Codon-optimized polynucleotide encoding blood coagulation factor ix, and raav-producing plasmid comprising same

Abstract

The present invention provides: a codon-optimized polynucleotide encoding factor IX (FIX); a recombinant AAV-producing plasmid comprising the polynucleotide; a recombinant AAV produced by the plasmid; and use of the recombinant AAV for treating hemophilia B. The plasmid comprising the polynucleotide can produce a recombinant AVV that exhibits high FIX expression and activity and has excellent productivity and high quality.

Description

Codon-optimized polynucleotide encoding blood coagulation factor IX and plasmid for producing rAAV comprising the same

[0001] The present invention relates to a codon-optimized polynucleotide encoding blood coagulation factor IX, a plasmid for producing a recombinant AAV comprising the polynucleotide, a recombinant AAV produced by the plasmid, and a use of the recombinant AAV for treating hemophilia B.

[0002] Hemophilia is a bleeding disorder caused by a deficiency of clotting factors in the blood due to an inherited mutation in a gene located on the X chromosome. Hemophilia A is caused by a deficiency of clotting factor VIII (FVIII), and hemophilia B is caused by a deficiency of clotting factor IX (FIX).

[0003] Current hemophilia treatment involves intravenous administration of recombinant clotting factors, depending on the severity of bleeding or as a preventative measure. However, this treatment approach has drawbacks, including a short half-life, the need for repeated infusions, treatment costs, and the potential risk of bleeding.

[0004] Due to these limitations, gene-based therapies for hemophilia are being developed, and adeno-associated virus (AAV) is emerging as the vector with the highest safety and efficacy for in vivo gene transfer. However, gene therapy using recombinant AAV (rAAV) presents the following challenges.

[0005] The first problem is low efficiency and productivity. Triple transfection for AAV production using HEK293 cells, a production cell line, requires three components: Rep / Cap, Helper, and Gene-of-Interest (GOI). All three components must be introduced simultaneously and in appropriate ratios into the production cell line to produce recombinant AAV with high efficiency. However, because each component operates independently during this introduction process, the rate of simultaneous injection of all three plasmids is low, at less than 30%. Furthermore, the ratios of the three plasmids injected into the cells cannot be precisely controlled, which can lead to differences in transfection efficiency and productivity across production batches, resulting in reduced productivity of recombinant AAV.

[0006] A second issue is the transfer of antibiotic resistance genes through reverse packaging. As pharmaceuticals, quality and safety are crucial for gene therapies. However, conventional triple-transfection technology has been known to produce AAVs with a %Full capsid ratio of less than 30%, and cases of recombinant AAVs producing less than 100% genome integrity have also been reported. Furthermore, empty capsids generated during AAV production or antibiotic resistance gene insertion through reverse packaging can trigger immune responses. Therefore, various high-purity recombinant AAV purification processes are being developed to overcome and minimize these quality limitations. While inserting a stuffer into the GOI AAV vector in a triple-transfection production system can reduce reverse packaging, it suffers from low productivity and the potential for immune responses due to incompletely removed stuffer genes (e.g., lambda stuffer). Due to these limitations, improvements in the triple-transfection system are required to improve the %Full capsid ratio in addition to the purification process.

[0007] A third challenge is low therapeutic gene expression and immune response. AAV gene therapy agents targeting hemophilia B have been developed with low therapeutic gene expression and immune response, resulting in reduced therapeutic efficacy. To overcome these challenges, the focus is on developing CpG-free therapeutic gene sequences, tissue-specific promoters, and capsids. However, this rational engineering approach is not yet mature, and the complexity of gene regulation in AAV vectors with comprehensive functional improvements in off-target tissues, overexpression toxicity, and innate immune responses must be assessed, and the stability of the translated therapeutic gene must be verified.

[0008] Therefore, there is a need to develop an AAV gene therapy for hemophilia B and a method for producing the same that can improve the problems described above.

[0009] The present disclosure relates to a codon-optimized polynucleotide capable of enhancing the expression and activity of FIX 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 hemophilia B with excellent productivity and high quality.

[0011] In addition, the present disclosure relates to scAAV for treating hemophilia B, which is produced by the above all-in-one vector system and has enhanced expression and activity of FIX.

[0012] Accordingly, one aspect is to provide a codon-optimized polynucleotide encoding Factor IX (FIX).

[0013] Another aspect is to provide plasmids for the production of recombinant adeno-associated virus (AAV) for treating hemophilia B.

[0014] Another aspect provides a method for producing recombinant AAV for treating hemophilia B.

[0015] Another aspect is to provide a recombinant AAV produced by a plasmid according to one aspect or by a method according to one aspect.

[0016] Another aspect is to provide recombinant AAV for treating hemophilia B.

[0017] Another aspect is to provide a pharmaceutical composition for preventing or treating hemophilia B, comprising a recombinant AAV produced by a plasmid according to one aspect or a recombinant AAV according to one aspect.

[0018] Another aspect provides a method of delivering FIX to a subject in need thereof, comprising administering to the subject an effective amount of a recombinant AAV produced by a plasmid according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.

[0019] Another aspect provides a method of treating hemophilia B, comprising administering to an individual an effective amount of a recombinant AAV produced by a plasmid according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.

[0020] Another aspect provides a use of the plasmid according to one aspect, the recombinant AAV produced by said plasmid, or the recombinant AAV according to one aspect, for the manufacture of a medicament for the treatment of hemophilia B.

[0021] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. Furthermore, while preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. Furthermore, numerical values ​​described herein are considered to include the meaning of "about," even if not explicitly stated. The contents of all publications cited as references herein are incorporated herein by reference in their entirety.

[0022] The terms "about" or "approximately" in this specification can generally be interpreted to mean a value or range that is within 10%, 5%, 4%, 3%, 2%, or 1% above or below a given value or range.

[0023]

[0024] One aspect provides a codon-optimized polynucleotide encoding Factor IX (FIX).

[0025] In this specification, the term "Factor IX (FIX)" may be used interchangeably with "blood coagulation factor IX" and "coagulation factor IX". Hemophilia B is caused by a deficiency of Factor IX. The FIX may be human FIX (hFIX).

[0026] The term "polynucleotide" can refer to any form of nucleic acid, including DNA and RNA, or oligonucleotides. Polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. The sequence or structure of a particular polynucleotide can be described according to the convention of providing the sequence in the 5' to 3' direction.

[0027] The "polypeptide," "protein," and "peptide" encoded by a polynucleotide sequence may include functional subsequences, modified forms, or sequence variants, as long as they retain the functionality of the native protein. The term "modified" or "variant" means that the sequence of the polynucleotide or polypeptide deviates from the reference sequence. Thus, the modified or variant sequence may have substantially the same activity or function as the reference sequence, or a greater or lesser activity or function, but retain at least a partial activity or function of the reference sequence.

[0028] Non-limiting examples of modifications include substitutions, insertions, and / or deletions of one or more nucleotides or amino acids. An example of an amino acid substitution is a conservative amino acid substitution. Examples of conservative amino acid substitutions are well known. A "conservative substitution" is the replacement of an amino acid with a biologically, chemically, or structurally similar residue. Biological similarity means that the substitution does not destroy biological activity. Structural similarity means that the amino acids have similar lengths (e.g., alanine, glycine, and serine) or similar sizes. Chemical similarity means that the residues have the same charge, or have the same hydrophilic or hydrophobic properties, or both. For example, conservative amino acid substitutions include substitutions within the following groups: glycine / alanine, valine / isoleucine / leucine, aspartic acid / glutamic acid, asparagine / glutamine, serine / threonine, lysine / arginine, and phenylalanine / tyrosine.

[0029] In the present specification, gene and protein variants having one or more biological activities (e.g., blood coagulation ability, etc.) may be included.

[0030] At the nucleotide sequence level, naturally occurring and non-naturally occurring variant genes can have at least 50%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with a reference gene.

[0031] At the amino acid sequence level, the naturally occurring and non-naturally occurring variant proteins can have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with the reference protein.

[0032] The term "sequence identity" refers to the degree of similarity in amino acid residues or bases between two sequences after aligning them to achieve maximum agreement over a specific comparison region. Sequence identity can be determined using methods known in the art. The percentage of sequence identity can be determined using known sequence comparison programs, such as NCBI's BLAST.

[0033] The codon-optimized polynucleotide encoding the FIX may comprise or consist of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 9 to 41.

[0034] The polynucleotide may comprise or consist of any one of the nucleotide sequences of SEQ ID NOs: 9 to 41.

[0035] The above FIX may be a FIX mutant or a wild-type FIX.

[0036] The above FIX variants may exhibit therapeutic activity comparable to or higher than wild-type FIX.

[0037] The above FIX may be a FIX variant comprising the R338L mutation (FIX-R338L); a FIX variant comprising the T148A and R338L mutations (FIX-T148A / R338L); or a wild-type FIX.

[0038] The FIX-R338L mutant is a mutant in which arginine is substituted with leucine at position 338 in the FIX protein, also known as the "FIX-Padua mutant." FIX-Padua has greater catalytic and coagulation activity compared to unmutated human FIX.

[0039] The FIX-T148A variant is a variant in which threonine at position 148 in the FIX protein is substituted with alanine, and is also known as the "FIX-Malmo variant."

[0040] The FIX-T148A / R338L mutant is a mutant that has both the T148A and R338L mutations in the FIX protein.

