B, B', C, C', and D regions. The ITR consists of two arm palindromes (BB' and C-C') embedded in a larger stem palindrome (A-A'). Consequently, the ITR has a T-shaped stem-loop structure. The ITR can have two configurations, namely flip and flop. The flip and flop configurations have the BB' and CC' palindromes closest to the 3' end, respectively. The two ITRs on either side are also referred to as the first ITR and the second ITR, or as the left (L)-ITR and the right (R)-ITR, or as the 5'-ITR and the 3'-ITR. The D region occurs only once at each terminus, so it remains single-stranded. The RBE is the region where the Rep78 and Rep68 proteins of AAV bind. The strand- and site-specific endonuclease catalytic domains of Rep78 and Rep68 introduce nicks into the terminal resolution site (trs). The structure and sequence of ITRs are known.

[0076] The ITR may be derived from a virus belonging to the genus Dependovirus of the family Parvoviridae. The ITR may be derived from AAV. The ITR may be derived from any AAV serotype. The ITR may be derived from at least one selected from, but not limited to, AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh39, AAVrh64R1, and AAVrh74. The AAV serotype may also include other AAV serotypes currently known or to be discovered in the future. The AAV serotype may also include artificial AAV serotypes. The two ITRs on both sides above may be derived from the same or different AAV serotypes.

[0077] In one specific example, the ITR may be derived from AAV2. The L-ITR and R-ITR may both be derived from AAV2.

[0078] The ITR may be a wild-type ITR or a variant thereof. The ITR may comprise all or part of a wild-type ITR sequence. The ITR may have a lower %GC content than the wild-type ITR. The ITR may be a synthetic ITR. The ITR may be an ITR derived from self-complementary AAV (scAAV). The L-ITR and R-ITR may be modified to enable the production of scAAV.

[0079] In one specific embodiment, either the L-ITR or the R-ITR can serve as a primer for DNA replication. The R-ITR can serve as a primer for DNA replication. Either the L-ITR or the R-ITR may not comprise trs. Either the L-ITR or the R-ITR may not comprise trs, and the other may be a wild-type ITR. The L-ITR may not comprise trs, and the R-ITR may be a wild-type ITR. Either the L-ITR or the R-ITR may not comprise trs and may comprise an RBE (Rep binding element). The L-ITR may not comprise trs and may comprise an RBE. Either the L-ITR or the R-ITR may not comprise trs and may have a hairpin structure. The L-ITR may not comprise trs and may have a hairpin structure. One of the above L-ITR and R-ITR may be an ITR mutant having a D region deleted, and the other may be a wild-type ITR. The L-ITR may be an ITR mutant having a D region deleted, and the R-ITR may be a wild-type ITR. The L-ITR may be composed of SEQ ID NO: 10, and the R-ITR may be composed of SEQ ID NO: 15. Therefore, the recombinant AAV produced by the above vector system may be a self-complementary AAV (scAAV).

[0080] The nucleic acid molecule may further comprise a sequence added to the ITR. As used herein, the term "sequence added to the ITR" may also be expressed as "ITR+." By further including the sequence added to the ITR, the %Full / empty capsid may be improved. In one embodiment, the sequence added to the ITR may be a non-ITR viral DNA sequence derived from the wild-type AAV genome. Specifically, the sequence added to the ITR may be a non-ITR viral DNA (46 bp) corresponding to wtAAV2 nt 4489-4534 derived from upstream of the 3'-ITR in the wild-type AAV2 genome. The sequence added to the ITR may be a sequence added to the 5'-end of the R-ITR based on the (+) strand. The sequence added to the ITR may be a sequence added upstream of the R-ITR.

[0081] The above vector system or the vectors included in the above system include one or more stuffers. Specifically, the one or more stuffers may be arranged in one or more vectors included in the vector system.

[0082] As used herein, the term "stuffer" may refer to an untranslated sequence of nucleic acid or a sequence that does not encode a protein. The stuffer may include a non-functional DNA sequence.

[0083] The number of stuffers included in the vector system or the vector of the system can be appropriately selected within a range that can maintain the function of the vector system for producing recombinant AAV while achieving the effect of including the stuffer, and is not limited to a specific number. The vector system can include one stuffer. The vector system can include two or more stuffers. The vector system can include three or more stuffers. The vector system can include 2 to 100, 2 to 50, 2 to 20, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 100, 3 to 50, 3 to 20, 3 to 10, 3 to 8, 3 to 6, or 3 to 4 stuffers. The vector system can include three stuffers. When there are two or more of the above stuffers, each stuffer may be referred to as a first stuffer, a second stuffer, etc.

[0084] The above stuffer may be arranged inside or outside the two ITRs. The above "inside the two ITRs" may mean arranged between or inside the two ITRs. The above "outside the two ITRs" may mean arranged outside or outside the two ITRs.

[0085] When the above vector system includes two or more stuffers, each stuffer can be independently arranged inside or outside the two ITRs. When the above vector system includes two or more stuffers, all stuffers can be arranged outside the two ITRs.

[0086] In the above vector system, the length of each stuffer can be from 0.1 kb to 20 kb. Specifically, the length of each stuffer is 0.1 kb to 20 kb, 0.1 kb to 15 kb, 0.1 kb to 10 kb, 0.1 kb to 9 kb, 0.1 kb to 8 kb, 0.1 kb to 7 kb, 0.1 kb to 6 kb, 0.1 kb to 5 kb, 0.1 kb to 4 kb, 0.1 kb to 3 kb, 0.1 kb to 2 kb, 0.5 kb to 20 kb, 0.5 kb to 15 kb, 0.5 kb to 10 kb, 0.5 kb to 9 kb, 0.5 kb to 8 kb, 0.5 kb to 7 kb, 0.5 kb to 6 kb, 0.5 kb to 5 kb, 0.5 kb to 4 kb, 0.5 kb to 3 kb, 0.5 kb to 2 kb, 1 kb to 20 kb, 1 kb to 15 kb, 1 kb to 10 kb, 1 kb to 9 kb, 1 kb to 8 kb, 1 kb to 7 kb, 1 kb to 6 kb, 1 kb to 5 kb, 1 kb to 4 kb, 1 kb to 3 kb, 1 kb to 2 kb, 2 kb to 20 kb, 2 kb to 15 kb, 2 kb to 10 kb, 2 kb to 9 kb, 2 kb to 8 kb, 2 kb to 7 kb, 2 kb to 6 kb, 2 kb to 5 kb, 2 kb to 4 kb, 2 kb to 3 kb, 3 kb to 20 kb, 3 kb to It can be 15 kb, 3 kb to 10 kb, 3 kb to 9 kb, 3 kb to 8 kb, 3 kb to 7 kb, 3 kb to 6 kb, 3 kb to 5 kb, 3 kb to 4 kb, 4 kb to 20 kb, 4 kb to 15 kb, 4 kb to 10 kb, 4 kb to 9 kb, 4 kb to 8 kb, 4 kb to 7 kb, 4 kb to 6 kb, 4 kb to 5 kb, 5 kb to 20 kb, 5 kb to 15 kb, 5 kb to 10 kb, 5 kb to 9 kb, 5 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb.

[0087] In the above vector system, the total length of all stuffers can be from 0.1 kb to 100 kb. Specifically, the total length of all stuffers is 0.1 kb to 100 kb, 0.1 kb to 90 kb, 0.1 kb to 80 kb, 0.1 kb to 70 kb, 0.1 kb to 60 kb, 0.1 kb to 56 kb, 0.1 kb to 55 kb, 0.1 kb to 50 kb, 0.1 kb to 40 kb, 0.1 kb to 30 kb, 0.1 kb to 20 kb, 0.1 kb to 10 kb, 0.1 kb to 8 kb, 0.5 kb to 100 kb, 0.5 kb to 90 kb, 0.5 kb to 80 kb, 0.5 kb to 70 kb, 0.5 kb to 60 kb, 0.5 kb to 56 kb, 0.5 kb to 55 kb, 0.5 kb to 50 kb, 0.5 kb to 40 kb, 0.5 kb to 30 kb, 0.5 kb to 20 kb, 0.5 kb to 10 kb, 0.5 kb to 8 kb, 1 kb to 100 kb, 1 kb to 90 kb, 1 kb to 80 kb, 1 kb to 70 kb, 1 kb to 60 kb, 1 kb to 56 kb, 1 kb to 55 kb, 1 kb to 50 kb, 1 kb to 40 kb, 1 kb to 30 kb, 1 kb to 20 kb, 1 kb to 10 kb, 1 kb to 8 kb, 1.5 kb to 100 kb, 1.5 kb to 90 kb, 1.5 kb to 80 kb, 1.5 kb to 70 kb, 1.5 kb to 60 kb, 1.5 kb to 56 kb, 1.5 kb to 55 kb, 1.5 kb to 50 kb, 1.5 kb to 40 kb, 1.5 kb to 30 kb, 1.5 kb to 20 kb, 1.5 kb to 10 kb, 1.5 kb to 8 kb, 3 kb to 100 kb, 3 kb to 90 kb, 3 kb to 80 kb, 3 kb to 70 kb, 3 kb to 60 kb, 3 kb to 56 kb, 3 kb to 55 kb, 3 kb to 50 kb, 3 kb to 40 kb, 3 kb to 30 kb, 3 kb to 20 kb, 3 kb to 10 kb, 3 kb to 8 kb, 5 kb to 100 kb, 5 kb to 90 kb, 5 kb to 80 kb, 5 kb to 70 kb, 5 kb to 60 kb, 5 kb to 56 kb, 5 kb to 55 kb, 5 kb to 50 kb, 5 kb to 40 kb, 5 kb to 30 kb, 5 kb to 20 kb, 5 kb to 10 kb, 5 kb to 8 kb, 7 kb to 100 kb, 7 kb to 90 kb, 7 kb to 80 kb, 7 kb to 70 kb, 7 kb to 60 kb, 7 kb to 56 kb, 7 kb to 55 kb, 7 kb to 50 kb, 7 kb to 40 kb, 7 kb to 30 kb, 7 kb to 20 kb, 7 kb to 10 kb, 7 kb to 8 kb, 7.5 kb to 100 kb, 7.5 kb to 90 kb, 7.5 kb to 80 kb, 7.5 kb to It may be 70 kb, 7.5 kb to 60 kb, 7.5 kb to 56 kb, 7.5 kb to 55 kb, 7.5 kb to 50 kb, 7.5 kb to 40 kb, 7.5 kb to 30 kb, 7.5 kb to 20 kb, 7.5 kb to 10 kb, or 7.5 kb to 8 kb.

[0088] The above vector system may include three stuffers. The stuffers may include a first stuffer, a second stuffer, and a third stuffer. All three stuffers may be arranged outside of the two ITRs.

[0089] The first stuffer may be arranged downstream of the R-ITR. Specifically, the first stuffer may be arranged after the 3' end of the R-ITR with respect to the (+) strand.

[0090] The length of the first stuffer may be from 0.1 kb to 20 kb. Specifically, the length of the first stuffer is 0.1 kb to 20 kb, 0.1 kb to 15 kb, 0.1 kb to 10 kb, 0.1 kb to 9 kb, 0.1 kb to 8 kb, 0.1 kb to 7 kb, 0.1 kb to 6 kb, 0.1 kb to 5 kb, 0.1 kb to 4 kb, 0.1 kb to 3 kb, 0.1 kb to 2 kb, 0.5 kb to 20 kb, 0.5 kb to 15 kb, 0.5 kb to 10 kb, 0.5 kb to 9 kb, 0.5 kb to 8 kb, 0.5 kb to 7 kb, 0.5 kb to 6 kb, 0.5 kb to 5 kb, 0.5 kb to 4 kb, 0.5 kb to 3 kb, 0.5 kb to 2 kb, 1 kb to 20 kb, 1 kb to 15 kb, 1 kb to 10 kb, 1 kb to 9 kb, 1 kb to 8 kb, 1 kb to 7 kb, 1 kb to 6 kb, 1 kb to 5 kb, 1 kb to 4 kb, 1 kb to 3 kb, 1 kb to 2 kb, 2 kb to 20 kb, 2 kb to 15 kb, 2 kb to 10 kb, 2 kb to 9 kb, 2 kb to 8 kb, 2 kb to 7 kb, 2 kb to 6 kb, 2 kb to 5 kb, 2 kb to 4 kb, 2 kb to 3 kb, 3 kb to 20 kb, 3 kb to It can be 15 kb, 3 kb to 10 kb, 3 kb to 9 kb, 3 kb to 8 kb, 3 kb to 7 kb, 3 kb to 6 kb, 3 kb to 5 kb, 3 kb to 4 kb, 4 kb to 20 kb, 4 kb to 15 kb, 4 kb to 10 kb, 4 kb to 9 kb, 4 kb to 8 kb, 4 kb to 7 kb, 4 kb to 6 kb, 4 kb to 5 kb, 5 kb to 20 kb, 5 kb to 15 kb, 5 kb to 10 kb, 5 kb to 9 kb, 5 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb.

[0091] The second stuffer may be arranged upstream of the Rep / Cap gene. Specifically, the second stuffer may be arranged before the 5' end of the Rep / Cap gene based on the (+) strand. More specifically, the second stuffer may be arranged (b) upstream of the Rep gene or (c) upstream of the Cap gene.

