Single vector system for recombinant AAV production

Abstract

Provided are a vector for producing recombinant adeno-associated virus (AAV) and a method for producing recombinant AAV using same. The vector for producing recombinant AAV comprises (a) helper virus genes, (b) AAV Rep genes, (c) AAV Cap genes, and (d) transgenes, arranged in a single nucleic acid molecule, and thus can produce high-quality recombinant AAV with high efficiency and high productivity.

Description

Single-vector system for recombinant AAV production

[0001] The present invention relates to a vector for producing a recombinant adeno-associated virus (AAV) 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. As of 2023, a total of six rAAV gene therapies have been approved, three of which were produced using triple transfection using HEK293 cells without the aid of helper viruses. Triple-transfection rAAV requires the simultaneous delivery of three plasmids to a single cell to produce normal AAV with transduction capabilities. 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 efficacy 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] The problems of conventional triple transfection are explained in detail as follows.

[0004] The first problem is low efficiency and productivity. To produce AAV using HEK293 cells, a production cell line, via triple transfection, three components are required: Rep / Cap, Helper, and a gene-of-interest (GOI). All three components must be introduced simultaneously and in appropriate ratios into the production cell line to produce recombinant AAV with high efficiency. However, because each component functions independently during this introduction process, the rate of simultaneous injection of all three plasmids is low, at less than 30%. Furthermore, the ratios of the three plasmids injected into the cells cannot be precisely controlled, which can lead to differences in transfection efficiency and productivity across production batches, resulting in reduced productivity of recombinant AAV.

[0005] The second issue is low quality. As pharmaceuticals, quality and safety are crucial for gene therapy. However, conventional triple-transfection technology has been known to produce AAVs with a %Full capsid content of less than 30%, and cases of recombinant AAVs producing less than 100% genome integrity have also been reported. Furthermore, empty capsids during AAV production can trigger immune responses. Therefore, various high-purity recombinant AAV purification processes are being developed to overcome and minimize these quality limitations. However, due to limitations in conventional technologies, improvements in the triple-transfection system are required to improve the %Full capsid content in addition to the purification process.

[0006] The third problem is the high production cost. Currently, Zolgensma, a gene therapy for spinal muscular atrophy (SMA), ®) is known to be mostly due to the cost of raw materials, which amounts to approximately KRW 2.8 billion. In the conventional triple transfection technology, the cost of producing three types of plasmids essential for recombinant AAV production accounts for up to 40%, and in particular, a process for producing high-quality plasmids is required.

[0007] Therefore, there is a need to improve the current three-type triple transfection process as well as to develop high-quality, high-productivity recombinant AAV production methods and processes.

[0008] The present disclosure provides a method for integrating Helper, Rep, Cap, and Gene-Of-Interest (GOI), which are essential elements for recombinant AAV virus production, into a single nucleic acid molecule or single vector system to enable improved production efficiency and robustness of the production process, improved %Full / Empty capsid ratio, reduced impurities, and reduced production costs.

[0009] One aspect is to provide a vector for producing recombinant adeno-associated virus (AAV).

[0010] Another aspect provides a method for producing recombinant AAV using the vector for producing recombinant AAV.

[0011] Another aspect is to provide a recombinant AAV produced by the above method.

[0012] Another aspect provides a composition comprising a recombinant AAV produced by the above method.

[0013] Another aspect provides a gene therapy method using a recombinant AAV produced by the above method.

[0014] Another aspect provides the use of the vector for producing the recombinant AAV or the recombinant AAV produced by the vector for the manufacture of a medicament for gene therapy.

[0015] 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.

[0016] 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.

[0017] One aspect provides a vector for producing recombinant adeno-associated virus (AAV). Specifically, an all-in-one vector system capable of producing recombinant AAV using a single vector is provided.

[0018] A vector according to the above aspect comprises the nucleotide sequence of the following genes arranged in a single nucleic acid molecule:

[0019] (a) Helper virus genes required for AAV production;

[0020] (b) Rep gene of AAV;

[0021] (c) Cap gene of AAV; and

[0022] (d) transgene.

[0023] Therefore, alternatively, one aspect provides a nucleic acid molecule comprising within a single molecule (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. More specifically, a nucleic acid molecule comprising within a single molecule (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.

[0024] In the above vector or nucleic acid molecule, (a), (b), (c), and (d) are all integrated together in a single nucleic acid molecule.

[0025] The following description applies commonly to vectors according to the above aspects or nucleic acid molecules according to the above aspects.

[0026] 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.

[0027] 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 mean a case where a polynucleotide that does not generally exist in the wild-type AAV genome has been inserted into the viral genome. Therefore, a recombinant AAV produced by the above recombinant AAV production vector may be applied as a gene therapy agent.

[0028] As used herein, the term "vector for recombinant AAV production" may refer to a vector capable of producing recombinant AAV in a host cell into which the vector has been introduced. The vector may refer to a plasmid vector. In one aspect, the vector for recombinant AAV production relates to vector technology that integrates all genes necessary for AAV production into a single vector. Therefore, as used herein, the term "vector for recombinant AAV production" may be used interchangeably with the term "all-in-one vector" or "AAV single vector system." The vector for recombinant AAV production may be a single plasmid.

[0029] 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.

[0030] 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."

[0031] 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.

[0032] The recombinant AAV produced by the above recombinant AAV production vector may be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV).

[0033] The nucleic acid molecule comprises (a) helper virus genes necessary for AAV production.

[0034] 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.

[0035] 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."

[0036] 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.

[0037] 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.

[0038] The above herpes simplex virus may be type 1 or type 2.

[0039] 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.

[0040] 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.

[0041] The above variant may be an engineered helper virus gene.

[0042] 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.

[0043] 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.

[0044] The nucleic acid molecule comprises (b) a Rep gene of AAV.

[0045] 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.

[0046] In one specific example, the nucleotide sequence of the Rep gene may be derived from AAV2.

[0047] The above Rep gene may include a gene encoding a known Rep protein or a variant thereof.

[0048] The Rep gene may include at least one selected from Rep78, Rep68, Rep52, Rep40, and variants thereof. The 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] The nucleic acid molecule comprises (c) the Cap gene of AAV.

[0053] The above Cap (Capsid) gene is a gene that encodes the viral capsid protein.

[0054] 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.

[0055] In one specific example, the nucleotide sequence of the Cap gene may be derived from AAV2.

[0056] In one specific example, the nucleotide sequence of the Cap gene may be derived from AAV5.

[0057] In one specific example, the nucleotide sequence of the Cap gene may be derived from AAV8.

[0058] The above Cap gene may include a gene encoding a known Cap protein or a variant thereof.

[0059] 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.

[0060] In one specific example, the Cap gene may be a wild-type Cap gene of AAV2.

[0061] 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.

[0062] The nucleotide sequences of the above Rep gene and Cap gene may be derived from the same or different AAV serotypes.

[0063] The nucleic acid molecule comprises (d) a transgene.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] The above transgene may be one or two or more.

[0073] 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.

[0074] 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.

[0075] The above ITR may be derived from a virus belonging to the genus Dependovirus of the family Parvoviridae. The above ITR may be derived from AAV. The above ITR may be derived from any AAV serotype. The above ITR 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 above AAV serotype may also include other AAV serotypes currently known or to be discovered in the future. The above 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.