[0041] The codon-optimized polynucleotide encoding the FIX-R338L may comprise or consist of any one nucleotide sequence selected from SEQ ID NOs: 9 to 18 and 39. In one specific embodiment, the codon-optimized polynucleotide encoding the FIX-R338L may comprise or consist of the nucleotide sequence of SEQ ID NO: 9 or 18.

[0042] The codon-optimized polynucleotide encoding the FIX-T148A / R338L may comprise or consist of any one nucleotide sequence selected from SEQ ID NOs: 19 to 28 and 40. In one specific embodiment, the codon-optimized polynucleotide encoding the FIX-T148A / R338L may comprise or consist of the nucleotide sequence of SEQ ID NO: 23 or 24.

[0043] The codon-optimized polynucleotide encoding the wild-type FIX may comprise or consist of any one nucleotide sequence selected from SEQ ID NOs: 29 to 38 and 41.

[0044] In one specific embodiment, the polynucleotide may comprise or consist of a nucleotide sequence of any one of SEQ ID NOs: 9, 18, 23, and 24.

[0045] The polynucleotide may have a reduced number of CpG dinucleotides compared to a non-codon-optimized wild-type sequence. The term "CpG dinucleotide" refers to a cytosine-guanine dinucleotide, and "p" represents a phosphate linkage between the two. The polynucleotide may have a reduced number of CpG dinucleotides by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% compared to a non-codon-optimized wild-type sequence. The 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.

[0046] In one specific example, the polynucleotide may be free of CpG dinucleotides. Accordingly, the polynucleotide may be one from which CpG dinucleotides have been completely removed.

[0047] The mRNA transcribed by the polynucleotide may have a stable secondary structure compared to the mRNA transcribed by the non-codon-optimized wild-type sequence. The mRNA transcribed by the polynucleotide may have an increased half-life compared to the mRNA transcribed by the non-codon-optimized wild-type sequence. The mRNA transcribed by the 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.

[0048] Non-limiting parameters that can predict the half-life of the mRNA include MFE (Minimum Free Energy), SUP (Total unpairing ratio), and DegScore.

[0049] The Minimum Free Energy (MFE) value refers to the minimum free energy required for folding, calculated based on the predicted secondary structure of mRNA. The MFE value can be calculated using known calculation models such as Genescript, Linear Fold, and Linear Partition, but is not limited thereto. A lower MFE value may indicate a more stable mRNA secondary structure, which may increase the half-life of the mRNA.

[0050] The Summed Unpaired Probability (SUP) and Average Unpaired Probability (AUP) values ​​are base pairing probabilities calculated using specific formulas. The SUP value can be calculated using known calculation models, such as Linear Partition, but is not limited thereto. A smaller SUP value may indicate a more stable mRNA secondary structure, which may increase the half-life of the mRNA.

[0051] The DegScore value is a result derived from the nucleotide sequence and predicted mRNA structure as input. The DegScore value can be calculated using the DegScore model. A lower DegScore value may indicate a more stable mRNA secondary structure, potentially increasing its half-life.

[0052] The correlation with the half-life of mRNA can be high in the order of DegScore, SUP, and MFE.

[0053] The MFE value of the above polynucleotide may be -440 kcal / mol or less, -450 kcal / mol or less, -455 kcal / mol or less, -460 kcal / mol or less, -465 kcal / mol or less, -470 kcal / mol or less, -475 kcal / mol or less, -480 kcal / mol or less, -485 kcal / mol or less, or -490 kcal / mol or less. The MFE value of the above polynucleotide may be -500 kcal / mol to -440 kcal / mol, -500 kcal / mol to -450 kcal / mol, -500 kcal / mol to -455 kcal / mol, -500 kcal / mol to -460 kcal / mol, -500 kcal / mol to -465 kcal / mol, -500 kcal / mol to -470 kcal / mol, or -500 kcal / mol to -475 kcal / mol.

[0054] The SUP value of the polynucleotide may be 560 or less, 555 or less, 550 or less, 545 or less, 540 or less, or 535 or less. The SUP value of the polynucleotide may be 530 to 560, 530 to 555, 530 to 550, 530 to 545, 530 to 540, or 530 to 535.

[0055] The DegScore value of the polynucleotide may be 450 or less, 445 or less, 440 or less, or 435 or less. The DegScore value of the polynucleotide may be 430 to 450, 430 to 445, 430 to 440, or 430 to 435.

[0056] The polynucleotide may have a cryptic splicing site removed. Removal of the cryptic splicing site eliminates the expression of variants that may arise from activation of the cryptic splicing site during mRNA transcription, thereby enabling stable expression of the transgene.

[0057] The polynucleotide may exhibit reduced immunotoxicity compared to the non-codon-optimized wild-type sequence. It is known that when CpG dinucleotides are abundant, hypomethylation of CpG motifs in the AAV genome activates the Toll-Like Receptor 9 (TLR9) pathway, thereby inducing an immune response. In one aspect, the polynucleotide may exhibit reduced immunotoxicity compared to the non-codon-optimized wild-type sequence due to the removal of CpG dinucleotides.

[0058] The term "immunotoxicity" may refer to an abnormal immune response of the immune system that occurs when exposed to a foreign substance. Previous clinical trials of AAV vectors for the treatment of hemophilia have been unsuccessful due to a strong immune response to the AAV capsid. Furthermore, FIX can also induce an immune response. Indicators of this abnormal immune response include, for example, anti-drug antibodies (ADAs) and neutralizing antibodies (Nabs) involved in the immune process. However, polynucleotides according to certain aspects may elicit minimal or no immune response over a significant period of time. This immune response may be an innate immune response, a humoral immune response, a cellular immune response, or a combination thereof.

[0059] The polynucleotide can be expressed at a higher level than a non-codon-optimized wild-type sequence. The polynucleotide can be expressed at a higher level than a conventional codon-optimized sequence. As used herein, increased expression may refer to an effect produced by a change in the codon sequence, rather than hyperactivity caused by an amino acid substitution (e.g., a Padua mutation) in the FIX protein.

[0060] The FIX protein expressed by the polynucleotide may exhibit a higher level of activity than the FIX protein expressed by the non-codon-optimized wild-type sequence. The FIX protein expressed by the polynucleotide may exhibit a higher level of activity than the FIX protein expressed by the existing codon-optimized sequence. The "activity" may refer to the activity of the protein, and may refer to, for example, the blood clotting ability of FIX. As used herein, the increased activity may refer to the effect produced by the change in the codon sequence rather than the hyperactivity caused by the amino acid substitution of the FIX protein (e.g., the Padua mutation).

[0061]

[0062] Another aspect provides a plasmid for producing a recombinant adeno-associated virus (AAV) for treating hemophilia B. Specifically, an all-in-one vector system capable of producing a recombinant AAV for treating hemophilia B using a single plasmid is provided.

[0063] The plasmid according to the above aspect comprises the nucleotide sequence of the following genes arranged in a single nucleic acid molecule:

[0064] (a) Helper virus genes required for AAV production;

[0065] (b) Rep gene of AAV;

[0066] (c) Cap gene of AAV; and

[0067] (d) A transgene comprising a codon-optimized polynucleotide encoding FIX according to one aspect.

[0068] Therefore, alternatively, one aspect provides a nucleic acid molecule comprising within a single molecule (a) a helper virus gene necessary for AAV production; (b) a Rep gene of AAV; (c) a Cap gene of AAV; and (d) a transgene comprising a codon-optimized polynucleotide encoding FIX according to one aspect. More specifically, one aspect provides a nucleic acid molecule comprising within a single molecule (a) a nucleotide sequence of a helper virus gene necessary for AAV production; (b) a nucleotide sequence of a Rep gene of AAV; (c) a nucleotide sequence of a Cap gene of AAV; and (d) a nucleotide sequence of a codon-optimized polynucleotide encoding FIX according to one aspect.

[0069] In the above plasmid or nucleic acid molecule, (a), (b), (c), and (d) are all integrated together into a single nucleic acid molecule.

[0070] The following description applies commonly to plasmids according to the above aspect or nucleic acid molecules according to the above aspect.

[0071] The above "adeno-associated virus (AAV)" is a virus belonging to the genus Dependovirus in the family Parvoviridae. It does not have the ability to replicate on its own and requires the coexistence of a helper virus for replication. The AAV has a single-stranded DNA (ssDNA) of approximately 4.7 kb.

[0072] The above "recombinant AAV (rAAV)" may be used interchangeably with "AAV vector", "AAV particle", "AAV vector particle", "rAAV particle", and "rAAV vector particle", and may refer to an AAV vector capable of expressing a target protein in a host cell. The term "recombinant" means that the AAV or sequence has been manipulated in a way that does not generally occur in nature. For example, a recombinant vector such as an AAV vector may refer to a case where a polynucleotide that does not generally exist in the wild-type AAV genome has been inserted into the viral genome. Therefore, the recombinant AAV produced by the plasmid for producing the recombinant AAV can be applied as a gene therapy agent. For example, the recombinant AAV produced by the plasmid can express the FIX protein, and thus can be used as a gene therapy agent for hemophilia B.

[0073] As used herein, the term "plasmid for recombinant AAV production" may refer to a plasmid capable of producing recombinant AAV in a host cell into which the plasmid has been introduced. The plasmid may refer to a plasmid vector. In one aspect, the plasmid for recombinant AAV production relates to vector technology that integrates all genes necessary for AAV production into a single plasmid. Therefore, as used herein, the term "plasmid for recombinant AAV production" may be used interchangeably with the term "all-in-one vector" or "AAV single vector system." The plasmid for recombinant AAV production may be a single plasmid.