[0092] In one embodiment, when the vector system or vector is an "All-in-one vector" or an "AAV single vector system", the second stuffer may be arranged between (i) whichever of the Rep gene and the Cap gene is closer to the helper virus gene and (ii) the helper virus gene. For example, the second stuffer may be arranged in the following order: (a) helper virus gene - second stuffer - (b) Rep gene; (b) Rep gene - second stuffer - (a) helper virus gene; (a) helper virus gene - second stuffer - (c) Cap gene; or (c) Cap gene - second stuffer - (a) helper virus gene.

[0093] The length of the second stuffer may be from 0.1 kb to 20 kb. Specifically, the length of the first stuffer is 0.1 kb to 20 kb, 0.1 kb to 15 kb, 0.1 kb to 10 kb, 0.1 kb to 9 kb, 0.1 kb to 8 kb, 0.1 kb to 7 kb, 0.1 kb to 6 kb, 0.1 kb to 5 kb, 0.1 kb to 4 kb, 0.1 kb to 3 kb, 0.1 kb to 2 kb, 0.5 kb to 20 kb, 0.5 kb to 15 kb, 0.5 kb to 10 kb, 0.5 kb to 9 kb, 0.5 kb to 8 kb, 0.5 kb to 7 kb, 0.5 kb to 6 kb, 0.5 kb to 5 kb, 0.5 kb to 4 kb, 0.5 kb to 3 kb, 0.5 kb to 2 kb, 1 kb to 20 kb, 1 kb to 15 kb, 1 kb to 10 kb, 1 kb to 9 kb, 1 kb to 8 kb, 1 kb to 7 kb, 1 kb to 6 kb, 1 kb to 5 kb, 1 kb to 4 kb, 1 kb to 3 kb, 1 kb to 2 kb, 2 kb to 20 kb, 2 kb to 15 kb, 2 kb to 10 kb, 2 kb to 9 kb, 2 kb to 8 kb, 2 kb to 7 kb, 2 kb to 6 kb, 2 kb to 5 kb, 2 kb to 4 kb, 2 kb to 3 kb, 3 kb to 20 kb, 3 kb to It can be 15 kb, 3 kb to 10 kb, 3 kb to 9 kb, 3 kb to 8 kb, 3 kb to 7 kb, 3 kb to 6 kb, 3 kb to 5 kb, 3 kb to 4 kb, 4 kb to 20 kb, 4 kb to 15 kb, 4 kb to 10 kb, 4 kb to 9 kb, 4 kb to 8 kb, 4 kb to 7 kb, 4 kb to 6 kb, 4 kb to 5 kb, 5 kb to 20 kb, 5 kb to 15 kb, 5 kb to 10 kb, 5 kb to 9 kb, 5 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb.

[0094] The third stuffer may be arranged upstream of the L-ITR. Specifically, the third stuffer may be arranged before the 5' end of the L-ITR with respect to the (+) strand.

[0095] The length of the third stuffer may be from 0.1 kb to 20 kb. Specifically, the length of the first stuffer is 0.1 kb to 20 kb, 0.1 kb to 15 kb, 0.1 kb to 10 kb, 0.1 kb to 9 kb, 0.1 kb to 8 kb, 0.1 kb to 7 kb, 0.1 kb to 6 kb, 0.1 kb to 5 kb, 0.1 kb to 4 kb, 0.1 kb to 3 kb, 0.1 kb to 2 kb, 0.5 kb to 20 kb, 0.5 kb to 15 kb, 0.5 kb to 10 kb, 0.5 kb to 9 kb, 0.5 kb to 8 kb, 0.5 kb to 7 kb, 0.5 kb to 6 kb, 0.5 kb to 5 kb, 0.5 kb to 4 kb, 0.5 kb to 3 kb, 0.5 kb to 2 kb, 1 kb to 20 kb, 1 kb to 15 kb, 1 kb to 10 kb, 1 kb to 9 kb, 1 kb to 8 kb, 1 kb to 7 kb, 1 kb to 6 kb, 1 kb to 5 kb, 1 kb to 4 kb, 1 kb to 3 kb, 1 kb to 2 kb, 2 kb to 20 kb, 2 kb to 15 kb, 2 kb to 10 kb, 2 kb to 9 kb, 2 kb to 8 kb, 2 kb to 7 kb, 2 kb to 6 kb, 2 kb to 5 kb, 2 kb to 4 kb, 2 kb to 3 kb, 3 kb to 20 kb, 3 kb to It can be 15 kb, 3 kb to 10 kb, 3 kb to 9 kb, 3 kb to 8 kb, 3 kb to 7 kb, 3 kb to 6 kb, 3 kb to 5 kb, 3 kb to 4 kb, 4 kb to 20 kb, 4 kb to 15 kb, 4 kb to 10 kb, 4 kb to 9 kb, 4 kb to 8 kb, 4 kb to 7 kb, 4 kb to 6 kb, 4 kb to 5 kb, 5 kb to 20 kb, 5 kb to 15 kb, 5 kb to 10 kb, 5 kb to 9 kb, 5 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb.

[0096] The above vector may include two stuffers. The stuffers may include a first stuffer and a third stuffer. Both of the stuffers may be arranged outside of the two ITRs.

[0097] The first stuffer may be arranged downstream of the R-ITR. Specifically, the first stuffer may be arranged after the 3' end of the R-ITR with respect to the (+) strand.

[0098] The third stuffer may be arranged upstream of the L-ITR. Specifically, the third stuffer may be arranged before the 5' end of the L-ITR with respect to the (+) strand.

[0099] The above stuffer can reduce impurities that may occur during the production of recombinant AAV using the vector system or vector.

[0100] The above impurity may be a DNA impurity.

[0101] As used herein, the term “impurity” may mean that a component other than a component (e.g., a transgene) present between two ITRs of a vector for recombinant AAV production is packaged into a recombinant AAV particle, an incomplete transgene is packaged into a recombinant AAV particle, or the vector is reverse packaged.

[0102] In one specific embodiment, when producing recombinant AAV using a single vector system, a portion of the Rep gene, Cap gene, or Helper gene sequence may be packaged within the recombinant AAV particle. Accordingly, the impurity may include a portion of the Rep gene, Cap gene, or Helper gene sequence packaged within the recombinant AAV particle.

[0103] For example, the impurity may include at least one selected from any sequence located outside of two ITRs in the vector system, such as all or part of a helper virus gene, all or part of a Rep gene, all or part of a Cap gene; part of a transgene; part of a genomic sequence of a host cell; and chimeric forms of two or more of these.

[0104] When Rep / Cap and Helper genes are inserted into recombinant particles, replication-competent AAV (rcAAV) can be formed. Wild-type AAV cannot autonomously replicate without a helper virus such as adenovirus. However, replication-competent rcAAV can induce an immune response clinically, and the presence of helper genes can lead to unintended AAV replication, which can affect the safety of gene therapy products. rcAAV can also be generated during AAV production, and because rcAAV cannot be isolated through purification processes, minimizing its formation is crucial.

[0105] Therefore, by lowering the content of the above impurities, a recombinant AAV with excellent safety and quality can be secured.

[0106] In one specific example, the stuffer may reduce reverse packaging of the vector. AAV packaging refers to the packaging of a structure present between two ITRs into an AAV particle. However, reverse packaging of AAV refers to the packaging of a recombinant AAV particle outwardly, rather than inwardly, of the two ITRs. Therefore, inhibiting reverse packaging of AAV can secure high-quality AAV. According to one aspect, a vector comprising a stuffer can reduce the content of impurities resulting from reverse packaging of the vector, thereby enabling the securement of high-quality recombinant AAV. Furthermore, it is expected that this can have an enhanced safety effect when applied as a therapeutic agent.

[0107] In one embodiment, the first stuffer arranged downstream of the R-ITR, the third stuffer arranged upstream of the L-ITR, or both can reduce reverse packaging of the vector.

[0108] In one specific example, the stuffer may reduce impurities that may occur during the recombinant AAV production process using a vector system for recombinant AAV production, specifically a single vector system.

[0109] In one specific embodiment, the second stuffer arranged upstream of (b) or upstream of (c) can reduce impurities in recombinant AAV particles produced by the vector system. The second stuffer can reduce impurities that may occur during the recombinant AAV production process using the vector system for recombinant AAV production, specifically, a single vector system.

[0110] For example, impurities that may occur during the production of recombinant AAV using a vector system for recombinant AAV production, specifically a single vector system, may include at least one selected from the group consisting of all or part of a helper virus gene; all or part of a Rep gene; all or part of a Cap gene; any sequence located outside of two ITRs in the vector; a portion of a genomic sequence of a host cell; and chimeric forms of two or more of these. In one specific embodiment, the impurity may include all or part of a helper virus gene.

[0111] The above stuffer may use a sequence having any one or more of the following characteristics: (i) being a human-derived DNA sequence; (ii) not encoding a protein; and (iii) not inhibiting the expression of a surrounding gene.

[0112] The above-mentioned stuffer may include one or more selected from among introns and partial sequences or modified sequences thereof. The intron refers to a nucleotide sequence that is removed from the final gene product by RNA splicing. The intron may be derived from the GOI gene or from another gene.

[0113] The above stuffer may include at least one selected from a barrier insulator and a partial sequence or modified sequence thereof. The barrier insulator refers to a DNA sequence that interferes with heterochromatin formation. The barrier insulator can maintain the expression of a peripheral gene by preventing gene silencing of the peripheral gene. The barrier insulator may include at least one selected from a matrix attachment region (MAR), a ubiquitous chromatin opening element (UCOE), a chromatin control element (CCE), a stabilizing and anti-repressor (STAR), a hypersensitive site 5 (5'HS5), and a partial sequence or modified sequence thereof.

[0114] As the above stuffer, a stuffer derived from lambda phage may not be suitable because it may reduce the expression of surrounding genes and cause an inflammatory response by expressing foreign proteins.

[0115] Non-limiting examples of the above stuffer may include all or part of the gene sequences selected from Table 1 below.

[0116] Sequence numberGene size (kb)16FIX-intron-1 (139,530,853-139,537,009 of NC_000023.11)6.217FIX-intron-2 (139,537,174-139,537,361 of NC_000023.11)0.218FIX-intron-3 (139,537,387-139,541,075 of NC_000023.11)3.719FIX-intron-4 (139,541,190-139,548,362 of NC_000023.11)7.220FIX-intron-5 (139,530,853-139,537,009 of NC_000023.11) 139,548,492-139,551,061)2.621FIX-intron-6 (139,551,265-139,560,740 in NC_000023.11)9.522FIX-intron-7 (in NC_000023.11) 139,560,856-139,561,523)0.71MAR (Matrix Attachment Regions)3.023UCOE (Ubiquitous Chromatin Opening Elements)4.15UCOE (Ubiquitous Chromatin Opening Elements) - partial1.4245'HS5 (Hypersensitive site 5)1.025STAR (Stabilizing Anti Repressor)1.09CCE (Chromatin Control Elements)3.6

[0117] The above stuffer may include at least one selected from the whole or part of the sequence of the MAR (Matrix Attachment Region), the whole or part of the sequence of the UCOE (Ubiquitous Chromatin Opening Element), and the whole or part of the sequence of the CCE (Chromatin Control Element).

[0118] The above stuffer may include a portion of a MAR (Matrix Attachment Region) sequence. The MAR is a type of barrier insulator that does not encode a protein and does not block the expression of surrounding genes. The MAR may be a human globin MAR. The above stuffer may include a polynucleotide comprising at least 100, at least 500, at least 1000, at least 1500, at least 2000, at least 2500, or at least 3000 consecutive nucleotides within the sequence of SEQ ID NO: 1. The above stuffer may include a polynucleotide of SEQ ID NO: 1.

[0119] The above stuffer may include a partial sequence of a Ubiquitous Chromatin Opening Element (UCOE). The UCOE is a type of barrier insulator that does not encode a protein and does not block the expression of surrounding genes. The UCOE may be the UCOE of the human HNRPA2B1 (heterogeneous nuclear ribonucleoproteins A2 / B1 gene). The above stuffer may include a polynucleotide comprising at least 100, at least 500, at least 1000, at least 1200, or at least 1400 consecutive nucleotides within the sequence of SEQ ID NO: 5. The above stuffer may include a polynucleotide of SEQ ID NO: 5.

[0120] The above stuffer may include a portion of a sequence of a Chromatin Control Element (CCE). The CCE is a type of barrier insulator that does not encode a protein and does not block the expression of surrounding genes. The CCE may be a human CCE. The above stuffer may include a polynucleotide comprising at least 100 consecutive nucleotides, at least 500 consecutive nucleotides, at least 1000 consecutive nucleotides, at least 1500 consecutive nucleotides, at least 2000 consecutive nucleotides, at least 2500 consecutive nucleotides, at least 3000 consecutive nucleotides, or at least 3500 consecutive nucleotides within the sequence of SEQ ID NO: 9. The above stuffer may include a polynucleotide of SEQ ID NO: 9.

[0121] In one specific example, the stuffer may include a first stuffer, a second stuffer, and a third stuffer, and the first stuffer, the second stuffer, and the third stuffer may each independently be any one selected from a portion of a MAR sequence, a portion of a UCOE sequence, and a portion of a CCE sequence.

[0122] In one specific example, the stuffer may include a first stuffer, a second stuffer, and a third stuffer, wherein the first stuffer may be a partial sequence of MAR, the second stuffer may be a partial sequence of UCOE, and the third stuffer may be a partial sequence of CCE.

[0123] In one embodiment, the stuffer may include:

[0124] A first stuffer comprising a polynucleotide of sequence number 1, arranged downstream of the R-ITR;

[0125] A second stuffer comprising a polynucleotide of sequence number 5, arranged upstream of (b) or upstream of (c); and

[0126] A third stuffer comprising a polynucleotide of sequence number 9, arranged upstream of the L-ITR.

