Codon-optimized polynucleotide encoding argininosuccinate synthase and plasmid for producing raav comprising same

Abstract

Provided are: a codon-optimized polynucleotide encoding an argininosuccinate synthase; a vector for producing a recombinant AAV, comprising the polynucleotide; a recombinant AAV produced by the vector; and use of the recombinant AAV for the treatment of citrullinemia type 1. The plasmid comprising the polynucleotide can produce a recombinant AVV exhibiting high expression and activity of an argininosuccinate synthase, with excellent productivity and high quality.

Description

Codon-optimized polynucleotide encoding argininosuccinate synthase and plasmid for rAAV production containing the same

[0001] The present invention relates to a codon-optimized polynucleotide encoding argininosuccinate synthase, a vector for producing recombinant AAV comprising said polynucleotide, recombinant AAV produced by said plasmid, and the use of said recombinant AAV for the treatment of type 1 citrullinemia.

[0002] Citrullinemia is a genetic disorder caused by a deficiency in the argininosuccinate synthethase (ASS) enzyme. The ASS enzyme is one of the five major enzymes involved in hepatic urea metabolism, and its deficiency leads to the accumulation of ammonia—a protein metabolite—and other toxic substances in the blood, as well as hypercitrullinemia and arginine deficiency. This results in neurological damage, such as severe vomiting, irritability, lethargy, and seizures, and can even lead to death. Citrullinemia is classified into Type 1 or Type 2; Type 1 citrullinemia occurs in newborns and infants and is caused by abnormalities in the ASS gene.

[0003] Previously, to treat type 1 citrullinemia, mild cases had to be treated with dietary therapy, while severe cases required liver transplantation. Consequently, patients faced difficulties as there were no other clear treatment options, and they had no choice but to follow a diet for the rest of their lives or wait for a liver transplant.

[0004] Due to these limitations, gene-based therapies for citrullinemia type 1 are being developed, and in particular, adeno-associated virus (AAV) is being selected as a vector with the highest safety and efficacy for in vivo gene delivery. However, gene therapies using recombinant AVV (rAAV) have the following problems.

[0005] The first problem is low efficiency and productivity. To produce AAV using the production cell line HEK293 via triple transfection, a total of three components are required, including Rep / Cap, Helper, and GOI (Gene-of-interest). Recombinant AAV can be produced with high efficiency only if all three components are introduced into the production cell line simultaneously in appropriate proportions. However, because this introduction process operates independently for each component, the rate at which all three plasmids are injected simultaneously is low, at less than 30%. Furthermore, the ratio of the three plasmids injected into the cells cannot be precisely controlled, which can lead to variations in transfection efficiency across production batches and result in reduced productivity and quality of recombinant AAV.

[0006] The second problem is the transfer of antibiotic resistance genes via reverse packaging. As gene therapy drugs, quality and safety are critical. However, it is known that the %Full capsid level of AAVs produced using conventional triple transfection technology is less than 30%, and there have been reports of cases where the genome within the produced recombinant AAV is not 100%. Furthermore, since the insertion of antibiotic resistance genes via empty capsids or reverse packaging during the AAV production process can trigger an immune response, various high-purity recombinant AAV purification processes are being developed to overcome and minimize these quality limitations.

[0007] The third problem is low therapeutic gene expression and immune response. Development candidates for AAV gene therapies targeting type 1 citrullinemia face issues such as low expression of therapeutic genes and reduced therapeutic efficacy due to the induction of immune responses. To overcome these problems, efforts are focused on developing therapeutic gene sequences with CpGs removed, as well as tissue-specific promoters and capsids. However, this rational engineering is not yet at a mature stage, and the complexity of gene regulation in functionally improved AAV vectors regarding off-target tissue, overexpression toxicity, and innate immune response must be evaluated, and the stability of the translated therapeutic gene must be verified.

[0008] Therefore, it is necessary to develop an AAV gene therapy for citrullinemia type 1 that can improve the aforementioned problems and a method for producing the same.

[0009] The present disclosure relates to a codon-optimized polynucleotide capable of enhancing the expression and activity of argininosuccinate synthase and minimizing the immune response.

[0010] In addition, the present disclosure relates to an all-in-one vector system capable of producing recombinant AAV for treating type 1 citrullinemia with excellent productivity and high quality.

[0011] In addition, the present disclosure relates to a recombinant AAV for treating type 1 citrullinemia, which is produced by the all-in-one vector system and has increased expression and activity of argininosuccinate synthase.

[0012] One aspect is to provide a codon-optimized polynucleotide encoding argininosuccinate synthase (ASS1).

[0013] Another aspect is providing a vector for producing recombinant adeno-associated virus (AAV) to treat type 1 citrullinemia.

[0014] Another aspect is to provide an expression cassette or a vector comprising said expression cassette, which includes a codon-optimized polynucleotide encoding argininosuccinate synthase (ASS1) and one or more expression regulatory elements.

[0015] Another aspect is to provide a method for producing recombinant AAV to treat type 1 citrullinemia.

[0016] Another aspect is to provide recombinant AAV produced by a vector according to one aspect or a method according to one aspect.

[0017] Another aspect is providing recombinant AAV to treat type 1 citrullinemia.

[0018] Another aspect is to provide a pharmaceutical composition for preventing or treating type 1 citrullinemia, comprising a vector according to one aspect, recombinant AAV produced by a vector according to one aspect, or recombinant AAV according to one aspect.

[0019] Another aspect provides a method for delivering argininosuccinate synthase to an individual in need of it, comprising the step of administering to the individual an effective amount of a vector according to one aspect, recombinant AAV produced by a vector according to one aspect, recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.

[0020] Another aspect provides a method for treating type 1 citrullinemia, comprising the step of administering to an individual an effective amount of a vector according to one aspect, a recombinant AAV produced by a vector according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.

[0021] Another aspect is to provide a vector according to one aspect, a recombinant AAV produced by said vector, or a use of a recombinant AAV according to one aspect for the manufacture of a therapeutic drug for citrullinemia type 1.

[0022] All technical terms used herein shall be used in the sense generally understood by those skilled in the art in the relevant field of the present invention, unless otherwise defined. Furthermore, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of the present invention. Additionally, numerical values ​​described herein are deemed to include the meaning of "approximately" unless explicitly stated otherwise. The contents of all publications cited as references in this specification are incorporated into this specification in their entirety by reference.

[0023] In this specification, the terms “about” or “approximately” may be interpreted to mean a value or range within 10%, 5%, 4%, 3%, 2%, or 1% above or below a given value or range.

[0024]

[0025] One aspect provides a codon-optimized polynucleotide encoding argininosuccinate synthase (ASS1).

[0026] In this specification, the term "argininosuccinate synthase" (argininosuccinate synthase or argininosuccinate synthetase, ASS or ASS1) is an enzyme that catalyzes the synthesis of argininosuccinate from citrulline and aspartic acid. In humans, argininosuccinate synthase is encoded by the ASS gene located on chromosome 9. A deficiency of said argininosuccinate synthase may result in citrullinemia type 1. said argininosuccinate synthase may be human argininosuccinate synthase.

[0027] The term "polynucleotide" may refer to any form of nucleic acid, including DNA and RNA, and oligonucleotides. Polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. The sequence or structure of a specific polynucleotide may be described according to the convention of providing the sequence in the 5' to 3' direction.

[0028] "Polypeptides," "proteins," and "peptides" encoded by polynucleotide sequences may include functional subsequences, modified forms, or sequence variants, provided that they retain the functionality of natural proteins. The terms "modified" or "variant" mean that the sequence of a polynucleotide or polypeptide deviates from the reference sequence. Thus, a modified sequence or variant sequence may have substantially the same activity or function as the reference sequence, or greater or less activity or function relative to it, but retain at least partial activity or function of the reference sequence.

[0029] Non-limiting examples of modifications include the substitution, insertion, and / or deletion 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 known. A "conservative substitution" is the replacement of a single amino acid by a biologically, chemically, or structurally similar residue. Biological similarity means that the substitution does not impair 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, 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, ceran / threonine, lysine / arginine, and phenylalanine / tyrosine.

[0030] In this specification, genes and protein variants having one or more biological activities (e.g., argininosuccinate synthase activity, etc.) may be included.

[0031] At the nucleotide sequence level, naturally occurring and non-naturally occurring variant genes may have at least 50%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with the reference gene.

[0032] At the amino acid sequence level, naturally occurring and non-naturally occurring variant proteins may have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with the reference protein.

[0033] The term "sequence identity" refers to the degree of similarity of amino acid residues or bases between sequences after aligning both sequences to match as closely as possible in a specific comparison region. Sequence identity can be verified according to methods known in the art. The percentage of sequence identity can be determined using known sequence comparison programs, such as NCBI's BLAST.

[0034] The codon-optimized polynucleotide encoding the above argininosuccinate synthase may include or be composed of a nucleotide sequence having sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or 100% with any one of SEQ ID NOs 1 to 3.

[0035] The above polynucleotide may include or be composed of any one of the nucleotide sequences of SEQ ID NOs 1 to 3.

[0036] The above argininosuccinate synthase may be a variant or a wild type.

[0037] The above argininosuccinate synthase variant can exhibit therapeutic activity comparable to or higher than that of the wild type.

[0038] In one embodiment, the codon-optimized polynucleotide encoding the argininosuccinate synthase may include or be composed of the nucleotide sequence of SEQ ID NO. 2 or 3.

[0039] In one embodiment, the codon-optimized polynucleotide encoding the argininosuccinate synthase may include or be composed of the nucleotide sequence of SEQ ID NO. 2.

[0040] The above polynucleotide may have a reduced number of CpG dinucleotides compared to a codon-unoptimized wild-type sequence. The term "CpG dinucleotide" refers to a cytosine-guanine dinucleotide, and "p" indicates a phosphate linkage between the two. The above polynucleotide may have a reduced number of CpG dinucleotides by 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% compared to a codon-unoptimized wild-type sequence. The above polynucleotide may have 100 or fewer, 90 or fewer, 80 or fewer, 70 or fewer, 60 or fewer, 50 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, 5 or fewer, 3 or fewer, 2 or fewer, 1 or fewer, or 0 CpG dinucleotides.

[0041] In one embodiment, the CpG dinucleotide may not be present in the polynucleotide. Accordingly, the polynucleotide may have the CpG dinucleotide completely removed.

[0042] The mRNA transcribed by the above polynucleotide may have a stable secondary structure compared to the mRNA transcribed by the codon-unoptimized wild-type sequence. The mRNA transcribed by the above polynucleotide may have an increased half-life compared to the mRNA transcribed by the codon-unoptimized wild-type sequence. For example, the mRNA transcribed by the above polynucleotide may have a half-life of 0.6 hours or more, 0.65 hours or more, 0.66 hours or more, 0.67 hours or more, 0.68 hours or more, 0.69 hours or more, or 0.7 hours or more.

[0043] Non-limiting parameters that can predict the half-life of the above mRNA include MFE (Minimum Free Energy), SUP (Total unpairing ratio), and DegScore.

[0044] The Minimum Free Energy (MFE) value refers to the minimum free energy value required for folding, calculated based on the predicted secondary structure of the mRNA. The MFE value can be calculated by known computational models, such as Genescript, Linear Fold, Linear Partition, etc., but is not limited thereto. A lower MFE value may indicate that the secondary structure of the mRNA is more stable, which may lead to an increased half-life of the mRNA.

[0045] The Summed Unpaired Probability (SUP) and Average Unpaired Probability (AUP) values ​​are base pairing probabilities calculated by a specific formula. The SUP value can be calculated by known computational models, such as Linear Partition, but is not limited thereto. A smaller SUP value may indicate that the mRNA secondary structure is more stable, which may increase the mRNA half-life.

[0046] The DegScore value is a result derived using the nucleotide sequence and the predicted mRNA structure as inputs. The DegScore value can be calculated by the DegScore model. A lower DegScore value may indicate that the mRNA secondary structure is stable, which could lead to an increased half-life of the mRNA.

[0047] The correlation with mRNA half-life can be highest in the order of DegScore, SUP, and MFE.

[0048] The above polynucleotide may have had its cryptotic splicing site removed. Since the removal of the cryptotic splicing site eliminates the expression of variants that may arise from the activation of the cryptotic splicing site during mRNA transcription, stable expression of the transgene is possible.

[0049] The above polynucleotide may exhibit reduced immunotoxicity compared to a codon-unoptimized wild-type sequence. It is known that when CpG dinucleotides are rich, hypomethylation of CpG motifs in the AAV genome activates the TLR9 (Toll-Like Receptor 9) pathway to induce an immune response. A polynucleotide according to one aspect may exhibit reduced immunotoxicity compared to a codon-unoptimized wild-type sequence due to the removal of CpG dinucleotides.

[0050] The term "immunotoxicity" may refer to an abnormal immune response of the immune system that occurs upon exposure to an external substance. In conventional clinical trials, AAV vectors for the treatment of type 1 citrullinemia have been unsuccessful due to a strong immune response to the AAV capsid. Additionally, argininosuccinate synthase can also induce an immune response. Indicators of such abnormal immune responses include, for example, anti-drug antibodies (ADA) and neutralizing antibodies (NAB) involved in the immune process. However, polynucleotides according to one aspect may induce or not induce a minimal immune response over a significant period. Such immune responses may be innate immune responses, humoral immune responses, cellular immune responses, or a combination thereof.

[0051] The above polynucleotide may be expressed at a high level compared to a non-codon-optimized wild-type sequence. The above polynucleotide may be expressed at a high level compared to an existing codon-optimized sequence. In this specification, increased expression may refer to an effect produced by a change in the codon sequence rather than hyperactivity caused by amino acid substitutions of the argininosuccinate synthase protein (e.g., Padua mutation).

[0052] The argininosuccinate synthase protein expressed by the above polynucleotide may exhibit a higher level of activity compared to the argininosuccinate synthase protein expressed by a wild-type sequence that is not codon-optimized. The argininosuccinate synthase protein expressed by the above polynucleotide may exhibit a higher level of activity compared to the argininosuccinate synthase protein expressed by a conventional codon-optimized sequence. The term "activity" may refer to the activity of a protein, for example, the enzymatic activity of the argininosuccinate synthase. In this specification, increased activity may refer to the effect produced by a change in the codon sequence rather than the hyperactivity caused by amino acid substitutions of the argininosuccinate synthase protein (e.g., Padua mutation).

[0053]

[0054] Another aspect provides a vector for producing recombinant adeno-associated virus (AAV). Specifically, it provides an all-in-one vector system capable of producing recombinant AAV using a single vector. The same parts described above apply equally to the vector system or the vector.

[0055] A vector according to the above aspect comprises the nucleotide sequence of the following gene arranged in a single nucleic acid molecule:

[0056] (a) Helper virus genes required for AAV production;

[0057] (b) AAV's Rep gene;

[0058] (c) Cap gene of AAV; and

[0059] (d) A transgene containing a codon-optimized polynucleotide encoding argininosuccinate synthase according to one pattern.

[0060] Therefore, alternatively, one aspect provides a nucleic acid molecule comprising, within a single molecule, (a) a helper virus gene required for AAV production; (b) a Rep gene of AAV; (c) a Cap gene of AAV; and (d) a transgene comprising a codon-optimized polynucleotide encoding argininosuccinate synthase according to one aspect. More specifically, the nucleic acid molecule comprises, within a single molecule, (a) a nucleotide sequence of a helper virus gene required for AAV production; (b) a nucleotide sequence of a Rep gene of AAV; (c) a nucleotide sequence of a Cap gene of AAV; and (d) a nucleotide sequence of a transgene comprising a codon-optimized polynucleotide encoding argininosuccinate synthase according to one aspect.

[0061] In one embodiment, the recombinant AAV may be intended to treat type 1 citrullinemia.