[0074] The term "vector" may refer to a vehicle capable of artificially transporting heterologous genetic material into another cell.

[0075] The nucleic acid molecule may be a DNA molecule. The nucleic acid molecule may be single-stranded (ss) or double-stranded (ds). The nucleic acid molecule may be a linear DNA molecule or a circular DNA molecule. The nucleic acid molecule may comprise the gene, specifically the sequence of the gene, more specifically the nucleotide sequence of the gene, and even more specifically the nucleotide sequence encoding the gene. The term "nucleotide sequence" may be used interchangeably with the terms "nucleic acid sequence" and "DNA sequence."

[0076] The nucleic acid molecule may exist in various forms. For example, the nucleic acid molecule may be linear or circular. Accordingly, the plasmid may be a linear plasmid or a circular plasmid. In certain embodiments, the plasmid may be a circular plasmid.

[0077] The nucleic acid molecule comprises (a) helper virus genes necessary for AAV production.

[0078] The above helper virus can refer to a virus that aids the replication of a virus that cannot replicate on its own through simultaneous infection. Because AAV lacks the ability to replicate on its own, it requires the genes of a helper virus for AAV replication.

[0079] 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."

[0080] The nucleotide sequence of the above helper virus gene may be derived from one or more selected from among adenovirus, herpes simplex virus (HSV), baculovirus, papillomavirus, and bocavirus, but is not limited thereto.

[0081] The adenovirus is known to have more than 50 serotypes. In one specific example, the nucleotide sequence of the helper virus gene may be derived from an adenovirus. The adenovirus may be selected from adenovirus 2 and adenovirus 5. The adenovirus may be adenovirus 2.

[0082] The above herpes simplex virus may be type 1 or type 2.

[0083] The above papillomavirus may be a human papillomavirus (HPV). More than 150 types of HPV are known. For example, the HPV may be, but is not limited to, HPV-16.

[0084] The helper virus gene may include at least one selected from E1, E2, E2a, E4, E4orf1, E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA (also referred to as “VA RNA gene”), DBP (DNA-binding protein), and variants thereof. The helper virus gene is a gene encoding the helper protein.

[0085] The above variant may be an engineered helper virus gene.

[0086] As used herein, the term "engineered" may mean that a gene has been intentionally modified and manipulated using genetic engineering techniques. Genetic engineering techniques for manipulating genes are well known. As used herein, "engineered A" may include a variant comprising one or more mutations in the wild-type sequence of A, a variant in which a portion of the sequence of A is truncated, etc. The mutations may be insertions, substitutions, deletions, or a combination thereof.

[0087] The helper virus genes may include helper virus genes of adenovirus 2. In one specific example, the helper virus genes may include E2a, E4, and VA.

[0088] The nucleic acid molecule comprises (b) a Rep gene of AAV.

[0089] The Rep (Replication) gene may be a gene required for AAV replication. The Rep gene may be derived from any AAV serotype. The nucleotide sequence of the Rep gene may be derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13, but is not limited thereto. When there are two or more Rep genes, each gene may be derived from the same or different AAV serotype. In one specific example, the nucleotide sequence of the Rep gene may be derived from AAV2.

[0090] The above Rep gene may include a gene encoding a known Rep protein or a variant thereof.

[0091] The Rep gene may include at least one selected from Rep78, Rep68, Rep52, Rep40, and variants thereof. The gene may include Rep68. The Rep gene may include Rep78 or Rep68. The Rep gene may include at least one selected from (i) Rep78, and (ii) Rep68, Rep52, and Rep40. The Rep gene may include at least one selected from (i) Rep68, and (ii) Rep78, Rep52, and Rep40. The Rep gene may include at least one selected from (i) Rep68, and (ii) Rep52 and Rep40. The Rep gene may include all of Rep78, Rep68, Rep52, and Rep40.

[0092] In one specific example, the Rep gene may include one or more (one, two, or three) selected from Rep68, Rep52, and Rep40. Rep78, when overexpressed, may increase cytotoxicity and decrease AAV productivity. Therefore, the Rep gene may include a Rep gene other than Rep78.

[0093] In one specific embodiment, the Rep gene may comprise a Rep gene of AAV2. In a specific specific embodiment, the Rep gene may comprise Rep78, Rep68, Rep52, and Rep40.

[0094] The above variant may be an engineered Rep (ERep) protein. The variant may be a variant having one or more mutations from the wild-type sequence, or a truncated variant.

[0095] The nucleic acid molecule comprises (c) the Cap gene of AAV.

[0096] The above Cap (Capsid) gene is a gene that encodes the viral capsid protein.

[0097] The Cap gene may be derived from any AAV serotype. The nucleotide sequence of the Cap gene may be derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13, but is not limited thereto. When there are two or more Cap genes, each gene may be derived from the same or different AAV serotype. In one specific example, the nucleotide sequence of the Cap gene may be derived from AAV8.

[0098] The above Cap gene may include a gene encoding a known Cap protein or a variant thereof.

[0099] The Cap gene may encode one or more selected from, but not limited to, a capsid protein, a VP1 protein, a VP2 protein, a VP3 protein, and variants thereof. The Cap gene may encode all of the VP1, VP2, and VP3 proteins.

[0100] The above variant may be an engineered Cap (ECap) protein. The variant may be a variant having one or more mutations from the wild-type sequence, or a truncated variant.

[0101] In one specific example, the Cap gene may comprise the wild-type Cap gene of AAV8.

[0102] The nucleotide sequences of the Rep gene and Cap gene may be derived from the same or different AAV serotypes. In one specific example, the Rep gene may be derived from AAV2, and the Cap gene may be derived from AAV8.

[0103] The nucleic acid molecule comprises (d) a transgene.

[0104] The transgene is a gene transferred from one organism to another. The transgene may be a heterologous polynucleotide. The transgene may be a gene of interest (GOI) to be packaged into a recombinant AAV capsid. The transgene may be a therapeutic gene. Therefore, the plasmid for producing the recombinant AAV can be used to produce a gene therapy clinical agent for treating a patient. The transgene may be one or two or more.

[0105] The transgene comprises a codon-optimized polynucleotide encoding FIX according to one aspect. Therefore, the plasmid for producing recombinant AAV can produce recombinant AAV that can be used as a gene therapy agent for treating hemophilia B.

[0106] The transgene may be arranged between Inverted Terminal Repeats (ITRs). The transgene may have ITRs arranged on both sides. The transgene may have two ITRs arranged on either side. The transgene may be arranged between L-ITRs and R-ITRs. For example, the L-ITR (first ITR), the transgene, and the R-ITR (second ITR) may be arranged sequentially in the 5' to 3' direction or in the 3' to 5' direction.

[0107] The above Inverted Terminal Repeat (ITR) is involved in the replication of the AAV genome and the packaging of AAV particles. The ITR includes the Rep binding element (RBE), RBE', A, A', B, B', C, C', and D regions. The ITR consists of two arm palindromes (BB' and C-C') embedded in a larger stem palindrome (A-A'). Consequently, the ITR has a T-shaped stem-loop structure. The ITR can have two configurations, namely flip and flop. The flip and flop configurations have the BB' and CC' palindromes closest to the 3' end, respectively. The two ITRs on either side are also referred to as the first ITR and the second ITR, or as the left (L)-ITR and the right (R)-ITR, or as the 5'-ITR and the 3'-ITR. The D region occurs only once at each terminus, so it remains single-stranded. The RBE is the region where the Rep78 and Rep68 proteins of AAV bind. The strand- and site-specific endonuclease catalytic domains of Rep78 and Rep68 introduce nicks into the terminal resolution site (trs). The structure and sequence of ITRs are known.

[0108] 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 at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13, but is not limited thereto. The above AAV serotype may also include other AAV serotypes currently known or to be discovered in the future. The above AAV serotype may also include an artificial AAV serotype. The two ITRs on both sides may be derived from the same or different AAV serotypes.

[0109] In one specific example, the ITR may be derived from AAV2. The L-ITR and R-ITR may both be derived from AAV2.

[0110] The ITR may be a wild-type ITR or a variant thereof. The ITR may comprise all or part of a wild-type ITR sequence. The ITR may have a lower %GC content than the wild-type ITR. The ITR may be a synthetic ITR. The ITR may be an ITR derived from self-complementary AAV (scAAV). The L-ITR and R-ITR may be modified to enable the production of scAAV.

[0111] In one specific embodiment, either the L-ITR or the R-ITR can serve as a primer for DNA replication. The R-ITR can serve as a primer for DNA replication. Either the L-ITR or the R-ITR may not comprise trs. Either the L-ITR or the R-ITR may not comprise trs, and the other may be a wild-type ITR. The L-ITR may not comprise trs, and the R-ITR may be a wild-type ITR. Either the L-ITR or the R-ITR may not comprise trs and may comprise an RBE (Rep binding element). The L-ITR may not comprise trs and may comprise an RBE. Either the L-ITR or the R-ITR may not comprise trs and may have a hairpin structure. The L-ITR may not comprise trs and may have a hairpin structure. One of the above L-ITR and R-ITR may be an ITR mutant having a D region deleted, and the other may be a wild-type ITR. The L-ITR may be an ITR mutant having a D region deleted, and the R-ITR may be a wild-type ITR. The L-ITR may be composed of SEQ ID NO: 3, and the R-ITR may be composed of SEQ ID NO: 8. Therefore, the recombinant AAV produced by the above plasmid may be a self-complementary AAV (scAAV).