[0076] In one specific example, the ITR may be derived from AAV2. The L-ITR and R-ITR may both be derived from AAV2.

[0077] 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.

[0078] 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 with a D region deleted, and the other may be a wild-type ITR. The L-ITR may be an ITR mutant with a D region deleted, and the R-ITR may be a wild-type ITR. Accordingly, the recombinant AAV produced by the above vector may be a self-complementary AAV (scAAV).

[0079] 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.

[0080] The nucleic acid molecule may further comprise additional components in addition to (a), (b), (c), and (d), which are essential components for recombinant AAV production.

[0081] The above additional components may have one or more functions selected from the following:

[0082] (i) increasing gene expression levels;

[0083] (ii) regulating the timing of gene expression;

[0084] (iii) regulates gene transcription;

[0085] (iv) stabilize the transcript for translation;

[0086] (v) reducing the content of impurities other than the transgene among the genes packaged in the recombinant AAV;

[0087] (vi) improving the productivity of recombinant AAV; and

[0088] (vii) Improves the %F / E capsid ratio of recombinant AAV.

[0089] 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. The impurity may be a DNA impurity.

[0090] When producing recombinant AAV using a single vector system, some sequences of the Rep gene, Cap gene, or Helper gene may be packaged within the recombinant AAV particle. Accordingly, the impurity may include some sequences of the Rep gene, Cap gene, or Helper gene packaged within the recombinant AAV particle.

[0091] For example, the impurity may include at least one selected from any sequence located outside of two ITRs in the vector, such as all or part of a helper virus gene, all or part of Rep, all or part of Cap; part of a transgene; part of a genomic sequence of a host cell; and chimeric forms of two or more of these.

[0092] 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.

[0093] Therefore, by lowering the content of the above impurities, a recombinant AAV with excellent safety and quality can be secured.

[0094] AAV packaging refers to the packaging of the structure located between the two ITRs into an AAV particle. However, AAV reverse packaging refers to the packaging of recombinant AAV particles outward, rather than inward, of the two ITRs. Therefore, inhibiting AAV reverse packaging can secure high-quality AAV. Vectors according to this aspect can reduce the content of impurities caused by reverse packaging, enabling the production of high-quality recombinant AAV, and furthermore, can be expected to have an enhanced safety effect when applied as a therapeutic.

[0095] In the above (vii), 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.

[0096] 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.

[0097] The nucleic acid molecule may further include at least one 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.

[0098] The nucleic acid molecule may further comprise one or more expression control elements operably linked to any one of (a), (b), (c), and (d).

[0099] The above expression control elements may include at least one selected from an enhancer, a promoter, a transcription factor, 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 (poly A) signal sequence, etc.

[0100] The term "enhancer" can refer to a regulatory region that promotes transcription of a gene.

[0101] Non-limiting examples of the above enhancers include the SV40 enhancer, the CMV (cytomegalovirus) enhancer, etc.

[0102] The term "promoter" can refer to a sequence that drives gene expression.

[0103] 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.

[0104] 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.

[0105] Non-limiting examples of the above introns may include minute virus of mice (MVM) intron, human 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.

[0106] The above "polyadenylation signal sequence" is also called "Poly A sequence" and refers to, for example, a polyadenylation signal sequence located 3' of a transgene, which allows the addition of a Poly A tail to the end of a nascent mRNA during transcription. The Poly A tail consists of up to 300 adenosine ribonucleotides that protect the mRNA from enzymatic degradation and aid in translation.

[0107] 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.

[0108] In one embodiment, the expression regulatory element may include one or more selected from an enhancer, a promoter, a silencer, an insulator, an intron, a UTR, a Kozak sequence, a signal peptide, and a polyadenylation signal sequence.

[0109] The above amino acids may include, but are not limited to, non-natural amino acids, non-canonical amino acids, or unnatural amino acids.

[0110] The above initiation codon may include, but is not limited to, a rare initiation codon or a non-ATG initiation codon.

[0111] The nucleic acid molecule may further comprise a promoter independently operably linked to (a), (b), (c), or (d). The promoter may control the amount and timing of expression of each gene. Generally, higher Rep / Cap expression increases AAV productivity, but certain Rep proteins may decrease productivity, and overexpression of the Cap gene may also increase empty particles (i.e., product-related impurities).

[0112] In one specific example, (i) when the amounts of Rep / Cap and GOI are fixed and the amount of Helper is increased, or (ii) when the amounts of Helper and GOI are fixed and the amount of Rep / Cap is increased, productivity and quality increase in a dose-dependent manner, but productivity and quality may decrease after a certain period. This suggests that fine control of Helper or Rep / Cap protein expression is important. Therefore, controlling the expression level of each gene within the vector may affect productivity and quality. Accordingly, by including the promoter, recombinant AAV productivity can be increased, %Full capsid can be increased, and %Empty capsid can be decreased, thereby producing high-quality recombinant AAV.

[0113] The nucleic acid molecule 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 the wild-type promoter.

[0114] The nucleic acid molecule 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.

[0115] The nucleic acid molecule 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 the wild-type promoter.

[0116] The nucleic acid molecule 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, a Kozak sequence, and a polyadenylation signal sequence.

[0117] 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.

[0118] 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.

[0119] In one embodiment, the nucleic acid molecule may further comprise an enhancer operably linked to (d). The enhancer may be an ocular tissue-specific enhancer.

[0120] In one embodiment, the nucleic acid molecule may further comprise a promoter operably linked to (d). The promoter may be an ocular tissue-specific promoter.

[0121] The nucleic acid molecule may further comprise a multi-cistronic element, a bi-cistronic element, a transposon, a transposase, or the like.

[0122] The nucleic acid molecule may comprise a vector backbone. The vector backbone may comprise one or more (one or two) selected from an origin of replication and a selection marker. The vector backbone may not comprise an origin of replication.

[0123] The nucleic acid molecule may not contain an origin of replication, or may additionally contain an origin of replication. The origin of replication may be a known origin of replication or a variant thereof. The origin of replication may be, but is not limited to, a pUC origin, a pBR322 origin, a pMB1 origin, a pSC101 origin, a p15A origin, a pJET1.2 origin, or a synthetic origin.

[0124] In one embodiment, the nucleic acid molecule may comprise a pBR322 origin.

[0125] In one embodiment, the nucleic acid molecule may comprise a pJET1.2 origin.

[0126] The nucleic acid molecule may further comprise a selectable marker or reporter capable of providing selection or identification of host cells into which the vector 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 nucleic acid molecule may further comprise a selectable marker.

[0127] In one embodiment, the nucleic acid molecule may further comprise ampicillin.

[0128] "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.

[0129] An "amplifiable selectable marker gene" may mean a gene that allows amplification of that gene under appropriate growth conditions.

[0130] In the nucleic acid molecule, (a), (b), (c), and (d) may be randomly arranged in the 5' to 3' direction or the 3' to 5' direction. In the nucleic acid molecule, (a), (b), (c), and (d) may be arranged in any order in the 5' to 3' direction or the 3' to 5' direction. In the nucleic acid molecule, (a), (b), (c), and (d) may be arranged in any order in the 5' to 3' direction.

[0131] In the above nucleic acid molecule, (a), (b), (c), and (d) may be independently arranged in any order in a forward orientation or a reverse orientation.