[0127] In one specific embodiment, the stuffer may include a first stuffer and a third stuffer, and the first stuffer and the third stuffer may each independently be any one selected from a portion of a MAR sequence, a portion of a UCOE sequence, and a portion of a CCE sequence.

[0128] In one specific example, the stuffer may comprise a first stuffer and a third stuffer, wherein the first stuffer may be a partial sequence of MAR, and the third stuffer may be a partial sequence of CCE.

[0129] In one embodiment, the stuffer may include:

[0130] A first stuffer comprising a polynucleotide of sequence number 1, arranged downstream of the R-ITR; and

[0131] A third stuffer comprising a polynucleotide of sequence number 9, arranged upstream of the L-ITR.

[0132] In one embodiment, the vector system may comprise a vector system for recombinant AAV production, comprising the following vectors and one or more stuffers:

[0133] (i) (a) a first-1 vector comprising a helper virus gene required for AAV production;

[0134] (ii) (b) a first-second vector comprising the Rep gene of AAV and (c) the Cap gene of AAV; and

[0135] (iii) (d) A vector 1-3 containing a transgene arranged between ITRs.

[0136] In a specific embodiment, one or more stuffers in the vector system may be arranged in one of the first-1 vector, the first-2 vector, and the first-3 vector, and may be arranged specifically as follows:

[0137] (i) the first stuffer is arranged downstream of the R-ITR of the 1-3 vector, or is not arranged;

[0138] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the first-second vector, or is not arranged; and

[0139] (iii) The third stuffer is arranged upstream of the L-ITR of the 1-3 vector, or is not arranged.

[0140] In a specific embodiment, two or more stuffers in the vector system may be arranged in one of the first-1 vector, the first-2 vector, and the first-3 vector, and may be arranged specifically as follows:

[0141] (i) The first stuffer is arranged downstream of the R-ITR of the 1-3 vector;

[0142] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the first-second vector, or is not arranged; and

[0143] (iii) The third stuffer is arranged upstream of the L-ITR of the 1-3 vector.

[0144] In one embodiment, the vector system may comprise a vector system for recombinant AAV production, comprising the following vectors and one or more stuffers:

[0145] (i) (a) a second-1 vector comprising a helper virus gene required for AAV production; and

[0146] (ii) A second-2 vector comprising a transgene arranged between (b) the Rep gene of AAV, (c) the Cap gene of AAV, and (d) the ITR.

[0147] In a specific embodiment, one or more stuffers in the vector system may be arranged in one of the second-1 vector and the second-2 vector, and may be arranged specifically as follows:

[0148] (i) the first stuffer is arranged downstream of the R-ITR of the second-2 vector, or is not arranged;

[0149] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the second-2 vector, or is not arranged; and

[0150] (iii) The third stuffer is arranged upstream of the L-ITR of the second-second vector, or is not arranged.

[0151] In a specific embodiment, two or more stuffers in the vector system may be arranged in one of the second-1 vector and the second-2 vector, and may be arranged specifically as follows:

[0152] (i) The first stuffer is arranged downstream of the R-ITR of the second-second vector;

[0153] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the second-2 vector, or is not arranged; and

[0154] (iii) The third stuffer is arranged upstream of the L-ITR of the 2-2 vector.

[0155] In one embodiment, the vector system may comprise a vector system for recombinant AAV production, comprising the following vectors and one or more stuffers:

[0156] (i) a third-1 vector comprising (a) a helper virus gene required for AAV production, (b) a Rep gene of AAV, and (c) a Cap gene of AAV; and

[0157] (ii) (d) A third-2 vector comprising a transgene arranged between ITRs.

[0158] In a specific embodiment, one or more stuffers in the vector system may be arranged in one of the third-1 vector and the third-2 vector, and may be arranged specifically as follows:

[0159] (i) the first stuffer is arranged downstream of the R-ITR of the 3-2 vector, or is not arranged;

[0160] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the third-1 vector, or is not arranged; and

[0161] (iii) The third stuffer is arranged upstream of the L-ITR of the third-2 vector, or is not arranged.

[0162] In a specific embodiment, two or more stuffers in the vector system may be arranged in one of the third-1 vector and the third-2 vector, and may be arranged specifically as follows:

[0163] (i) The first stuffer is arranged downstream of the R-ITR of the 3-2 vector;

[0164] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the third-1 vector, or is not arranged; and

[0165] (iii) The third stuffer is arranged upstream of the L-ITR of the third-2 vector.

[0166] In one embodiment, the vector system may comprise a vector system for recombinant AAV production, comprising the following vectors and one or more stuffers:

[0167] (i) a 4-1 vector comprising (a) a helper virus gene required for AAV production, and (d) a transgene arranged between the ITRs; and

[0168] (ii) (b) A 4-2 vector comprising the Rep gene of AAV, and (c) the Cap gene of AAV.

[0169] In a specific embodiment, one or more stuffers in the vector system may be arranged in one of the 4-1 vector and the 4-2 vector, and may be arranged specifically as follows:

[0170] (i) the first stuffer is arranged downstream of the R-ITR of the 4-1 vector, or is not arranged;

[0171] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the 4-2 vector, or is not arranged; and

[0172] (iii) The third stuffer is arranged upstream of the L-ITR of the 4-1 vector, or is not arranged.

[0173] In a specific embodiment, two or more stuffers in the vector system may be arranged in one of the 4-1 vector and the 4-2 vector, and may be arranged specifically as follows:

[0174] (i) The first stuffer is arranged downstream of the R-ITR of the 4-1 vector;

[0175] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the 4-2 vector, or is not arranged; and

[0176] (iii) The third stuffer is arranged upstream of the L-ITR of the 4-1 vector.

[0177] In one embodiment, the vector system may comprise a vector system for recombinant AAV production, comprising the following vectors and one or more stuffers:

[0178] A fifth vector comprising (a) a helper virus gene required for AAV production, (b) a Rep gene of AAV, (c) a Cap gene of AAV, and (d) a transgene arranged between ITRs.

[0179] In certain embodiments, the vector system comprising the fifth vector may be an "AAV single vector system," whereby the fifth vector may be an "all-in-one vector" as a single nucleic acid molecule. Accordingly, the genes of (a), (b), (c), and (d) and one or more stuffers may be arranged in a single nucleic acid molecule.

[0180] In certain embodiments, one or more stuffers in the vector system may be arranged in the fifth vector, and may be arranged specifically as follows:

[0181] (i) the first stuffer is arranged downstream of the R-ITR of the fifth vector, or is not arranged;

[0182] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the fifth vector, or is not arranged; and

[0183] (iii) The third stuffer is arranged upstream of the L-ITR of the fifth vector, or is not arranged.

[0184] In certain embodiments, two or more stuffers in the vector system may be arranged in the fifth vector, and may be arranged specifically as follows:

[0185] (i) the first stuffer is arranged downstream of the R-ITR of the fifth vector;

[0186] (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the fifth vector, or is not arranged; and

[0187] (iii) The third stuffer is arranged upstream of the L-ITR of the fifth vector.

[0188] The above vector system or the vector included in the above system may further include additional components in addition to (a), (b), (c), and (d), which are essential components for recombinant AAV production.

[0189] The above additional elements may have one or more functions selected from the following:

[0190] (i) increasing gene expression levels;

[0191] (ii) regulating the timing of gene expression;

[0192] (iii) regulates gene transcription;

[0193] (iv) stabilize the transcript for translation;

[0194] (v) improving the productivity of recombinant AAV; and

[0195] (vi) Improves the %F / E capsid ratio of recombinant AAV.

[0196] In the above (vi), the improvement in the %F / E capsid ratio may be due to not only increasing the production of full capsids but also decreasing the production of empty capsids.

[0197] A person skilled in the art will appreciate that the nucleotide sequences of (a), (b), (c), and (d) above can each be operably linked to a suitable control sequence. For example, the nucleotide sequence can be operably linked to a transcription / translation control element, which can include, for example, a transcription / translation control signal, an origin of replication, a polyadenylation signal, an internal ribosome entry site (IRES), a furin, a 2A peptide, a promoter, and / or an enhancer.

[0198] The above vector system or the vector included in the above system may additionally include one or more selected from a stabilizer, a costabilizer, an activator, a coactivator, a repressor, a corepressor, an epigenetic regulatory element, a co-epigenetic regulatory element, a regulatory element, a co-regulatory element, etc.

[0199] The above vector system or the vector included in the above system may additionally comprise one or more expression control elements operably linked to any one of (a), (b), (c), and (d).

[0200] The above expression control elements may include at least one selected from a promoter, a transcription factor, an enhancer, a silencer, an insulator, an intron, a splicing donor and acceptor, an engineered splicing donor and acceptor, a riboswitch, an amino acid, a miRNA (microRNA), a shRNA (short hairpin RNA), a 5'- or 3'-UTR (untranslated region), a Kozak sequence, an initiation codon, a GOI codon, a signal peptide, a polyadenylation signal sequence, etc.

[0201] The term "enhancer" can refer to a regulatory region that promotes transcription of a gene.

[0202] Non-limiting examples of the above enhancers include the apolipoprotein (ApoE) HCR-1 and HCR-2 enhancers, and the mouse transthyretin (mTTR) enhancer.

[0203] The term "promoter" can refer to a sequence that drives gene expression.

[0204] The promoter may include, but is not limited to, an early promoter, a late promoter, a ubiquitous promoter, or a controllable promoter. Non-limiting examples of the promoter may include a P5 promoter, an SV40 promoter, a CMV (cytomegalovirus) promoter, a SFFV (spleen focus-forming virus) promoter, a RSV (Rous sarcoma virus) promoter, or a pEF (elongation factor 1-alpha) promoter.

[0205] The above "intron" sequence refers to a nucleotide sequence that is removed from the final gene product by RNA splicing. Using introns downstream of enhancer / promoter regions and upstream of cDNA inserts can increase gene expression levels.

[0206] Non-limiting examples of the above introns may include minute virus of mice (MVM) intron, beta-globin intron, FIX intron A, SV40 intron, modified SV40 intron, beta-actin intron, or human ctEF1 (C-terminal EF-1a) first intron.

[0207] The above "polyadenylation signal sequence", also called "Poly A sequence", refers to, for example, a polyadenylation signal sequence located 3' of a transgene, which allows the addition of a polyadenylation signal sequence to the end of a nascent mRNA during transcription. The polyadenylation signal sequence consists of up to 300 adenosine ribonucleotides that protect the mRNA from enzymatic degradation and aid in translation.

[0208] Non-limiting examples of the above polyadenylation signal sequences may include the SV40 polyadenylation signal, the bovine growth hormone (BGH) polyadenylation signal, the rabbit globin polyadenylation signal, and the like.

[0209] The above vector system or the vector included in the above system may further include a promoter independently operably linked to (a), (b), (c), or (d). The promoter may control the expression level and timing of each gene. Controlling the expression level of each gene within the vector may affect productivity and quality. Therefore, by including the promoter, the productivity of recombinant AAV can be increased, the %Full capsid can be increased, and the %Empty capsid can be decreased, thereby producing high-quality recombinant AAV.

[0210] The above vector system or the vector included in the above system may further comprise a promoter operably linked to (a). The promoter may be a helper gene promoter derived from a helper virus or a variant thereof. The variant may be an engineered promoter, a truncated promoter, or a promoter having a lower GC content than a wild-type promoter.

[0211] The vector system or the vector included in the system may further comprise a promoter operably linked to (b). The promoter may be a Rep gene promoter or a variant thereof. The variant may be an engineered promoter, a truncated promoter, or a promoter having a lower GC content than the wild-type promoter. In one specific example, the promoter may be, but is not limited to, a P5 promoter. The P5 promoter may comprise or consist of SEQ ID NO: 6.

[0212] The above vector system or the vector included in the above system may further comprise a promoter operably linked to (c). The promoter may be a Cap gene promoter or a variant thereof. The variant may be an engineered promoter, a truncated promoter, or a promoter having a lower GC content than a wild-type promoter.

[0213] The above vector system or the vector included in the above system may further comprise an expression control element operably linked to (d). In one embodiment, the expression control element may comprise one or more selected from an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

[0214] The above expression regulatory element may be a tissue-specific expression regulatory element. A tissue-specific expression regulatory element is active in a specific cell, tissue, or organ. The specific cell, tissue, or organ may include the liver, brain, central nervous system, spinal cord, eye, retina, bone, muscle, lung, pancreas, heart, kidney, etc. For example, 'A tissue-specific' refers to preferential or dominant in vivo expression of a specific gene in tissue A compared to other tissues. 'A tissue-specific expression' may mean that greater than or equal to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of all expression of a specific gene occurs within tissue A of an individual.

[0215] The tissue-specific expression regulatory element may include at least one selected from an enhancer and a promoter. The tissue-specific expression regulatory element may include an enhancer and a promoter.

[0216] In one specific embodiment, the vector system or the vector included in the system may further comprise an enhancer operably linked to (d). The enhancer may be a liver tissue-specific enhancer. The enhancer may be an mTTR enhancer or a variant thereof.

[0217] In one embodiment, the vector system or the vector included in the system may further comprise a promoter operably linked to (d). The promoter may be a liver tissue-specific promoter. Non-limiting examples of liver tissue-specific promoters include the human alpha 1-antitrypsin (hAAT) promoter, albumin, the hepatitis B virus core promoter, alpha-fetoprotein (AFP), the mTTR promoter, and the like. The promoter may be the mTTR promoter or a variant thereof.