[0112] The nucleic acid molecule may further comprise a sequence added to the ITR. As used herein, "sequence added to the ITR" may also be expressed as "ITR+." By further 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 the wild-type AAV genome. Specifically, the sequence added to the ITR may be a non-ITR viral DNA corresponding to wtAAV2 nt 4489-4534 derived from upstream of the 3'-ITR in the wild-type AAV2 genome. For example, the sequence added to the ITR may be a nucleotide sequence (46 bp) consisting of SEQ ID NO: 7. The sequence added to the ITR may be a sequence added to the 5'-end of the R-ITR based on the (+) strand. In one specific embodiment, the nucleic acid molecule may further comprise a nucleotide sequence comprising SEQ ID NO: 7 upstream of the R-ITR.

[0113] In addition to (a), (b), (c), and (d), which are essential elements for recombinant AAV production, the nucleic acid molecule may further comprise a flanking sequence, an engineered flanking sequence, or additional components.

[0114] The above-mentioned peripheral sequence or engineered peripheral sequence may be the peripheral sequence of an ITR or an engineered peripheral sequence. Specifically, the above-mentioned peripheral sequence may be the 5'-peripheral sequence of an L-ITR or the 3'-peripheral sequence of an R-ITR based on the (+) strand. By including the above-mentioned peripheral sequence or engineered peripheral sequence, the impurity content of the gene packaged into AAV can be reduced, thereby securing high-quality AAV, and further, an increased safety effect can be expected when applied as a therapeutic agent.

[0115] The surrounding sequence or engineered surrounding sequence of the above ITR may include a sequence with a high AT content (i.e., an AT-rich sequence). The AT-rich sequence may inhibit reverse packaging of AAV, thereby enabling the acquisition of high-quality recombinant AAV.

[0116] The flanking sequence, engineered flanking sequence, or additional components may have one or more functions selected from the following:

[0117] (i) increasing gene expression levels;

[0118] (ii) regulating the timing of gene expression;

[0119] (iii) regulates gene transcription;

[0120] (iv) stabilize the transcript for translation;

[0121] (v) reducing the content of impurities other than the transgene among the genes packaged in the recombinant AAV;

[0122] (vi) inhibits reverse packaging of AAV;

[0123] (vii) improving the productivity of recombinant AAV; and

[0124] (viii) Improves the %F / E capsid ratio of recombinant AAV.

[0125] As used herein, the term "impurity" may mean that a component other than the component (e.g., a transgene) present between two ITRs of a plasmid for recombinant AAV production is packaged into a recombinant AAV particle, or that an incomplete transgene is packaged into a recombinant AAV particle. For example, the impurity may include all or part of a helper virus gene, all or part of Rep, all or part of Cap, part of a transgene, a genomic sequence of a host cell, all or part of a Rep gene promoter (derived from an AAV virus or another virus), and a chimeric form of two or more of all the impurities listed above. Therefore, by reducing the content of the impurities, a recombinant AAV with excellent safety and quality can be secured.

[0126] AAV packaging refers to the packaging of the structure located between the two ITRs into an AAV particle. However, AAV reverse packaging refers to the packaging of recombinant AAV particles outward from the two ITRs, rather than inward. Therefore, inhibiting AAV reverse packaging can secure high-quality AAV.

[0127] The nucleic acid molecule may not contain a stuffer. The nucleic acid molecule may not contain a lambda stuffer. Even without a stuffer, the reverse packaging of AAV may be inhibited by the use of an all-in-one vector system. Furthermore, because the nucleic acid molecule does not contain a stuffer, the problems of low productivity and induction of an immune response due to the use of a stuffer (e.g., a lambda stuffer) may not occur.

[0128] In the above (viii), the improvement in the %F / E capsid ratio may be due to not only increasing the production of full capsids but also decreasing the production of empty capsids.

[0129] Those skilled in the art will appreciate that the nucleotide sequences of (a), (b), (c), and (d) above can each be operably linked to an appropriate control sequence. For example, the nucleotide sequence can be operably linked to a transcription / translation control element (e.g., a transcription / translation control signal, an origin of replication, a polyadenylation signal, an internal ribosome entry site (IRES), a Furin, a 2A peptide, a promoter, and / or an enhancer, etc.).

[0130] The nucleic acid molecule may further comprise at least one selected from a stabilizer, a costabilizer, an activator, a coactivator, a repressor, a corepressor, an epigenetic regulatory element, a co-epigenetic regulatory element, a regulatory element, a co-regulatory element, etc.

[0131] The above regulatory element may be an element involved in the regulation of gene expression. The above regulatory element may include at least one selected from a promoter, a transcription factor, an enhancer, a silencer, an insulator, an intron, a splicing donor and acceptor, an engineered splicing donor and acceptor, a riboswitch, an amino acid, a miRNA (microRNA), a shRNA (short hairpin RNA), a 5'- or 3'-UTR (untranslated region), a Kozak sequence, an initiation codon, a GOI codon, a signal peptide, a polyadenylation signal sequence, and the like.

[0132] In one specific embodiment, the regulatory element may include one or more selected from an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

[0133] The nucleic acid molecule may further comprise an expression control element operably linked to (d).

[0134] The expression regulatory element may be a tissue-specific expression regulatory element. A tissue-specific expression regulatory element is active in a specific cell, tissue, or organ. The specific cell, tissue, or organ may include the liver, brain, central nervous system, spinal cord, eye, retina, bone, muscle, lung, pancreas, heart, kidney, etc. In a specific embodiment, the expression regulatory element may be a liver tissue-specific expression regulatory element. The term "liver tissue-specific" refers to preferential or dominant in vivo expression of a specific gene (e.g., a codon-optimized polynucleotide encoding FIX) in liver tissue compared to other tissues. Liver tissue-specific expression can mean that more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of all expression of a particular gene occurs within the liver tissue of an individual.

[0135] The above expression control element may include at least one selected from an enhancer and a promoter. The above expression control element may include an enhancer and a promoter.

[0136] The term "enhancer" can refer to a regulatory region that promotes transcription of a gene.

[0137] The term "promoter" can refer to a sequence that drives gene expression.

[0138] The above enhancer may be a liver tissue-specific enhancer. Non-limiting examples of liver tissue-specific enhancers include the apolipoprotein (ApoE) HCR-1 and HCR-2 enhancers, and the mouse transthyretin (mTTR) enhancer. In one specific embodiment, the enhancer may be an mTTR enhancer or a variant thereof.

[0139] The promoter may be a liver tissue-specific promoter. Non-limiting examples of liver tissue-specific promoters include the human alpha 1-antitrypsin (hAAT) promoter, albumin, the hepatitis B virus core promoter, alpha-fetoprotein (AFP), and the mTTR promoter. In one embodiment, the promoter may be the mTTR promoter or a variant thereof.

[0140] In one specific embodiment, the enhancer and promoter may be a variant of the mTTR enhancer and promoter. The enhancer and promoter may have 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity with the sequence of the mTTR enhancer and promoter. The variant of the mTTR enhancer and promoter may have nucleotides at positions 1 to 22 and 124 to 138 deleted from the nucleotide sequence of the wild-type mTTR enhancer and promoter, and ACAGGA inserted after position 361. The variant of the mTTR enhancer and promoter may comprise or consist of SEQ ID NO: 4.

[0141] The nucleic acid molecule may additionally comprise an intron.

[0142] The above "intron" sequence refers to a nucleotide sequence that is removed from the final gene product by RNA splicing. Using introns downstream of enhancer / promoter regions and upstream of cDNA inserts can increase gene expression levels.

[0143] Non-limiting examples of the above introns may include minute virus of mice (MVM) intron, beta-globin intron, FIX intron A, SV40 intron, altered SV40 intron, beta-actin intron, and the like.

[0144] In one specific example, the intron may be a modified SV40 intron. The modified SV40 intron may comprise or consist of SEQ ID NO: 5.

[0145] The nucleic acid molecule may additionally comprise a polyadenylation (Poly A) signal sequence.

[0146] The above "polyadenylation signal sequence", also called "Poly A sequence", refers to, for example, a polyadenylation signal sequence located 3' of a transgene, which allows the addition of a polyadenylation signal sequence to the end of a nascent mRNA during transcription. The polyadenylation signal sequence consists of up to 300 adenosine ribonucleotides that protect the mRNA from enzymatic degradation and aid in translation.

[0147] Non-limiting examples of the above polyadenylation signal sequences may include the SV40 polyadenylation signal, the bovine growth hormone (BGH) polyadenylation signal, the rabbit globin polyadenylation signal, and the like.

[0148] In one specific embodiment, the polyadenylation signal sequence may be a rabbit globin poly A signal sequence. The rabbit globin polyadenylation signal sequence may be a partial sequence of the rabbit beta-globin gene (GenBank No. V00882.1). The rabbit globin polyadenylation signal sequence may comprise or consist of SEQ ID NO: 6.

[0149] The nucleic acid molecule may further comprise a multi-cistronic element, a bi-cistronic element, a transposon, a transposase, or the like.

[0150] The nucleic acid molecule may further comprise a promoter independently operably linked to (a), (b), or (c). The promoter may control the amount and timing of expression of each gene.