[0132] The above forward orientation may mean that the gene is inserted in the 5' to 3' direction in the 5'→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.

[0133] 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 be independently forward oriented or reverse oriented:

[0134] 1) (d) - (b) - (c) - (a);

[0135] 2) (d) - (c) - (b) - (a);

[0136] 3) (d) - (a) - (b) - (c);

[0137] 4) (d) - (a) - (c) - (b);

[0138] 5) (b) - (c) - (d) - (a);

[0139] 6) (c) - (b) - (d) - (a);

[0140] 7) (b) - (c) - (a) - (d);

[0141] 8) (c) - (b) - (a) - (d);

[0142] 9) (a) - (b) - (c) - (d);

[0143] 10) (a) - (c) - (b) - (d);

[0144] 11) (a) - (d) - (b) - (c); or

[0145] 12) (a) - (d) - (c) - (b).

[0146] In one specific example, (a) is oriented normally; and (b), (c), and (d) can each independently be oriented normally or reversely.

[0147] In one specific example, (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.

[0148] In one specific example, (a), (b), and (c) are oriented normally; and (d) may be oriented normally or reversely.

[0149] In certain embodiments, (a), (b), (c), and (d) may all be oriented in the right direction.

[0150] In certain embodiments, (a), (b), and (c) may be oriented normally; and (d) may be oriented in reverse.

[0151] In one specific embodiment, two ITRs may be arranged on either side of (d). Accordingly, in the nucleic acid molecule, (a), (b), (c), (d), and ITRs may be arranged in the following order:

[0152] 1') L-ITR - (d) - R-ITR - (b) - (c) - (a);

[0153] 2') L-ITR - (d) - R-ITR - (c) - (b) - (a);

[0154] 3') L-ITR - (d) - R-ITR - (a) - (b) - (c);

[0155] 4') L-ITR - (d) - R-ITR - (a) - (c) - (b);

[0156] 5') (b) - (c) - L-ITR - (d) - R-ITR - (a);

[0157] 6') (c) - (b) - L-ITR - (d) - R-ITR - (a);

[0158] 7') (b) - (c) - (a) - L-ITR - (d) - R-ITR;

[0159] 8') (c) - (b) - (a) - L-ITR - (d) - R-ITR;

[0160] 9') (a) - (b) - (c) - L-ITR - (d) - R-ITR;

[0161] 10') (a) - (c) - (b) - L-ITR - (d) - R-ITR;

[0162] 11') (a) - L-ITR - (d) - R-ITR - (b) - (c); or

[0163] 12') (a) - L-ITR - (d) - R-ITR - (c) - (b).

[0164] In one embodiment, the vector backbone may be arranged in any order from 1) to 12). In another embodiment, the vector backbone may be arranged after the last order from 1) to 12), or before the first order. In yet another embodiment, the vector backbone may be arranged after the last order from 1) to 12). For example, in 5), the arrangement order may be (b) - (c) - (d) - (a) - backbone. Specifically, in 5'), the arrangement order may be (b) - (c) - L-ITR - (d) - R-ITR - (a) - backbone.

[0165] The above vector backbone may include one or more selected from a replication origin and a selection marker.

[0166] According to one aspect, the 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.

[0167] In one specific example, the components in the nucleic acid molecule may have the following arrangement and orientation (normal orientation is abbreviated as 'F' and reverse orientation is abbreviated as 'R'):

[0168] (i) positively oriented (a) - backbone - positively oriented (b) - positively oriented (c) - positively oriented (d) (i.e., helper (F) - backbone - Rep (F) - Cap (F) - transgene (F));

[0169] (ii) positively oriented (a) - backbone - positively oriented (c) - positively oriented (b) - positively oriented (d) (i.e., helper (F) - backbone - Cap (F) - Rep (F) - transgene (F));

[0170] (iii) positively oriented (b) - positively oriented (c) - positively oriented (a) - backbone - positively oriented (d) (i.e., Rep(F) - Cap(F) - helper(F) - backbone - transgene(F));

[0171] (iv) Oriented (a) - Backbone - Oriented (b) - Oriented (c) - Anti-orientated (d) (i.e., Helper (F) - Backbone - Rep (F) - Cap (F) - Transgene (R));

[0172] (v) positively oriented (a) - backbone - positively oriented (c) - positively oriented (b) - anti-oriented (d) (i.e., helper (F) - backbone - Cap (F) - Rep (F) - transgene (R));

[0173] (vi) positively oriented (b) - positively oriented (c) - positively oriented (a) - backbone - reversely oriented (d) (i.e., Rep(F) - Cap(F) - helper(F) - backbone - transgene(R));

[0174] (vii) backbone - positively oriented (b) - positively oriented (c) - positively oriented (a) - anti-oriented (d) (i.e., backbone - Rep(F) - Cap(F) - helper(F) - transgene(R)); or

[0175] (viii) Oriented (a) - Oriented (b) - Oriented (c) - Backbone - Oriented (d) (i.e., Helper (F) - Rep (F) - Cap (F) - Backbone - Transgene (F)).

[0176] In one specific example, the `(d) transgene` among the components in the nucleic acid molecule may be in reverse orientation.

[0177] In one specific example, among the components in the nucleic acid molecule, `(b) Rep gene of AAV` and `(c) Cap gene of AAV` may be arranged in the order of `(c)-(b)`, and specifically, may be arranged in the order of `normally oriented (c) - normally oriented (b)`.

[0178] In one specific example, the components in the nucleic acid molecule may have the following arrangement and orientation (normal orientation is abbreviated as 'F' and reverse orientation is abbreviated as 'R'):

[0179] (ii) positively oriented (a) - backbone - positively oriented (c) - positively oriented (b) - positively oriented (d) (i.e., helper (F) - backbone - Cap (F) - Rep (F) - transgene (F));

[0180] (iii) positively oriented (b) - positively oriented (c) - positively oriented (a) - backbone - positively oriented (d) (i.e., Rep(F) - Cap(F) - helper(F) - backbone - transgene(F));

[0181] (iv) positively oriented (a) - backbone - positively oriented (b) - positively oriented (c) - anti-oriented (d) (i.e., helper (F) - backbone - Rep (F) - Cap (F) - transgene (R)); or

[0182] (v) Normally oriented (a) - backbone - Normally oriented (c) - Normally oriented (b) - Anti-oriented (d) (i.e., helper (F) - backbone - Cap (F) - Rep (F) - transgene (R)).

[0183] In certain embodiments, the components in the nucleic acid molecule may have the following arrangement and orientation (normal orientation is abbreviated as 'F' and reverse orientation is abbreviated as 'R'):

[0184] (v) Normally oriented (a) - backbone - Normally oriented (c) - Normally oriented (b) - Anti-oriented (d) (i.e., helper (F) - backbone - Cap (F) - Rep (F) - transgene (R)).

[0185] In the above (i) to (viii), two ITRs can be arranged on both sides of the above (d).

[0186] The above nucleic acid molecules, plasmids and vectors can be prepared by any suitable technique, which techniques are well known in the art.

[0187] The above recombinant AAV production vector comprises all components required for recombinant AAV production (i.e., (a), (b), (c), and (d)) linked within a single molecule, so that each component can be introduced into a host cell at an equal 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 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.