[0218] In one specific embodiment, the enhancer and promoter may be a variant of the mTTR enhancer and promoter. The enhancer and promoter may have 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the sequence of the mTTR enhancer and promoter. The variant of the mTTR enhancer and promoter may have nucleotides at positions 1 to 22 and 124 to 138 deleted from the nucleotide sequence of the wild-type mTTR enhancer and promoter, and ACAGGA inserted after position 361. The variant of the mTTR enhancer and promoter may comprise or consist of SEQ ID NO: 11.

[0219] In one specific embodiment, the vector system or a vector included in the system may further comprise an intron operably linked to (d). The intron may be a modified SV40 intron. The modified SV40 intron may comprise or consist of SEQ ID NO: 12.

[0220] In one embodiment, the vector system or the vector included in the system may further comprise a polyadenylation signal sequence operably linked to (d). The polyadenylation signal sequence may be a rabbit globin poly A signal sequence. The rabbit globin polyadenylation signal sequence may be a partial sequence of the rabbit beta-globin gene (GenBank No. V00882.1). The rabbit globin polyadenylation signal sequence may comprise or consist of SEQ ID NO: 14.

[0221] The above vector system or the vector included in the above system may additionally include a multi-cistronic element, a bi-cistronic element, a transposon, a transposase, etc.

[0222] The above vector system or the vector included in the above system may include a vector backbone. The "vector backbone" refers to a non-genomic portion, which is important for cloning and amplification of the vector, a process necessary for propagation and production of recombinant viruses, but is not packaged or encapsidated into viral particles. The vector backbone may include one or more (one or two) selected from an origin of replication and a selection marker. The vector backbone may not include an origin of replication.

[0223] The vector system or the vector included in the system may not include a replication origin, or may additionally include a replication origin. The replication origin may be a known replication origin or a variant thereof. The replication origin may be, but is not limited to, a pUC origin, a pBR322 origin, a pMB1 origin, a pSC101 origin, a p15A origin, or a synthetic origin.

[0224] In one embodiment, the vector system or a vector included in the system may comprise a pBR322 origin.

[0225] The vector system or the vector included in the system may further comprise a selectable marker or reporter that can provide for selection or identification of a host cell into which the vector system has been introduced. Selectable markers or reporters are known in the art. Non-limiting examples of the selectable marker include genes that provide resistance to ampicillin, streptavidin, kanamycin, hygromycin, neomycin, puromycin, blasticidin, zeocin, and the like. Non-limiting examples of the reporter include luciferase, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and the like. In one embodiment, the vector system or the vector included in the system may further comprise a selectable marker.

[0226] In one specific embodiment, the vector system or the vector included in the system may further comprise at least one of neomycin and kanamycin.

[0227] "Gene amplification" may refer to the process by which a particular DNA sequence (i.e., a gene) in a genome is replicated disproportionately relative to other sequences in the genome, such that the amplified DNA sequence is present in a higher copy number than originally present. As used herein, "amplified" or "amplification" may refer to a gene or nucleic acid sequence that is present in two or more copies in a host cell due to gene amplification.

[0228] An "amplifiable selectable marker gene" may mean a gene that allows amplification of that gene under appropriate growth conditions.

[0229] In one specific example, the vector system comprising the fifth vector (AAV single vector system) comprises a single nucleic acid molecule, in which (a), (b), (c), and (d) may be randomly arranged in the 5' to 3' direction or the 3' to 5' direction. In which (a), (b), (c), and (d) may be arranged in any order in the 5' to 3' direction or the 3' to 5' direction. In which (a), (b), (c), and (d) may be arranged in any order in the 5' to 3' direction. In which (a), (b), (c), and (d) may be independently arranged in any order in the forward orientation or the reverse orientation. The forward orientation may mean that the gene is inserted in the 5' to 3' direction in the 5' to 3' strand ((+) strand). The above reverse orientation may mean that the gene is inserted in the 3' to 5' direction in the 5'→3' strand.

[0230] In certain specific embodiments, in the nucleic acid molecule, (a), (b), (c), and (d) may be arranged in the following order, wherein (a), (b), (c), and (d) may each independently be forward oriented or reverse oriented:

[0231] 1) (d) - (b) - (c) - (a);

[0232] 2) (d) - (c) - (b) - (a);

[0233] 3) (d) - (a) - (b) - (c);

[0234] 4) (d) - (a) - (c) - (b);

[0235] 5) (b) - (c) - (d) - (a);

[0236] 6) (c) - (b) - (d) - (a);

[0237] 7) (b) - (c) - (a) - (d);

[0238] 8) (c) - (b) - (a) - (d);

[0239] 9) (a) - (b) - (c) - (d);

[0240] 10) (a) - (c) - (b) - (d);

[0241] 11) (a) - (d) - (b) - (c); or

[0242] 12) (a) - (d) - (c) - (b).

[0243] In certain embodiments, (a) is oriented normally; and (b), (c), and (d) can each independently be oriented normally or reversely.

[0244] In certain embodiments, (a) is oriented normally; (b) and (c) are both oriented normally, or both are reversely oriented; and (d) can be either oriented normally or reversely oriented.

[0245] In certain embodiments, (a), (b), and (c) are oriented normally; and (d) may be oriented normally or reversely.

[0246] In a specific embodiment, (a), (b), (c), and (d) are arranged in the order of (a) - (b) - (c) - (d), wherein (a), (b), (c), and (d) can each independently be oriented normally or in reverse orientation.

[0247] In a specific embodiment, (a), (b), (c), and (d) are arranged in the order of (a) - (b) - (c) - (d), wherein (a), (b), (c), and (d) may all be oriented. Here, in (a), E2a may be reversely oriented, E4 may be normal oriented, and VA may be reversely oriented.

[0248] In certain embodiments, the vector backbone may be arranged in any order from 1) to 12). In other embodiments, the vector backbone may be arranged after the last order from 1) to 12), or before the first order.

[0249] In certain embodiments, the vector backbone may be arranged between (a) and (b). Accordingly, the sequence of components in the nucleic acid molecule may be (a) - vector backbone - (b) - (c) - (d).

[0250] The above vector backbone may include one or more selected from a replication origin and a selection marker.

[0251] In one specific example, a vector according to a vector system (AAV single vector system) including the fifth vector can improve the %Full / Empty capsid ratio of the produced recombinant AAV through a combination of optimized arrangements and orientations of components (e.g., (a), (b), (c), (d), backbone). In particular, through the combination of the optimized arrangements and orientations, it is possible to not only increase the %Full capsid but also decrease the %Empty capsid.

[0252] In certain embodiments, the components in the nucleic acid molecule may be arranged in the following order: (a) - vector backbone - (b) - (c) - L-ITR - enhancer and promoter - intron - transgene - polyadenylation signal sequence - R-ITR.

[0253] In certain embodiments, the components in the nucleic acid molecule may be arranged in the following order: first stuffer - (a) - vector backbone - second stuffer - (b) - (c) - third stuffer - L-ITR - enhancer and promoter - intron - transgene - polyadenylation signal sequence - R-ITR.

[0254] In certain embodiments, the components in the nucleic acid molecule may be arranged in the following order: first stuffer - (a) - vector backbone - (b) - (c) - third stuffer - L-ITR - enhancer and promoter - intron - transgene - polyadenylation signal sequence - R-ITR.

[0255] The above nucleic acid molecules, plasmids and vectors can be prepared by any suitable technique, which techniques are well known in the art.

[0256] In one specific example, the vector for producing a recombinant AAV included in the vector system (AAV single vector system) comprising the fifth vector comprises all components necessary for producing a recombinant AAV (i.e., (a), (b), (c), and (d)) linked within a single molecule, so that each component can be introduced into a host cell at the same ratio. A host cell into which a single nucleic acid molecule has been introduced can exhibit balanced gene expression. Balanced gene expression can enhance recombinant AAV productivity and / or increase the %F / E capsid ratio of the recombinant AAV. The enhancement of recombinant AAV productivity can reduce unit production costs. Specifically, the enhancement of recombinant AAV productivity can improve the overall yield of the purification process. In addition, since the all-in-one vector system requires the production of only one single vector instead of the independent production of three conventional vectors, the cost of raw materials can be reduced. In addition, since the above all-in-one vector system only requires the introduction of a single vector without the need to introduce three vectors separately, the amount of individual vector input can be reduced by more than 50%.

[0257] In one embodiment, compared to a control triple transfection comprising a helper vector, a Rep-Cap expression vector, and a GOI vector, an all-in-one vector containing a stuffer was found to exhibit improved recombinant AAV productivity when using the same amount of plasmid DNA (pDNA).

[0258] In one embodiment, the recombinant AAV produced by the all-in-one vector including the stuffer was confirmed to exhibit GOI expression and activity at an equivalent level or greater than that of the recombinant AAV produced by the control triple-transfection method comprising a helper vector, a Rep-Cap expression vector, and a GOI vector, or by the all-in-one vector control not including the stuffer. Therefore, the all-in-one vector including the stuffer can produce recombinant AAV at an equivalent level or greater than that of the recombinant AAV produced by the conventional triple-transfection method or the recombinant AAV produced by the all-in-one vector not including the stuffer in terms of transduction ability and GOI functionality.

[0259]

[0260] Another aspect provides a method for producing a recombinant adeno-associated virus (AAV). The method may utilize a vector system for producing a recombinant AAV according to one aspect.

[0261] The method comprises the steps of introducing a vector system for producing recombinant AAV according to one aspect into a host cell; and the step of isolating recombinant AAV from the host cell.

[0262] The host cell may be a mammalian cell. The mammalian cell may include a cell derived from any organ or tissue of a human, mouse, rat, hamster, monkey, rabbit, donkey, horse, sheep, cow, or ape. The mammalian cell may be selected from, but is not limited to, HEK293 cells, CHO cells, Jurkat cells, KS62 cells, PerC6 cells, HeLa cells, MDCK cells, C127 cells, A549 cells, Vero cells, WI38 cells, MRC5 cells, HT1080 cells, or derivatives or functional equivalents thereof.

[0263] In one specific embodiment, the mammalian cell may be selected from HEK293 cells, HEK293F cells, HEK293T cells, and cells derived therefrom. The HEK293 cells are a human embryonic kidney 293 cell line commonly used in biotechnology. 'HeK293 cells, HEK293F cells, or cells derived from HEK293T cells' refers to cells derived from the parental cell lines HEK293 cells, HEK293F cells, or HEK293T cells, and may include both commercially available cells and cells to be developed in the future. Exemplary cells derived from the HEK293 parental cell line include Expi293F (manufactured by ThermoFisher), HEK293F (manufactured by ThermoFisher), HEK293.2 (ATCC), etc. Exemplary cells derived from the HEK293T parental cell line include HEK293FT (manufactured by ThermoFisher). Exemplary cells derived from the HEK293F parental cell line include Viral Production Cells 1.0 (VPC1.0) (manufactured by ThermoFisher), Viral Production Cells 2.0 (VPC2.0) (manufactured by ThermoFisher). VPC2.0 is a clonal cell line derived from the HEK293F parental cell line and is a host cell suitable for AAV production. In certain embodiments, the mammalian cell may be a HEK293F cell or a cell derived therefrom.

[0264] The host cell may be an insect cell. The insect cell may include a cell derived from the tropical armyworm (Spodoptera frugiperda) or the cabbage moth (Trichoplusia ni). The insect cell may be selected from, but is not limited to, Sf9 cells, Sf21 cells, TN-5B1-4 cells, High Five cells, or derivatives or functional equivalents thereof.

[0265] The above introduction can be used without limitation as long as it is a known method that can insert a vector system for recombinant AAV production or a vector included in the system into a host cell. The introduction may be by transfection, transformation, or transduction. The terms "transfection," "transformation," and "transduction" may be used to describe the insertion of a non-mammalian vector or a viral vector into a target cell. Insertion of a vector is generally referred to as transformation for bacterial cells and transfection for eukaryotic cells, and insertion of a viral vector is also referred to as transduction. A person skilled in the art can introduce the vector into a host cell using a known method. Non-limiting examples of such introduction include, but are not limited to, physical methods (e.g., electroporation, cell compression, sonication, optical transfection, protoplast fusion, impalfection, magnetofection, gene gun, or particle bombardment), chemical reagents (e.g., calcium phosphate, highly branched organic compounds, or cationic polymers), or cationic lipids (e.g., lipofection). Such cationic polymers include, but are not limited to, polyethyleneimine (PEI). Transfection methods may additionally require contacting cells with a solution of plasmid DNA, followed by growth and selection using marker gene expression.

[0266] The above method may further include a step of culturing cells after the introducing step.

[0267] The above culture may involve culturing host cells under conditions that allow for the production of recombinant AAV. Appropriate culture methods are well known to those skilled in the art. For example, the cells may be cultured in suspension and / or under animal component-free conditions.

[0268] The above method may further include, after the culturing step, a step of selecting cells into which the vector has been introduced using a selection marker.

[0269] The separation can be performed by a known method. In one specific example, the separation can be performed using centrifugation or chromatography.

[0270] The above centrifugation method may be, but is not limited to, a cesium chloride (CsCl)-based ultra-high-speed centrifugation method.

[0271] The above chromatography may be at least one selected from, but is not limited to, affinity chromatography, ion exchange chromatography, column chromatography, gel-filtration chromatography, thin-layer chromatography, radial flow chromatography, interference chromatography, and reverse phase chromatography.