[0151] The nucleic acid molecule may further comprise a promoter operably linked to (a). The promoter may be a helper gene promoter derived from a helper virus or a variant thereof. The variant may be an engineered promoter, a truncated promoter, or a promoter having a lower GC content than the wild-type promoter. The promoter may be an early promoter, a late promoter, a ubiquitous promoter, or a regulatable promoter. The promoter may be a CMV promoter, an SFFV promoter, an RSV promoter, or a pEF promoter.

[0152] The nucleic acid molecule may further comprise a promoter operably linked to (b). The promoter may be a Rep gene promoter or a variant thereof. The variant may be an engineered promoter, a truncated promoter, or a promoter having a lower GC content than the wild-type promoter. The promoter may be an early promoter, a late promoter, a ubiquitous promoter, or a regulatable promoter. The promoter may be a CMV promoter, an SFFV promoter, an RSV promoter, or a pEF promoter.

[0153] The nucleic acid molecule may further comprise a promoter operably linked to (c). The promoter may be a Cap gene promoter or a variant thereof. The variant may be an engineered promoter, a truncated promoter, or a promoter having a lower GC content than the wild-type promoter. The promoter may be an early promoter, a late promoter, a ubiquitous promoter, or a regulatable promoter. The promoter may be a CMV promoter, an SFFV promoter, an RSV promoter, or a pEF promoter.

[0154] The nucleic acid molecule may comprise a plasmid backbone. The term "plasmid backbone" refers to a non-genomic portion that is essential for cloning and amplification of plasmids, a process necessary for propagation and production of recombinant viruses, but is not packaged or encapsidated into viral particles. The plasmid backbone may comprise one or more (one or two) selected from an origin of replication and a selection marker. The plasmid backbone may not comprise an origin of replication.

[0155] The nucleic acid molecule may not contain an origin of replication, or may additionally contain an origin of replication. The origin of replication may be a known origin of replication or a variant thereof. The origin of replication may be, but is not limited to, a pUC origin, a pBR322 origin, a pMB1 origin, a pSC101 origin, a p15A origin, or a synthetic origin.

[0156] In one embodiment, the nucleic acid molecule may comprise a pBR322 origin.

[0157] The nucleic acid molecule may further comprise a selectable marker or reporter capable of providing selection or identification of host cells into which the plasmid has been introduced. Selectable markers or reporters are known in the art. Non-limiting examples of the selectable marker include genes that provide resistance to ampicillin, streptavidin, kanamycin, hygromycin, neomycin, puromycin, blasticidin, zeocin, and the like. Non-limiting examples of the reporter include luciferase, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), and the like. In one embodiment, the nucleic acid molecule may further comprise a selectable marker.

[0158] In one embodiment, the nucleic acid molecule may further comprise kanamycin.

[0159] "Gene amplification" may refer to the process by which a particular DNA sequence (i.e., a gene) in a genome is replicated disproportionately relative to other sequences in the genome, such that the amplified DNA sequence is present in a higher copy number than originally present. As used herein, "amplified" or "amplification" may refer to a gene or nucleic acid sequence that is present in two or more copies in a host cell due to gene amplification.

[0160] An "amplifiable selectable marker gene" may mean a gene that allows amplification of that gene under appropriate growth conditions.

[0161] In the nucleic acid molecule, (a), (b), (c), and (d) may be randomly arranged in the 5' to 3' direction or the 3' to 5' direction. In the nucleic acid molecule, (a), (b), (c), and (d) may be arranged in any order in the 5' to 3' direction or the 3' to 5' direction. In the nucleic acid molecule, (a), (b), (c), and (d) may be arranged in any order in the 5' to 3' direction.

[0162] In the above nucleic acid molecule, (a), (b), (c), and (d) may be independently arranged in any order in a forward orientation or a reverse orientation.

[0163] The above forward orientation may mean that the gene is inserted in the 5' to 3' direction in the 5'→3' strand ((+) strand). The above reverse orientation may mean that the gene is inserted in the 3' to 5' direction in the 5'→3' strand.

[0164] In the nucleic acid molecule, (a), (b), (c), and (d) may be arranged in the following order, wherein (a), (b), (c), and (d) may each be independently forward oriented or reverse oriented:

[0165] 1) (d) - (b) - (c) - (a);

[0166] 2) (d) - (c) - (b) - (a);

[0167] 3) (d) - (a) - (b) - (c);

[0168] 4) (d) - (a) - (c) - (b);

[0169] 5) (b) - (c) - (d) - (a);

[0170] 6) (c) - (b) - (d) - (a);

[0171] 7) (b) - (c) - (a) - (d);

[0172] 8) (c) - (b) - (a) - (d);

[0173] 9) (a) - (b) - (c) - (d);

[0174] 10) (a) - (c) - (b) - (d);

[0175] 11) (a) - (d) - (b) - (c); or

[0176] 12) (a) - (d) - (c) - (b).

[0177] In one specific example, (a), (b), (c), and (d) are arranged in the order of (a) - (b) - (c) - (d), wherein (a), (b), (c), and (d) may each be independently oriented or reversely oriented.

[0178] In one specific example, (a), (b), (c), and (d) are arranged in the order of (a) - (b) - (c) - (d), wherein (a), (b), (c), and (d) may all be oriented normally. Here, in (a), E2a may be reversely oriented, E4 may be normal oriented, and VA may be reversely oriented.

[0179] In one specific embodiment, two ITRs may be arranged on either side of (d). Accordingly, in the nucleic acid molecule, (a), (b), (c), (d), and ITRs may be arranged in the following order: (a) - (b) - (c) - L-ITR - (d) - R-ITR.

[0180] In one specific embodiment, the plasmid backbone may be arranged in any order from 1) to 12). In another specific embodiment, the plasmid backbone may be arranged after the last order from 1) to 12), or before the first order.

[0181] In certain embodiments, the plasmid backbone may be arranged between (a) and (b). Thus, the sequence of components in the nucleic acid molecule may be (a) - plasmid backbone - (b) - (c) - L-ITR - (d) - R-ITR.

[0182] The above plasmid backbone may comprise one or more selected from an origin of replication and a selection marker.

[0183] Plasmids according to one aspect can improve the %Full / Empty capsid ratio of the produced recombinant AAV through a combination of optimized arrangements and orientations of components (e.g., (a), (b), (c), (d), backbone). In particular, through a combination of the above optimized arrangements and orientations, it is possible to not only increase the %Full capsid but also decrease the %Empty capsid.

[0184] In certain embodiments, the components in the nucleic acid molecule may be arranged in the following order: (a) - plasmid backbone - (b) - (c) - L-ITR - enhancer and promoter - intron - (d) - polyadenylation signal sequence - R-ITR.

[0185] Additionally, a sequence added to the ITR may be included between the polyadenylation signal sequence and the R-ITR. Details regarding the sequence added to the ITR are as described above.

[0186] In certain embodiments, the components in the nucleic acid molecule may be arranged in the following order: (a) - plasmid backbone - (b) - (c) - L-ITR - enhancer and promoter - intron - (d) - polyadenylation signal sequence - sequence added to ITR - R-ITR.

[0187] The above nucleic acid molecules, plasmids and vectors can be prepared by any suitable technique, which techniques are well known in the art.

[0188] The plasmid for producing the above recombinant AAV comprises all components required for recombinant AAV production (i.e., (a), (b), (c), and (d)) linked within a single molecule, so that each component can be introduced into a host cell at an equal ratio. A host cell into which a single nucleic acid molecule has been introduced can exhibit balanced gene expression. Balanced gene expression can enhance recombinant AAV productivity and / or increase the %F / E capsid ratio of recombinant AAV. The enhancement of recombinant AAV productivity can reduce unit production costs. Specifically, the enhancement of recombinant AAV productivity can improve the overall yield of the purification process, which accounts for more than 60% of the cost of pharmaceutical production.

[0189] Furthermore, the use of the above all-in-one vector system reduces the cost of raw materials by producing only one single plasmid instead of the three separate plasmids required in the past. Furthermore, the all-in-one vector system allows the introduction of a single plasmid instead of the three separate plasmids, thereby reducing the amount of individual plasmid input by more than 50%.

[0190] In one embodiment, the all-in-one vector comprises a specific combination of components between L-ITR and R-ITR, such as a specific combination of a specific type of L-ITR, an enhancer and promoter, an intron, a codon-optimized polynucleotide of FIX, a polyadenylation signal sequence, a sequence added to the ITR, and an R-ITR, thereby improving the expression and activity (blood coagulation ability) of the FIX protein compared to cases where the all-in-one vector comprises a combination of different types of components.

[0191] In one embodiment, compared to a control triple transfection comprising a helper vector, a Rep / Cap expression vector, and a GOI expression vector, the all-in-one vector was found to exhibit improved %Full / Empty capsid ratio and productivity when the same amount of plasmid DNA (pDNA) was used. Specifically, the all-in-one vector was found to be capable of producing high-quality recombinant AAV with higher productivity compared to the triple transfection.

[0192] In one embodiment, the recombinant AAV produced by the all-in-one vector was confirmed to have improved expression and activity (blood coagulation ability) of the FIX protein by including a specific combination of components between L-ITR and R-ITR, such as a specific combination of a specific type of L-ITR, an enhancer and a promoter, an intron, a codon-optimized polynucleotide encoding FIX, a polyadenylation signal sequence, and an R-ITR, compared to cases where the recombinant AAV produced by the all-in-one vector includes a combination of other types of components. That is, the FIX protein expressed in the recombinant AAV produced by the all-in-one vector was confirmed to have improved biological activity (blood coagulation ability), and both expression and activity of the FIX protein were improved compared to existing AAV-based gene therapy for hemophilia B.