[0062] In the above vector or nucleic acid molecule, (a), (b), (c), and (d) are all integrated together in a single nucleic acid molecule.

[0063] The following description applies commonly to a vector according to the above-mentioned aspect or a nucleic acid molecule according to the above-mentioned aspect.

[0064] The above "adeno-associated virus (AAV)" is a virus belonging to the genus Dependovirus of the family Parvoviridae. It does not have the ability to self-replicate on its own, so the coexistence of a helper virus is required for replication. The above 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.

[0065] 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 host cells. The term "recombinant" means that the AAV or sequence has been manipulated in a manner that does not typically occur in nature. For example, a recombinant vector, such as an AAV vector, may refer to a case where a polynucleotide not typically present in the wild-type AAV genome is inserted into the viral genome. Therefore, the recombinant AAV produced by the above recombinant AAV production vector can be applied as a gene therapy. For example, since the recombinant AAV produced by the above plasmid can express the argininosuccinate synthase protein, it can be used as a gene therapy for citrullinemia type 1.

[0066] In this specification, the term "recombinant AAV production vector" may mean a vector capable of producing recombinant AAV in a host cell into which said vector has been introduced. said vector may mean a plasmid vector. A recombinant AAV production vector according to one aspect relates to a vector technology in which all genes required for AAV production are integrated into a single vector. Accordingly, in this specification, the term "recombinant AAV production vector" may be used interchangeably with the terms "all-in-one vector" or "AAV single-vector system." said recombinant AAV production vector may be a single plasmid.

[0067] The term "vector" may refer to a vehicle capable of artificially transporting heterologous genetic material to another cell. Examples of such vectors include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. The plasmids may include plasmids derived from E. coli (pBR322, pBR325, pUC118, pUC119, pET21a(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5), and plasmids derived from yeast (YEp13, YEp24, and YCp50). The viruses may include animal viruses such as retroviruses, adenoviruses, or vaccinia viruses; insect viruses such as baculoviruses.

[0068] 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 include 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."

[0069] 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 a specific embodiment, the vector may be a circular vector.

[0070] The recombinant AAV produced by the above recombinant AAV production vector may be a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

[0071] The nucleic acid molecule above contains (a) a helper virus gene required for AAV production.

[0072] The aforementioned helper virus may refer to a virus that assists in replication through co-infection with a virus that cannot replicate through a single infection. Since AAV lacks the ability to replicate on its own, it requires the genes of a helper virus necessary for AAV replication.

[0073] 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."

[0074] The nucleotide sequence of the above helper virus gene may be derived from one or more selected from adenovirus, herpes simplex virus (HSV), baculovirus, papillomavirus, and bocavirus, but is not limited thereto.

[0075] More than 50 serotypes of the above adenovirus are known. In one embodiment, 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.

[0076] The above herpes simplex virus may be type 1 or type 2.

[0077] The above papilloma virus may be a papilloma virus or a human papilloma virus (HPV). More than 150 types of the above HPV are known. For example, the above HPV may be HPV-16, but is not limited thereto.

[0078] The above helper virus gene may include one or more selected from E1, E2, E2a, E4, E4orf1, E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA (or also called "VA RNA gene"), DBP (DNA-binding protein), and variants thereof. The above helper virus gene is a gene that encodes the above helper protein.

[0079] The above variant may be an engineered helper virus gene.

[0080] In this specification, 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 known. In this specification, "engineered A" may include a variant containing one or more variations in the wild-type sequence of A, a variant in which a portion of the sequence of A has been truncated, etc. The variations may be insertions, substitutions, deletions, or combinations thereof.

[0081] The nucleotide sequence of the helper virus gene may be derived from adenovirus 2. In one embodiment, the helper virus gene may include E2a, E4, and VA. The helper virus gene may include E2a and E4.

[0082] In one embodiment, the E4 may include or be composed of a nucleotide sequence having 90% or more sequence identity with SEQ ID NO. 13.

[0083] In one embodiment, the E2a may include or be composed of a nucleotide sequence having 90% or more sequence identity with SEQ ID NO. 14.

[0084] In one embodiment, the VA may include or be composed of a nucleotide sequence having 90% or more sequence identity with SEQ ID NO. 15 or SEQ ID NO. 16. Additionally, the VA may include one or more selected from the group consisting of SEQ ID NO. 15 and SEQ ID NO. 16.

[0085] The nucleic acid molecule above contains (b) the Rep gene of AAV.

[0086] The above Rep (Replication) gene may be a gene required for the replication of AAV. The above Rep gene may be derived from any AAV serotype. The nucleotide sequence of the above 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 above AAV serotypes may include other AAV serotypes currently known or to be discovered in the future. The above AAV serotypes may also include artificial AAV serotypes. If there are two or more Rep genes, each gene may be derived from the same or different AAV serotypes.

[0087] In one embodiment, the nucleotide sequence of the Rep gene may be derived from AAV2.

[0088] The above Rep gene may include a gene encoding a known Rep protein or a variant thereof.

[0089] The Rep gene may include one or more 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 (i) Rep78, and (ii) one or more selected from Rep68, Rep52, and Rep40. The Rep gene may include (i) Rep68, and (ii) one or more selected from Rep78, Rep52, and Rep40. The Rep gene may include (i) Rep68, and (ii) one or more selected from Rep52 and Rep40. The Rep gene may include all of Rep78, Rep68, Rep52, and Rep40.

[0090] In one embodiment, the Rep gene may include one or more (one, two, or three) selected from Rep68, Rep52, and Rep40. If Rep78 is overexpressed, cytotoxicity may increase and AAV productivity may decrease. Therefore, the Rep gene may include Rep genes excluding Rep78.

[0091] The variant may be an engineered Rep (ERep) protein. The variant may be a variant having one or more variations in the wild-type sequence, or a truncated variant.

[0092] In one embodiment, the Rep gene may include the Rep gene of AAV2. In a specific embodiment, the Rep gene may include Rep78, Rep68, Rep52, and Rep40.

[0093] In one embodiment, the Rep gene may include or be composed of a nucleotide sequence having 90% or more sequence identity with SEQ ID NO. 12.

[0094] The above nucleic acid molecule contains (c) the Cap gene of AAV.

[0095] The above Cap (Capsid) gene is a gene that encodes the viral capsid protein.

[0096] The above Cap gene may be derived from any AAV serotype. The nucleotide sequence of the above Cap 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 above AAV serotype may include other AAV serotypes currently known or to be discovered later. The above AAV serotype may also include artificial AAV serotypes. If there are two or more Cap genes, each gene may be derived from the same or different AAV serotypes.

[0097] In one embodiment, the nucleotide sequence of the Cap gene may be derived from AAV2.

[0098] In one embodiment, the nucleotide sequence of the Cap gene may be derived from AAV5.

[0099] In one embodiment, the nucleotide sequence of the Cap gene may be derived from AAV8.

[0100] The above Cap gene may include a gene encoding a known Cap protein or a variant thereof.

[0101] The above Cap gene may encode one or more selected from capsid protein, VP1 protein, VP2 protein, VP3 protein, and variants thereof, but is not limited thereto. The above Cap gene may encode all of VP1, VP2, and VP3 proteins.

[0102] 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 variations in the wild-type sequence, or a truncated variant.

[0103] In one embodiment, the Cap gene may be the wild-type Cap gene of AAV8.

[0104] In one embodiment, the Cap gene may include or be composed of a nucleotide sequence having 90% or more sequence identity with SEQ ID NO. 11.

[0105] The nucleotide sequences of the Rep gene and Cap gene may be derived from the same or different AAV serotypes. In one embodiment, the Rep gene may be derived from AAV2, and the Cap gene may be derived from AAV8.

[0106] The above nucleic acid molecule includes (d) a transgene.

[0107] The above transgene is a gene transferred from one organism to another. The above transgene may be a heterogeneous polynucleotide. The above transgene may be a gene-of-interest (GOI) to be packaged in a recombinant AAV capsid. The above transgene may be a therapeutic gene. Therefore, the above recombinant AAV production plasmid may be used to produce clinical material for gene therapy for the treatment of patients. The above transgene may be one or two or more.

[0108] The above-mentioned transgene comprises a codon-optimized polynucleotide encoding an argininosuccinate synthase according to one aspect. Accordingly, the vector for producing the recombinant AAV can produce a recombinant AAV that can be used as a gene therapy for treating type 1 citrullinemia.

[0109] The above transgenic gene may be arranged between ITRs (Inverted Terminal Repeats). The above transgenic gene may have ITRs arranged on both sides. The above transgenic gene may have two ITRs arranged on the sides. The above transgenic gene may be arranged between L-ITR and R-ITR. For example, L-ITR (first ITR), transgenic gene, and R-ITR (second ITR) may be arranged sequentially in the 5' to 3' direction or in the 3' to 5' direction.

[0110] 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 within a larger stem palindrome (A-A'). Consequently, the ITR has a T-shaped stem-loop structure. The ITR can have two configurations: flip and flop. The flip and flop configurations each have the BB' and CC' palindromes closest to the 3' end, respectively. Additionally, the two ITRs on both sides are referred to as the first ITR and the second ITR, or as the L(left)-ITR and R(right)-ITR, or as the 5'-ITR and 3'-ITR. The D region exists only once at each end and 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 trs (terminal resolution sites). The structure and sequence of the ITRs are known.

[0111] 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 serotypes may include other AAV serotypes currently known or to be discovered in the future. The above AAV serotypes may also include artificial AAV serotypes. The two ITRs on both sides mentioned above may originate from the same or different AAV serotypes.

[0112] In one embodiment, the ITR may be derived from AAV2. The L-ITR and R-ITR may both be derived from AAV2.

[0113] The above ITR may be a wild-type ITR or a variant thereof. The above ITR may contain all or part of the wild-type ITR sequence. The above ITR may have a lower %GC content than the wild-type ITR. The above ITR may be a synthetic ITR. The above ITR may be an ITR derived from a self-complementary AAV (scAAV). The above L-ITR and R-ITR may be modified to generate scAAV.

[0114] In one embodiment, either of the L-ITR and R-ITR may serve as a primer for DNA replication. The R-ITR may serve as a primer for DNA replication. Either of the L-ITR and R-ITR may not contain trs. Either of the L-ITR and R-ITR may not contain trs, and the other may be a wild-type ITR. The L-ITR may not contain trs, and the R-ITR may be a wild-type ITR. Either of the L-ITR and R-ITR may not contain trs and may contain an RBE (Rep binding element). The L-ITR may not contain trs and may contain an RBE. Either of the L-ITR and R-ITR may not contain trs and may have a hairpin structure. The L-ITR may not contain trs and may have a hairpin structure. Either of the above L-ITR and R-ITR may be an ITR variant with a deleted D region, and the other may be a wild-type ITR. The above L-ITR may be an ITR variant with a deleted D region, and the above R-ITR may be a wild-type ITR. Therefore, the recombinant AAV produced by the above vector may be a self-complementary AAV (scAAV).

[0115] In one embodiment, the L-ITR may include or be composed of a nucleotide sequence having 90% or more sequence identity with SEQ ID NO. 17.

[0116] In one embodiment, the R-ITR may include or be composed of a nucleotide sequence having 90% or more sequence identity with SEQ ID NO. 18.

[0117] The nucleic acid molecule may additionally include a sequence added to the ITR. In this specification, "sequence added to the ITR" may also be expressed as "ITR+". By additionally including the sequence added to the ITR, the %Full / empty capsid can be improved. In one embodiment, the sequence added to the ITR may be a non-ITR viral DNA sequence derived from a wild-type AAV genome. Specifically, the sequence added to the ITR may be non-ITR viral DNA (46 bp) corresponding to wtAAV2 nt 4489-4534 derived from the 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 with respect to the (+) strand. The sequence added to the ITR may be a sequence added upstream of the R-ITR.

[0118] In addition to (a), (b), (c), and (d), which are essential components for recombinant AAV production, the nucleic acid molecule may additionally include a flanking sequence, an engineered flanking sequence, and / or additional components.

[0119] The above peripheral sequence or manipulated peripheral sequence may be a peripheral sequence of an ITR or a manipulated peripheral sequence. Specifically, the above peripheral sequence may be a 5'-peripheral sequence of an L-ITR or a 3'-peripheral sequence of an R-ITR based on the (+) strand. By including the above peripheral sequence or manipulated peripheral sequence, it is possible to obtain high-quality AAV by lowering the impurity content of the gene packaged in AAV, and furthermore, an enhanced safety effect can be expected when applied as a therapeutic agent.

[0120] The surrounding sequence of the above ITR or the manipulated surrounding sequence may include a sequence with a high AT content (i.e., an AT-rich sequence). The AT-rich sequence may inhibit the reverse packaging of AAV, thereby enabling the acquisition of high-quality recombinant AAV.

[0121] The above flanking sequence, engineered flanking sequence, or additional components may have one or more functions selected from the following:

[0122] (i) Increase gene expression levels;

[0123] (ii) Regulating the timing of gene expression;

[0124] (iii) Regulates gene transcription;

[0125] (iv) Stabilizing the transcriptome for translation;

[0126] (v) Reduces the content of impurities other than transgenic genes among the genes packaged in recombinant AAV;

[0127] (vi) Inhibits reverse packaging of AAV;

[0128] (vii) improving the productivity of recombinant AAV; and

[0129] (viii) Improves the %F / E capsid ratio of recombinant AAV.

[0130] In this specification, the term "impurity" may mean that a component other than a component existing between two ITRs (e.g., a transposable gene) of a recombinant AAV production vector is packaged within the recombinant AAV particle, an incomplete transposable gene is packaged within the recombinant AAV particle, or the vector has been reverse-packaged. The impurity may be a DNA impurity.

[0131] 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. Therefore, the impurity may include some sequences of the Rep gene, Cap gene, or Helper gene packaged within the recombinant AAV particle.

[0132] For example, the impurity may include any sequence located outside two ITRs in the vector, e.g., all or part of a helper virus gene, all or part of Rep, all or part of Cap; part of a transposable gene; part of a host cell genome sequence; and one or more selected from two or more chimeric forms among these.

[0133] Replication-competent AAV (rcAAV) can be formed when Rep / Cap or Helper genes are inserted into recombinant particles. Wild-type AAVs cannot replicate autonomously without helper viruses such as adenoviruses. However, replication-competent rcAAV can clinically induce an immune response, and since unintended AAV replication may occur depending on the presence of helper genes, it can affect the safety of gene therapy drugs. rcAAV can also occur during the AAV production process, and since rcAAV cannot be isolated by the purification process, it is important to minimize its formation.

[0134] Therefore, by lowering the content of the aforementioned impurities, it is possible to secure recombinant AAV with excellent safety and quality.

[0135] AAV packaging refers to the packaging of the components existing between two ITRs within the AAV particle. However, AAV reverse packaging refers to the packaging of the recombinant AAV particle outwardly rather than inwardly between the two ITRs. Therefore, inhibiting AAV reverse packaging allows for the securing of high-quality AAV. A vector according to one aspect can reduce the impurity content caused by vector reverse packaging, thereby enabling the securing of high-quality recombinant AAV; furthermore, when applied as a therapeutic agent, enhanced safety can be expected.

[0136] In (viii) above, the improvement in the %F / E capsid ratio may be due to not only increasing the production of full capsid but also decreasing the production of empty capsid.

[0137] A person skilled in the art will understand that the nucleotide sequences of (a), (b), (c), and (d) above may each be operablely associated with an appropriate control sequence. For example, the nucleotide sequence may be operablely linked to a transcription / translation control element, and the transcription / translation control element may include, for example, a transcription / translation control signal, an origin of replication, a polyadenylation signal, an IRES (internal ribosome entry site), a furin, a 2A peptide, a promoter and / or an enhancer.