[0272] In one specific example, when the method utilizes an "all-in-one vector" or an "AAV single vector system," the method can be easily scaled up to industrial production because it only requires a single transfection of a host cell with a vector system for producing a recombinant AAV with excellent recombinant AAV productivity. According to the method, unlike the conventional triple transfection method using three vectors, only a single vector is required to be introduced, thereby simplifying the recombinant AAV production process and reducing costs. In addition, unlike the triple transfection method in which the ratios of the three vectors injected into the cell cannot be precisely controlled, according to the method according to the above aspect, since each component is integrated into a single vector, each component can be introduced at the same ratio. Accordingly, the productivity of the recombinant AAV can be improved. The recombinant AAV produced by the method can exhibit increased transduction ability and GOI functionality compared to the recombinant AAV produced by triple transfection.

[0273]

[0274] Another aspect provides a recombinant AAV produced by the above method.

[0275] The recombinant AAV described above may have a low impurity content. Therefore, the recombinant AAV may have excellent quality and safety.

[0276] In one specific embodiment, the recombinant AAV may be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV).

[0277] In one specific example, the recombinant AAV may be a self-complementary AAV (scAAV). Accordingly, the recombinant AAV may increase the expression level of the GOI.

[0278] The above recombinant AAV may have both increased expression and activity of GOI.

[0279] The above recombinant AAV can be used in a method of gene therapy.

[0280] The above recombinant AAV can be used for mass production of gene therapy clinical materials for treating patients.

[0281] The above recombinant AAV comprises an AAV capsid and genome.

[0282] The AAV capsid may be a wild-type capsid or a variant thereof. The AAV capsid may be derived from any AAV serotype. The AAV capsid may be any one AAV capsid selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh39, AAVrh64R1, and AAVrh74. The AAV serotype may also include other AAV serotypes currently known or later discovered. The AAV serotype may also include artificial AAV serotypes. The variant may include an engineered capsid.

[0283] In one specific example, the AAV capsid may be a wild-type AAV8 capsid.

[0284] The above AAV capsid may comprise a VP1 protein, a VP2 protein, and a VP3 protein.

[0285] The "genome" of the above AAV refers to the sequences that are ultimately packaged or encapsidated to form viral particles.

[0286] The genome may be linear single-stranded DNA.

[0287] The above genome contains a transgene.

[0288] The transgene may be arranged between ITRs. The transgene may be arranged between L-ITRs and R-ITRs.

[0289] The ITR may be derived from any AAV serotype. The ITR may be derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh39, AAVrh64R1, and AAVrh74. The AAV serotype may also include other AAV serotypes currently known or later discovered. The AAV serotype may also include artificial AAV serotypes. In one specific embodiment, the ITR may be derived from AAV2.

[0290] The above ITR may be a wild-type ITR or a mutant thereof.

[0291]

[0292] Another aspect provides a composition comprising a recombinant AAV produced by the above method.

[0293] The above composition may be a pharmaceutical composition.

[0294] The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0295] The composition may be used for gene therapy. The composition may be a composition for therapeutic gene delivery for gene therapy.

[0296] The term "gene therapy" refers to the use of genes to treat or prevent disease. Recombinant AAVs, which deliver therapeutic genes into cells, can be used as gene therapy. Diseases for which gene therapy is applicable include, but are not limited to, diseases caused by defects in a single gene.

[0297] The above pharmaceutically acceptable carrier is used to mean an excipient, diluent, or adjuvant. The carrier may be one suitable for delivering recombinant AAV into a living body. Specifically, the carrier may be selected to be suitable for formulation as a parenteral formulation (e.g., an injectable formulation). For example, the carrier may be selected to be suitable for formulation as an intravenous formulation. The carrier may be an aqueous solution, such as water or a buffered saline solution.

[0298] The pharmaceutical composition may be prepared in any dosage form according to conventional methods. The pharmaceutical composition may be formulated in a form suitable for delivering recombinant AAV to a subject. The composition may be formulated in an aqueous solution, for example, in water or a buffered saline solution. The pharmaceutical composition may be formulated in an injectable dosage form suitable for administration by any suitable route, such as intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, or intrathecal. The pharmaceutical composition may be prepared in a systemic dosage form or a topical dosage form.

[0299] The pharmaceutical composition, when used for gene therapy for a specific disease, may further comprise an additional second therapeutic agent having a preventive or therapeutic effect for the disease. The pharmaceutical composition may be a single composition or individual compositions.

[0300] The above pharmaceutical composition may contain the recombinant AAV in a pharmaceutically effective amount. The effective amount can be appropriately selected by a person skilled in the art depending on the individual.

[0301]

[0302] Another aspect provides a gene therapy method using a recombinant AAV produced by the above method.

[0303] Specifically, the above aspect provides a gene therapy method comprising the step of administering a recombinant AAV produced by the above method or a pharmaceutical composition comprising the same to a subject in need thereof.

[0304] The subject may be an individual requiring expression of a transgene delivered by a recombinant AAV. The subject may be an individual suffering from, or at high risk of suffering from, a disease amenable to gene therapy. The subject may be an individual suffering from, or at high risk of suffering from, a disease amenable to treatment by expression of a transgene delivered by a recombinant AAV.

[0305] The above object may be a mammal. The mammal may include, but is not limited to, a human, a mouse, a rat, a hamster, a monkey, a rabbit, a donkey, a horse, a sheep, a cow, and the like.

[0306] The route of administration can be determined by those skilled in the art and may include, for example, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. Two or more routes of administration may be combined, if necessary. In one embodiment, the administration may be intravenous.

[0307] The above administration may be administered in an amount sufficient to infect the subject and in an amount sufficient to provide a sufficient level of introduction and expression of the transgene.

[0308] The term "effective amount" may mean "therapeutically effective amount" and refers to the dosage administered to achieve a therapeutic effect.

[0309] The dose to achieve a therapeutic effect, e.g., the dose in vector genomes per kilogram of body weight (vg / kg), may vary based on several factors, including the route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease being treated, any host immune response by the viral vector, the host immune response to the heterologous polynucleotide or the expression product (protein), and the stability of the expressed protein. One skilled in the art can determine a range of rAAV doses to treat a patient with a particular disease or disorder based on the factors mentioned above as well as other factors. Typically, the dose is 1x10 per kilogram of body weight of the subject to achieve a therapeutic effect. 7　 or 1x10 8　 or 1x10 9　 or 1x10 10　 or 1x10 11　 or 1x10 12　 or 1x10 13　 or 1x10 14　 or 1x10 15　 The vector genome (vg / kg) can be in the range of . The expression level of the heterologous polynucleotide can be monitored to determine the method and frequency of administration.

[0310]

[0311] Another aspect provides the use of the vector for producing said recombinant AAV or the recombinant AAV produced by said vector for the manufacture of a medicament for gene therapy.

[0312]

[0313] Duplicate content is omitted in consideration of the complexity of this specification, and terms not otherwise defined herein have the meanings commonly used in the technical field to which the present invention belongs.

[0314] A vector system for producing recombinant AAV according to one aspect can reduce impurities or impurities resulting from reverse packaging during recombinant AAV production by including a stuffer. Furthermore, the vector system can produce recombinant AAV with the same or improved productivity and efficiency compared to a vector system that does not include a stuffer.

[0315] According to a method for producing recombinant AAV according to one aspect, recombinant AAV can be produced by introducing a single vector, thereby reducing production costs. Specifically, the single vector system, which is an example of the vector system, has all components linked on a single molecule, so they can be introduced into cells at the same ratio, and thus, cells transfected with the vector can exhibit balanced gene expression. Balanced gene expression can improve the productivity of recombinant AAV and increase the %Full capsid. The increased productivity of recombinant AAV can reduce unit production costs and improve the overall yield of the purification process. Furthermore, unlike the existing triple transfection method that produces three vectors independently, the cost of raw materials can be reduced because a single vector is produced. Furthermore, since all components are combined into a single vector, the amount of single vector input used for transfection can be reduced by more than 50%.

[0316] Recombinant scAAV produced using a vector system for recombinant AAV production according to the daily aspect can have increased expression and activity of therapeutic genes, and has excellent safety due to reduced impurities, so it can be applied in various fields of gene therapy.

[0317] Figure 1 is a vector map of the All-in-One stuffer-scAAV-FIX vector according to one embodiment.

[0318] Figure 2 is a vector map of All-in-One stuffer-1 to 6 vectors according to one embodiment.

[0319] Figure 3 is a graph showing the FIX expression level (ng / mL) of recombinant AAV produced by the all-in-one stuffer vector (AiO-stuffer-SBEFIX), all-in-one vector (AiO-SBEFIX), or triple vector (3TF-SBEFIX).

[0320] Figure 4 is a graph showing the FIX activity (mIU / mL) of recombinant AAV produced by the all-in-one stuffer vector (AiO-stuffer-SBEFIX), the all-in-one vector (AiO-SBEFIX), or the triple vector (3TF-SBEFIX).

[0321] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0322]

[0323] Example 1. Production of an all-in-one vector including a stuffer.

[0324] An all-in-one stuffer vector was constructed by inserting three types of non-functional DNA sequences as stuffers into an all-in-one vector for recombinant AAV production containing the GOI (gene-of-interest) and Rep, Cap, Helper, and ITR genes, which are components necessary for adeno-associated virus (AAV) production.

[0325] The three types of non-functional DNA sequences used were MAR (Matrix Attachment Region), UCOE (Ubiquitous Chromatin Opening Element), and CCE (Chromatin Control Element). As the GOI, the gene encoding codon-optimized blood coagulation factor IX (Factor IX, FIX) was used as an example.

[0326] Specifically, the genetic sequence information of each component of the all-in-one stuffer vector was obtained based on existing literature.

[0327] Helper E2A, E4, and VA genes were obtained from Adenovirus 2 (Gene Bank Accession No. NC_001405.1).

[0328] Rep, and L-ITR and R-ITR genes were obtained from Adeno-associated virus serotype 2 (Gene Bank Accession No. NC_001401.2). For L-ITR, a L-ITR mutant lacking the terminal resolution site (trs) was used by deleting the D region from the AAV2 wild-type L-ITR. By using the L-ITR mutant lacking trs and the wild-type R-ITR, self-complementary AAV (scAAV) was produced by the all-in-one vector.

[0329] The Cap gene was obtained from Adeno-associated virus serotype 8 (Gene Bank Accession No. NC_006261.1).

[0330] The GOI (gene-of-interest) used as a transgene used the codon-optimized sequence of the FIX mutant.

[0331] The MAR gene was obtained from the Homo sapiens beta-globin cluster gene, enhancers with repeat region (Gene Bank Accession No. L22754.1).

[0332] The UCOE gene was obtained from Homo sapiens heterogeneous nuclear ribonucleoprotein A2 / B1 (HNRNPA2B1), RefSeqGene on chromosome 7 (Gene Bank Accession No. NG_029680.1).

[0333] The CCE gene was obtained from human DNA sequence from clone RP3-368A4 on chromosome Xq12, complete sequence (Gene Bank Accession No. Z83843.3).

[0334] Based on the sequence information of the corresponding genes of each component, primers were designed and PCR amplified to obtain DNA fragments, which were then inserted into the vector one copy at a time. The expression vector was constructed using molecular genetic techniques according to Ausubel et al. (Ausubel et al. (ed.), J. Wiley & Sons, 1997, Curr. Protocols of Molecular Biology.). The primers used for gene and PCR amplification were synthesized and manufactured by Macrogen Co., Ltd. (Seoul, Korea). The genetic sequence of the cloned expression vector was confirmed by request to Macrogen Co., Ltd. (Seoul, Korea).

[0335] The produced vector was named “All-in-One stuffer-scAAV-FIX vector” (also abbreviated as “Stuffer-scAIO-FIX”).

[0336] Figure 1 is a vector map of the All-in-One stuffer-scAAV-FIX vector according to one embodiment.

[0337] The All-in-One stuffer-scAAV-FIX vector of FIG. 1 comprises each component in the following order: a first stuffer, a helper gene, a vector backbone, a second stuffer, a promoter operably linked to Rep, a Rep gene, a Cap gene, a third stuffer, an L-ITR, an enhancer and promoter, a large T antigen (fragment), a GOI, a polyadenylation signal sequence, and an R-ITR.

[0338] In the All-in-One stuffer-scAAV-FIX vector of FIG. 1, MAR (SEQ ID NO: 1) is used as a first stuffer; E2A, E4, and VA of Adenovirus 2 (SEQ ID NOs: 2 to 4) are used as helper genes; UCOE (SEQ ID NO: 5) is used as a second stuffer; P5 promoter (SEQ ID NO: 6) is used as a promoter operably linked to Rep; Rep2 (SEQ ID NO: 7) is used as a Rep gene; wild-type AAV8 Cap (SEQ ID NO: 8) is used as a Cap gene; CCE (SEQ ID NO: 9) is used as a third stuffer; AAV2 L-ITR variant with the D region deleted from the wild-type sequence (SEQ ID NO: 10); variant of mTTR enhancer / promoter (SEQ ID NO: 11); large T antigen (fragment) (SEQ ID NO: 12); codon-optimized FIX variant (SEQ ID NO: 13) is used as a GOI; Rabbit globin polyadenylation signal sequence (rabbit globin pA) (SEQ ID NO: 14); and wild-type R-ITR of AAV2 (SEQ ID NO: 15) were used.

[0339] The mutant of mTTR enhancer / promoter is a liver tissue-specific enhancer / promoter, and a sequence (330 bp) was used in which the sequences at positions 1 to 22 and 124 to 138 (37 bp) were deleted from the wild-type mTTR enhancer / promoter (361 bp), and ACAGGA (6 bp) was inserted after position 361.