[0188] Furthermore, the all-in-one vector system reduces raw material costs by producing only one vector instead of the three separate vectors required in the past. Furthermore, the all-in-one vector system allows for the introduction of a single vector instead of the three separate vectors, reducing the amount of individual vector input by more than 50%.

[0189] In one example, compared to a control triple transfection of a GOI expression vector, a Rep / Cap expression vector, and a helper vector, the all-in-one vector was shown to exhibit high recombinant AAV productivity even with a lower amount of plasmid DNA (pDNA).

[0190] In one embodiment, compared to a control triple-transfected with a GOI expression vector, a Rep / Cap expression vector, and a helper vector, it was confirmed that recombinant AAV produced by the all-in-one vector exhibited a high %Full / Empty capsid ratio even with a small amount of pDNA. Specifically, it was confirmed that the %F / E capsid ratio before purification of AAV produced by the all-in-one vector was at least 31% or higher and at most 47.6%. Therefore, the all-in-one vector can produce recombinant AAV with improved quality due to a high proportion of full capsids containing the therapeutic gene.

[0191] In one embodiment, it was confirmed that the recombinant AAV produced by the all-in-one vector exhibited equivalent protein expression levels compared to the recombinant AAV produced by the control triple-transfection method using the GOI expression vector, the Rep / Cap expression vector, and the helper vector. Therefore, the all-in-one vector can produce recombinant AAV at levels equivalent to those of recombinant AAV produced by the conventional triple-transfection method in terms of infectivity and gene expression function.

[0192] In one embodiment, the transgene (e.g., aflibercept) expressed in the recombinant AAV produced by the all-in-one vector was confirmed to possess biological activity, thereby demonstrating its potential as a therapeutic agent. Therefore, the all-in-one vector was confirmed to be immediately applicable to therapeutic agent development.

[0193] Another aspect provides a method for producing a recombinant adeno-associated virus (AAV). The method may be a method using a vector for producing a recombinant AAV according to one aspect.

[0194] The method comprises the steps of introducing a vector for producing a recombinant AAV according to one aspect into a host cell; and the step of isolating the recombinant AAV from the host cell.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] The above introduction can be used without limitation as long as it is a known method that can insert a vector for recombinant AAV production 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.

[0199] The above method may further include a step of culturing cells after the introducing step.

[0200] 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.

[0201] 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.

[0202] The separation can be performed by a known method. In one specific example, the separation can be performed using centrifugation or chromatography.

[0203] The above centrifugation method may be, but is not limited to, a cesium chloride (CsCl)-based ultra-high-speed centrifugation method.

[0204] 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.

[0205] The method can also be easily scaled up to industrial production because it requires only a single transfection of the host cell with a vector for recombinant AAV production with excellent recombinant AAV productivity.

[0206] In one embodiment, it was confirmed that the productivity and quality of recombinant AAV were excellent not only at the flask scale but also at the 2 L reactor scale. Therefore, it was confirmed that the method for producing recombinant AAV using the vector for recombinant AAV production according to the above aspect is capable of scale-up and immediate process application.

[0207] According to the above method, unlike the existing triple transfection method using three vectors, only a single vector is required, simplifying the recombinant AAV production process and reducing costs. Furthermore, unlike the triple transfection method, where the ratios of the three vectors injected into cells cannot be precisely controlled, the method according to the above aspect integrates all components into a single vector, allowing each component to be introduced at the same ratio. Accordingly, the productivity of recombinant AAV can be improved.

[0208] In addition, according to the above method, recombinant AAV having a high %Full capsid ratio can be produced, so that high-quality recombinant AAV can be produced.

[0209] Recombinant AAV produced by the above method can exhibit transduction ability and GOI functionality at an equivalent level to recombinant AAV produced by triple transfection.

[0210] Another aspect provides a recombinant AAV produced by the above method.

[0211] The recombinant AAV described above may have a high %Full / Empty capsid ratio. Furthermore, the recombinant AAV may have a low impurity content. Therefore, the recombinant AAV may have excellent quality and safety.

[0212] In one specific embodiment, the recombinant AAV may be single-stranded AAV (ssAAV) or self-complementary AAV (scAAV).

[0213] The above recombinant AAV can be used in a method of gene therapy.

[0214] The above recombinant AAV can be used for mass production of gene therapy clinical materials for treating patients.

[0215] The above recombinant AAV comprises an AAV capsid and genome.

[0216] 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.

[0217] In one specific example, the AAV capsid may be a wild-type AAV2 capsid.

[0218] The above AAV capsid may comprise a VP1 protein, a VP2 protein, and a VP3 protein.

[0219] The "genome" of the above AAV refers to the sequences that are ultimately packaged or encapsidated to form viral particles.

[0220] The genome may be linear single-stranded DNA.

[0221] The above genome contains a transgene.

[0222] The transgene may be arranged between ITRs. The transgene may be arranged between L-ITRs and R-ITRs.

[0223] The ITR may be derived from any AAV serotype. The ITR 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. 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.

[0224] The above ITR may be a wild-type ITR or a mutant thereof.

[0225] Another aspect provides a composition comprising a recombinant AAV produced by the above method.

[0226] The above composition may be a pharmaceutical composition.

[0227] The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0228] The composition may be used for gene therapy. The composition may be a composition for therapeutic gene delivery for gene therapy.

[0229] 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.

[0230] 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.

[0231] 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, intrathecal, or intravitreal. The pharmaceutical composition may be prepared in a systemic dosage form or a topical dosage form.

[0232] 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.

[0233] 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.

[0234] Another aspect provides a gene therapy method using a recombinant AAV produced by the above method.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] The route of administration can be determined by a person skilled in the art and may include, for example, intranasal, intravenous, intramuscular, subcutaneous, intradermal, intravitreal, oral, and other parenteral routes of administration. Two or more routes of administration may be combined, if necessary.

[0239] The above administration may be performed 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] According to the vector for recombinant AAV production according to one aspect, the components essential for recombinant AAV production are integrated into a single vector system, thereby enabling the production of high-quality recombinant AAV with high efficiency and productivity. Furthermore, since each component is positioned in a combination of optimized arrangements and orientations, the vector has the effects of improving AAV productivity, increasing the %Full / Empty capsid ratio, and reducing impurities.

[0244] The method for producing recombinant AAV according to one aspect allows for the production of recombinant AAV through the introduction of a single vector, thereby reducing production costs. Furthermore, the method can be easily scaled up to industrial production and can be immediately applied to the process.

[0245] Recombinant AAV produced using a vector for recombinant AAV production according to the daily aspect has excellent safety due to reduced impurities, and thus can be applied in various fields of gene therapy.

[0246] Figure 1 illustrates the structure of an all-in-one vector for recombinant AAV production according to one embodiment.

[0247] Figure 2 is a vector map of an all-in-one vector for recombinant AAV production according to one embodiment.

[0248] Figure 3 shows the arrangement order and orientation of each gene in an all-in-one vector for recombinant AAV production according to one embodiment.

[0249] Figure 4 shows the results showing the AAV productivity (relative viral genome) of the All-in-One vector and the control (Triple) at the flask scale.

[0250] Figure 5 shows the results showing the relative full / empty capsid ratios of the All-in-One vector and the control group (Triple) at the flask scale.

[0251] Figure 6 shows the results showing AAV productivity of the All-in-One vector and the control group (Triple) 72 hours (3 days) after transfection at a 2 L scale.