[0138] The above nucleic acid molecule may additionally include one or more selected from stabilizers, costabilizers, activators, coactivators, repressors, corepressors, epigenetic regulatory elements, co-epigenetic regulatory elements, regulatory elements, and co-regulatory elements.

[0139] The nucleic acid molecule may additionally include one or more expression regulatory elements operably linked to any one of (a), (b), (c), and (d).

[0140] The above expression regulatory elements may include one or more selected from enhancers, promoters, transcription factors, silencers, insulators, introns, splicing donors and receptors, engineered splicing donors and receptors, riboswitches, amino acids, miRNA (microRNA), shRNA (short hairpin RNA), 5'- or 3'-UTR (untranslated region), Kozak sequences, start codons, GOI codons, signal peptides, polyadenylation (poly A) signal sequences, etc.

[0141] In one embodiment, the expression regulating element may include one or more selected from enhancers, promoters, introns, and polyadenylation signal sequences.

[0142] The nucleic acid molecule may further include an expression regulatory element operably linked to (d). In one embodiment, the expression regulatory element may include one or more selected from enhancers, promoters, introns, Kozak sequences, and polyadenylation signal sequences.

[0143] The term "enhancer" may refer to a regulatory region that promotes gene transcription. Non-limiting examples of such enhancers include the SV40 enhancer and the CMV (cytomegalovirus) enhancer.

[0144] In one embodiment, the enhancer may comprise one or more selected from the group consisting of AMBP (alpha-1-microglobulin / bikunin precursor) enhancers, ApoE1 enhancers, and variants thereof, and specifically may be an AMBP enhancer or a variant thereof.

[0145] In one embodiment, the AMBP enhancer or a variant thereof may include the 1st to 100th nucleotide sequence of SEQ ID NO. 5 one or more times, specifically, the 1st to 100th nucleotide sequence of SEQ ID NO. 5 may be repeated two or more times, and more specifically, the 1st to 100th nucleotide sequence of SEQ ID NO. 5 may be repeated twice by being connected by 'agatcc'.

[0146] In one embodiment, the enhancer may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 4 or 5. Specifically, the enhancer may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 5.

[0147] The term "promoter" may mean a sequence that drives gene expression. 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, an SFFV (spleen focus-forming virus) promoter, an RSV (Rous sarcoma virus) promoter, or a pEF (elongation factor 1-alpha) promoter.

[0148] In one embodiment, the promoter may include one or more selected from the group consisting of an A1AT promoter, a TBG promoter, and variants thereof, and specifically may be a TBG promoter or a variant thereof.

[0149] In one embodiment, the promoter may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 6 or 7. Specifically, the promoter may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 7.

[0150] In one embodiment, the promoter may be a liver tissue-specific promoter.

[0151] The above "intron" sequence refers to a nucleotide sequence that is removed from the final gene product by RNA splicing. Using introns downstream of the enhancer / promoter region and upstream of the cDNA insert can increase the level of gene expression. Non-limiting examples of the above introns may include minute virus of mice (MVM) introns, human MVM introns, beta-globin introns, ASS introns, SV40 introns, modified SV40 introns, beta-actin introns, or human ctEF1 (C-terminal EF-1a) first introns, etc.

[0152] In one embodiment, the intron may comprise one or more selected from the group consisting of SV40 introns, beta-globin introns, and variants thereof, and specifically may be an SV40 intron or a variant thereof.

[0153] In one embodiment, the intron may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 8 or 9. Specifically, the intron may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 9.

[0154] The above "polyadenylation signal sequence" is also referred to as a "Poly A sequence" and means, for example, a polyadenylation signal sequence placed at the 3' end of a transposable gene, which allows a Poly A tail to be added to the end of the initial 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. Non-limiting examples of the above polyadenylation signal sequence may include the SV40 polyadenylation signal, bovine growth hormone (BGH) polyadenylation signal, human growth hormone (hGH) polyadenylation signal, rabbit globin polyadenylation signal, etc.

[0155] In one embodiment, the Poly A sequence may include a human growth hormone (Hgh) polyadenylation signal sequence or a variant thereof.

[0156] In one embodiment, the Poly A sequence may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 10.

[0157] In one embodiment, the expression regulating element may include one or more selected from enhancers, promoters, silencers, insulators, introns, UTRs, Kozak sequences, signal peptides, and polyadenylated signal sequences.

[0158] The above amino acids may include, but are not limited to, non-natural amino acids, non-canonical amino acids, or unnatural amino acids.

[0159] The above start codon may include, but is not limited to, a rare start codon or a non-ATG start codon.

[0160] The nucleic acid molecule may additionally include a promoter independently operably linked to (a), (b), (c), or (d), respectively. The promoter can regulate the expression level and timing of each gene. Generally, higher Rep / Cap expression increases AAV productivity, but specific Rep proteins can decrease productivity, and overexpression of the Cap gene can also increase empty particles (i.e., product-related impurities).

[0161] In one embodiment, when (i) the amounts of Rep / Cap and GOI are fixed and the amount of Helper is increased, or (ii) 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-tuning of Helper or Rep / Cap protein expression is important. Therefore, the regulation of the expression levels of each gene within the vector can affect productivity and quality. Accordingly, by including the promoter, it is possible to increase the productivity of recombinant AAV, increase the %Full capsid while simultaneously decreasing the %Empty capsid, and thereby produce high-quality recombinant AAV.

[0162] 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 a wild-type promoter. The promoter may be an early promoter, a late promoter, a ubiquitous promoter, or a modifiable promoter. The promoter may be a CMV promoter, an SFFV promoter, an RSV promoter, or a pEF promoter.

[0163] 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 a wild-type promoter. The promoter may be an early promoter, a late promoter, a ubiquitous promoter, or a modifiable promoter. The promoter may be a CMV promoter, an SFFV promoter, an RSV promoter, or a pEF promoter.

[0164] 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 a wild-type promoter. The promoter may be an early promoter, a late promoter, a ubiquitous promoter, or a modifiable promoter. The promoter may be a CMV promoter, an SFFV promoter, an RSV promoter, or a pEF promoter.

[0165] 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 above 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 the preferential or dominant in vivo expression of a specific gene in tissue A compared to other tissues. 'A tissue-specific expression' may mean that 50% or more, 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. In one embodiment, the above expression regulatory element may be a liver tissue-specific expression regulatory element.

[0166] The above tissue-specific expression regulatory element may include one or more selected from enhancers and promoters. The above tissue-specific expression regulatory element may include enhancers and promoters.

[0167] The above nucleic acid molecule may additionally include multi-cistronic factors, bi-cistronic factors, transposons, transposases, etc.

[0168] The nucleic acid molecule may include a vector backbone. The vector backbone may include one or more (one or two) selected from a replication origin and a selection marker. The vector backbone may not include a replication origin.

[0169] The nucleic acid molecule may not include a replication origin, or may additionally include a replication origin. The replication origin may be a known replication origin or a variant thereof. The replication origin may be, but is not limited to, a pUC origin, a pBR322 origin, a pMB1 origin, a pSC101 origin, a p15A origin, a pJET1.2 origin, or a synthetic origin.

[0170] In one embodiment, the nucleic acid molecule may include a pBR322 origin.

[0171] In one embodiment, the replication origin may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 22.

[0172] The nucleic acid molecule may further comprise a selectable marker or reporter capable of providing selection or identification of the host cell 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, etc. Non-limiting examples of the reporter include luciferase, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), etc. In one embodiment, the nucleic acid molecule may further comprise a selectable marker.

[0173] In one embodiment, the nucleic acid molecule may additionally include a kanamycin selection marker.

[0174] In one embodiment, the kanamycin selection marker may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 23.

[0175] "Gene amplification" may refer to a process in which a specific DNA sequence (i.e., a gene) of a genome is replicated disproportionately in relation to other sequences of the genome, so that the amplified DNA sequence exists at a higher copy number than was originally present. As used herein, "amplified" or "amplified" may refer to a gene or nucleic acid sequence that exists in two or more copies in a host cell by gene amplification.

[0176] "Amplifiable selection marker gene" may refer to a gene that allows amplification of the corresponding gene under appropriate growth conditions.

[0177] In the nucleic acid molecule, (a), (b), (c), and (d) may be arranged randomly 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.

[0178] In the nucleic acid molecule above, (a), (b), (c), and (d) may each be independently arranged in any order with forward orientation or reverse orientation.

[0179] The above forward orientation may mean that the gene is inserted from the 5' to the 3' direction in the 5' → 3' strand ((+) strand). The above reverse orientation may mean that the gene is inserted from the 3' to the 5' to the 3' strand in the 5' → 3' strand.

[0180] In the above 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:

[0181] 1) (d) - (b) - (c) - (a);

[0182] 2) (d) - (c) - (b) - (a);

[0183] 3) (d) - (a) - (b) - (c);

[0184] 4) (d) - (a) - (c) - (b);

[0185] 5) (b) - (c) - (d) - (a);

[0186] 6) (c) - (b) - (d) - (a);

[0187] 7) (b) - (c) - (a) - (d);

[0188] 8) (c) - (b) - (a) - (d);

[0189] 9) (a) - (b) - (c) - (d);

[0190] 10) (a) - (c) - (b) - (d);

[0191] 11) (a) - (d) - (b) - (c); or

[0192] 12) (a) - (d) - (c) - (b).

[0193] In one embodiment, (a) is oriented in the positive direction; (b), (c), and (d) may each be independently oriented in the positive direction or in the negative direction.

[0194] In one embodiment, (a) is oriented positively; (b) and (c) are both oriented positively or both are oriented negatively; and (d) may be oriented positively or negatively oriented.

[0195] In one embodiment, (a), (b), and (c) are oriented in the forward direction; (d) may be oriented in the forward direction or reverse direction.

[0196] In certain embodiments, (a), (b), (c), and (d) may all be oriented.

[0197] In certain embodiments, (a), (b), and (c) are oriented in the forward direction; and (d) may be oriented in the reverse direction.

[0198] In one embodiment, two ITRs may be arranged on both sides of (d). Thus, in the nucleic acid molecule, (a), (b), (c), (d), and ITRs may be arranged in the following order:

[0199] 1') L-ITR - (d) - R-ITR - (b) - (c) - (a);

[0200] 2') L-ITR - (d) - R-ITR - (c) - (b) - (a);

[0201] 3') L-ITR - (d) - R-ITR - (a) - (b) - (c);

[0202] 4') L-ITR - (d) - R-ITR - (a) - (c) - (b);

[0203] 5') (b) - (c) - L-ITR - (d) - R-ITR - (a);

[0204] 6') (c) - (b) - L-ITR - (d) - R-ITR - (a);

[0205] 7') (b) - (c) - (a) - L-ITR - (d) - R-ITR;

[0206] 8') (c) - (b) - (a) - L-ITR - (d) - R-ITR;

[0207] 9') (a) - (b) - (c) - L-ITR - (d) - R-ITR;

[0208] 10') (a) - (c) - (b) - L-ITR - (d) - R-ITR;

[0209] 11') (a) - L-ITR - (d) - R-ITR - (b) - (c); or

[0210] 12') (a) - L-ITR - (d) - R-ITR - (c) - (b).

[0211] In one embodiment, the vector backbones may be arranged in any order in 1) to 12). In another embodiment, the vector backbones may be arranged after the last order in 1) to 12) or before the first order. In yet another embodiment, the vector backbones may be arranged after the last order in 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.

[0212] The above vector backbone may include one or more selected from replication origins and selection markers.

[0213] A vector according to one aspect can improve the %Full / Empty capsid ratio of the produced recombinant AAV through a combination of optimized arrangement and orientation of components (e.g., (a), (b), (c), (d), backbone). In particular, through the combination of the optimized arrangement and orientation, it is possible not only to increase the %Full capsid but also to decrease the %Empty capsid.

[0214] In one embodiment, the components of the nucleic acid molecule may have the following arrangement and orientation (the forward orientation is abbreviated as 'F' and the reverse orientation as 'R'):

[0215] (i) Oriented (a) - Backbone - Oriented (b) - Oriented (c) - Oriented (d) (i.e., Helper(F) - Backbone - Rep(F) - Cap(F) - Transposable(F));

[0216] (ii) Oriented (a) - Backbone - Oriented (c) - Oriented (b) - Oriented (d) (i.e., Helper(F) - Backbone - Cap(F) - Rep(F) - Transposable (F));

[0217] (iii) Oriented (b) - Oriented (c) - Oriented (a) - Backbone - Oriented (d) (i.e., Rep(F) - Cap(F) - Helper(F) - Backbone - Transposable(F));

[0218] (iv) Oriented (a) - Backbone - Oriented (b) - Oriented (c) - Inversely oriented (d) (i.e., Helper(F) - Backbone - Rep(F) - Cap(F) - Transposable(R));

[0219] (v) Oriented (a) - Backbone - Oriented (c) - Oriented (b) - Inversely oriented (d) (i.e., Helper(F) - Backbone - Cap(F) - Rep(F) - Transposable(R));

[0220] (vi) Oriented (b) - Oriented (c) - Oriented (a) - Backbone - Inversely oriented (d) (i.e., Rep(F) - Cap(F) - Helper(F) - Backbone - Transposable(R));

[0221] (vii) Backbone - Oriented (b) - Oriented (c) - Oriented (a) - Back-oriented (d) (i.e., Backbone - Rep(F) - Cap(F) - Helper(F) - Transposable(R)); or

[0222] (viii) Oriented (a) - Oriented (b) - Oriented (c) - Backbone - Oriented (d) (i.e., Helper(F) - Rep(F) - Cap(F) - Backbone - Transposable(F)).

[0223] In one embodiment, among the components of the nucleic acid molecule, `(d) transfer gene` may be inversely oriented.

[0224] In one embodiment, among the components of the nucleic acid molecule, `(b) the Rep gene of AAV` and `(c) the Cap gene of AAV` may be arranged in the order `(c)-(b)`, and specifically, may be arranged in the order `correctly oriented (c) - correctly oriented (b)`.

[0225] In one embodiment, the components of the nucleic acid molecule may have the following arrangement and orientation (the forward orientation is abbreviated as 'F' and the reverse orientation as 'R'):

[0226] (ii) Oriented (a) - Backbone - Oriented (c) - Oriented (b) - Oriented (d) (i.e., Helper(F) - Backbone - Cap(F) - Rep(F) - Transposable (F));

[0227] (iii) Oriented (b) - Oriented (c) - Oriented (a) - Backbone - Oriented (d) (i.e., Rep(F) - Cap(F) - Helper(F) - Backbone - Transposable(F));

[0228] (iv) Oriented (a) - Backbone - Oriented (b) - Oriented (c) - Backward-oriented (d) (i.e., Helper (F) - Backbone - Rep (F) - Cap (F) - Transposable (R)); or

[0229] (v) Oriented (a) - Backbone - Oriented (c) - Oriented (b) - Inversely oriented (d) (i.e., Helper(F) - Backbone - Cap(F) - Rep(F) - Transposable(R)).

[0230] In certain embodiments, the components of the nucleic acid molecule may have the following arrangement and orientation (forward orientation abbreviated as 'F' and reverse orientation as 'R'):

[0231] (v) Oriented (a) - Backbone - Oriented (c) - Oriented (b) - Inversely oriented (d) (i.e., Helper(F) - Backbone - Cap(F) - Rep(F) - Transposable(R)).

[0232] In (i) to (viii) above, two ITRs may be arranged on both sides of (d).

[0233] The above nucleic acid molecules, plasmids, and vectors can be manufactured by any suitable technique, and such techniques are widely known in the art.