[0340] The sequences used to construct the All-in-One stuffer-scAAV-FIX vector are shown in Table 2 below.

[0341] Sequence number Name Description Size 1 MAR First stuffer: human globin MAR (Matrix Attachment Region) 3,009 bp 2 Adeno E2 E2 of AAV2 A5,336 bp 3 Adeno E4 E4 of Adenovirus 2 3,200 bp 4 Adeno VAA VA745 bp of AAV2 5 UCOE Second stuffer: Part of the UCOE located in human HNRPA2 B1 (heterogeneous nuclear ribonucleoproteins A2 / B1 gene) 1,405 bp 6 Promoter operably linked to Rep 164 bp 7 AAV2 Rep Rep of AAV2 (Rep78, Rep68, Rep52, and Rep40) 1,866 bp 8 wtAAV Cap 8 Wild-type Cap2 of AAV8 217 bp 9 CCE Third stuffer 3,556 bp10L-ITR (trs deletion) L-ITR mutant with deletion of the D region in the L-ITR of AAV2121 bp11MtTR enhancer / promoter mutant37 bp deletion and 6 bp insertion in the mTTR enhancer / promoter330 bp12Modified SV40 intronModified SV40 intron94 bp13FIXCodon-optimized Factor IX mutant (T148A / R338L)1389 bp14Rabbit globin pARabbit globin polyadenylation signal sequence127 bp15Wt R-ITR of AAV2145 bp

[0342]

[0343] Example 2. Production of vectors according to positional combinations of stuffer sequences.

[0344] An all-in-one stuffer vector was constructed by inserting one or two of three types of non-functional DNA sequences as stuffers into various positions of an all-in-one vector for recombinant AAV production containing the GOI (gene-of-interest) and Rep, Cap, Helper, and ITR genes, which are components necessary for adeno-associated virus (AAV) production.

[0345] The three types of non-functional DNA sequences used were MAR (Matrix Attachment Region), UCOE (Ubiquitous Chromatin Opening Element), and CCE (Chromatin Control Element). As the GOI, the gene encoding codon-optimized blood coagulation factor IX (Factor IX, FIX) was used as an example.

[0346] Figure 2 is a vector map of the stuffer introduction location.

[0347] Table 3 shows the configuration of an all-in-one stuffer vector manufactured with different numbers and locations of stuffer introductions.

[0348] No. Name Stuffer insertion number Stuffer insertion position / sequenceSize (bp)Stuffer 1Stuffer 2Stuffer 3(3'of R-ITR)(5'of p5 promoter)(5'of L-ITR)1All-in-One stuffer-11MARXX21,3492All-in-One stuffer-21XUCOEX19,7453All-in-One stuffer-31XXCCE21,8964All-in-One stuffer-42MARUCOEX22,7625All-in-One stuffer-52MARXCCE24,9136All-in-One stuffer-62XUCOECCE23,309

[0349] Table 4 shows the composition of vectors produced with different numbers and positions of stuffer introductions, including Rep / Cap and Helper or Rep / Cap and GOI.

[0350] No NameVector ClassificationStuffer insertion locationSize (kb)Stuffer 1Stuffer 2Stuffer 3(3' of R-ITR)(5' of p5 promoter)(5' of L-ITR)1GR-1GOI+Rep / capMARUCOECCE17.22GR-2GOI+Rep / capMARXCCE15.63GR-3GOI+Rep / capXXX9.24RH-1Rep / cap+HelperN / AUCOEN / A17.45RH-2Rep / cap+HelperN / AXN / A16

[0351] Table 5 is a composition of vectors that contain one of Rep / Cap, Helper, and GOI, and are produced with different numbers and locations of stuffer introductions.

[0352] No NameVector ClassificationStuffer Insertion PositionSize(kb)Stuffer 1Stuffer 2Stuffer 3(3' of R-ITR)(5' of p5 promoter)(5' of L-ITR)1GOI-1GOI vectorMARN / ACCE11.12GOI-2GOI vectorXN / AX4.53Rep / cap-1Rep / Cap vectorN / AUCOEN / A8.34Rep / cap-2Rep / Cap vectorN / AXN / A6.9

[0353]

[0354] Example 3. AAV production of vectors containing stuffers

[0355] AAV production of a vector system for AAV production including stuffers was performed at flask scale.

[0356] The cells used in this example were suspension HEK293F cells, and the experimental conditions were 37°C, 8% CO2, and 80% humidity. The medium used to evaluate AAV production was SFM (Serum-free media).

[0357] AAV production was performed using triple transfection (GOI vector, Rep / Cap vector, Helper vector), dual transfection (GOI-Rep / Cap vector, Rep / Cap-Helper vector), and all-in-one vector.

[0358] The All-in-One stuffer-scAAV-FIX vector of Example 1 and the vectors constructed for stuffer evaluation in Tables 3, 4, and 5 were used. A vector without a stuffer insert was used as a control. The vector systems according to the vector combination for production are as shown in Table 6.

[0359] Platform NameStuffer 1Stuffer 2Stuffer 33' of R-ITR5' of p5promoter5' of R-ITRAiO VectorTransfectionAiO-1XN / AXAiO-2MARN / AXAiO-3XUCOEXAiO-4XN / ACCEAiO-5MARUCOEXAiO-6MARN / ACCEAiO-7XUCOECCEAiO-8MARUCOECCEDual Transfection (RC-GOI / Helper)DGR-1XN / AXDGR-2MARN / ACCEDGR-3MARUCOECCEDual Transfection (RC-Helper / GOI)DRH-1XN / AXDRH-2MARN / ACCEDRH-3MARUCOECCETriple TransfectionTRI-1XN / AXTRI-2MARN / ACCETRI-3MARUCOECCE

[0360]

[0361] Example 4. Analysis of DNA impurities in recombinant scAAV produced by an all-in-one vector containing a stuffer.

[0362] In order to determine the level of DNA impurity suppression according to the inclusion of a stuffer in the All-in-One stuffer vector, the following experiments were performed.

[0363] In this example, the All-in-One stuffer-scAAV-FIX vector of Example 1 was used as the experimental group. As a control group, the All-in-One scAAV-FIX vector, which is identical to the All-in-One stuffer-scAAV-FIX vector of Example 1 except that it does not contain a stuffer, was used; or a triple vector for triple transfection, i.e., a GOI vector, a Rep / Cap vector, and a Helper vector (pALD-HELP, Aldevron) was used.

[0364] Specifically, both the experimental and control groups were transiently transfected with PEI (Polyethylenimine) at 3E06 cells / mL (30 mL, 125 mL flask). 72 hours after transfection, cells were lysed to extract recombinant AAV. All extracted recombinant AAV was first purified using the AKTA purification system, and then separated using CsCl ultracentrifugation to obtain high-purity recombinant AAV. DNA within the AAV particles was isolated from the obtained AAV using the QIAquick® Gel Extraction Kit. Genome analysis of the isolated DNA was performed at Macrogen (Seoul, Korea) using the PacBio SequelII (long-read) sequencing or Novaseq (short-read) sequencing platform. In addition, genome analysis was performed at Advanced Medicine Partners (AMP) using the Nanopore (short-read) sequencing platform, and the results were obtained.

[0365] Table 7 shows the results of genome analysis of recombinant AAV produced by the all-in-one stuffer vector (AiO-stuffer-SBEFIX), all-in-one vector (AiO-SBEFIX), or triple vector (3TF-SBEFIX) using the PacBio SequelII sequencing platform.

[0366] Table 8 shows the results of genome analysis of recombinant AAV produced by the all-in-one stuffer vector (AiO-stuffer-SBEFIX), all-in-one vector (AiO-SBEFIX), or triple vector (3TF-SBEFIX) using the Novaseq sequencing platform.

[0367] Table 9 shows the results of genome analysis of recombinant AAV produced by the all-in-one stuffer vector (AiO-stuffer-SBEFIX), all-in-one vector (AiO-SBEFIX), or triple vector (3TF-SBEFIX) using the Nanopore sequencing platform.

[0368] SamplePacBio SequelIICalculation (%)AAVImpurity3TF-SBEFIX98.471.53AiO-SBEFIX98.941.06AiO-stuffer-SBEFIX99.360.64

[0369] SampleNova SeqCalculation (%)AAVImpurity3TF-SBEFIX93.056.95AiO-SBEFIX98.961.04AiO-stuffer-SBEFIX99.620.38

[0370] SampleNanoporeCalculation (%)AAVImpurity3TF-SBEFIX95.054.95AiO-SBEFIX98.441.56AiO-stuffer-SBEFIX98.881.12

[0371] As shown in Table 7 above, the PacBio SequelII analysis results classify the genome into AAV sequences containing ITRs in the form of ssAAV and scAAV, Other (sequences that are mapped to the GOI but cannot be classified as ssAAV or scAAV due to different directionality or form), Chimeric (genomes that are mapped to two or more (e.g., vector and host; Helper and Rep / Cap, etc.)), Rep / Cap (Rep / Cap plasmid), Helper (Helper plasmid), Host (e.g., host cell genome), backbone sequences, and forms other than ssAAV and scAAV are classified as impurities.

[0372] As a result, we confirmed low impurity (inserted genome excluding AAV GOI) results in the all-in-one vector and all-in-one stuffer vector compared to the triple vector (triple vector: 1.53%, all-in-one vector: 1.06%, all-in-one stuffer vector: 0.64%).

[0373] Next, as shown in Table 8 above, the Nova Seq analysis results classify the genome into AAV sequences containing ITRs, Rep / Cap, Helper, backbone (sequences of the vector excluding GOI, Rep / Cap, and Helper sequences), etc., and classify the forms excluding AAV sequences containing ITRs as impurities.

[0374] As a result, the results were the same as those in Table 7, and low impurity results were confirmed in the all-in-one vector and all-in-one stuffer vector compared to the triple vector (triple vector: 6.95%, all-in-one vector: 1.04%, all-in-one stuffer vector: 0.38%).

[0375] As shown in Table 9 above, the Nanopore analysis results classify the genome into AAV sequences containing ITRs, Rep / Cap, Helper, backbone (sequences of the vector excluding GOI, Rep / Cap, Helper, and host sequences), Host (e.g., host cell genome), etc., and classify the form excluding AAV sequences containing ITRs as impurities.

[0376] As a result, similar to the results in Tables 7 and 8, low impurity results were confirmed in the all-in-one vector and all-in-one stuffer vector compared to the triple vector (triple vector: 4.95%, all-in-one vector: 1.56%, all-in-one stuffer vector: 1.12%).

[0377] When producing recombinant AAV using an all-in-one vector with Rep / Cap and helper inserted into a single vector, there is a concern that the insertion of Rep / Cap and helper into the recombinant AAV particle may lead to the formation of replication-competent AAV (rcAAV). Therefore, an all-in-one stuffer vector was produced with stuffers inserted on both sides of the ITR and between Rep / Cap and the helper. As a result, the proportion of impurities, including Rep / Cap and helper, was reduced in the all-in-one stuffer vector compared to the single vector. This result confirms that the risk of reverse packaging that can occur in the all-in-one vector is reduced by positioning the stuffers on both sides of the ITR and in front of the P5 promoter.

[0378]

[0379] Example 5. Comparison of DNA impurity analysis according to positional combinations of all-in-one stuffer sequences.

[0380] In order to determine the level of DNA impurity suppression according to the insertion position of the stuffer sequence in the All-in-One stuffer vector, the following experiments were performed.

[0381] In this example, the All-in-One stuffer-1 to 6 vectors of Example 2 were used as the experimental group. As the control group, the All-in-One stuffer-scAAV-FIX vector of Example 1 and the All-in-One scAAV-FIX vector, which is identical to the All-in-One stuffer-scAAV-FIX vector of Example 1 except that it does not contain the stuffer, were used.

[0382] Both the experimental and control groups were transiently transfected with PEI (Polyethylenimine) at 3E06 cells / mL (300 mL, 1 L flask). Seventy-two hours after transfection, cells were lysed to extract recombinant AAV. All extracted recombinant AAV was first purified using the AKTA purification system, and then separated using CsCl ultracentrifugation to obtain high-purity recombinant AAV. DNA within the AAV particles was isolated from the obtained AAV using the QIAquick® Gel Extraction Kit. Genome analysis of the isolated DNA was performed using the Novaseq (short-read) sequencing platform at Macrogen Co., Ltd. (Seoul, Korea).

[0383] Table 10 shows the results of genome analysis of recombinant AAV produced by the all-in-one stuffer vector (AiO-stuffer-SBEFIX), the all-in-one vector (AiO-SBEFIX), or six types of all-in-one stuffer site combination vectors (All-in-One stuffer-1 to 6) using the Novaseq sequencing platform.

[0384] IDStuffer 1Stuffer 2Stuffer 3Assigned TypeCalculation (%)3 ofR-ITR5'of p5 promoter5'of L-ITRGOIRep / CapHelperBacboneMARUCOECCEAAVImpurityAiO-1N / AN / AN / A99.0370.0650.6770.222-- - 99.0370.963AiO-2MARN / AN / A99.530.0390.0480.2350.148- - 99.5300.470AiO-3N / AUCOEN / A99.580.0210.2850.058-0.056- 99.5800.420AiO-4N / AN / ACCE99.5030.0080.3390.146-- 0.00399.5030.497AiO-5MARUCOEN / A99.4350.060.020.1420.1840.159- 99.4350.565AiO-6MARN / ACCE99.6450.0070.040.1730.106- 0.02999.6450.355AiO-7N / AUCOECCE99.0640.0140.6240.145-0.1470.00699.06 40.936AiO-8MARUCOECCE99.6030.0080.0160.1060.1480.1120.00799.6030.397

[0385] As shown in Table 10 above, Nova Seq analysis results confirmed low impurity in all vectors with stuffer insertions compared to the all-in-one vector. This result confirms that the stuffer insertions at all locations flanking the ITR and preceding the P5 promoter reduce the risk of reverse packaging that can occur in all-in-one vectors.