[0252] Figure 7 shows the results of AAV productivity (vg / L, N=4) of the All-in-One vector and the control group (Triple) at 24 hours (day 1), 48 hours (day 2), and 72 hours (day 3) after transfection in a 2 L scale.

[0253] Figure 8 shows the results of the quality (VG / VP ratio, N=4) of AAV produced by the All-in-One vector and the control group (Triple) 24 hours (day 1), 48 hours (day 2), and 72 hours (day 3) after transfection at a 2 L scale.

[0254] Figure 9 shows the results showing the amount of transduction gene expression of rAAV produced from the All-in-One vector and the control group (Triple).

[0255] Figure 10 shows the results of confirming the cell proliferation inhibition ability of aflibercept expressed after transduction of recombinant AAV produced from the All-in-One vector and the control group (Triple).

[0256] Figure 11 is a graph showing the cell proliferation inhibition ability (% relative potency) of aflibercept expressed after transduction of recombinant AAV produced from the All-in-One vector and the control group (Triple).

[0257] Figure 12 is a graph showing the VEGF neutralizing ability (% relative potency) of aflibercept expressed after transduction of recombinant AAV produced from the All-in-One vector and the control group (Triple).

[0258] Figure 13 illustrates the process for creating an all-in-one vector with different order and orientation of components.

[0259] Figure 14 is a diagram showing the results of evaluating the AAV productivity of eight all-in-one vectors.

[0260] Figure 15 is a diagram showing the results of evaluating the AAV quality (VG / VP ratio) of eight all-in-one vectors.

[0261] 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.

[0262] Example 1. Creation of an all-in-one vector

[0263] An all-in-one vector for recombinant AAV production containing the gene-of-interest (GOI) and Rep, Cap, and Helper genes, which are components required for adeno-associated virus (AAV) production, was constructed.

[0264] Specifically, the genetic sequence information of each component of the all-in-one vector was obtained based on existing literature.

[0265] Helper E2A, E4, and VA genes were obtained from Adenovirus 2 (Gene Bank Accession No. NC_001405.1).

[0266] Rep, Cap, and L-ITR, R-ITR genes were obtained from Adeno-associated virus serotype 2 (Gene Bank Accession No. NC_001401.2).

[0267] As a GOI, the aflibercept gene is Eylea ® (PMDA) was used.

[0268] The backbone vector to be used in the expression vector was obtained by synthesizing the pBR322 origin and ampicillin resistance gene based on previously known literature (Bolivar et al., 1977).

[0269] To construct an all-in-one vector, the restriction enzyme sites of BamHI (NEB), NotI (NEB), PmeI (NEB), and PacI (NEB) were made 5' to 3' or 3' to 5', respectively, to enable the construction of a bidirectional vector. Primers were designed based on the sequence information of the corresponding genes of each component, and the components were obtained by PCR amplification and then inserted into the vector one copy at a time. The genetic sequence of the cloned expression vector was confirmed by request to Macrogen Co., Ltd. (Seoul, Korea).

[0270] The components were inserted in various orders into vectors each having restriction enzymes BamHI (NEB), NotI (NEB), PmeI (NEB), and PacI (NEB), as shown in Figure 3.

[0271] Figure 1 illustrates the structure of an all-in-one vector for recombinant AAV production according to one embodiment.

[0272] Figure 2 is a vector map of an all-in-one vector for recombinant AAV production according to one embodiment. The vector of Figure 2 according to one embodiment uses E2A (SEQ ID NO: 2), E4 (SEQ ID NO: 3), and VA (SEQ ID NO: 5) as helper genes; Rep2 (SEQ ID NO: 6) as a Rep gene; Cap2 (SEQ ID NO: 7) as a Cap gene; and L-ITR (SEQ ID NO: 8), R-ITR (SEQ ID NO: 9), and Eylea cassete (SEQ ID NO: 10).

[0273] Figure 3 shows the arrangement order and orientation of each gene in an all-in-one vector for recombinant AAV production according to one embodiment.

[0274] The sequences used to create the all-in-one vector and their sequence information are shown in Table 1 below.

[0275] Sequence number name description 1 All-in-One vector 2 Sequence of the all-in-one vector 2 E2 AAV2-derived helper gene 3 E4 Adenovirus 2-derived helper gene 4 E4 orf 6 Adenovirus-derived helper gene 5 VAA Adenovirus 2-derived helper gene 6 Rep2 AAV2-derived Rep gene 7 Cap2 AAV2-derived Cap gene 8 L-ITRAAV2-derived L-ITR gene 9 R-ITRAAV2-derived R-ITR gene 10 Eylea cassette Transgene cassette (including enhancer, promoter, intron, Kozak sequence, aflibercept, and poly A sequence)

[0276]

[0277] Example 2. Confirmation of AAV productivity of the all-in-one vector (flask scale)

[0278] We conducted feasibility experiments for AAV productivity of the all-in-one vector at flask scale.

[0279] 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).

[0280] The all-in-one vector of Fig. 2 of Example 1 was used as the experimental group. The triple vector for triple transfection, i.e., the GOI vector, the Rep / Cap vector, and the Helper vector, was used as the control group. Since the all-in-one vector has 1 copy of each of the GOI, Rep / Cap, and Helper genes inserted and each gene is processed with the same copy number, in the case of the control group (triple vector), the GOI vector, the Rep / Cap vector, and the Helper vector were triple transfected at a molar ratio of 1:1:1 to compare the productivity. In the case of the all-in-one vector, the experiment was conducted under various pDNA conditions from 33% to 100% compared to the control pDNA (plasmid DNA).

[0281] Both the control and experimental groups were transiently transfected with polyethylenimine (PEI) at 3E06 cells / mL (30 mL, 125 mL flask) (N=4). Forty-eight hours after transfection, cells were lysed to determine AAV productivity, and the produced AAV viral genome (VG) was measured by qPCR using ITR-specific primers using the AAVpro® Titration Kit (TAKARA).

[0282] Figure 4 shows the results showing the AAV productivity (relative viral genome) of the All-in-One vector and the control (Triple) at the flask scale.

[0283] As a result, as shown in Fig. 4, the AAV productivity of the all-in-one vector showed a sequential increase as the transfected plasmid DNA concentration increased from 33% to 100% pDNA compared to the control group. In addition, the all-in-one vector showed an AAV productivity of 96.8%, which was equivalent to the control group, under the 33% pDNA condition, and showed an approximately 1.9-fold improvement in AAV productivity under the 83% pDNA condition.

[0284] Therefore, it was confirmed that the All-in-One vector exhibited high recombinant AAV productivity using less pDNA than the three existing triple transfections, indicating that it had improved recombinant AAV productivity.

[0285] Example 3. Effect of All-in-One Vector on AAV Quality Improvement (flask scale)

[0286] To determine the effectiveness of the all-in-one vector in improving AAV quality, a flask-scale experiment was conducted to measure the full / empty capsid ratio of the all-in-one vector. A full capsid represents a capsid containing genetic material, while an empty capsid represents a capsid containing only a portion of the genetic material. Therefore, a higher full / empty capsid ratio indicates better AAV quality.

[0287] Specifically, AAV produced in a flask scale according to Example 2 was purified using an AAV affinity resin, and then the full / empty capsid ratio of the virus was measured using anion exchange-high-performance liquid chromatography (AEX-HPLC) analysis (N=4).