[0234] Since the above recombinant AAV production vector contains all components necessary for recombinant AAV production (i.e., (a), (b), (c), and (d)) linked within a single molecule, each component can be introduced into host cells in equal proportions. Host cells into which a single nucleic acid molecule has been introduced enable balanced gene expression. Balanced gene expression can improve recombinant AAV productivity and / or increase the %F / E capsid ratio of recombinant AAV. Improved recombinant AAV productivity can reduce unit production costs. Specifically, improved recombinant AAV productivity makes it possible to improve the total yield of the purification process.

[0235] In addition, by using the above all-in-one vector system, the cost of raw materials can be reduced because only one single vector needs to be produced instead of the existing three vectors independently. Furthermore, since the above all-in-one vector system only requires the introduction of a single vector without the need to introduce three vectors separately, the input amount of individual vectors can be reduced by more than 50%.

[0236] In one embodiment, compared to a control group that triple transfected a GOI expression vector, a Rep / Cap expression vector, and a helper vector, it was confirmed that the all-in-one vector exhibited high recombinant AAV productivity with a small amount of plasmid DNA (pDNA).

[0237] In one embodiment, compared to a control group triple transfected with a GOI expression vector, a Rep / Cap expression vector, and a helper vector, it was confirmed that the recombinant AAV produced by the all-in-one vector exhibited a high %Full / Empty capsid ratio with a small amount of pDNA. Therefore, the all-in-one vector can produce recombinant AAV of improved quality with a high ratio of full capsids containing therapeutic genes.

[0238] In one embodiment, it was confirmed that the recombinant AAV produced by the all-in-one vector exhibited equivalent levels of protein expression compared to the recombinant AAV produced by a control group that triple transfected the GOI expression vector, the Rep / Cap expression vector, and the helper vector. Therefore, the all-in-one vector can produce recombinant AAV equivalent to the recombinant AAV produced by the conventional triple transfection method in terms of infectivity and gene expression function.

[0239] In one embodiment, it was confirmed that the argininosuccinate synthase expressed in the recombinant AAV produced by the all-in-one vector possesses biological activity, thus confirming that it has function as a therapeutic agent. Therefore, it was confirmed that the all-in-one vector can be immediately applied to the development of a treatment for type 1 citrullinemia.

[0240] The nucleic acid molecule may additionally include (e) one or more stuffers.

[0241] In this specification, the term "stuffer" may mean an untranslated sequence of nucleic acid or a sequence that does not encode a protein. The stuffer may include a non-functional DNA sequence.

[0242] The number of stuffers included in the above vector can be appropriately selected within a range that maintains the function of the recombinant AAV production vector while achieving the effect of including the stuffers, and is not limited to a specific number. The above vector may include one stuffer. The above vector may include two or more stuffers. The above vector may include three or more stuffers. The above vector may include 2 to 100, 2 to 50, 2 to 20, 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 100, 3 to 50, 3 to 20, 3 to 10, 3 to 8, 3 to 6, or 3 to 4 stuffers. The above vector may include three stuffers. If there are two or more of the above-mentioned stuffers, each stuffer may be referred to as the first stuffer, the second stuffer, etc.

[0243] The above stuffer may be arranged inside or outside of two ITRs. The term 'inside of two ITRs' may mean arranged between or inside the two ITRs. The term 'outside of two ITRs' may mean arranged in the outer direction or outside of the two ITRs.

[0244] If the above vector includes two or more stuffers, each stuffer may be independently arranged inside or outside the two ITRs. If the above vector includes two or more stuffers, all stuffers may be arranged outside the two ITRs.

[0245] In the above vector, the length of each stuffer can be from 0.1 kb to 20 kb. Specifically, the length of each stuffer is 0.1 kb to 20 kb, 0.1 kb to 15 kb, 0.1 kb to 10 kb, 0.1 kb to 9 kb, 0.1 kb to 8 kb, 0.1 kb to 7 kb, 0.1 kb to 6 kb, 0.1 kb to 5 kb, 0.1 kb to 4 kb, 0.1 kb to 3 kb, 0.1 kb to 2 kb, 0.5 kb to 20 kb, 0.5 kb to 15 kb, 0.5 kb to 10 kb, 0.5 kb to 9 kb, 0.5 kb to 8 kb, 0.5 kb to 7 kb, 0.5 kb to 6 kb, 0.5 kb to 5 kb, 0.5 kb to 4 kb, 0.5 kb to 3 kb, 0.5 kb to 2 kb, 1 kb to 20 kb, 1 kb to 15 kb, 1 kb to 10 kb, 1 kb to 9 kb, 1 kb to 8 kb, 1 kb to 7 kb, 1 kb to 6 kb, 1 kb to 5 kb, 1 kb to 4 kb, 1 kb to 3 kb, 1 kb to 2 kb, 2 kb to 20 kb, 2 kb to 15 kb, 2 kb to 10 kb, 2 kb to 9 kb, 2 kb to 8 kb, 2 kb to 7 kb, 2 kb to 6 kb, 2 kb to 5 kb, 2 kb to 4 kb, 2 kb to 3 kb, 3 kb to 20 kb, 3 kb to 15 It may be kb, 3 kb to 10 kb, 3 kb to 9 kb, 3 kb to 8 kb, 3 kb to 7 kb, 3 kb to 6 kb, 3 kb to 5 kb, 3 kb to 4 kb, 4 kb to 20 kb, 4 kb to 15 kb, 4 kb to 10 kb, 4 kb to 9 kb, 4 kb to 8 kb, 4 kb to 7 kb, 4 kb to 6 kb, 4 kb to 5 kb, 5 kb to 20 kb, 5 kb to 15 kb, 5 kb to 10 kb, 5 kb to 9 kb, 5 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb.

[0246] In the above vector, the total length of all stuffers can be from 0.1 kb to 100 kb. Specifically, the total length of all stuffers is 0.1 kb to 100 kb, 0.1 kb to 90 kb, 0.1 kb to 80 kb, 0.1 kb to 70 kb, 0.1 kb to 60 kb, 0.1 kb to 56 kb, 0.1 kb to 55 kb, 0.1 kb to 50 kb, 0.1 kb to 40 kb, 0.1 kb to 30 kb, 0.1 kb to 20 kb, 0.1 kb to 10 kb, 0.1 kb to 8 kb, 0.5 kb to 100 kb, 0.5 kb to 90 kb, 0.5 kb to 80 kb, 0.5 kb to 70 kb, 0.5 kb to 60 kb, 0.5 kb to 56 kb, 0.5 kb to 55 kb, 0.5 kb to 50 kb, 0.5 kb to 40 kb, 0.5 kb to 30 kb, 0.5 kb to 20 kb, 0.5 kb to 10 kb, 0.5 kb to 8 kb, 1 kb to 100 kb, 1 kb to 90 kb, 1 kb to 80 kb, 1 kb to 70 kb, 1 kb to 60 kb, 1 kb to 56 kb, 1 kb to 55 kb, 1 kb to 50 kb, 1 kb to 40 kb, 1 kb to 30 kb, 1 kb to 20 kb, 1 kb to 10 kb, 1 kb to 8 kb, 1.5 kb to 100 kb, 1.5 kb to 90 kb, 1.5 kb to 80 kb, 1.5 kb to 70 kb, 1.5 kb to 60 kb, 1.5 kb to 56 kb, 1.5 kb to 55 kb, 1.5 kb to 50 kb, 1.5 kb to 40 kb, 1.5 kb to 30 kb, 1.5 kb to 20 kb, 1.5 kb to 10 kb, 1.5 kb to 8 kb, 3 kb to 100 kb, 3 kb to 90 kb, 3 kb to 80 kb, 3 kb to 70 kb, 3 kb to 60 kb, 3 kb to 56 kb, 3 kb to 55 kb, 3 kb to 50 kb, 3 kb to 40 kb, 3 kb to 30 kb, 3 kb to 20 kb, 3 kb to 10 kb, 3 kb to 8 kb, 5 kb to 100 kb, 5 kb to 90 kb, 5 kb to 80 kb, 5 kb to 70 kb, 5 kb to 60 kb, 5 kb to 56 kb, 5 kb to 55 kb, 5 kb to 50 kb, 5 kb to 40 kb, 5 kb to 30 kb, 5 kb to 20 kb, 5 kb to 10 kb, 5 kb to 8 kb, 7 kb to 100 kb, 7 kb to 90 kb, 7 kb to 80 kb, 7 kb to 70 kb, 7 kb to 60 kb, 7 kb to 56 kb, 7 kb to 55 kb, 7 kb to 50 kb, 7 kb to 40 kb, 7 kb to 30 kb, 7 kb to 20 kb, 7 kb to 10 kb, 7 kb to 8 kb, 7.5 kb to 100 kb, 7.5 kb to 90 kb, 7.5 kb to 80 kb, 7.5 kb to 70 kb, It may be 7.5 kb to 60 kb, 7.5 kb to 56 kb, 7.5 kb to 55 kb, 7.5 kb to 50 kb, 7.5 kb to 40 kb, 7.5 kb to 30 kb, 7.5 kb to 20 kb, 7.5 kb to 10 kb, or 7.5 kb to 8 kb.

[0247] The above vector may include three stuffers. The stuffers may include a first stuffer, a second stuffer, and a third stuffer. All three stuffers may be arranged outside of two ITRs.

[0248] The first stuffer may be arranged downstream of the R-ITR. Specifically, the first stuffer may be arranged after the 3' end of the R-ITR with respect to the (+) strand.

[0249] The length of the first stuffer above may be 0.1 kb to 20 kb. Specifically, the length of the first stuffer is 0.1 kb to 20 kb, 0.1 kb to 15 kb, 0.1 kb to 10 kb, 0.1 kb to 9 kb, 0.1 kb to 8 kb, 0.1 kb to 7 kb, 0.1 kb to 6 kb, 0.1 kb to 5 kb, 0.1 kb to 4 kb, 0.1 kb to 3 kb, 0.1 kb to 2 kb, 0.5 kb to 20 kb, 0.5 kb to 15 kb, 0.5 kb to 10 kb, 0.5 kb to 9 kb, 0.5 kb to 8 kb, 0.5 kb to 7 kb, 0.5 kb to 6 kb, 0.5 kb to 5 kb, 0.5 kb to 4 kb, 0.5 kb to 3 kb, 0.5 kb to 2 kb, 1 kb to 20 kb, 1 kb to 15 kb, 1 kb to 10 kb, 1 kb to 9 kb, 1 kb to 8 kb, 1 kb to 7 kb, 1 kb to 6 kb, 1 kb to 5 kb, 1 kb to 4 kb, 1 kb to 3 kb, 1 kb to 2 kb, 2 kb to 20 kb, 2 kb to 15 kb, 2 kb to 10 kb, 2 kb to 9 kb, 2 kb to 8 kb, 2 kb to 7 kb, 2 kb to 6 kb, 2 kb to 5 kb, 2 kb to 4 kb, 2 kb to 3 kb, 3 kb to 20 kb, 3 kb to 15 It may be kb, 3 kb to 10 kb, 3 kb to 9 kb, 3 kb to 8 kb, 3 kb to 7 kb, 3 kb to 6 kb, 3 kb to 5 kb, 3 kb to 4 kb, 4 kb to 20 kb, 4 kb to 15 kb, 4 kb to 10 kb, 4 kb to 9 kb, 4 kb to 8 kb, 4 kb to 7 kb, 4 kb to 6 kb, 4 kb to 5 kb, 5 kb to 20 kb, 5 kb to 15 kb, 5 kb to 10 kb, 5 kb to 9 kb, 5 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb.

[0250] The second stuffer may be arranged between the helper virus gene and the Rep / Cap gene. Specifically, the second stuffer may be arranged between (a) the helper virus gene and (b) the Rep gene, or between (a) the helper virus gene and (c) the Cap gene. The second stuffer may be arranged between (i) the Rep gene and the Cap gene that is closer to the helper virus gene and (ii) the helper virus gene. For example, it may be arranged in the order of (a) helper virus gene - second stuffer - (b) Rep gene; (b) Rep gene - second stuffer - (a) helper virus gene; (a) helper virus gene - second stuffer - (c) Cap gene; or (c) Cap gene - second stuffer - (a) helper virus gene.

[0251] The length of the second stuffer above may be 0.1 kb to 20 kb. Specifically, the length of the first stuffer is 0.1 kb to 20 kb, 0.1 kb to 15 kb, 0.1 kb to 10 kb, 0.1 kb to 9 kb, 0.1 kb to 8 kb, 0.1 kb to 7 kb, 0.1 kb to 6 kb, 0.1 kb to 5 kb, 0.1 kb to 4 kb, 0.1 kb to 3 kb, 0.1 kb to 2 kb, 0.5 kb to 20 kb, 0.5 kb to 15 kb, 0.5 kb to 10 kb, 0.5 kb to 9 kb, 0.5 kb to 8 kb, 0.5 kb to 7 kb, 0.5 kb to 6 kb, 0.5 kb to 5 kb, 0.5 kb to 4 kb, 0.5 kb to 3 kb, 0.5 kb to 2 kb, 1 kb to 20 kb, 1 kb to 15 kb, 1 kb to 10 kb, 1 kb to 9 kb, 1 kb to 8 kb, 1 kb to 7 kb, 1 kb to 6 kb, 1 kb to 5 kb, 1 kb to 4 kb, 1 kb to 3 kb, 1 kb to 2 kb, 2 kb to 20 kb, 2 kb to 15 kb, 2 kb to 10 kb, 2 kb to 9 kb, 2 kb to 8 kb, 2 kb to 7 kb, 2 kb to 6 kb, 2 kb to 5 kb, 2 kb to 4 kb, 2 kb to 3 kb, 3 kb to 20 kb, 3 kb to 15 It may be kb, 3 kb to 10 kb, 3 kb to 9 kb, 3 kb to 8 kb, 3 kb to 7 kb, 3 kb to 6 kb, 3 kb to 5 kb, 3 kb to 4 kb, 4 kb to 20 kb, 4 kb to 15 kb, 4 kb to 10 kb, 4 kb to 9 kb, 4 kb to 8 kb, 4 kb to 7 kb, 4 kb to 6 kb, 4 kb to 5 kb, 5 kb to 20 kb, 5 kb to 15 kb, 5 kb to 10 kb, 5 kb to 9 kb, 5 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb.

[0252] The third stuffer may be arranged upstream of the L-ITR. Specifically, the third stuffer may be arranged before the 5' end of the L-ITR with respect to the (+) strand.

[0253] The length of the third stuffer above may be 0.1 kb to 20 kb. Specifically, the length of the first stuffer is 0.1 kb to 20 kb, 0.1 kb to 15 kb, 0.1 kb to 10 kb, 0.1 kb to 9 kb, 0.1 kb to 8 kb, 0.1 kb to 7 kb, 0.1 kb to 6 kb, 0.1 kb to 5 kb, 0.1 kb to 4 kb, 0.1 kb to 3 kb, 0.1 kb to 2 kb, 0.5 kb to 20 kb, 0.5 kb to 15 kb, 0.5 kb to 10 kb, 0.5 kb to 9 kb, 0.5 kb to 8 kb, 0.5 kb to 7 kb, 0.5 kb to 6 kb, 0.5 kb to 5 kb, 0.5 kb to 4 kb, 0.5 kb to 3 kb, 0.5 kb to 2 kb, 1 kb to 20 kb, 1 kb to 15 kb, 1 kb to 10 kb, 1 kb to 9 kb, 1 kb to 8 kb, 1 kb to 7 kb, 1 kb to 6 kb, 1 kb to 5 kb, 1 kb to 4 kb, 1 kb to 3 kb, 1 kb to 2 kb, 2 kb to 20 kb, 2 kb to 15 kb, 2 kb to 10 kb, 2 kb to 9 kb, 2 kb to 8 kb, 2 kb to 7 kb, 2 kb to 6 kb, 2 kb to 5 kb, 2 kb to 4 kb, 2 kb to 3 kb, 3 kb to 20 kb, 3 kb to 15 It may be kb, 3 kb to 10 kb, 3 kb to 9 kb, 3 kb to 8 kb, 3 kb to 7 kb, 3 kb to 6 kb, 3 kb to 5 kb, 3 kb to 4 kb, 4 kb to 20 kb, 4 kb to 15 kb, 4 kb to 10 kb, 4 kb to 9 kb, 4 kb to 8 kb, 4 kb to 7 kb, 4 kb to 6 kb, 4 kb to 5 kb, 5 kb to 20 kb, 5 kb to 15 kb, 5 kb to 10 kb, 5 kb to 9 kb, 5 kb to 8 kb, 5 kb to 7 kb, or 5 kb to 6 kb.