[0386] In addition, looking at Table 10 above, it was confirmed that All-in-One stuffer-6, in which the stuffer was inserted on both sides of the ITR but not in front of the P5 promoter, showed a significantly lower impurity content compared to other experimental groups.

[0387]

[0388] Example 6. Comparison of DNA impurity analysis according to position combination of dual vector stuffer sequences.

[0389] In order to determine the level of DNA impurity suppression according to the insertion position of the stuffer sequence in the dual stuffer vector, the following experiment was performed.

[0390] Specifically, the impurity inhibition level of the stuffer combination (AiO-6 and AiO-8), which showed remarkably excellent low impurity results in Example 5, was confirmed even under dual vector conditions. In this example, the vectors in Table 4 were used, and a control group was produced using one that did not include a stuffer. Transient transfection using PEI (Polyethylenimine) was performed at 3E06 cells / mL (300 mL, 1 L flask) for both the experimental and control groups. 72 hours after transfection, cells were lysed to extract recombinant AAV. All extracted recombinant AAV was first purified using an AKTA purification system, and then separated using CsCl ultracentrifugation to obtain high-purity recombinant AAV. DNA within the AAV particles was isolated from the obtained AAV using a QIAquick® Gel Extraction Kit. Genome analysis was performed on the isolated DNA using the Novaseq (short-read) sequencing platform at Macrogen Co., Ltd. (Seoul, Korea).

[0391] Table 11 shows the results of genome analysis of recombinant AAV using the Novaseq sequencing platform.

[0392] IDAssigned TypeCalculation (%)GOIRep / CapHelperBackboneMARUCOECCEAAVImpurityDGR-193.7200.6800.0095.591--- 93.7206.280DGR-299.6500.0130.0030.1040.222- 0.00899.6500.350DGR-399.4030.0290.0090.0790.3210.1480.01199.4030.597DR H-195.0090.0330.0094.949---95.0094.991DRH-299.7280.0110.0130.0790.152- 0.01899.7280.272DRH-399.7770.0070.0030.0250.1400.0370.01299.7770.223

[0393] As shown in Table 11 above, based on the Nova Seq analysis results, we first confirmed the results of improving impurities by stuffers according to the dual combination vectors (DGR-1, DRH-1). This showed that the types of impurities varied depending on the combination of sequences linked to the vector containing the ITR, and in the results of the DGR combination vector linked to the GOI, we confirmed that the risk of reverse packaging of sequences linked to all positions on both sides of the ITR and in front of the P5 promoter was reduced. This result was more effective when it was in the same vector as the GOI, and it was confirmed that the effect was effective when stuffer sequences were present on both sides of the ITR.

[0394]

[0395] Example 7. Comparison of DNA impurity analysis according to positional combinations of triple vector stuffer sequences.

[0396] In order to determine the level of DNA impurity suppression according to the insertion position of the stuffer sequence in the triple stuffer vector, the following experiment was performed.

[0397] Specifically, the impurity suppression level of the stuffer combination (AiO-6 and AiO-8) that showed remarkably excellent low impurity results in Example 5 was also confirmed under triple vector conditions. In this Example, the vectors in Table 5 were used, and a control group was produced using one that did not include a stuffer.

[0398] Both the experimental and control groups were transiently transfected with PEI (Polyethylenimine) at 3E06 cells / mL (300 mL, 1 L flask). Seventy-two hours after transfection, cells were lysed to extract recombinant AAV. All extracted recombinant AAV was first purified using the AKTA purification system, and then separated using CsCl ultracentrifugation to obtain high-purity recombinant AAV. DNA within the AAV particles was isolated from the obtained AAV using the QIAquick® Gel Extraction Kit. Genome analysis of the isolated DNA was performed using the Novaseq (short-read) sequencing platform at Macrogen Co., Ltd. (Seoul, Korea).

[0399] Table 12 shows the results of genome analysis of recombinant AAV using the Novaseq sequencing platform.

[0400] IDAssigned TypeCalculation (%)GOIRep / CapHelperBackboneMARUCOECCEAAVImpurityTRI-193.9000.1870.0005.913- - - 93.9006.100TRI-298.9480.1280.0020.4500.455- 0.01698.9481.052TRI-399.2110.0730.0010.1390.2740.2910.01199.2110.789

[0401] As shown in Table 12 above, the Nova Seq analysis results confirmed that impurity improvement was achieved by stuffers arranged on both sides of the ITR and in front of the P5 promoter.

[0402]

[0403] Example 8. Test of rcAAV production using an all-in-one vector including a sputterer.

[0404] To determine whether rcAAV production is inhibited by suppression of DNA impurities in the All-in-One stuffer vector, the following experiments were performed.

[0405] The rcAAV (replication-competent AAV) test is an important safety test item for gene therapy to eliminate the risk of potential viral replication in the vector injected into the patient, in cases where the rep and cap genes of AAV are unintentionally recombined to create replication-competent AAV.

[0406] In this example, AAV produced using the All-in-One stuffer-scAAV-FIX vector of Example 1 was cultured after infection with Ad5 virus in HEK293 cells as an experimental group, and the presence of rcAAV was measured through qPCR analysis. The experiment was performed at Charles River (CRL, USA). The positive criterion for rcAAV is determined when the Ct value is less than 35.

[0407] Table 13 shows the rcAAV test results for AiO-stuffer-FIX.

[0408] PassageCT ValuesUn-infected controlAd5 Only controlAiO-stufferPositive Control (AAV8)AiO-stuffer + AAV8 SpikePassage 0UndeterminedUndetermined351819Passage 1UndeterminedUndetermined381111Passage 2UndeterminedUndeterminedUndetermined1010

[0409] As shown in Table 13 above, the rcAAV analysis results confirmed that rcAAV was not detected in the all-in-one stuffer vector.

[0410]

[0411] Example 9. Verification of transduction ability and GOI functionality of recombinant scAAV produced by an all-in-one vector containing a stuffer.

[0412] To verify the transducing capability and GOI functionality of recombinant scAAV produced by the all-in-one stuffer vector, the expression of FIX protein and blood coagulation ability were confirmed after scAAV injection into target cells.

[0413] Specifically, the target cells used were Huh7 cells, and the cells were cultured in DMEM medium supplemented with 10% FBS (fetal bovine serum) at 37°C and 5% CO2 conditions. One day before treatment with the recombinant virus, Huh7 cells were plated on a 48-well plate at 2.5E04 cells / well. Both the recombinant virus produced by the all-in-one stuffer vector of Example 1 and the recombinant virus produced by the control vector were obtained as highly pure recombinant AAV viruses using the AKTA purification system and CsCl density-gradient ultracentrifuge (CsCl-DGUC). The control vector was the all-in-one vector or triple transfection vector used as a control in Example 2. Next, the obtained viruses were diluted in FBS-free DMEM medium to treat with a multiplicity of infection (MOI) of 3.3E05, 1E06, or 3E06 vg / cell. The viruses were added to 48-well plates seeded with Huh7 cells from which the culture medium had been completely removed, and the treatment was performed by adding the viruses to DMEM medium without FBS and supplemented with 350 ng / mL vitamin K3. Four days after virus treatment, Huh7 cultures were harvested and the FIX expression level was measured using a Human Factor IX ELISA Kit (ELABSCIENCE) or Human Factor IX ELISA (Capture ab:anti-FIX, Abcam / Detection ab:FIX-HRP, Affinity biologicals). In addition, the FIX coagulability was analyzed using a Factor IX chromogenic assay kit (BIOPHEN).

[0414] Figure 3 is a graph showing the FIX expression level (ng / mL) of recombinant AAV produced by the all-in-one stuffer vector (AiO-stuffer-SBEFIX), all-in-one vector (AiO-SBEFIX), or triple vector (3TF-SBEFIX).

[0415] Figure 4 is a graph showing the FIX activity (mIU / mL) of recombinant AAV produced by the all-in-one stuffer vector (AiO-stuffer-SBEFIX), the all-in-one vector (AiO-SBEFIX), or the triple vector (3TF-SBEFIX).

[0416] As a result, as shown in Figs. 3 and 4, 1Х10 5 1Х10 7 When introduced into cells at an MOI of 1.0 vg / cell, the recombinant AAV produced by the all-in-one stuffer vector showed similar levels of FIX expression and activity to the recombinant AAV produced by the all-in-one vector, confirming that there was no functional difference. In addition, the recombinant AAV produced by the all-in-one stuffer vector showed an MOI of 1.0 x 10 5 1Х10 7 When introduced into cells at an MOI of vg / cell, it was confirmed that the GOI protein expression level and activity were increased compared to when recombinant AAV produced by triple transfection was introduced at the same MOI.

[0417]

[0418] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A vector system for producing a recombinant adeno-associated virus (AAV) comprising the nucleotide sequence of the following gene and one or more stuffers, A vector system for producing recombinant AAV, wherein the vector system comprises one or more vectors: (a) Helper virus genes required for AAV production; (b) Rep gene of AAV; (c) Cap gene of AAV; and (d) Transgene arranged between ITRs (Inverted Terminal Repeats).

2. A vector system for producing recombinant AAV, wherein the transgene is arranged between L-ITR and R-ITR in claim 1.

3. A vector system for producing recombinant AAV, wherein the vector system according to claim 1 comprises two or more stuffers.

4. A vector system for producing recombinant AAV, wherein the vector system comprises three or more stuffers according to claim 1.

5. A vector for producing a recombinant AAV, wherein the vector comprises three stuffers according to claim 1.

6. A vector system for producing recombinant AAV, wherein the stuffer is arranged inside or outside two ITRs according to claim 1.

7. A vector system for producing recombinant AAV, wherein the vector system according to claim 1 comprises two or more stuffers, each stuffer independently arranged inside or outside two ITRs.

8. A vector system for producing recombinant AAV, wherein the vector system according to claim 1 comprises two or more stuffers, and all stuffers are arranged outside of two ITRs.

9. A vector system for producing recombinant AAV, wherein the length of each stuffer is 0.1 kb to 20 kb in claim 1.

10. A vector system for producing recombinant AAV, wherein the total length of all stuffers in claim 1 is 0.1 kb to 100 kb.

11. A vector system for producing a recombinant AAV according to claim 1, wherein the at least one stuffer comprises at least one of a first stuffer, a second stuffer, and a third stuffer.

12. A vector system for producing recombinant AAV, wherein the first stuffer is arranged downstream of the R-ITR in claim 11.

13. A vector system for producing a recombinant AAV, wherein the length of the first stuffer is 0.1 kb to 20 kb in claim 11.

14. A vector system for producing recombinant AAV, wherein the second stuffer is arranged upstream of (b) or upstream of (c) in claim 11.

15. A vector system for producing a recombinant AAV, wherein the second stuffer has a length of 0.1 kb to 20 kb in claim 11.

16. A vector system for producing recombinant AAV, wherein the third stuffer is arranged upstream of the L-ITR in claim 11.

17. A vector system for producing a recombinant AAV, wherein the length of the third stuffer is 0.1 kb to 20 kb in claim 11.

18. A vector system for producing recombinant AAV, wherein the stuffer reduces impurities that may occur during the recombinant AAV production process using the vector system according to claim 1.

19. A vector system for producing a recombinant AAV, wherein the impurity in claim 18 is a DNA impurity.

20. A vector system for producing a recombinant AAV, wherein the first stuffer arranged downstream of the R-ITR, the third stuffer arranged upstream of the L-ITR, or both, reduces reverse packaging of the vector.

21. A vector system for producing recombinant AAV, wherein the second stuffer arranged upstream of (b) or upstream of (c) in claim 11 reduces impurities in recombinant AAV particles produced by the vector system.

22. A vector system for producing a recombinant AAV, wherein the impurity comprises at least one selected from the group consisting of all or part of a helper virus gene; all or part of a Rep gene; all or part of a Cap gene; any sequence located outside of two ITRs in the vector; a portion of a genomic sequence of a host cell; and two or more chimeric forms thereof.

23. A vector system for producing a recombinant AAV, wherein the impurity comprises all or part of a helper virus gene according to claim 21.

24. A vector system for producing a recombinant AAV, wherein the stuffer comprises at least one selected from among introns and partial sequences or modified sequences thereof, according to claim 1.

25. A vector system for producing a recombinant AAV, wherein the stuffer comprises at least one selected from among a barrier insulator and a partial sequence or a modified sequence thereof, according to claim 1.

26. A vector system for producing a recombinant AAV, wherein the barrier insulator comprises a MAR (Matrix Attachment Region), a UCOE (Ubiquitous Chromatin Opening Element), a CCE (Chromatin Control Element), a STAR (Stabilizing and Anti-Repressor), or a 5'HS5 (Hypersensitive site 5) in claim 25.

27. A vector system for producing a recombinant AAV, wherein the stuffer comprises at least one selected from the whole or part of a MAR (Matrix Attachment Region) sequence, the whole or part of a UCOE (Ubiquitous Chromatin Opening Element) sequence, and the whole or part of a CCE (Chromatin Control Element) sequence.