[0193]

[0194] Another aspect provides a method for producing a recombinant adeno-associated virus (AAV) for treating hemophilia B. The method may be a method using a plasmid for producing a recombinant AAV for treating hemophilia B according to one aspect.

[0195] The method comprises the steps of introducing a plasmid for producing a recombinant AAV for treating hemophilia B according to one aspect into a host cell; and the step of isolating the recombinant AAV from the host cell.

[0196] In the above method, 0.25×10 6 Dog or 3×10 6 The amount of plasmid DNA (pDNA) introduced per host cell may be, but is not limited to, about 0.1 to about 10 μg, about 0.1 to about 5 μg, about 0.1 to about 4 μg, about 0.1 to about 3 μg, about 0.5 to about 10 μg, about 0.5 to about 5 μg, about 0.5 to about 4 μg, about 0.5 to about 3 μg, about 1 to about 10 μg, about 1 to about 5 μg, about 1 to about 4 μg, or about 1 to about 3 μg.

[0197] In one specific example, 3×10 6 The amount of plasmid DNA (pDNA) introduced per host cell may be, but is not limited to, about 0.1 to about 10 μg, about 0.1 to about 5 μg, about 0.1 to about 4 μg, about 0.1 to about 3 μg, about 0.5 to about 10 μg, about 0.5 to about 5 μg, about 0.5 to about 4 μg, about 0.5 to about 3 μg, about 1 to about 10 μg, about 1 to about 5 μg, about 1 to about 4 μg, or about 1 to about 3 μg.

[0198] The host cell may be a mammalian cell. The mammalian cell may include a cell derived from any organ or tissue of a human, mouse, rat, hamster, monkey, rabbit, donkey, horse, sheep, cow, or ape. The mammalian cell may be selected from, but is not limited to, HEK293 cells, CHO cells, Jurkat cells, KS62 cells, PerC6 cells, HeLa cells, MDCK cells, C127 cells, A549 cells, Vero cells, WI38 cells, MRC5 cells, HT1080 cells, or derivatives or functional equivalents thereof.

[0199] In one specific embodiment, the mammalian cell may be selected from HEK293 cells, HEK293F cells, HEK293T cells, and cells derived therefrom. The HEK293 cells are a human embryonic kidney 293 cell line commonly used in biotechnology. 'HeK293 cells, HEK293F cells, or cells derived from HEK293T cells' refers to cells derived from the parental cell lines HEK293 cells, HEK293F cells, or HEK293T cells, and may include both commercially available cells and cells to be developed in the future. Exemplary cells derived from the HEK293 parental cell line include Expi293F (manufactured by ThermoFisher), HEK293F (manufactured by ThermoFisher), HEK293.2 (ATCC), etc. Exemplary cells derived from the HEK293T parental cell line include HEK293FT (manufactured by ThermoFisher). Exemplary cells derived from the HEK293F parental cell line include Viral Production Cells 1.0 (VPC1.0) (manufactured by ThermoFisher), Viral Production Cells 2.0 (VPC2.0) (manufactured by ThermoFisher). VPC2.0 is a clonal cell line derived from the HEK293F parental cell line and is a host cell suitable for AAV production. In certain embodiments, the mammalian cell may be a HEK293F cell or a cell derived therefrom.

[0200] The host cell may be an insect cell. The insect cell may include a cell derived from the tropical armyworm (Spodoptera frugiperda) or the cabbage moth (Trichoplusia ni). The insect cell may be selected from, but is not limited to, Sf9 cells, Sf21 cells, TN-5B1-4 cells, High Five cells, or derivatives or functional equivalents thereof.

[0201] The above introduction can be performed by any known method capable of inserting a plasmid for recombinant AAV production into a host cell without limitation. The introduction may be by transfection, transformation, or transduction. The terms "transfection," "transformation," and "transduction" may be used to describe the insertion of a non-mammalian vector or a viral vector into a target cell. Insertion of a vector is generally referred to as transformation for bacterial cells and transfection for eukaryotic cells, and insertion of a viral vector is also referred to as transduction. A person skilled in the art can introduce the plasmid into a host cell using any known method. Non-limiting examples of such introduction include, but are not limited to, physical methods (e.g., electroporation, cell compression, sonication, optical transfection, protoplast fusion, impalfection, magnetofection, gene gun, or particle bombardment), chemical reagents (e.g., calcium phosphate, highly branched organic compounds, or cationic polymers), or cationic lipids (e.g., lipofection). Such cationic polymers include, but are not limited to, polyethyleneimine (PEI). Transfection methods may additionally require contacting cells with a solution of plasmid DNA, followed by growth and selection using marker gene expression.

[0202] The above method may further include a step of culturing cells after the introducing step.

[0203] The above culture may involve culturing host cells under conditions that allow for the production of recombinant AAV. Appropriate culture methods are well known to those skilled in the art. For example, the cells may be cultured in suspension and / or under animal component-free conditions.

[0204] The above method may further include, after the culturing step, a step of selecting cells into which a plasmid has been introduced using a selection marker.

[0205] The separation can be performed by a known method. In one specific example, the separation can be performed using centrifugation or chromatography.

[0206] The above centrifugation method may be, but is not limited to, a cesium chloride (CsCl)-based ultra-high-speed centrifugation method.

[0207] The above chromatography may be at least one selected from, but is not limited to, affinity chromatography, ion exchange chromatography, column chromatography, gel-filtration chromatography, thin-layer chromatography, radial flow chromatography, interference chromatography, and reverse phase chromatography.

[0208] The method can also be easily scaled up to industrial production because it requires only a single transfection of the host cell with a plasmid for recombinant AAV production with excellent recombinant AAV productivity.

[0209] According to the above method, unlike the existing triple transfection method using three plasmids, only a single plasmid is required, simplifying the recombinant AAV production process and reducing costs. Furthermore, unlike the triple transfection method, where the ratios of the three plasmids injected into cells cannot be precisely controlled, the method according to the above aspect integrates all components into a single plasmid, allowing each component to be introduced at the same ratio. Accordingly, the productivity of recombinant AAV can be improved.

[0210] Recombinant AAV produced by the above method may have an increased Full capsid ratio (%Full capsid) compared to recombinant AAV produced by triple transfection. Therefore, the above method can produce high-quality recombinant AAV.

[0211] Recombinant AAV produced by the above method can exhibit increased FIX expression levels compared to recombinant AAV produced by triple transfection.

[0212] Recombinant AAV produced by the above method can exhibit increased FIX blood coagulation ability compared to recombinant AAV produced by triple transfection.

[0213] In one embodiment, the recombinant AAV produced by the method is 1×10 5 1×10 6 , specifically, 1.1×10 5 1×10 6 When introduced into cells at a multiplicity of infection (MOI) of vg / cell, it was confirmed that FIX expression was 1.3 to 1.6 times higher and FIX coagulation activity was 1.2 to 1.6 times higher compared to when recombinant AAV produced by triple transfection was introduced at the same MOI.

[0214]

[0215] Another aspect provides a recombinant AAV produced by a plasmid according to one aspect or by a method according to one aspect.

[0216] The above recombinant AAV may be a recombinant AAV for treating hemophilia B. Therefore, the above recombinant AAV may be used in a method of gene therapy for hemophilia B.

[0217] The above recombinant AAV may be a self-complementary AAV (scAAV). Therefore, the recombinant AAV may increase the expression level of the FIX protein.

[0218] The recombinant AAV described above may have a high Full capsid ratio (%Full capsid). Furthermore, the recombinant AAV may have a low impurity content. Therefore, the recombinant AAV may have excellent quality and safety.

[0219] The above recombinant AAV may have both increased expression and activity of the FIX protein.

[0220]

[0221] Another aspect provides recombinant AAV for treating hemophilia B.

[0222] The above recombinant AAV comprises an AAV capsid and genome.

[0223] The above genome contains a transgene.

[0224] The transgene comprises a codon-optimized polynucleotide encoding FIX according to the above aspect.

[0225] The above AAV capsid may be any one AAV capsid selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.

[0226] In one specific example, the AAV capsid may be a wild-type AAV8 capsid.

[0227] The above AAV capsid may comprise a VP1 protein, a VP2 protein, and a VP3 protein.

[0228] The "genome" of the above AAV refers to the sequences that are ultimately packaged or encapsidated to form viral particles.

[0229] The genome may be linear single-stranded DNA.

[0230] The transgene may be arranged between ITRs. The transgene may be arranged between L-ITRs and R-ITRs.

[0231] The ITR may be derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13. In one specific example, the ITR may be derived from AAV2.

[0232] The above ITR may be a wild-type ITR or a mutant thereof. The ITR may comprise all or part of a wild-type ITR sequence.

[0233] In one specific embodiment, one of the L-ITR and the R-ITR may not comprise trs. One of the L-ITR and the R-ITR may not comprise trs, and the other may be a wild-type ITR. The L-ITR may not comprise trs, and the R-ITR may be a wild-type ITR. One of the L-ITR and the R-ITR may be an ITR variant having a D region deleted, and the other may be a wild-type ITR. The L-ITR may be an ITR variant having a D region deleted, and the R-ITR may be a wild-type ITR. The L-ITR may be composed of SEQ ID NO: 3, and the R-ITR may be composed of SEQ ID NO: 8. Therefore, the recombinant AAV may be a self-complementary AAV (scAAV).