[0254] The above stuffer can reduce impurities that may occur during the recombinant AAV production process by the above vector.

[0255] In one embodiment, the stuffer may reduce reverse packaging of the vector. Packaging of AAV means that the configuration existing between two ITRs is packaged within the AAV particle. However, reverse packaging of AAV means that the recombinant AAV particle is packaged outward rather than inwardly towards the two ITRs. Therefore, by inhibiting reverse packaging of AAV, high-quality AAV can be obtained. A vector according to one aspect can enable the obtaining of high-quality recombinant AAV by including a stuffer to reduce the impurity content caused by reverse packaging of the vector, and furthermore, an effect of increased safety can be expected when applied as a therapeutic agent.

[0256] In one embodiment, a first stuffer arranged downstream of the R-ITR, a third stuffer arranged upstream of the L-ITR, or both can reduce the reverse packaging of the vector.

[0257] In one embodiment, the stuffer may reduce impurities that may occur during the production process of recombinant AAV using a single vector system.

[0258] In one embodiment, a second stuffer arranged between (a) and (b) or between (a) and (c) can reduce impurities in the recombinant AAV particles produced by the vector. Specifically, the second stuffer can reduce impurities that may occur during the recombinant AAV production process using a single vector system.

[0259] For example, impurities that may occur during the production of recombinant AAV using a single vector system may include one or more selected from: all or part of the helper virus gene; all or part of the Rep gene; all or part of the Cap gene; any sequence located outside two ITRs in the vector; part of the host cell genome sequence; and two or more chimeric forms among these. In one embodiment, the impurities may include all or part of the helper virus gene.

[0260] The above stuffer may use a sequence having one or more of the following features: (i) it is a human-derived DNA sequence; (ii) it does not encode a protein; and (iii) it does not inhibit the expression of surrounding genes.

[0261] The above stuffer may include one or more selected from introns and some or modified sequences thereof. The above intron refers to a nucleotide sequence removed from the final gene product by RNA splicing. The above intron may be an intron derived from a gene of GOI or an intron derived from other genes.

[0262] The above stuffer may include one or more selected from barrier insulators and some or modified sequences thereof. The barrier insulator refers to a DNA sequence that interferes with heterochromatin formation. The barrier insulator can prevent gene silencing of surrounding genes and maintain the expression of surrounding genes. The barrier insulator may include one or more selected from MAR (Matrix Attachment Region), UCOE (Ubiquitous Chromatin Opening Element), CCE (Chromatin Control Element), STAR (Stabilizing and Anti-Repressor), 5'HS5 (Hypersensitive site 5), and some or modified sequences thereof.

[0263] As the above stuffer, a stuffer derived from lambda phage may not be suitable because it can reduce the expression of surrounding genes and cause an inflammatory response by expressing foreign proteins.

[0264] The above stuffer may include one or more selected from all or part of the sequence of a MAR (Matrix Attachment Region), all or part of the sequence of a UCOE (Ubiquitous Chromatin Opening Element), and all or part of the sequence of a CCE (Chromatin Control Element).

[0265] The above stuffer may include a portion of the sequence of a Matrix Attachment Region (MAR). The MAR is a type of barrier insulator that does not encode a protein and does not block the expression of surrounding genes. The MAR may be a human globin MAR. The above stuffer may include a polynucleotide containing 100 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, or 3000 or more consecutive nucleotides within the sequence of SEQ ID NO. 1.

[0266] In one embodiment, the stuffer may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 20.

[0267] The above stuffer may include a portion of the sequence of a Ubiquitous Chromatin Opening Element (UCOE). The UCOE is a type of barrier insulator that does not encode a protein and does not block the expression of surrounding genes. The UCOE may be the UCOE of the human HNRPA2B1 (heterogeneous nuclear ribonucleoproteins A2 / B1 gene). The above stuffer may include a polynucleotide containing 100 or more, 500 or more, 1000 or more, 1200 or more, or 1400 or more consecutive nucleotides within the sequence of SEQ ID NO. 5.

[0268] In one embodiment, the stuffer may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 21.

[0269] The above stuffer may include a portion of the sequence of a Chromatin Control Element (CCE). The above CCE is a type of barrier insulator that does not encode a protein and does not block the expression of surrounding genes. The above CCE may be a human CCE. The above stuffer may include a polynucleotide comprising 100 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, or 3500 or more consecutive nucleotides within the sequence of SEQ ID NO. 9.

[0270] In one embodiment, the stuffer may include or be composed of a nucleotide sequence having 90% or more sequence identity with the sequence of SEQ ID NO. 19.

[0271] In one embodiment, the stuffer may include a first stuffer, a second stuffer, and a third stuffer, and the first stuffer, the second stuffer, and the third stuffer may each be independently selected from CCE or a sequence thereof, MAR or a sequence thereof, and UCOE or a sequence thereof.

[0272] In one embodiment, the stuffer may include a first stuffer, a second stuffer, and a third stuffer, wherein the first stuffer is CCE or a sequence thereof, the second stuffer is MAR or a sequence thereof, and the third stuffer is UCOE or a sequence thereof.

[0273] In one embodiment, the stuffer may include the following:

[0274] A first stuffer comprising the polynucleotide of SEQ ID NO. 19 arranged downstream of the R-ITR;

[0275] A second stuffer comprising a polynucleotide of SEQ ID NO. 20 arranged between (a) and (b) or between (a) and (c); and

[0276] A third stuffer comprising the polynucleotide of SEQ ID NO. 21 arranged upstream of the L-ITR.

[0277] In one embodiment, compared to a control group triple transfecting a helper vector, a Rep-Cap expression vector, and a GOI vector, it was confirmed that an all-in-one vector containing a stuffer exhibited improved recombinant AAV productivity when using the same amount of plasmid DNA (pDNA).

[0278] In one embodiment, it was confirmed that the recombinant AAV produced by an all-in-one vector containing a stuffer exhibited GOI expression and activity at an equivalent or higher level compared to a control group that triple transfected a helper vector, a Rep-Cap expression vector, and a GOI vector, or an all-in-one vector control group that did not contain a stuffer. Therefore, an all-in-one vector containing a stuffer can produce recombinant AAV at an equivalent or higher level than that produced by the conventional triple transfection method or by an all-in-one vector that did not contain a stuffer in terms of transduction ability and GOI functionality.

[0279]

[0280] Another aspect provides a vector system for producing recombinant adeno-associated virus (AAV) comprising the nucleotide sequence of the following gene, wherein the vector system comprises one or more vectors:

[0281] (a) Helper virus genes required for AAV production;

[0282] (b) AAV's Rep gene;

[0283] (c) Cap gene of AAV; and

[0284] (d) a transgene arranged between ITRs (Inverted Terminal Repeats), wherein the transgene comprises a codon-optimized polynucleotide encoding argininosuccinate synthase (ASS1) having a nucleotide sequence having 90% or more sequence identity with any one of SEQ ID NOs 1 to 3.

[0285] The same parts as those described above also apply to the above system.

[0286]

[0287] Another aspect provides a method for producing recombinant adeno-associated virus (AAV). The method may be a method using a vector for producing recombinant AAV according to one aspect. The same parts as described above apply equally to the method.

[0288] In one embodiment, the method may be a method for producing recombinant AAV to treat type 1 citrullinemia.

[0289] The above method comprises the steps of: introducing a recombinant AAV production vector according to one aspect into a host cell; and isolating the recombinant AAV from the host cell.

[0290] The host cell may be a mammalian cell. The mammalian cell may include cells derived from any organ or tissue of humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cattle, and primates. 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.

[0291] In one embodiment, the mammalian cell may be selected from HEK293 cells, HEK293F cells, HEK293T cells, and cells derived therefrom. The HEK293 cell is a Human Embryonic Kidney 293 cell line commonly used in biotechnology. 'Cells derived from HEK293 cells, HEK293F cells, or HEK293T cells' refers to cells derived from the parent 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 parent cell line include Expi293F (Manufacturer: ThermoFisher), HEK293F (Manufacturer: ThermoFisher), HEK293.2 (ATCC), etc. Exemplary cells derived from the HEK293T parent cell line include HEK293FT (Manufacturer: ThermoFisher). Exemplary cells derived from the HEK293F parent cell line include Viral Production Cells 1.0 (VPC1.0) (Manufacturer: ThermoFisher) and Viral Production Cells 2.0 (VPC2.0) (Manufacturer: ThermoFisher). VPC2.0 is a clonal cell line derived from the HEK293F parent cell line and is a host cell suitable for AAV production. In certain embodiments, the mammalian cell may be an HEK293F cell or a cell derived therefrom.

[0292] The host cell may be an insect cell. The insect cell may include cells derived from the fall armyworm (Spodoptera frugiperda) or the cabbage silver 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.

[0293] The above introduction may be used without limitation as long as it is a known method capable of inserting a recombinant AAV production vector into a host cell. The above 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. The insertion of a vector is generally referred to as transformation in the case of bacterial cells and transfection in the case of eukaryotic cells, and the insertion of a viral vector is also referred to as transduction. A person skilled in the art may introduce the above vector into a host cell using a known method. Non-limiting examples of the above 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 impaction), chemical reagents (e.g., calcium phosphate, highly branched organic compounds, or cationic polymers), or cationic lipids (e.g., lipofection). The cationic polymer includes, but is not limited to, polyethyleneimine (PEI). The transfection method may additionally require contacting a solution of plasmid DNA with cells, growing them, and selecting them using marker gene expression.

[0294] The above method may additionally include a step of culturing cells after the introduction step.

[0295] The above culture may involve culturing host cells under conditions in which recombinant AAV can be produced. Suitable culture methods are well known to those skilled in the art. For example, the cells may be cultured in a suspension and / or under animal component-free conditions.

[0296] The above method may additionally include a step of selecting cells into which the vector has been introduced using a selection marker after the above-mentioned culture step.

[0297] The above separation may be performed by known methods. In one embodiment, the separation may be performed using centrifugation or chromatography.

[0298] The above centrifugation method may be a cesium chloride (CsCl)-based ultra-high-speed centrifugation method, but is not limited thereto.

[0299] The above chromatography may be one or more selected from affinity chromatography, ion exchange chromatography, column chromatography, gel-filtration chromatography, thin-layer chromatography, radial flow chromatography, interference chromatography, and reverse phase chromatography, but is not limited thereto.

[0300] The above method can also be easily scaled up for industrial production because it requires only a single transfection of host cells with a recombinant AAV production vector that has excellent recombinant AAV productivity.

[0301] According to the above method, unlike the conventional triple transfection method using three types of vectors, only the introduction of a single vector is required, so the recombinant AAV production process is simplified, thereby reducing costs. In addition, unlike the triple transfection method where the ratio of the three types of vectors injected into the cell cannot be accurately controlled, according to the method according to the above aspect, since each component is integrated into a single vector, each component can be introduced in equal proportions. Accordingly, the productivity of recombinant AAV can be improved.

[0302] In addition, according to the above method, since recombinant AAV with a high %Full capsid ratio can be produced, high-quality recombinant AAV can be produced.

[0303] The recombinant AAV produced by the above method can exhibit increased transduction ability and GOI functionality compared to the recombinant AAV produced by triple transfection.

[0304] The recombinant AAV produced by the above method may exhibit increased argininosuccinate synthase expression levels compared to the recombinant AAV produced by triple transfection.

[0305] Recombinant AAV produced by the above method may exhibit increased argininosuccinate synthase activity compared to recombinant AAV produced by triple transfection.

[0306]

[0307] Another aspect provides a recombinant AAV produced by a vector according to one aspect or by a method according to one aspect. The same parts described above apply equally to the recombinant AAV.

[0308] The above recombinant AAV may be a recombinant AAV for treating citrullinemia type 1. Therefore, the above recombinant AAV may be used in a method of gene therapy for citrullinemia type 1.

[0309] The above recombinant AAV may have a high %Full / Empty capsid ratio. In addition, the above recombinant AAV may have a low impurity content. Therefore, the above recombinant AAV may have excellent quality and safety.

[0310] In one embodiment, the recombinant AAV may be a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV).

[0311] The above recombinant AAV may have increased expression and activity of GOI.

[0312] The above recombinant AAV can be used in gene therapy methods.

[0313] The above recombinant AAV can be used for the mass production of clinical materials for gene therapy for the treatment of patients.

[0314] The above recombinant AAV includes an AAV capsid and a genome.

[0315] The above AAV capsid may be a wild-type capsid or a variant thereof. The above AAV capsid may be derived from any AAV serotype. The above AAV capsid may be any one of 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 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 above variant may include an engineered capsid.

[0316] In one embodiment, the AAV capsid may be a wild-type AAV8 capsid.

[0317] The above AAV capsid may include VP1 protein, VP2 protein, and VP3 protein.

[0318] The "genome" of the above AAV refers to the sequence that is finally packaged or capsidized to form a viral particle.

[0319] The above genome may be linear single-stranded DNA.

[0320] The above genome includes a transgene.

[0321] The above-mentioned transgene comprises a codon-optimized polynucleotide encoding an argininosuccinate synthase according to the above-mentioned aspect.

[0322] The above-mentioned transgenic gene may be arranged between ITRs. The above-mentioned transgenic gene may be arranged between L-ITR and R-ITR.

[0323] 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. The above AAV serotypes may also include other AAV serotypes currently known or to be discovered later. The above AAV serotypes may also include artificial AAV serotypes. In one embodiment, the above ITR may be derived from AAV2.

[0324] The above ITR may be a wild-type ITR or a variant thereof.

[0325]

[0326] Another aspect provides an expression cassette or a vector comprising said expression cassette, comprising a codon-optimized polynucleotide encoding argininosuccinate synthase (ASS1) and one or more expression regulatory elements. The same parts described above apply equally to said expression cassette or vector.

[0327] The codon-optimized polynucleotide encoding the above argininosuccinate synthase may include or be composed of a nucleotide sequence having sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or 100% with any one of SEQ ID NOs 1 to 3.

[0328] The above expression cassette or vector may additionally contain an ITR.

[0329] The codon-optimized polynucleotide encoding the argininosuccinate synthase may be arranged between Inverted Terminal Repeats (ITRs). The codon-optimized polynucleotide encoding the argininosuccinate synthase may have ITRs arranged on both sides. The codon-optimized polynucleotide encoding the argininosuccinate synthase may have two ITRs arranged on the sides. The codon-optimized polynucleotide encoding the argininosuccinate synthase may be arranged between L-ITRs and R-ITRs. For example, L-ITRs (first ITRs), the codon-optimized polynucleotide encoding the argininosuccinate synthase, and R-ITRs (second ITRs) may be arranged sequentially in the 5' to 3' direction or the 3' to 5' direction.

[0330] The above expression regulatory elements may include one or more selected from enhancers, promoters, transcription factors, silencers, insulators, introns, splicing donors and receptors, engineered splicing donors and receptors, riboswitches, amino acids, miRNA (microRNA), shRNA (short hairpin RNA), 5'- or 3'-UTR (untranslated region), Kozak sequences, start codons, GOI codons, signal peptides, polyadenylation (poly A) signal sequences, etc.