28. A vector system for producing a recombinant AAV, wherein the first stuffer comprises a partial sequence of a MAR (Matrix Attachment Region) according to claim 11.

29. A vector system for producing a recombinant AAV, wherein the MAR according to claim 28 is a human globin MAR.

30. A vector system for producing a recombinant AAV, wherein the first stuffer comprises a polynucleotide comprising at least 100 consecutive nucleotides within the sequence of SEQ ID NO:

1.

31. A vector system for producing a recombinant AAV, wherein the first stuffer comprises a polynucleotide of sequence number 1, according to claim 28.

32. A vector system for producing a recombinant AAV, wherein the second stuffer comprises a partial sequence of UCOE (Ubiquitous Chromatin Opening Element) according to claim 11.

33. A vector system for producing a recombinant AAV, wherein the UCOE of claim 32 is a UCOE of human HNRPA2B1 (heterogeneous nuclear ribonucleoproteins A2 / B1 gene).

34. A vector system for producing a recombinant AAV, wherein the second stuffer comprises a polynucleotide comprising at least 100 consecutive nucleotides within the sequence of SEQ ID NO:

5.

35. A vector system for producing a recombinant AAV, wherein the second stuffer comprises a polynucleotide of sequence number 5, according to claim 32.

36. A vector system for producing a recombinant AAV, wherein the third stuffer comprises a partial sequence of a CCE (Chromatin Control Element) according to claim 11.

37. A vector system for producing recombinant AAV, wherein the CCE is a human CCE, according to claim 36.

38. A vector system for producing a recombinant AAV, wherein the third stuffer comprises a polynucleotide comprising at least 100 consecutive nucleotides within the sequence of SEQ ID NO:

9.

39. A vector system for producing a recombinant AAV, wherein the third stuffer comprises a polynucleotide of sequence number 9, according to claim 36.

40. A vector system for producing a recombinant AAV, wherein the stuffer comprises: A first stuffer comprising a polynucleotide of sequence number 1, arranged downstream of the R-ITR; A second stuffer comprising a polynucleotide of sequence number 5, arranged upstream of (b) or upstream of (c); and A third stuffer comprising a polynucleotide of sequence number 9, arranged upstream of the L-ITR.

41. A vector system for producing a recombinant AAV, wherein the vector system comprises the following vector and one or more stuffers according to claim 1: (i) (a) a first-1 vector comprising a helper virus gene required for AAV production; (ii) (b) a first-second vector comprising the Rep gene of AAV and (c) the Cap gene of AAV; and (iii) (d) A vector 1-3 containing a transgene arranged between ITRs.

42. A vector system for producing a recombinant AAV, wherein the at least one stuffer is arranged as follows in claim 41: (i) the first stuffer is arranged downstream of the R-ITR of the 1-3 vector, or is not arranged; (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the first-second vector, or is not arranged; and (iii) The third stuffer is arranged upstream of the L-ITR of the 1-3 vector, or is not arranged.

43. A vector system for producing a recombinant AAV, wherein the vector system comprises the following vector and one or more stuffers according to claim 1: (i) (a) a second-1 vector comprising a helper virus gene required for AAV production; and (ii) A second-2 vector comprising a transgene arranged between (b) the Rep gene of AAV, (c) the Cap gene of AAV, and (d) the ITR.

44. A vector system for producing a recombinant AAV, wherein the at least one stuffer is arranged as follows in claim 43: (i) the first stuffer is arranged downstream of the R-ITR of the second-2 vector, or is not arranged; (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the second-2 vector, or is not arranged; and (iii) The third stuffer is arranged upstream of the L-ITR of the second-second vector, or is not arranged.

45. A vector system for producing a recombinant AAV, wherein the vector system comprises the following vector and one or more stuffers according to claim 1: (i) a third-1 vector comprising (a) a helper virus gene required for AAV production, (b) a Rep gene of AAV, and (c) a Cap gene of AAV; and (ii) (d) A third-2 vector comprising a transgene arranged between ITRs.

46. ​​A vector system for producing a recombinant AAV, wherein the at least one stuffer is arranged as follows in claim 45: (i) the first stuffer is arranged downstream of the R-ITR of the 3-2 vector, or is not arranged; (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the third-1 vector, or is not arranged; and (iii) The third stuffer is arranged upstream of the L-ITR of the third-2 vector, or is not arranged.

47. A vector system for producing a recombinant AAV, wherein the vector system comprises the following vector and one or more stuffers according to claim 1: (i) a 4-1 vector comprising (a) a helper virus gene required for AAV production, and (d) a transgene arranged between the ITRs; and (ii) (b) A 4-2 vector comprising the Rep gene of AAV, and (c) the Cap gene of AAV.

48. A vector system for producing a recombinant AAV, wherein the at least one stuffer is arranged as follows in claim 47: (i) the first stuffer is arranged downstream of the R-ITR of the 4-1 vector, or is not arranged; (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the 4-2 vector, or is not arranged; and (iii) The third stuffer is arranged upstream of the L-ITR of the 4-1 vector, or is not arranged.

49. A vector system for producing a recombinant AAV, wherein the vector system comprises the following vector and one or more stuffers according to claim 1: A fifth vector comprising (a) a helper virus gene required for AAV production, (b) a Rep gene of AAV, (c) a Cap gene of AAV, and (d) a transgene arranged between ITRs.

50. A vector system for producing a recombinant AAV, wherein the at least one stuffer is arranged as follows in claim 49: (i) the first stuffer is arranged downstream of the R-ITR of the fifth vector, or is not arranged; (ii) the second stuffer is arranged upstream of (b) or upstream of (c) of the fifth vector, or is not arranged; and (iii) The third stuffer is arranged upstream of the L-ITR of the fifth vector, or is not arranged.

51. A vector system for producing recombinant AAV, wherein the recombinant AAV produced by the vector system according to claim 1 is single-stranded AAV (ssAAV) or self-complementary AAV (scAAV).

52. A vector system for producing a recombinant AAV, wherein the nucleotide sequence of the helper virus gene according to claim 1 is derived from at least one selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus.

53. A vector system for producing a recombinant AAV, wherein the nucleotide sequence of the helper virus gene according to claim 1 is derived from an adenovirus.

54. A vector system for producing a recombinant AAV, wherein the nucleotide sequence of the helper virus gene in claim 1 is derived from adenovirus 2.

55. A vector system for producing a recombinant AAV, wherein the helper virus gene of claim 1 comprises at least one selected from E1, E2, E2a, E4, E4orf1, E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DBP (DNA-binding protein), and variants thereof.

56. A vector system for producing a recombinant AAV according to claim 1, wherein the helper virus genes include E2a, E4, and VA.

57. A vector system for producing a recombinant AAV, wherein the nucleotide sequence of the Rep gene in claim 1 is derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.

58. A vector system for producing a recombinant AAV, wherein the nucleotide sequence of the Rep gene according to claim 1 is derived from AAV2.

59. A vector system for producing a recombinant AAV, wherein the Rep gene according to claim 1 comprises at least one selected from Rep78, Rep68, Rep52, Rep40, and variants thereof.

60. A vector system for producing a recombinant AAV, wherein the Rep gene comprises Rep78, Rep68, Rep52, and Rep40, according to claim 1.

61. A vector system for producing a recombinant AAV, wherein the nucleotide sequence of the Cap gene in claim 1 is derived from at least one selected from among AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh39, AAVrh64R1, and AAVrh74.

62. A vector system for producing a recombinant AAV, wherein the nucleotide sequence of the Cap gene according to claim 1 is derived from AAV8.

63. A vector system for producing a recombinant AAV, wherein the Cap gene encodes at least one selected from capsid protein, VP1 protein, VP2 protein, VP3 protein, and variants thereof, according to claim 1.

64. A vector system for producing a recombinant AAV according to claim 63, wherein the variant comprises an engineered capsid protein.

65. A vector system for producing a recombinant AAV, wherein the Cap gene according to claim 1 is a wild-type Cap gene of AAV8.

66. A vector system for producing a recombinant AAV, wherein the transgene is a therapeutic gene according to claim 1.

67. A vector system for producing a recombinant AAV, wherein the ITR in claim 1 is derived from at least one selected from among AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh32.33, AAVrh39, AAVrh64R1, and AAVrh74.

68. A vector system for producing a recombinant AAV, wherein the ITR according to claim 1 is derived from AAV2.

69. A vector system for producing a recombinant AAV, wherein the ITR of claim 1 is a wild-type ITR or a mutant thereof.

70. A vector system for producing a recombinant AAV, wherein the ITR comprises all or part of a wild-type ITR sequence according to claim 1.

71. A vector system for producing a recombinant AAV according to claim 2, wherein either the L-ITR or the R-ITR does not contain a trs (terminal resolution site).

72. A vector system for producing a recombinant AAV according to claim 2, wherein one of the L-ITR and the R-ITR does not contain trs, and the other is a wild-type ITR.

73. A vector system for producing a recombinant AAV according to claim 2, wherein one of the L-ITR and the R-ITR is an ITR mutant having a D region deleted, and the other is a wild-type ITR.

74. A vector system for producing a recombinant AAV, wherein the L-ITR is composed of sequence number 10 and the R-ITR is composed of sequence number 15 in claim 2.

75. A vector system for producing a recombinant AAV, according to any one of claims 71 to 74, wherein the recombinant AAV produced by the vector system is a self-complementary AAV (scAAV).

76. A vector system for producing a recombinant AAV, wherein the vector system of claim 1 further comprises at least one expression control element operably linked to at least one of (a), (b), (c), and (d).

77. A vector system for producing a recombinant AAV, wherein the expression control element according to claim 76 comprises at least one selected from an enhancer, a promoter, an intron, and a polyadenylation (poly A) signal sequence.

78. A vector system for producing a recombinant AAV, wherein the vector system further comprises a promoter operably linked to (b) in claim 76.

79. A vector system for producing a recombinant AAV according to claim 1, wherein at least one of the vectors further comprises a replication origin.

80. A vector system for producing a recombinant AAV, wherein the replication origin is a pUC origin, a pBR322 origin, a pMB1 origin, a pSC101 origin, a p15A origin, or a synthetic origin, according to claim 79.

81. A vector for producing a recombinant AAV according to claim 1, wherein at least one of the vectors further comprises a selectable marker.

82. A vector system for producing a recombinant AAV, wherein the selection marker is at least one selected from ampicillin, streptavidin, kanamycin, hygromycin, neomycin, puromycin, blasticidin, and zeocin, according to claim 81.

83. A vector system for producing recombinant AAV, wherein in claim 49, (a), (b), (c), and (d) in the vector system are each independently arranged in a forward orientation or a reverse orientation in any order.

84. In claim 49, a vector system for producing recombinant AAV, wherein (a), (b), (c), and (d) are arranged in the following order, wherein (a), (b), (c), and (d) are each independently in a forward or reverse orientation: 1) (d) - (b) - (c) - (a); 2) (d) - (c) - (b) - (a); 3) (d) - (a) - (b) - (c); 4) (d) - (a) - (c) - (b); 5) (b) - (c) - (d) - (a); 6) (c) - (b) - (d) - (a); 7) (b) - (c) - (a) - (d); 8) (c) - (b) - (a) - (d); 9) (a) - (b) - (c) - (d); 10) (a) - (c) - (b) - (d); 11) (a) - (d) - (b) - (c); or 12) (a) - (d) - (c) - (b).

85. A vector system for producing recombinant AAV, wherein the vector backbone is arranged in any order from 1) to 12) in claim 84.

86. A vector system for producing recombinant AAV, wherein the vector backbone is arranged after the last sequence in 1) to 12) above, or before the first sequence.

87. A vector system for producing a recombinant AAV, wherein the vector backbone comprises at least one selected from a replication origin and a selection marker according to claim 85 or 86.

88. A step of introducing a vector system for producing a recombinant AAV according to any one of claims 1 to 87 into a host cell; and Comprising the step of isolating recombinant AAV from the host cell, A method for producing a recombinant adeno-associated virus (AAV).

89. A method for producing a recombinant AAV according to claim 88, wherein the host cell is a mammalian cell or an insect cell.

90. A method for producing a recombinant AAV according to claim 82, wherein the mammalian cell is selected from HEK293 cells, HEK293F cells, HEK293T cells, and cells derived therefrom.

91. A method for producing a recombinant AAV according to claim 89, wherein the insect cell is an Sf9 cell, an Sf21 cell, a TN-5B1-4 cell, or a High Five cell.

92. A method for producing a recombinant AAV according to claim 88, wherein the introduction is selected from physical methods including electroporation, cell compression, sonication, optical transfection, protoplast fusion, impalfection, magnetofection, gene gun, and particle bombardment; methods using a cationic polymer such as polyethyleneimine (PEI) and a chemical reagent including calcium phosphate; and methods using a cationic lipid such as lipofection.

93. A method for producing a recombinant AAV according to claim 88, wherein the separation is performed using centrifugation or chromatography.

94. A method for producing recombinant AAV according to claim 93, wherein the centrifugation method is a cesium chloride-based ultra-high-speed centrifugation method.

95. A method for producing a recombinant AAV according to claim 93, wherein the chromatography is at least one selected from affinity chromatography, ion exchange chromatography, column chromatography, gel-filtration chromatography, thin layer chromatography, radial flow chromatography, interference chromatography, and reverse phase chromatography.

96. A recombinant AAV produced by the vector system of any one of claims 1 to 87.

97. In claim 96, the recombinant AAV is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

98. A composition comprising the recombinant AAV of claim 96.

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