[0234] The genome may further comprise a sequence added to the ITR. Details regarding the sequence added to the ITR are as described above. In one embodiment, the genome may further comprise a sequence added to the ITR upstream of the R-ITR. The sequence added to the ITR may comprise or consist of SEQ ID NO: 7. The genome may further comprise an expression regulatory element operably linked to the transgene. The expression regulatory element may be a tissue-specific expression regulatory element. The expression regulatory element may comprise at least one selected from an enhancer and a promoter.

[0235] The above enhancer may be a liver tissue-specific enhancer, and the promoter may be a liver tissue-specific promoter. The enhancer may be an mTTR enhancer or a variant thereof, and the promoter may be an mTTR promoter or a variant thereof. The above enhancer and promoter may be a variant of the mTTR enhancer and promoter in which nucleotides at positions 1 to 22 and 124 to 138 are deleted from the nucleotide sequence of the wild-type mTTR enhancer and promoter, and ACAGGA is inserted after position 361. The variant of the mTTR enhancer and promoter may comprise or consist of SEQ ID NO: 4.

[0236] The genome may further comprise an intron. The intron may be a modified SV40 intron. The modified SV40 intron may comprise or consist of SEQ ID NO: 5.

[0237] The genome may further comprise a polyadenylation (poly A) signal sequence. The polyadenylation signal sequence may be a rabbit globin polyadenylation signal sequence. The rabbit globin polyadenylation signal sequence may comprise or consist of SEQ ID NO: 6.

[0238] The genome may comprise, in 5' to 3' order, an L-ITR, an enhancer, a promoter, an intron, a codon-optimized polynucleotide encoding FIX according to one aspect, a polyadenylation signal sequence, and an R-ITR.

[0239] The genome may comprise, in 5' to 3' order, an L-ITR, an enhancer, a promoter, an intron, a codon-optimized polynucleotide encoding FIX according to one aspect, a polyadenylation signal sequence, a sequence appended to the ITR, and an R-ITR.

[0240] In one specific embodiment, the L-ITR is an ITR variant of AAV2, wherein the variant does not include trs; the enhancer and promoter are variants of an mTTR enhancer and promoter, wherein the variant has nucleotides deleted at positions 1 to 22 and 124 to 138 from the nucleotide sequence of a wild-type mTTR enhancer and promoter, and ACAGGA inserted after position 361; the intron is a modified SV40 intron; the polyadenylation signal sequence is a rabbit globin polyadenylation signal sequence; and the R-ITR may be a wild-type ITR of AAV2. The sequence added to the ITR may be a non-ITR viral DNA sequence derived from a wild-type AAV2 genome.

[0241] In one specific example, the L-ITR may be composed of SEQ ID NO: 3; the enhancer and promoter may be composed of SEQ ID NO: 4; the intron may be composed of SEQ ID NO: 5; the polyadenylation signal sequence may be composed of SEQ ID NO: 6; and the R-ITR may be composed of SEQ ID NO: 8.

[0242] In one specific example, the L-ITR may be composed of SEQ ID NO: 3; the enhancer and promoter may be composed of SEQ ID NO: 4; the intron may be composed of SEQ ID NO: 5; the polyadenylation signal sequence may be composed of SEQ ID NO: 6; the sequence added to the ITR may be composed of SEQ ID NO: 7; and the R-ITR may be composed of SEQ ID NO: 8.

[0243]

[0244] Another aspect provides a pharmaceutical composition for preventing or treating hemophilia B, comprising a recombinant AAV produced by a plasmid according to one aspect or a recombinant AAV according to one aspect.

[0245] The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.

[0246] The pharmaceutical composition may be used for gene therapy for hemophilia B. The pharmaceutical composition may be a composition for delivering FIX protein for gene therapy for hemophilia B.

[0247] The term "hemophilia B" can be caused by a deficiency or reduction of factor IX (FIX), or by a loss or reduction of the clotting function of FIX. Hemophilia B is an X-linked disorder. According to the National Hemophilia Foundation, an individual is classified as having mild hemophilia B when their plasma contains 6% to 49% of the FIX activity of normal human plasma, moderate hemophilia B when it contains 1% to 5%, and severe hemophilia B when it contains less than 1%.

[0248] In one embodiment, normal human plasma is defined as containing 1 IU of FIX activity per mL. Thus, plasma of an individual classified as having mild hemophilia B may contain 0.05 to 0.49 IU of FIX activity per mL, plasma of an individual classified as having moderate hemophilia B may contain 0.01 to 0.05 IU of FIX activity per mL, and plasma of an individual classified as having severe hemophilia B may contain less than 0.01 IU of FIX activity per mL.

[0249] The term "gene therapy" refers to the use of genes to treat or prevent disease. Recombinant AAVs, which deliver therapeutic genes into cells, can be used as gene therapy. Diseases for which gene therapy is applicable include, but are not limited to, diseases caused by defects in a single gene.

[0250] The above pharmaceutically acceptable carrier is used to mean an excipient, diluent, or adjuvant. The carrier may be one suitable for delivering recombinant AAV into a living body. Specifically, the carrier may be selected to be suitable for formulation as a parenteral formulation (e.g., an injectable formulation). For example, the carrier may be selected to be suitable for formulation as an intravenous formulation. The carrier may be an aqueous solution, such as water or a buffered saline solution.

[0251] The pharmaceutical composition may be prepared in any dosage form according to conventional methods. The pharmaceutical composition may be formulated in a form suitable for delivering recombinant AAV to a subject. The composition may be formulated in an aqueous solution, for example, in water or a buffered saline solution. The pharmaceutical composition may be formulated in an injectable dosage form suitable for administration by any suitable route, such as intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, or intrathecal. The pharmaceutical composition may be prepared in a systemic dosage form or a topical dosage form.

[0252] The pharmaceutical composition may further comprise an additional second therapeutic agent having a preventive or therapeutic effect on hemophilia B. The pharmaceutical composition may be a single composition or individual compositions.

[0253] The above pharmaceutical composition may contain the recombinant AAV in a pharmaceutically effective amount. The effective amount can be appropriately selected by a person skilled in the art depending on the individual.

[0254]

[0255] Another aspect provides a method of delivering Factor IX (FIX) to a subject in need thereof, comprising administering to the subject an effective amount of a recombinant AAV produced by a plasmid according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.

[0256] Another aspect provides a method of treating hemophilia B, comprising administering to an individual an effective amount of a recombinant AAV produced by a plasmid according to one aspect, a recombinant AAV according to one aspect, or a pharmaceutical composition according to one aspect.

[0257] In the above aspects, the subject may be an individual requiring expression of FIX delivered by a recombinant AAV. The subject may be an individual suffering from or likely to suffer from hemophilia B. The subject may be an individual suffering from or likely to suffer from a disease that can be treated by expression of FIX delivered by a recombinant AAV. The subject may be a hemophilia B patient. The subject may be a mammal. The mammal may include, but is not limited to, a human, a mouse, a rat, a hamster, a monkey, a rabbit, a donkey, a horse, a sheep, a cow, and the like.

[0258] In one specific example, the subject may be a human. The human may include a fetus, a newborn, an infant, an adolescent, or an adult.

[0259] The route of administration can be determined by those skilled in the art and may include, for example, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. Two or more routes of administration may be combined, if necessary. In one embodiment, the administration may be intravenous.

[0260] The above administration may be administered in an amount sufficient to infect the subject and in an amount sufficient to provide a sufficient level of introduction and expression of FIX.

[0261] The term "effective amount" may mean "therapeutically effective amount" and refers to the dosage administered to achieve a therapeutic effect.

[0262] The dose to achieve a therapeutic effect, e.g., the dose in vector genomes per kilogram of body weight (vg / kg), may vary based on several factors, including the route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease being treated, any host immune response by the viral vector, the host immune response to the heterologous polynucleotide or the expression product (protein), and the stability of the expressed protein. One skilled in the art can determine a range of rAAV doses to treat a patient with a particular disease or disorder based on the factors mentioned above as well as other factors. Typically, the dose is 1x10 per kilogram of body weight of the subject to achieve a therapeutic effect. 7　 or 1x10 8　 or 1x10 9　 or 1x10 10　 or 1x10 11　 or 1x10 12　 or 1x10 13　 or 1x10 14　 or 1x10 15　 The vector genome (vg / kg) can be in the range of . The expression level of the heterologous polynucleotide can be monitored to determine the method and frequency of administration.

[0263] A therapeutically effective amount of rAAV may mean an amount sufficient to convert severe hemophilia B to moderate or mild hemophilia B, or to completely cure hemophilia B, when administered to an individual with hemophilia B.

[0264] A therapeutically effective amount of rAAV may mean an amount sufficient to achieve plasma FIX activity that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more of normal FIX activity when administered to an individual with severe, moderate, or mild hemophilia B.

[0265] A therapeutically effective amount of rAAV can mean an amount that, when administered to an individual with hemophilia B, is sufficient to maintain adequate hemostasis by reducing or even eliminating the individual's need for recombinant FIX replacement therapy. Thus, a therapeutically effective amount of rAAV can reduce the frequency with which an average human individual with moderate or severe hemophilia B requires FIX replacement therapy to maintain adequate hemostasis by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0266] A therapeutically effective amount of rAAV may mean an amount that, when administered to a human subject with severe hemophilia B, is sufficient to reduce or even eliminate spontaneous bleeding into a joint. Thus, a therapeutically effective amount of rAAV may reduce the frequency of spontaneous bleeding into a joint in a human subject with severe hemophilia B by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an average human subject with severe hemophilia B.