[0331] In one embodiment, the expression regulating element may include one or more selected from enhancers, promoters, introns, and polyadenylation signal sequences.

[0332] In one embodiment, the expression cassette or the vector containing the expression cassette may be for producing AAV.

[0333]

[0334] Another aspect provides a recombinant AAV comprising an AAV capsid and a genome, wherein the genome comprises a polynucleotide encoding argininosuccinate synthase (ASS1) having a nucleotide sequence having 90% or more sequence identity with any one of SEQ ID NOs 1 to 3. The same parts as described above apply equally to the recombinant AAV.

[0335] The codon-optimized polynucleotide encoding the above argininosuccinate synthase may include or be composed of a nucleotide sequence having sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.9% or more, or 100% with any one of SEQ ID NOs 1 to 3.

[0336] The above AAV capsid may be expressed by the Cap (Capsid) gene.

[0337] In one embodiment, the genome may comprise an expression cassette comprising a polynucleotide encoding an argininosuccinate synthase having a nucleotide sequence having 90% or more sequence identity with any one of SEQ ID NOs 1 to 3, and one or more expression regulatory elements.

[0338] The polynucleotide encoding the argininosuccinate synthase may be arranged between ITRs. The polynucleotide encoding the argininosuccinate synthase may be arranged between L-ITR and R-ITR.

[0339] The above recombinant AAV may be intended to prevent or treat type 1 citrullinemia.

[0340]

[0341] Another aspect provides a pharmaceutical composition for preventing or treating type 1 citrullinemia, comprising a vector according to one aspect, recombinant AAV produced by a vector according to one aspect, or recombinant AAV according to one aspect. The same parts as described above apply equally to the composition.

[0342] The above recombinant AAV may comprise a polynucleotide encoding argininosuccinate synthase (ASS1) comprising a nucleotide sequence having 90% or more sequence identity with any one of SEQ ID NOs 1 to 3.

[0343] The above pharmaceutical composition may be for gene therapy for citrullinemia type 1. The above pharmaceutical composition may be a composition for delivering an argininosuccinate synthase protein or a gene encoding it for gene therapy of citrullinemia type 1.

[0344] The term "Citrullinaemia type 1 (CTLN1)" refers to a condition that may result from a deficiency or reduction in argininosuccinate synthase, or a loss or reduction in its activity. Citrullinaemia type 1 occurs in newborns and infants; although the infant may appear normal at birth, symptoms such as increased intracranial pressure, increased neuromuscular tone, rigidity, intermittent convulsions, seizures, and loss of consciousness appear upon the onset of breastfeeding due to hyperammonemia and the accumulation of toxic metabolites. Without immediate intervention, it can lead to death. The incidence rate is 1 in 57,000 newborns.

[0345] The term "gene therapy" refers to a treatment that utilizes genes to treat or prevent disease. Recombinant AAVs used to deliver therapeutic genes into cells can be used as gene therapy agents. Diseases to which gene therapy is applicable include, but are not limited to, those caused by defects in a single gene.

[0346] The term "treatment" in this specification refers to any act of improving or beneficially altering the symptoms of citrullinemia type 1 by administering the composition of the present invention.

[0347] The term "prevention" in this specification refers to any act in which the possibility of developing type 1 citrullinemia is suppressed or delayed by the administration of the composition of the present invention.

[0348] In one embodiment, the vector, the recombinant AAV, or the composition containing the same may be capable of improving the survival rate of an individual with citrullinemia type 1.

[0349] In one embodiment, the vector, the recombinant AAV, or a composition comprising the same may be capable of inhibiting weight loss in an individual with type 1 citrullinemia or recovering the reduced weight; specifically, it may be capable of recovering weight to a level of about 95% or more, about 90% or more, about 85% or more, about 80% or more, about 75% or more, or about 70% or more relative to the weight of a normal individual without type 1 citrullinemia; more specifically, it may be capable of recovering weight to about 70% to about 100%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 75% to about 100%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 80% to about 100%, or about 80% to about 80% relative to the weight of a normal individual. It may be possible to recover body weight to 95%, about 80% to about 90%, or about 80% to about 85%.

[0350] In one embodiment, the vector, the recombinant AAV, or a composition containing the same may be capable of inhibiting an increase in blood ammonia concentration in an individual with citrullinemia type 1, and / or reducing an increased blood ammonia concentration. Specifically, the recombinant AAV or a composition containing the same may be administered to reduce the blood ammonia concentration in an individual with type 1 citrullinemia to a level of about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, or about 30% or less relative to the blood ammonia concentration in an individual with type 1 citrullinemia who has not been administered the recombinant AAV or a composition containing the same; more specifically, the reduction may be about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, or about 10% to about 30%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, or about It may be reduced to a level of 20% to about 50%, about 20% to about 40%, or about 20% to about 30%.

[0351] Additionally, specifically, the vector, the recombinant AAV, or a composition containing the same may reduce the blood ammonia concentration of a normal individual not suffering from citrullinemia type 1 to about 150% or less, about 140% or less, about 130% or less, about 120% or less, about 110% or less, or about 100% or less; more specifically, to about 50% to about 150%, about 50% to about 140%, about 50% to about 130%, about 50% to about 120%, about 50% to about 110%, about 50% to about 100%, about 70% to about 150%, about 70% to about 140%, about 70% to about 130%, about 70% to about 120%, about 70% to about 110%, or about 70% to about 110%. It could be to reduce it to a 100% level.

[0352] The above pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "pharmaceuticalally acceptable carrier" may refer to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the injected compound. Here, "pharmaceuticalally acceptable" means that the target of application (prescription) does not possess toxicity beyond an tolerable level without inhibiting the activity of the active ingredient. Any type of carrier that is commonly used in the relevant technical field and is pharmaceutically acceptable may be used in the above pharmaceutical composition. Non-limiting examples of the above carriers include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. These may be used alone or in a mixture of two or more. The above pharmaceutical composition may be prepared into an oral or parenteral formulation according to the route of administration by conventional methods known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The above pharmaceutical compositions may each be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions according to conventional methods.

[0353] The above-mentioned pharmaceutically acceptable carrier is used in the sense of including excipients, diluents, or adjuvants. The carrier may be suitable for delivering recombinant AAV in vivo. Specifically, the carrier may be selected to be suitable for formulation into a parenteral formulation (e.g., an injectable formulation). For example, the carrier may be selected to be suitable for formulation into an intravenous formulation. The carrier may be an aqueous solution, e.g., water or a buffered saline solution.

[0354] The above pharmaceutical composition may be prepared in any formulation according to conventional methods. The above pharmaceutical composition may be formulated in a form suitable for delivering recombinant AAV to an individual. The composition may be formulated in an aqueous solution, for example, in water or a buffered saline solution. The above pharmaceutical composition may be formulated in an injectable formulation suitable for administration via any suitable route, such as intravenous, intra-arterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intra-articular, or intravertebral. The above pharmaceutical composition may be prepared as a systemic formulation or a local formulation.

[0355] When formulating the above pharmaceutical composition, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, or surfactants, but is not limited thereto.

[0356] When the above pharmaceutical composition is prepared as an oral formulation, it may be prepared in the form of powder, granules, tablets, pills, coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., in accordance with methods known in the art together with a suitable carrier. Examples of pharmaceutically acceptable suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinylpyrrolidone; water; methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate; mineral oil; malt; gelatin; talc; polyols; vegetable oils, etc. In the case of formulation, the formulation may include diluents and / or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants as needed.

[0357] When the above pharmaceutical composition is prepared as a parenteral formulation, it may be formulated in the form of an injectable, transdermal, nasal inhalant, and suppository according to methods known in the art with a suitable carrier. When formulated as an injectable, suitable carriers may include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof; preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose, etc. When formulated as a transdermal formulation, it may be formulated in the form of an ointment, cream, lotion, gel, topical solution, paste, liniment, aerosol, etc. In the case of nasal inhalers, they can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide, and when formulated as suppositories, the base may be Witepsol, Tween 61, polyethylene glycols, cocoa starch, laurin starch, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate, and sorbitan fatty acid esters.

[0358] The above pharmaceutical composition may further include an additional second therapeutic agent having a preventive or therapeutic effect for citrullinemia type 1. The above pharmaceutical composition may be a single composition or individual compositions.

[0359] The above pharmaceutical composition may contain the recombinant AAV in a pharmaceutically effective amount. The effective amount may be appropriately selected by a person skilled in the art depending on the individual.

[0360] The above pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level may be determined based on factors including the severity of the disease, drug activity, patient's age, weight, health, gender, patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a component known to exhibit therapeutic effects for known diseases. It is important to administer an amount that obtains maximum effect with minimum amount without side effects, taking all of the above factors into consideration.

[0361] The dosage of the above pharmaceutical composition may be determined by a person skilled in the art by taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, gender, medical history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at a dose of about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg per adult, and although the frequency of administration of the composition of the present invention is not particularly limited thereto, it may be administered once a day or administered several times by dividing the dose. The above dosage or frequency of administration does not limit the scope of the present invention in any way.

[0362]

[0363] Another aspect provides a method for delivering argininosuccinate synthase to an individual in need thereof, comprising the step of administering to the individual an effective amount of a vector according to one aspect, recombinant AAV produced by a vector according to one aspect, recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect. The same parts as described above apply equally to the method.

[0364]

[0365] Another aspect provides a method for treating type 1 citrullinemia, comprising the step of administering to an individual an effective amount of a vector according to one aspect, a recombinant AAV produced by a vector according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect. The same parts as described above apply equally to the method.

[0366] In the above aspects, the individual may be an individual requiring the expression of argininosuccinate synthase delivered by recombinant AAV. The individual may be an individual suffering from or likely to suffer from citrullinemia type 1. The individual may be an individual suffering from or likely to suffer from a disease that can be treated by the expression of argininosuccinate synthase delivered by recombinant AAV. The individual may be a patient with citrullinemia type 1.

[0367] The above-mentioned individual may be a mammal. The above-mentioned mammal may include, but is not limited to, humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cattle, etc.

[0368] In one embodiment, the entity may be a human. The human may include a fetus, a newborn, an infant, an adolescent, or an adult.

[0369] The above pharmaceutical composition may be administered as a single or multiple doses in pharmaceutically effective amounts. In this case, the composition may be administered in the form of a liquid, powder, aerosol, injection, intravenous fluid (Ringer), capsule, pill, tablet, suppository, or patch. The route of administration of the above pharmaceutical composition for the prevention or treatment of disease may be any general route as long as it can reach the target tissue.

[0370] The route of administration can be determined by a person skilled in the art and may include, for example, intranasal, intravenous, subcutaneous, intradermal, oral, and other parenteral administration routes. Two or more routes of administration may be combined if necessary. In one embodiment, the administration may be intravenous.

[0371] The above administration can be administered in an amount sufficient to infect the individual, and in an amount sufficient to provide a sufficient level of introduction and expression of argininosuccinate synthase.

[0372] The term "effective dose" may mean "therapeutic effective dose" and refers to the dosage administered to achieve a therapeutic effect.

[0373] The dose required to achieve a therapeutic effect, e.g., the dose of vector genome / kilogram of body weight (vg / kg), may vary based on several factors including the route of administration, the level of heterogeneous polynucleotide expression required to achieve a therapeutic effect, the specific disease being treated, any host immune response to the viral vector, the host immune response to the heterogeneous polynucleotide or the expression product (protein), and the stability of the expressed protein. A person skilled in the art may determine the rAAV dose range for treating patients with a specific disease or disorder based on factors other than those mentioned above. Generally, the dose is 1 x 10⁶ per kilogram of the individual's body weight to achieve a therapeutic effect. 7 or 1x10 8 or 1x10 9or 1x10 10 or 1x10 11 or 1x10 12 or 1x10 13 or 1x10 14 or 1x10 15 It may be within the range of the vector genome (vg / kg). The method and frequency of administration can be determined by monitoring the expression level of heterogeneous polynucleotides.

[0374] The therapeutically effective dose of rAAV may mean an amount sufficient to convert severe citrullinemia type 1 into moderate or mild citrullinemia type 1, or to completely cure citrullinemia type 1, when administered to an individual with type 1 citrullinemia.

[0375] A therapeutically effective dose of rAAV may mean an amount sufficient to achieve plasma argininosuccinate synthase activity of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more than normal argininosuccinate synthase activity when administered to an individual with severe, moderate, or mild citrullinemia type 1.

[0376]

[0377] Another aspect provides a vector according to one aspect, a recombinant AAV produced by said vector, or a recombinant AAV according to one aspect for the manufacture of a therapeutic drug for citrullinemia type 1. The same parts as described above apply equally to said uses.

[0378]

[0379] Another aspect provides a use for the prevention or treatment of type 1 citrullinemia of a vector according to one aspect, a recombinant AAV produced by a vector according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect. The same parts as described above apply equally to the said use.

[0380]

[0381] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0382] According to a codon-optimized polynucleotide based on a specific pattern, the expression and activity of argininosuccinate synthase in the individual are enhanced, and the immune response can be minimized.

[0383] According to the vector for recombinant AAV production based on a specific aspect, the elements 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 the components of the vector are positioned in a combination of optimized arrangement and orientation, it offers effects such as improved AAV productivity, an increased %Full / Empty capsid ratio, and reduced impurities.

[0384] A vector for producing recombinant AAV according to one aspect includes a stuffer, thereby reducing impurities caused by the use of a single vector system or by reverse packaging during recombinant AAV production. In addition, the vector can produce recombinant AAV with the same or improved level of productivity and efficiency compared to a single vector system that incorporates elements essential for recombinant AAV production.

[0385] According to the method for producing recombinant AAV based on a single aspect, recombinant AAV can be produced by introducing a single vector, thereby reducing production costs. Furthermore, the method can be easily scaled up for industrial production and can be immediately applied to the process.

[0386] Recombinant AAV produced using a vector for recombinant AAV production according to a specific pattern has excellent safety due to reduced impurities, so it can be applied in various ways in the field of gene therapy.

[0387] Recombinant AAV produced using a recombinant AAV production vector according to one aspect can increase the expression and activity of argininosuccinate synthase.

[0388] FIG. 1 is a diagram illustrating the configuration of a vector for producing recombinant AAV according to one embodiment.

[0389] FIG. 2 is a diagram showing a vector map of an all-in-one vector for recombinant AAV production according to one embodiment.

[0390] FIG. 3 is a diagram showing a vector map of an all-in-one vector for recombinant AAV production including a stuffer sequence according to one embodiment.

[0391] FIG. 4 is a diagram showing a vector map of a triple vector for conventional triple transfection according to one embodiment.

[0392] Figure 5 is a diagram showing the AAV productivity (viral genome) of an all-in-one vector and a control group (Triple) according to one embodiment.

[0393] Figure 6 is a diagram showing the VP (Viral particle) of an all-in-one vector and a control group (Triple) according to one embodiment.

[0394] Figure 7 is a diagram showing the quality (VG / VP ratio) of AAV produced by an all-in-one vector and a control group (Triple) according to one embodiment.

[0395] FIG. 8 is a figure showing the expression level of argininosuccinate synthase of a recombinant AAV virus produced by an all-in-one vector according to one embodiment.

[0396] FIG. 9 is a diagram showing the activity level of argininosuccinate synthase of a recombinant AAV virus produced by an all-in-one vector according to one embodiment.

[0397] Figure 10 is a diagram showing the change in body weight following the administration of a recombinant AAV virus produced by an all-in-one vector according to one embodiment.

[0398] Figure 11 is a diagram showing blood ammonia levels following the administration of a recombinant AAV virus produced by an all-in-one vector according to one embodiment.

[0399] One aspect is described in more detail below through examples. However, these examples are intended to illustrate one aspect and the scope of one aspect is not limited to these examples; the examples of one aspect are provided to more completely explain one aspect to those with average knowledge in the art.