[0288] Figure 5 shows the results showing the relative full / empty capsid ratios of the All-in-One vector and the control group (Triple) at the flask scale.

[0289] As a result, as shown in Fig. 5, the %Full / empty capsid ratio of AAV was confirmed to have an AAV quality improvement effect of about 2 times higher than that of the control group (Triple) under the All-in-One vector pDNA 33% condition. In addition, the %full / empty capsid ratio decreased as the transfected plasmid DNA concentration increased from the pDNA 33% to 100% condition, but the AAV quality improvement effect of about 1.3 times higher than that of the control group was confirmed even under the pDNA 100% condition compared to the control group.

[0290] Therefore, it was confirmed that the All-in-One vector had the effect of improving AAV quality by improving the ratio of full particles containing therapeutic genes using less pDNA than AAV produced through the existing three-type triple transfection.

[0291] Example 4. Evaluation of AAV productivity and AAV quality of the all-in-one vector (2 L scale)

[0292] Experiments were conducted to evaluate the AAV productivity and AAV quality of the all-in-one vector at 2 L scale.

[0293] AAV productivity was evaluated using the same method as in Example 2, except that the experiment was conducted under conditions of 67% compared to the control pDNA and cells were lysed to check AAV productivity 24, 48, and 72 hours after transfection. Specifically, VPM liquid medium (Viral Production Medium, manufacturer: Thermo Fisher scientific, catalog number: A4817901) was used as the medium. The pDNA concentration was 2 mg / L for the all-in-one vector and 3 mg / L (1.0 μg / 1E06 viable cells) for the triple vector. In addition, the AAV quality was evaluated by measuring the full / empty capsid ratio (VG / VP ratio) for the produced AAV using the same method as in Example 3 (N=4).

[0294] Figure 6 shows the results showing AAV productivity of the All-in-One vector and the control group (Triple) 72 hours (3 days) after transfection at a 2 L scale.

[0295] Figure 7 shows the results of AAV productivity (vg / L, N=4) of the All-in-One vector and the control group (Triple) at 24 hours (day 1), 48 hours (day 2), and 72 hours (day 3) after transfection in a 2 L scale.

[0296] Figure 8 shows the results of the quality (VG / VP ratio, N=4) of AAV produced by the All-in-One vector and the control group (Triple) 24 hours (day 1), 48 hours (day 2), and 72 hours (day 3) after transfection at a 2 L scale.

[0297] As a result, as shown in Figures 6 and 7, it was confirmed that the productivity of the recombinant AAV virus was improved by approximately 1.5 times even under the all-in-one vector condition at 67% of the pDNA level compared to the control group.

[0298] In addition, as shown in Fig. 8, AAV produced by the all-in-one vector on the third day after transfection had a VG / VP ratio of 5.23% (SD: 0.23), and it was confirmed that AAV could be produced with a higher VG / VP ratio even with 67% of the amount of pDNA compared to the control group.

[0299] Therefore, it was confirmed that the all-in-one vector can produce recombinant AAV with significantly improved quality with significantly improved recombinant AAV productivity even using a smaller amount of pDNA compared to the triple vector.

[0300] Example 5. Evaluation of gene expression function of recombinant AAV produced using an all-in-one vector.

[0301] To evaluate the transducing capability of recombinant AAV produced by the All-in-One vector, an experiment was conducted to compare the amount of gene expression after rAAV injection into target cells.

[0302] Specifically, the target cells used were ARPE-19 (human retinal pigment epithelia) cells, and the cells were cultured in DMEM / F12 medium supplemented with 10% FBS (fetal bovine serum) at 37°C and 5% CO2 conditions. The day before rAAV injection, ARPE-19 cells were cultured at 1.0E4 cell / cm 2The viruses of the control group (Triple) and the all-in-one test group produced in Example 4 were both prepared using CsCl ultracentrifugation to obtain highly pure AAV viruses. Then, the obtained viruses were diluted in DMEM / F12 medium without FBS to treat with multiplicities of infection (MOI) of 1.5E02, 4.6E02, 1.4E03, and 4.1E03 vg / cell. The viruses were treated in 96-well plates coated with ARPE-19 cells from which the culture medium had been completely removed. Two hours after the virus treatment, DMEM / F12 medium containing 10% FBS was added to the 96-well plates treated with the viruses. After 72 hours of virus treatment, ARPE-19 cultures were harvested and the expression levels of proteins expressed by the AAV virus were analyzed using the ECL (Electrochemiluminescence) assay.

[0303] Figure 9 shows the results showing the amount of transduction gene expression of rAAV produced from the All-in-One vector and the control group (Triple).

[0304] As a result, as shown in Fig. 9, it was confirmed that rAAV produced by the all-in-one vector and rAAV produced by the triple vector had equivalent GOI protein expression levels. This confirmed that rAAV produced by the all-in-one vector can produce rAAV equivalent to rAAV produced by the triple vector in terms of infectivity and gene expression function.

[0305] Example 6. Confirmation of the cell proliferation inhibitory ability of aflibercept expressed after transduction of recombinant AAV produced using an all-in-one vector.

[0306] To verify the GOI functionality of rAAV produced by the all-in-one vector, the biological activity of aflibercept expressed after transduction of the recombinant AAV obtained in Example 5 was evaluated, including its ability to inhibit cell proliferation.

[0307] The GOI used as an example in this example is Eylea (active ingredient: aflibercept). The aflibercept protein is a recombinant fusion protein consisting of a portion of the extracellular domains of human VEGF receptor 1 and 2 fused to the Fc portion of human IgG1. Aflibercept binds VEGF and consequently prevents angiogenesis. Therefore, to confirm the biological activity of aflibercept in inhibiting cell proliferation, the VEGF-dependent proliferation inhibitory effect on human umbilical vein endothelial cells (HUVECs) was measured using the Cell Titer-Blue® system. Since when aflibercept is treated on HUVECs, the fluorescence signal appears to be proportional or inversely proportional to the antibody concentration as an indicator of proliferation inhibition, the relative anti-proliferation ability of aflibercept compared to the reference was confirmed using the Cell Titer-Blue® system to confirm the biological activity of cell proliferation inhibition.

[0308] Figure 10 shows the results of confirming the cell proliferation inhibition ability of aflibercept expressed after transduction of recombinant AAV produced from the All-in-One vector and the control group (Triple).

[0309] Figure 11 is a graph showing the cell proliferation inhibition ability (% relative potency) of aflibercept expressed after transduction of recombinant AAV produced from the All-in-One vector and the control group (Triple).

[0310] As a result, as shown in FIGS. 10 and 11, it was confirmed that rAAV produced by the All-in-One vector had the biological activity of aflibercept that inhibits cell proliferation.

[0311] Example 7. Confirmation of the cell signal neutralizing ability of aflibercept expressed after transduction of recombinant AAV produced using an all-in-one vector.

[0312] To verify the GOI functionality of rAAV produced by the all-in-one vector, the biological activity of aflibercept expressed after transduction of the recombinant AAV obtained in Example 5 was evaluated, including its ability to neutralize cell signals.