[0400]

[0401] Example 1: Construction of a codon-optimized sequence encoding argininosuccinate synthase

[0402] Codon optimization sequences for argininosuccinate synthase (ASS1) were constructed. Specifically, based on the sequence of SEQ ID NO. 1, the sequences ASS-GA_CpG and ASS-GS_CpG, which are optimized sequences with reduced CpG, were constructed. The specific sequences are described below.

[0403] Name Sequence Number ASS (SB) (1,239 bp)1ASS-GA_CpG (1,239 bp)2ASS-GS_CpG (1,239 bp)3

[0404]

[0405] Example 2: Preparation of a vector for producing rAAV to treat type 1 citrullinemia

[0406] A vector for producing recombinant AAV containing a polynucleotide encoding argininosuccinate synthase (ASS1) was constructed.

[0407] Specifically, an all-in-one vector for recombinant AAV production and an all-in-one vector containing stuffer sequences were constructed, and as a comparative example, a triple vector for conventional triple transfection was constructed. Information regarding the composition included in the above-mentioned vectors is described in Fig. 1.

[0408] In addition, the vector map of the all-in-one vector APL106 is shown in FIG. 2, the vector map of the all-in-one vector APL303 containing a stuffer sequence is shown in FIG. 3, and the vector map of APL102 as an example of a triple vector is shown in FIG. 4.

[0409] Meanwhile, the sequence information of the enhancer used in the above vector production is listed in the table below.

[0410] Name Sequence Number ApoE1 enhancer (322 bp)4AMBP enhancer (206 bp)5

[0411]

[0412] The sequence information of the promoter used to construct the above vector is listed in the table below.

[0413] Name Sequence Number A1AT promoter (735bp)6TBG promoter(477bp)7

[0414]

[0415] The sequence information of the introns used to construct the above vector is listed in the table below.

[0416] Name Sequence Number βglobin IVS2 intron (572 bp)8SV40 intron (133 bp)9

[0417]

[0418] The sequence information of the Poly-A used in the above vector construction is listed in the table below.

[0419] Name Sequence Number hGH (479 bp)10

[0420]

[0421] The sequence information of the capsid (Capside, Cap) used in the above vector construction is listed in the table below.

[0422] Name Sequence Number AAV8(WT) Origin Cap11

[0423]

[0424] The sequence information of Rep used in the above vector construction is listed in the table below.

[0425] Name Sequence Number AAV2 (WT) Origin Rep12

[0426]

[0427] The sequence information of the helper used in the above vector construction is listed in the table below.

[0428] Name Sequence Number Adenovirus Type 2 E4 Wildtype13 Adenovirus Type 2 E2A Wildtype14VA RNA 1_Adenovirus Type 2 Wildtype15VA RNA 2_Adenovirus Type 2 Wildtype16

[0429]

[0430] The sequence information of the ITR used in the above vector construction is listed in the table below.

[0431] Name Sequence Number L-ITR (AVA2 derived) 17R-ITR (AVA2 derived) 18

[0432]

[0433] The sequence information of the Stuffer used to construct the above vector is listed in the table below.

[0434] Name Sequence Number CCE19MAR20UCOE21

[0435]

[0436] The sequence information of the replication origin used to construct the above vector is listed in the table below.

[0437] Name Sequence Number pBR322 Replication Origin22

[0438]

[0439] The sequence information of the selection markers used in the above vector construction is listed in the table below.

[0440] Name Sequence Number Kanamycin Selection Marker 23

[0441]

[0442] Example 3: Evaluation of Productivity of ASS1-All-in-One AAV Vector

[0443] To verify the AAV productivity of an all-in-one vector for recombinant AAV production containing a polynucleotide (Sequence No. 2) encoding argininosuccinate synthase (ASS1), the following experiment was performed.

[0444] Specifically, the all-in-one vector for recombinant AAV production containing a polynucleotide encoding argininosuccinate synthase and the all-in-one vector containing a stuffer prepared in Example 2 above were transiently transfected into HEK293 cell lines, and the AAV viral genome (VG) produced was measured by qPCR, and the AAV viral protein (VP) was measured using AAV Titration ELISA (PROGEN). In addition, the quality (%Full / empty) of the produced AAV particles was confirmed through the measured VG / VP ratio value. Meanwhile, a triple vector was used as a control.

[0445] As a result, the productivity results through VG or VP confirmed that APL106 using the all-in-one vector showed excellent productivity with 7.11E14 VG / L (Figs. 5 and 6).

[0446] In addition, regarding the quality of AAV particles measured by the VG / VP ratio value, APL303, an all-in-one vector containing a stuffer, showed excellent results with a value of 13.44 (Fig. 7).

[0447]

[0448] Example 4: Evaluation of expression and activity of argininosuccinate synthase of AAV produced from ASS1-All-in-One AAV vector

[0449] To verify the transducing capability and GOI functionality of recombinant AAV viruses produced by an ASS1-all-in-one AAV vector for recombinant AAV production containing a polynucleotide (SEQ No. 2) encoding argininosuccinate synthase (ASS1), recombinant AAV was injected into target cells (Huh7 cells) to evaluate the expression and enzymatic activity of the argininosuccinate synthase protein.

[0450] Specifically, target cells (Huh7 cells) were seeded 24 hours prior to treatment with recombinant AAV. The target cells (Huh7 cells) were transduced with recombinant AAV according to MOI (Multiplicity of Index, number of recombinant AAVs per cell). Target cells were harvested 72 hours after treatment with recombinant AAV, and the expression and activity of argininosuccinate synthase were evaluated using the harvested cells. For expression evaluation, the concentration of argininosuccinate synthase was measured using an ELISA-based assay. For activity evaluation, an in-vitro enzyme activity assay was performed. An enzymatic reaction was induced by mixing the reaction precursor of argininosuccinate synthase with the lysate obtained from the target cell harvest. The activity of argininosuccinate synthase in the lysate was determined by measuring the amount of ATP consumed during the enzymatic reaction.

[0451] As a result, it was confirmed that the expression level and activity of argininosuccinate synthase were excellent when using a recombinant AAV virus produced from an ASS1-all-in-one AAV vector containing an ASS1-all-in-one AAV vector and a stuffer (Figs. 8 and 9).

[0452]

[0453] Example 5: Evaluation of the therapeutic efficacy of AAV generated from the ASS1-All-in-One AAV Vector for type 1 citrullinemia

[0454] To evaluate the therapeutic efficacy of recombinant AAV viruses produced by an ASS1-all-in-one-stuffer AAV vector for recombinant AAV production containing a polynucleotide (SEQ No. 2) encoding argininosuccinate synthase (ASS1) for type 1 citrullinemia, the following experiment was performed.

[0455] Specifically, to construct an animal model of citrullinemia type 1, an appropriate number of heterozygous mice were obtained by thawing embryos of the ASS1 fold / fold mouse (B6Ei.P-Ass1 / GrsrJ; Strain #: 006449) (https: / www.jax.org / strain / 006449), a recessive model with low ASS1 activity, from Jackson Laboratory in the United States. Subsequently, the mice were shipped to a Contract Research Organization (CRO) to conduct additional breeding rounds, thereby obtaining homozygous mice with low or almost no ASS1 activity, which were established as the animal model for citrullinemia type 1. Information on the above animal model is listed in Table 13 below.

[0456] CategoryDetailsStrain NameB6Ei.P-Ass1 fold / GrsrJMutation TypeSpontaneous autosomal recessive mutationPhenotypeAbnormal hair growth, wrinkled skin at 2 weeks, sparse coat, smaller sizeLifespanMost homozygotes die at 3 weeks of ageGenotypeHomozygous, heterozygous, or wildtype for Ass1Gene MutationC to T transition in exon 15 of the Ass1 gene (T389I amino acid substitution)Genetic BackgroundC57BL / 6JEiOriginArised spontaneously in 1996 on the P / J strain at The Jackson LaboratoryBreeding StrategyHeterozygous x wild-type or C57BL / 6JEiJUse in ResearchModel for citrullinemia type I and other hyperammonemic syndromesReferencesOriginating article: Harris BS et al. (2007), Perez CJ et al. (2010)

[0457]

[0458] Next, AAVs produced using the recombinant AAV production vector of the present invention were observed for 12 weeks after a single administration. AAV administration was performed by intravenous injection (IV injection) to 3-week-old mice at a standard volume of 5 ml / kg (maximum concentration 10 ml / kg). Additionally, the AAV dose was administered at 1.0E14 vg / kg or 2.0E14 vg / kg.

[0459]

[0460] 5.1: Evaluation of Survival Rate Improvement Efficacy

[0461] The efficacy of improving the survival rate of a type 1 citrullinemia animal model following administration of the recombinant AAV virus of the present invention was evaluated.

[0462] As a result, it was confirmed that in the animal model with type 1 citrullinemia (KO) administered only the vehicle as a control, all died within 12 weeks, whereas in the animal model administered the AAV of the present invention, all survived for 12 weeks (Table 14).

[0463] Test System WT KODose (vg / kg) N / A E14 Number of administered individuals (N) 1065 Number of deceased individuals (N) 060 Number of deceased individuals (after administration) N / A 2 individuals (Week 7) 2 individuals (Week 8) 2 individuals (Week 10) N / A Survival rate (%) 1000 100

[0464]

[0465] 5.2: Evaluation of Weight Recovery Efficacy

[0466] The efficacy of weight recovery in a type 1 citrullinemia animal model following administration of the recombinant AAV virus of the present invention was evaluated.

[0467] As a result, compared to the case where only the vehicle was administered as a control to an animal model (KO) with induced citrullinemia type 1, it was confirmed that the body weight of the animal model administered with the AAV of the present invention recovered to the level of a normal mouse (WT) (Fig. 10).

[0468]

[0469] 5.3: Assessment of Blood Ammonia Levels

[0470] The blood ammonia (NH3) level of a type 1 citrullinemia animal model following administration of the recombinant AAV virus of the present invention was evaluated.

[0471] As a result, compared to the case where only the vehicle was administered as a control to an animal model (KO) with induced citrullinemia type 1, it was confirmed that the blood ammonia level in the animal model administered the AAV of the present invention was restored to the level of a normal mouse (WT) (Fig. 11).

[0472]

[0473] Based on the above results, it can be seen that the recombinant AAV virus produced by the ASS1-all-in-one AAV vector for recombinant AAV production containing a polynucleotide (SEQ No. 2) encoding argininosuccinate synthase (ASS1) has excellent therapeutic efficacy against citrullinemia type 1 disease, specifically, it can improve the survival rate of individuals with citrullinemia type 1, suppress weight loss or restore weight to a normal level, and / or suppress the increase in blood ammonia concentration or restore it to a normal level.

[0474]

[0475] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

A codon-optimized polynucleotide encoding argininosuccinate synthase (ASS1), comprising a nucleotide sequence having 90% or more sequence identity with any one of SEQ ID NOs 1 to 3. A codon-optimized polynucleotide encoding argininosuccinate synthase, wherein the polynucleotide comprises a nucleotide sequence of any one of SEQ ID NOs 1 to 3. A codon-optimized polynucleotide encoding argininosuccinate synthase, wherein the polynucleotide comprises the nucleotide sequence of either SEQ ID NO. 2 or 3. A codon-optimized polynucleotide encoding argininosuccinate synthase, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO.

2. A codon-optimized polynucleotide encoding argininosuccinate synthase, wherein the polynucleotide has a reduced number of CpG dinucleotides compared to a non-codon-optimized wild-type sequence, in any one of claims 1 to 4. A codon-optimized polynucleotide encoding argininosuccinate synthase, wherein the polynucleotide exhibits reduced immunotoxicity compared to a non-codon-optimized wild-type sequence, in any one of claims 1 to 4. A codon-optimized polynucleotide encoding argininosuccinate synthase, wherein the polynucleotide is expressed at a high level relative to a non-codon-optimized wild-type sequence, in any one of claims 1 to 4. A codon-optimized polynucleotide encoding an argininosuccinate synthase, wherein, in any one of claims 1 to 4, the argininosuccinate synthase protein expressed by the polynucleotide exhibits a higher level of activity compared to the argininosuccinate synthase protein expressed by a non-codon-optimized wild-type sequence. A vector for producing recombinant adeno-associated virus (AAV) comprising the nucleotide sequence of the following gene arranged in a single nucleic acid molecule: (a) Helper virus genes required for AAV production; (b) AAV's Rep gene; (c) Cap gene of AAV; and (d) A transgene comprising a codon-optimized polynucleotide encoding the argininosuccinate synthase of any one of claims 1 to 8. A vector for producing recombinant AAV according to claim 9, wherein the recombinant AAV is intended to treat type 1 citrullinemia. A vector for producing recombinant AAV according to claim 9, wherein the transgenic gene is arranged between ITRs (Inverted Terminal Repeats). A vector for producing recombinant AAV according to claim 9, wherein the transfer gene is arranged between L-ITR and R-ITR. A vector for producing recombinant AAV according to claim 9, wherein the nucleic acid molecule is linear or circular. A vector for producing recombinant AAV according to claim 9, wherein the recombinant AAV produced by the vector is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV). A vector for producing recombinant AAV according to claim 9, wherein the nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus. A vector for producing recombinant AAV according to claim 9, wherein the nucleotide sequence of the helper virus gene is derived from an adenovirus. A vector for producing recombinant AAV according to claim 9, wherein the nucleotide sequence of the helper virus gene is derived from adenovirus 2. A vector for producing recombinant AAV according to claim 9, wherein the helper virus gene comprises one or more selected from E1, E2, E2a, E4, E4orf1, E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DBP (DNA-binding protein), and variants thereof. A vector for producing recombinant AAV according to claim 9, wherein the helper virus gene comprises E2A, E4, and VA. A vector for producing recombinant AAV according to claim 18, wherein the variant is an engineered helper virus gene. A recombinant AAV production vector according to claim 9, wherein the nucleotide sequence of the Rep gene is 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. A vector for producing recombinant AAV according to claim 9, wherein the nucleotide sequence of the Rep gene is derived from AAV2. A vector for producing recombinant AAV according to claim 9, wherein the Rep gene comprises one or more selected from Rep78, Rep68, Rep52, Rep40, and variants thereof. A vector for producing recombinant AAV according to claim 9, wherein the Rep genes comprise Rep78, Rep68, Rep52, and Rep40. A vector for producing recombinant AAV according to claim 23, wherein the variant is a variant having one or more variations in the wild-type sequence or a truncated variant. A recombinant AAV production vector according to claim 9, wherein the nucleotide sequence of the Cap gene is 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. A vector for producing recombinant AAV according to claim 9, wherein the nucleotide sequence of the Cap gene is derived from AAV2, AAV5, or AAV8. A vector for producing recombinant AAV according to claim 9, wherein the Cap gene encodes one or more selected from capsid protein, VP1 protein, VP2 protein, VP3 protein, and variants thereof. A vector for producing recombinant AAV according to claim 28, wherein the variant is a variant having one or more variations in the wild-type sequence or a truncated variant. A vector for producing recombinant AAV according to claim 11, wherein the ITR is 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. A vector for producing recombinant AAV according to claim 11, wherein the ITR is derived from AAV2. A vector for producing recombinant AAV according to claim 11, wherein the ITR is a wild-type ITR or a variant thereof. A vector for producing recombinant AAV according to claim 11, wherein the ITR comprises all or part of a wild-type ITR sequence. A vector for producing recombinant AAV according to claim 32, wherein the variant has a lower GC content than wild-type ITR. A vector for producing recombinant AAV according to claim 12, wherein the L-ITR and R-ITR are modified to produce scAAV. A vector for producing recombinant AAV according to claim 12, wherein the L-ITR comprises the sequence of SEQ ID NO.