[0313] NFAT-RE-luc2P / KDR HEK293 (KDR293) cells were transduced with recombinant AAV (AAV2-Aflibercept) produced by an all-in-one vector to measure the biological efficacy of aflibercept in culture. KDR293 cells are a genetically modified cell line containing two gene cassettes. One cassette ensures constitutive expression of VEGFR2 (KDR) to open the response to VEGF-A in KDR293 cells, and the other cassette allows inducible expression of luciferase by the NFAT response element when the VEGFR2 signaling pathway is activated. Consequently, the amount of luminescence was quantitatively measured in proportion to the level of activation of the VEGFR2 signaling pathway by exogenous VEGF.

[0314] Figure 12 is a graph showing the VEGF neutralizing ability (% relative potency) of aflibercept expressed after transduction of recombinant AAV produced from the All-in-One vector and the control group (Triple).

[0315] As a result, as shown in Fig. 12, it was confirmed that the AAV virus produced with the all-in-one vector had the biological activity of aflibercept, which neutralizes VEGF.

[0316] Example 8. Impurity analysis of recombinant AAV produced using an all-in-one vector

[0317] An experiment was conducted to analyze impurities in rAAV produced by the all-in-one vector manufactured in Figure 2 of Example 1.

[0318] rAAV produced by triple vector, which was triple transfected with GOI vector, Rep / Cap vector, and Helper vector at a molar ratio of 1:2.5:0.5 or 1:1:1, was used as a control.

[0319] High-purity rAAV was obtained by isolating rAAV produced by all-in-one or triple vectors using CsCl ultracentrifugation. DNA within the AAV particles was isolated from the obtained AAV using the QIAquick® Gel Extraction Kit. The isolated DNA was subjected to genome analysis using the PacBio SequelII (long-read) sequencing platform at Macrogen (Seoul, Korea).

[0320] Table 2 shows the results of genome analysis of rAAV produced by all-in-one vector or triple vector using the PacBio SequelII sequencing platform.

[0321] SampleAssigned TypeCalculation (%)ssAAVscAAVotherchimericRep / CaphelperhostunmappedAAVImpurityTriple (1:2.5:0.5)83.1711.34.160.490.40.320.15094.475.52Triple (1:1:1)87.858.383.120.260.180.140.07096.233.77All-in-One 60ug86.929.61.890.510.680.360.02096.523.46

[0322] PacBio SequelII analysis results classify the genome into GOI sequences containing ITRs in the form of ssAAV or scAAV, Other (sequences that map to the GOI but cannot be classified as ssAAV or scAAV due to different orientation or form), Chimeric (two or more genomes mapped (e.g., chimeric forms of vector and host; chimeric forms of Helper and Rep / Cap, etc.)), Rep / Cap (Rep / Cap plasmid), Helper (Helper plasmid), and Host (e.g., host cell genome).

[0323] As a result, as shown in Table 2, the impurity level of rAAV produced by the all-in-one vector was 3.46%, which was confirmed to be significantly lower than the impurity level of rAAV produced by the triple vector.

[0324] Example 9. Production of various all-in-one vectors

[0325] To derive the optimal vector for AAV productivity, eight all-in-one vectors were created by varying the order and orientation of the components of the all-in-one vector, Rep, Cap, Helper, and GOI (Transgene).

[0326] Specifically, all-in-one vectors were constructed using the same method as in Example 1, except that the pBR322-Kan vector was used as a backbone vector and PmeI, PacI, and AsiSI restriction enzyme sites were inserted to facilitate replacement of each component. The order and orientation of each component in the eight constructed all-in-one vectors are shown in Table 3 below. The all-in-one vector AiO0165 has the same order and orientation of the components as the all-in-one vector of Fig. 2 constructed in Example 1. In addition, the eight vectors were constructed based on AiO0165, and all sequences are identical except for the direction and arrangement of the components and the restriction enzyme sites for cloning.

[0327] Figure 13 illustrates the process for creating an all-in-one vector with different order and orientation of components.

[0328] No. Order of names of all-in-one vector components (5'→3') and orientation (Forward: F, Reverse: R) 1 AiO0165 Helper (F) - backbone - Rep (F) - Cap (F) - GOI (F) 2 AiO0166 Helper (F) - backbone - Cap (F) - Rep (F) - GOI (F) 3 AiO0167 Rep (F) - Cap (F) - Helper (F) - backbone - GOI (F) 4 AiO0169 Helper (F) - backbone - Rep (F) - Cap (F) - GOI (R) 5 AiO0170 Helper (F) - backbone - Cap (F) - Rep (F) - GOI (R) 6 AiO0171 Rep (F) - Cap (F) - Helper (F) - backbone - GOI (R)7AiO0172Backbone - Rep (F) - Cap (F) - Helper (F) - GOI (R)8AiO0173Helper (F) - Rep (F) - Cap (F) - backbone - GOI (F)

[0329]

[0330] Example 10. Evaluation of AAV productivity and AAV quality according to the order and orientation of components of the all-in-one vector.

[0331] An experiment was conducted to evaluate AAV productivity and AAV quality according to the order and orientation of Helper, Rep, Cap, and GOI, which are components of the all-in-one vector.

[0332] Specifically, for the eight all-in-one vectors produced in Example 9, AAV productivity and AAV quality were evaluated using the same method as in Examples 2 and 3.

[0333] First, as a result of evaluating the AAV productivity of the above 8 all-in-one vectors, it was confirmed that AiO0170 showed the best productivity, showing 3.2 times the productivity compared to AiO0165 (Fig. 14). It can be seen that AiO0170 is a vector in which the GoI direction is reversed and the order of Rep / Cap is reversed compared to the control group AiO0165. Meanwhile, AiO0169, in which only the GoI direction is reversed compared to the control group (AiO0165), showed an improvement in productivity of approximately 1.6 times, and AiO0166, in which only the order of Rep / Cap is reversed compared to the control group (AiO0165), showed an improvement in productivity of approximately 2.2 times.

[0334] Next, the quality of AAVs produced with the above eight all-in-one vectors was evaluated through VG / VP ratio analysis. As a result, AiO0170, which showed the highest productivity, also showed a significantly improved VG / VP ratio, indicating superior quality (Fig. 15). Meanwhile, the VG / VP ratios of all other candidates, which showed increased productivity compared to the control group, did not significantly improve compared to the control group (AiO0165).

[0335] In summary, among the eight all-in-one vectors with different component orders and orientations, AiO0170 has the highest AAV productivity and AAV quality.

[0336]

[0337] 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 for producing a recombinant adeno-associated virus (AAV) comprising the nucleotide sequence of the following genes arranged in a single nucleic acid molecule: (a) Helper virus genes required for AAV production; (b) Rep gene of AAV; (c) Cap gene of AAV; and (d) transgene.

2. A vector for producing a recombinant AAV, wherein the transgene is arranged between ITRs (Inverted Terminal Repeats) according to claim 1.

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

4. A vector for producing a recombinant AAV, wherein the nucleic acid molecule according to claim 1 is linear or circular.

5. A vector for producing a recombinant AAV, wherein the recombinant AAV produced by the vector in claim 1 is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

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

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

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

9. A vector 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.

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

11. A vector for producing a recombinant AAV, wherein the variant is an engineered helper virus gene according to claim 9.

12. A vector for producing a recombinant AAV, wherein the nucleotide sequence of the Rep 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.

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

14. A vector 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.

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

16. A vector for producing a recombinant AAV according to claim 14, wherein the variant is a variant having one or more mutations in the wild-type sequence, or a truncated variant.

17. A vector 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.

18. A vector for producing a recombinant AAV, wherein the nucleotide sequence of the Cap gene according to claim 1 is derived from AAV2, AAV5, or AAV8.

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

20. A vector for producing a recombinant AAV, wherein the variant is a variant having one or more mutations in the wild-type sequence, or a truncated variant, according to claim 19.

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

22. A vector for producing a recombinant AAV, wherein the ITR in claim 2 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.

23. A vector for producing a recombinant AAV, wherein the ITR according to claim 2 is derived from AAV2.

24. A vector for producing a recombinant AAV, wherein the ITR in claim 2 is a wild-type ITR or a mutant thereof.

25. A vector for producing a recombinant AAV, wherein the ITR comprises all or part of a wild-type ITR sequence according to claim 2.

26. A vector for producing a recombinant AAV, wherein the variant has a lower GC content than the wild-type ITR according to claim 24.

27. A vector for producing a recombinant AAV, wherein the L-ITR and R-ITR in claim 3 are modified to be capable of producing scAAV.

28. A vector for producing a recombinant AAV, wherein the nucleic acid molecule of claim 1 further comprises at least one 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, and a co-regulatory element.

29. A vector for producing a recombinant AAV, wherein the nucleic acid molecule further comprises at least one expression control element operably linked to any one of (a), (b), (c), and (d) according to claim 1.

30. A vector for producing a recombinant AAV, wherein the expression control element according to claim 29 comprises at least one selected from an enhancer, a promoter, a silencer, an insulator, an intron, an untranslated region (UTR), a Kozak sequence, a signal peptide, and a polyadenylation signal sequence.

31. A vector for producing a recombinant AAV according to claim 1, wherein the nucleic acid molecule further comprises at least one promoter operably linked to any one of (a), (b), and (c).

32. A vector for producing a recombinant AAV, wherein the nucleic acid molecule further comprises an expression control element operably linked to (d) in claim 1.

33. A vector for producing a recombinant AAV, wherein the expression control element according to claim 32 comprises at least one selected from an enhancer, a promoter, an intron, a Kozak sequence, and a polyadenylation signal sequence.

34. A vector for producing a recombinant AAV according to any one of claims 1 to 33, wherein the nucleic acid molecule does not contain a replication origin or additionally contains a replication origin.

35. A vector for producing a recombinant AAV according to claim 34, wherein the replication origin is a pUC origin, a pBR322 origin, a pMB1 origin, a pSC101 origin, a p15A origin, a pJET1.2 origin, or a synthetic origin.

36. A vector for producing a recombinant AAV according to any one of claims 1 to 35, wherein the nucleic acid molecule further comprises a selectable marker.

37. A vector for producing a recombinant AAV, wherein the selection marker according to claim 36 is at least one selected from ampicillin, streptavidin, kanamycin, hygromycin, neomycin, puromycin, blasticidin, and zeocin.

38. A vector for producing a recombinant AAV, wherein in the nucleic acid molecule of claim 1, (a), (b), (c), and (d) are each independently arranged in a forward orientation or a reverse orientation in any order.

39. A vector for producing a recombinant AAV, wherein in the nucleic acid molecule of claim 1, (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).

40. A vector for producing a recombinant AAV, wherein in claim 38 or 39, (a), (b), (c), and (d) are all oriented in the right direction.

41. A vector for producing a recombinant AAV according to claim 38 or 39, wherein (a), (b), and (c) are oriented normally; and (d) is oriented in reverse.

42. A vector for producing a recombinant AAV, according to any one of claims 38 to 41, wherein the ITR is arranged on both sides of (d).

43. A vector for producing a recombinant AAV, wherein the vector backbone is arranged in any order from 1) to 12) in claim 39.

44. A vector for producing a recombinant AAV, wherein the vector backbone is arranged after the last order in 1) to 12) above, or before the first order in claim 39.

45. A vector for producing a recombinant AAV according to claim 43 or 44, wherein the vector backbone comprises at least one selected from a replication origin and a selection marker.

46. ​​A vector for producing a recombinant AAV, wherein in the nucleic acid molecule of claim 1, (a), (b), (c), and (d) have the following arrangement and orientation: (i) Oriented (a) - Backbone - Oriented (b) - Oriented (c) - Oriented (d); (ii) Oriented (a) - backbone - Oriented (c) - Oriented (b) - Oriented (d); (iii) Oriented (b) - Oriented (c) - Oriented (a) - Backbone - Oriented (d); (iv) Oriented (a) - backbone - Oriented (b) - Oriented (c) - reverse oriented (d); (v) Oriented (a) - backbone - Oriented (c) - Oriented (b) - reverse oriented (d); (vi) Oriented (b) - Oriented (c) - Oriented (a) - Backbone - Reverse Oriented (d); (vii) backbone - positively oriented (b) - positively oriented (c) - positively oriented (a) - reversely oriented (d); or (viii) Oriented (a) - Oriented (b) - Oriented (c) - Backbone - Oriented (d).

47. A vector for producing a recombinant AAV, wherein in the nucleic acid molecule of claim 1, (b) and (c) are arranged in the order of (c) - (b).

48. A vector for producing a recombinant AAV, wherein (d) in the nucleic acid molecule is arranged in a reverse orientation in claim 1.

49. A vector for producing a recombinant AAV, wherein in the nucleic acid molecule of claim 1, (a), (b), (c), and (d) have the following arrangement and orientation: (ii) Oriented (a) - backbone - Oriented (c) - Oriented (b) - Oriented (d); (iii) Oriented (b) - Oriented (c) - Oriented (a) - Backbone - Oriented (d); (iv) oriented (a) - backbone - oriented (b) - oriented (c) - reverse oriented (d); or (v) Oriented (a) - backbone - Oriented (c) - Oriented (b) - reverse oriented (d).

50. A step of introducing a vector for producing a recombinant AAV according to any one of claims 1 to 49 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).

51. A method for producing a recombinant AAV according to claim 50, wherein the host cell is a mammalian cell or an insect cell.

52. A method for producing a recombinant AAV according to claim 51, wherein the mammalian cell is selected from HEK293 cells, HEK293F cells, HEK293T cells, and cells derived therefrom.

53. A method for producing a recombinant AAV according to claim 51, wherein the insect cell is an Sf9 cell, an Sf21 cell, a TN-5B1-4 cell, or a High Five cell.

54. A method for producing a recombinant AAV according to claim 50, 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.

55. A method for producing a recombinant AAV according to claim 50, wherein the separation is performed using centrifugation or chromatography.

56. A method for producing a recombinant AAV, wherein the centrifugation method according to claim 55 is a cesium chloride-based ultra-high-speed centrifugation method.

57. A method for producing a recombinant AAV according to claim 55, 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.

58. A recombinant AAV produced by the vector of any one of claims 1 to 49.

59. In claim 58, the recombinant AAV is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

60. A composition comprising the recombinant AAV of claim 58.

61. A gene therapy method comprising administering to a subject in need thereof a recombinant AAV of claim 58 or a pharmaceutical composition comprising the same.

62. Use of a vector for producing a recombinant AAV according to any one of claims 1 to 49, or a recombinant AAV produced by said vector, for the manufacture of a medicament for gene therapy.

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