17. A vector for producing recombinant AAV according to claim 12, wherein the R-ITR comprises the sequence of SEQ ID NO.

18. A vector for producing recombinant AAV according to claim 9, wherein the nucleic acid molecule further comprises one or more selected from a stabilizer, a costabilizer, an activator, a coactivator, a repressor, a corepressor, an epigenetic regulatory element, a co-epigenetic regulatory element, a regulatory element, and a co-regulatory element. A vector for producing recombinant AAV according to claim 9, wherein the nucleic acid molecule further comprises one or more expression regulatory elements operably linked to any one of (a), (b), (c), and (d). A vector for producing recombinant AAV according to claim 39, wherein the expression control element comprises one or more selected from enhancers, promoters, silencers, insulators, introns, UTRs (untranslated regions), Kozak sequences, signal peptides, and polyadenylation signal sequences. A vector for producing recombinant AAV according to claim 9, wherein the nucleic acid molecule further comprises one or more promoters operably linked to any one of (a), (b), and (c). A vector for producing recombinant AAV according to claim 9, wherein the nucleic acid molecule further comprises an expression regulatory element operably linked to (d). A vector for producing recombinant AAV, wherein, in any one of claims 39 to 42, the expression control element comprises one or more selected from enhancers, promoters, introns, Kozak sequences, and polyadenylation signal sequences. A vector for producing recombinant AAV according to claim 43, wherein the enhancer is an AMBP (alpha-1-microglobulin / bikunin precursor) enhancer or a variant thereof. A vector for producing recombinant AAV according to claim 43, wherein the enhancer comprises the sequence of SEQ ID NO.

5. A vector for producing recombinant AAV according to claim 43, wherein the promoter is a TBG promoter or a variant thereof. A vector for producing recombinant AAV according to claim 43, wherein the promoter comprises the sequence of SEQ ID NO.

7. A vector for producing recombinant AAV according to claim 43, wherein the intron is an SV40 intron or a variant thereof. A vector for producing recombinant AAV according to claim 43, wherein the intron comprises the sequence of SEQ ID NO.

9. A vector for producing recombinant AAV according to claim 43, wherein the polyadenylation signal sequence is a human growth hormone (Hgh) polyadenylation signal sequence or a variant thereof. A vector for producing recombinant AAV according to claim 43, wherein the polyadenylation signal sequence comprises the sequence of SEQ ID NO.

10. A vector for producing recombinant AAV, wherein, in any one of claims 9 to 51, the nucleic acid molecule does not include a replication origin or additionally includes a replication origin. A vector for producing recombinant AAV according to claim 52, 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. A vector for producing recombinant AAV, wherein, in any one of claims 9 to 53, the nucleic acid molecule further comprises a selectable marker. A vector for producing recombinant AAV according to claim 54, wherein the selection marker is one or more selected from ampicillin, streptavidin, kanamycin, hygromycin, neomycin, puromycin, blasticidin, and zeosin. A vector for producing recombinant AAV according to claim 9, wherein (a), (b), (c), and (d) in the nucleic acid molecule are each independently arranged in any order in forward orientation or reverse orientation. A vector for producing recombinant AAV according to claim 9, wherein (a), (b), (c), and (d) in the nucleic acid molecule are arranged in the following order, wherein (a), (b), (c), and (d) are each independently oriented forward or backward: 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). A vector for producing recombinant AAV according to claim 56 or 57, wherein (a), (b), (c), and (d) are all oriented. A vector for producing recombinant AAV according to claim 56 or 57, wherein (a), (b), and (c) are positively oriented; and (d) is negatively oriented. A vector for producing recombinant AAV, wherein, in any one of claims 56 to 59, the ITR is arranged on both sides of (d). A vector for producing recombinant AAV according to claim 57, wherein the vector backbone is arranged in any order in 1) to 12). A vector for producing recombinant AAV according to claim 57, wherein the vector backbone is arranged after the last order or before the first order in 1) to 12). A vector for producing recombinant AAV according to claim 61 or 62, wherein the vector backbone comprises one or more selected from a replication origin and a selection marker. A vector for producing recombinant AAV according to claim 9, wherein (a), (b), (c), and (d) in the nucleic acid molecule 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) - Inversely oriented (d); (v) Oriented (a) - Backbone - Oriented (c) - Oriented (b) - Inversely oriented (d); (vi) Oriented (b) - Oriented (c) - Oriented (a) - Backbone - Inversely oriented (d); (vii) Backbone - positively oriented (b) - positively oriented (c) - positively oriented (a) - negatively oriented (d); or viii) Oriented (a) - Oriented (b) - Oriented (c) - Backbone - Oriented (d). A vector for producing recombinant AAV according to claim 9, wherein (b) and (c) in the nucleic acid molecule are arranged in the order of (c) - (b). A vector for producing recombinant AAV according to claim 9, wherein (d) in the nucleic acid molecule is arranged in a reverse orientation. A recombinant AAV production vector according to claim 9, wherein the recombinant AAV production vector further comprises the nucleotide sequence of the following gene: (e) 1 or more stuffers. A vector for producing recombinant AAV according to claim 67, wherein the vector comprises two or more stuffers. A vector for producing recombinant AAV according to claim 67, wherein the vector comprises three or more stuffers. A vector for producing recombinant AAV according to claim 67, wherein the vector comprises three stuffers. A vector for producing recombinant AAV according to claim 67, wherein the stuffer is arranged inside or outside of two ITRs. A vector for producing recombinant AAV according to claim 67, wherein the vector comprises two or more stuffers, each stuffer being independently arranged inside or outside two ITRs. A vector for producing recombinant AAV according to claim 67, wherein the vector comprises two or more stuffers, and all stuffers are arranged outside of two ITRs. A vector for producing recombinant AAV according to claim 67, wherein the length of each stuffer is 0.1 kb to 20 kb. A vector for producing recombinant AAV according to claim 67, wherein the total length of all stuffers is 0.1 kb to 100 kb. A vector for producing recombinant AAV according to claim 67, wherein the stuffer comprises a first stuffer, a second stuffer, and a third stuffer. A vector for producing recombinant AAV according to claim 76, wherein the first stuffer is arranged downstream of the R-ITR. A vector for producing recombinant AAV according to claim 76, wherein the length of the first stuffer is 0.1 kb to 20 kb. A vector for producing recombinant AAV according to claim 76, wherein the second stuffer is arranged between (a) and (b) or between (a) and (c). A vector for producing recombinant AAV according to claim 76, wherein the length of the second stuffer is 0.1 kb to 20 kb. A vector for producing recombinant AAV according to claim 76, wherein the third stuffer is arranged upstream of the L-ITR. A vector for producing recombinant AAV according to claim 76, wherein the length of the third stuffer is 0.1 kb to 20 kb. A vector for producing recombinant AAV according to claim 67, wherein the stuffer reduces impurities that may occur during the process of producing recombinant AAV by the vector. A vector for producing recombinant AAV according to claim 83, wherein the impurity is a DNA impurity. A vector for producing recombinant AAV according to claim 76, wherein a first stuffer arranged downstream of the R-ITR, a third stuffer arranged upstream of the L-ITR, or both reduce reverse packaging of the vector. A vector for producing recombinant AAV according to claim 76, wherein a second stuffer arranged between (a) and (b) or between (a) and (c) reduces impurities in the recombinant AAV particles produced by the vector. A vector for producing recombinant AAV according to claim 86, wherein the impurity comprises one or more selected from: all or part of a helper virus gene; all or part of a Rep gene; all or part of a Cap gene; any sequence located outside two ITRs in the vector; part of the host cell genome sequence; and two or more chimeric forms of these. A vector for producing recombinant AAV according to claim 86, wherein the impurity comprises all or part of a helper virus gene. A vector for producing recombinant AAV according to claim 67, wherein the stuffer comprises one or more selected from introns and some sequences or modified sequences thereof. A vector for producing recombinant AAV according to claim 67, wherein the stuffer comprises one or more selected from a barrier insulator and some sequences or modified sequences thereof. A vector for producing recombinant AAV according to claim 90, wherein the barrier insulator comprises MAR (Matrix Attachment Region), UCOE (Ubiquitous Chromatin Opening Element), CCE (Chromatin Control Element), STAR (Stabilizing and Anti-Repressor), or 5'HS5 (Hypersensitive site 5). A vector for producing recombinant AAV according to claim 67, wherein the stuffer comprises one or more selected from the sequence of a Matrix Attachment Region (MAR), the sequence of a Ubiquitous Chromatin Opening Element (UCOE), and the sequence of a Chromatin Control Element (CCE). A vector for producing recombinant AAV according to claim 67, wherein the stuffer comprises a sequence of a Matrix Attachment Region (MAR). A vector for producing recombinant AAV according to claim 93, wherein the MAR is a human globin MAR. A vector for producing recombinant AAV according to claim 93, wherein the stuffer comprises a polynucleotide comprising 100 or more consecutive nucleotides within the sequence of SEQ ID NO.

20. A vector for producing recombinant AAV according to claim 93, wherein the stuffer comprises the polynucleotide of SEQ ID NO.

20. A vector for producing recombinant AAV according to claim 67, wherein the stuffer comprises a partial sequence of UCOE (Ubiquitous Chromatin Opening Element). A vector for producing recombinant AAV according to claim 97, wherein the UCOE is the UCOE of human HNRPA2B1 (heterogeneous nuclear ribonucleoproteins A2 / B1 gene). A vector for producing recombinant AAV according to claim 97, wherein the stuffer comprises a polynucleotide comprising 100 or more consecutive nucleotides within the sequence of SEQ ID NO.

21. A vector for producing recombinant AAV according to claim 97, wherein the stuffer comprises the polynucleotide of SEQ ID NO.

21. A vector for producing recombinant AAV according to claim 67, wherein the stuffer comprises a partial sequence of a Chromatin Control Element (CCE). A vector for producing recombinant AAV according to claim 101, wherein the CCE is a human CCE. A vector for producing recombinant AAV according to claim 101, wherein the stuffer comprises a polynucleotide comprising 100 or more consecutive nucleotides within the sequence of SEQ ID NO.

19. A vector for producing recombinant AAV according to claim 101, wherein the stuffer comprises the polynucleotide of SEQ ID NO.

19. A vector for producing recombinant AAV according to claim 67, wherein the stuffer comprises the following: A first stuffer comprising the polynucleotide of SEQ ID NO. 19 arranged downstream of the R-ITR; A second stuffer comprising a polynucleotide of SEQ ID NO. 20 arranged between (a) and (b) or between (a) and (c); and A third stuffer comprising the polynucleotide of SEQ ID NO. 21 arranged upstream of the L-ITR. A step of introducing a recombinant AAV production vector of any one of claims 1 to 105 into a host cell; and A step comprising isolating recombinant AAV from the host cell, Method for producing recombinant adeno-associated virus (AAV). A method for producing recombinant AAV according to claim 106, wherein the host cell is a mammalian cell or an insect cell. A method for producing recombinant AAV according to claim 107, wherein the mammalian cell is selected from HEK293 cells, HEK293F cells, HEK293T cells, and cells derived therefrom. A method for producing recombinant AAV according to claim 107, wherein the insect cell is an Sf9 cell, an Sf21 cell, a TN-5B1-4 cell, or a High Five cell. A method for producing recombinant AAV according to claim 106, wherein the introduction is selected from physical methods including electroporation, cell compression, ultrasonic perforation, optical transfection, protoplast fusion, impalfection, magnetofection, gene gun, and particle impaction; methods using chemical reagents including a cationic polymer such as polyethyleneimine (PEI) and calcium phosphate; and methods using cationic lipids such as lipofection. A method for producing recombinant AAV according to claim 106, wherein the separation is performed using centrifugation or chromatography. A method for producing recombinant AAV according to claim 111, wherein the centrifugation method is a cesium chloride-based ultra-high-speed centrifugation method. A method for producing recombinant AAV according to claim 111, wherein the chromatography is one or more selected from affinity chromatography, ion exchange chromatography, column chromatography, gel-filtration chromatography, thin-layer chromatography, radial flow chromatography, interference chromatography, and reverse-phase chromatography. A method for producing recombinant AAV according to any one of claims 106 to 113, wherein the recombinant AAV produced by the method has an increased Full capsid ratio (%Full capsid) compared to recombinant AAV produced by triple transfection. A method for producing recombinant AAV according to any one of claims 106 to 113, wherein the recombinant AAV produced by the method exhibits an increased argininosuccinate synthase expression level compared to recombinant AAV produced by triple transfection. A method for producing recombinant AAV according to any one of claims 106 to 113, wherein the recombinant AAV produced by the method exhibits increased argininosuccinate synthase activity compared to recombinant AAV produced by triple transfection. A method for producing recombinant AAV according to any one of claims 106 to 113, wherein the method is for producing recombinant AAV for treating type 1 citrullinemia. Recombinant AAV produced by the vector of any one of claims 9 to 105. In claim 118, the recombinant AAV is a single-stranded AAV (ssAAV) or a self-complementary AAV (scAAV). The recombinant AAV of claim 118, wherein the recombinant AAV is intended to treat or prevent citrullinemia type 1. An expression cassette comprising a polynucleotide encoding argininosuccinate synthase (ASS1) having a nucleotide sequence having 90% or more sequence identity with any one of SEQ ID NOs 1 to 3, and one or more expression regulatory elements. An expression cassette according to claim 121, wherein the expression cassette further comprises an ITR. As a combination AAV comprising an AAV capsid and a genome, A recombinant AAV in which the genome comprises a polynucleotide encoding argininosuccinate synthase (ASS1) comprising a nucleotide sequence having 90% or more sequence identity with any one of SEQ ID NOs 1 to 3. The recombinant AAV of claim 123, wherein the polynucleotide encoding the argininosuccinate synthase is arranged between ITRs. A vector of any one of claims 9 to 104, a recombinant AAV produced by the vector of any one of claims 9 to 104, a recombinant AAV of any one of claims 118 to 120, or a recombinant AAV of any one of claims 123 to 124; and containing a pharmaceutically acceptable carrier A pharmaceutical composition for preventing or treating type 1 citrullinemia. A pharmaceutical composition according to claim 125, wherein the composition is capable of improving the survival rate of an individual with citrullinemia type 1. A pharmaceutical composition according to claim 125, wherein the composition is capable of inhibiting weight loss in an individual with citrullinemia type 1 or restoring lost weight. A pharmaceutical composition according to claim 125, wherein the composition is capable of inhibiting an increase in blood ammonia concentration in an individual with citrullinemia type 1 or reducing an increased blood ammonia concentration. A method for delivering an argininosuccinate synthase or a gene encoding the same to an individual in need of the same, comprising the step of administering to the individual an effective amount of a vector of any one of claims 9 to 104, a recombinant AAV produced by the vector of any one of claims 9 to 104, a recombinant AAV of any one of claims 118 to 120, a recombinant AAV of any one of claims 123 to 124, or a pharmaceutical composition of claim 125. A method of delivering argininosuccinate synthase or a gene encoding it to an individual in need of it, wherein the individual is a patient with type 1 citrullinemia. A method for treating type 1 citrullinemia, comprising the step of administering to an individual an effective amount of a vector of any one of claims 9 to 104, a recombinant AAV produced by the vector of any one of claims 9 to 104, a recombinant AAV of any one of claims 118 to 120, a recombinant AAV of any one of claims 123 to 124, or a pharmaceutical composition of claim 125. A method for treating type 1 citrullinemia according to claim 131, wherein the individual is a mammal. A method for treating type 1 citrullinemia according to claim 132, wherein the mammal is a human. Use of a vector of any one of claims 9 to 104, a recombinant AAV produced by said vector, a recombinant AAV of any one of claims 118 to 120, or a recombinant AAV of any one of claims 123 to 124 for the manufacture of a therapeutic drug for citrullinemia type 1.

Images