[0267] A therapeutically effective dose of rAAV may be defined as an amount that induces adequate FIX activity to maintain hemostasis in an individual with hemophilia B, while inducing minimal or no humoral immune response to the components of AAV, namely the capsid, genome and / or FIX protein.

[0268]

[0269] Another aspect provides a use of a plasmid according to one aspect, a recombinant AAV produced by said plasmid, or a recombinant AAV according to one aspect, for the manufacture of a medicament for the treatment of hemophilia B.

[0270]

[0271] Duplicate content is omitted in consideration of the complexity of this specification, and terms not otherwise defined herein have the meanings commonly used in the technical field to which the present invention belongs.

[0272] According to the codon-optimized polypeptide according to the aspect, the expression and activity of FIX in an organism can be increased, and the immune response can be minimized.

[0273] According to a plasmid for recombinant AAV production according to one aspect, since the essential elements for recombinant AAV production are integrated into a single vector system, high-quality recombinant AAV can be produced with high efficiency and high productivity. Since all components of the plasmid are linked on a single molecule, they can be introduced into cells at the same ratio, and thus, cells transfected with the plasmid can exhibit balanced gene expression. Balanced gene expression can improve the productivity of recombinant AAV and increase the %Full capsid. The increased productivity of recombinant AAV can reduce unit production costs and improve the overall yield of the purification process, which accounts for more than 60% of the cost of pharmaceutical production. Furthermore, unlike the existing triple transfection method that produces three plasmids independently, a single plasmid is produced, thereby reducing the cost of raw materials. Furthermore, since all components are combined into a single plasmid, the amount of single plasmid input used for transfection can be reduced by more than 50%. Additionally, reverse packaging may be reduced because a single plasmid replaces the stuffer function.

[0274] Recombinant scAAV produced using a plasmid for recombinant AAV production according to one aspect can have enhanced expression and activity of FIX.

[0275] Figure 1 is a vector map of an All-in-One scAAV-FIX vector according to one embodiment.

[0276] Figure 2 is a graph showing FIX protein expression and activity (blood coagulation ability) to evaluate the GOI functionality of the All-in-One scAAV-FIX vector.

[0277] Figure 3 shows the results showing the AAV productivity (viral genome / L) and quality of produced AAV particles (%Full / empty) of the All-in-One scAAV-FIX vector and the triple vector at the flask scale.

[0278] Figure 4 is a graph showing the FIX expression level of recombinant AAV viruses produced by All-in-One scAAV-FIX vector or triple transfection.

[0279] Figure 5 is a graph showing the FIX activity of recombinant AAV viruses produced by the All-in-One scAAV-FIX vector or triple transfection.

[0280] Figure 6 is a graph showing the FIX activity of recombinant AAV viruses produced in the All-in-One vector system according to the type of GOI.

[0281] Figure 7 is a graph showing FIX protein expression and activity (blood coagulation ability) to evaluate GOI functionality according to the components and codon-optimized sequences between 5'-ITR and 3'ITR of the All-in-One scAAV-FIX vector.

[0282] Figure 8 is a graph showing the expression and activity of FIX protein (blood coagulation ability) in plasma after intravenous administration of a recombinant AAV virus produced by the All-in-One scAAV-FIX (scAIO-FIX-3) vector to a mouse model.

[0283] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0284]

[0285] Example 1. Production of an all-in-one vector for producing rAAV to treat hemophilia B.

[0286] An all-in-one vector for producing recombinant AAV for treating hemophilia B, comprising a polynucleotide encoding factor IX (FIX), was constructed. The all-in-one vector was designed to contain a helper virus gene, a Rep gene, a Cap gene, and a transgene in a single vector.

[0287] The genetic sequence information of each component of the all-in-one vector was obtained based on existing literature.

[0288] Helper E2A, E4, and VA genes were obtained from Adenovirus 2 (Gene Bank Accession No. NC_001405.1).

[0289] Rep, and L-ITR and R-ITR genes were obtained from Adeno-associated virus serotype 2 (Gene Bank Accession No. NC_001401.2). For L-ITR, a L-ITR mutant lacking the terminal resolution site (trs) was used by deleting the D region from the AAV2 wild-type L-ITR. By using the L-ITR lacking trs and the wild-type R-ITR, self-complementary AAV (scAAV) was produced by the all-in-one vector.

[0290] The Cap gene was obtained from Adeno-associated virus serotype 8 (Gene Bank Accession No. NC_006261.1).

[0291] The GOI (gene-of-interest) used as a transgene used the codon-optimized base sequence of FIX produced in Example 2 below.

[0292] Based on the sequence information of the corresponding genes of each component, primers were designed and PCR amplified to secure the components, and then 1 copy each was inserted into the pMG-Kan vector (Macrogen). The expression vector was constructed using molecular genetic technology according to Ausubel et al. (Ausubel et al. (ed.), J. Wiley & Sons, 1997, Curr. Protocols of Molecular Biology.). The primers used for genes and PCR amplification were synthesized and manufactured by Macrogen Co., Ltd. (Seoul, Korea). The genetic sequence of the cloned expression vector was confirmed by requesting Macrogen Co., Ltd. (Seoul, Korea). The constructed vector was named "All-in-One scAAV-FIX vector" (abbreviated as "scAIO-FIX").

[0293] Figure 1 is a vector map of an All-in-One scAAV-FIX vector according to one embodiment.

[0294] The All-in-One scAAV-FIX vector of Figure 1 contains each component in the following order: helper gene, plasmid backbone, Rep gene, Cap gene, L-ITR, liver tissue-specific enhancer, liver tissue-specific promoter, intron, GOI, polyadenylation signal sequence, ITR+, R-ITR.

[0295] In the All-in-One scAAV-FIX vector of Figure 1, E2A, E4, and VA of Adenovirus 2 (SEQ ID NO: 1) were used as helper genes; Rep2 as Rep gene and wild-type AAV8 Cap (SEQ ID NO: 2) as Cap gene; AAV2 L-ITR with the D region deleted from the wild-type sequence (SEQ ID NO: 3); a mutant of the mTTR enhancer / promoter (SEQ ID NO: 4); a modified SV40 intron (SEQ ID NO: 5); a rabbit globin polyadenylation signal sequence (rabbit globin pA) (SEQ ID NO: 6); a non-ITR viral DNA sequence corresponding to wtAAV2 nt 4489-4534 derived from the upstream of the 3' ITR in the wild-type AAV2 genome (SEQ ID NO: 7), and the wild-type R-ITR of AAV2 (SEQ ID NO: 8) were used.

[0296] The liver tissue-specific enhancer / promoter used was an mTTR enhancer / promoter mutant (330 bp) in which the sequences at positions 1 to 22 and 124 to 138 (37 bp) were deleted from the wild-type mTTR enhancer / promoter (361 bp) and ACAGGA (6 bp) was inserted after position 361.

[0297] The sequence information used to construct the All-in-One scAAV-FIX vector is shown in Table 1 below.

[0298] Sequence number Name Description Size 1 E2A, E4, and VA of Helper Adenovirus 2 9279 bp 2 Rep and CapAAV2 Rep and AAV8 Cap 4099 bp 3 D region deleted in L-ITR of ...

[0299]

[0300] Example 2. Codon Optimization of Factor IX

[0301] Codon-optimized sequences of Factor IX mutants or wild-type (WT) Factor IX were generated using the GenSmart Codon Optimization Tool (Genescript) or GeneArt Codon Optimization Tool (GeneArt). Factor IX mutants contain the R338L mutation or the T148A / R338L mutation in the wild-type sequence. The generated codon-optimized sequences were used as GOIs.

[0302] The optimized GOI was designed to optimize transcription, protein refolding, translation, gene synthesis, and minimize immunotoxicity.

[0303] Specifically, codon optimization included the following parameters:

[0304] 1) Removal of CpG dinucleotides;

[0305] 2) Selection of sequences with low MFE (Minimum Free Energy), low SUP, and high DegScore to increase mRNA half-life; and

[0306] 3) Removal of cryptic splicing sites.

[0307] Information about the produced codon-optimized FIX sequence is shown in Table 2 below.

[0308] Codon Optimization Tool FIX Name Sequence Number Genscript GenSmart FIX Padua (R338L) HEY0025-019 HEY0025-0210 HEY0025-0311 HEY0025-0412 HEY0025-0513 HEY0025-0614 HEY0025-0715 HEY0025-0816 HEY0025-0917 HEY0025-1018 FIX Padua (R338L, T148A)HEY0028-0119HEY0028-0220HEY0028-0321HEY0028-0422HEY0028-0523HEY0028-0624HEY0028-0725HEY0028-0826HEY0028-0927HEY0028-1028FIX WTHEY0031-0129HEY0031-0230HEY0031-0331HEY0031-0432HEY0031-0533HEY0031-0634HEY0031-0735HEY0031-0836HEY0031-0937HEY0031-1038GeneArtFIX Padua (R338L)HEY0025-1139FIX Padua (R338L, T148A)HEY0028-1140FIX WTHEY0031-1141

[0309] Through in silico evaluation, four sequences were selected from the above-mentioned codon-optimized FIX sequences that showed high mRNA stability scores. The parameter results for codon optimization of the four selected sequences are shown in Table 3 below.

[0310] ToolGenescriptLinear FoldLinear PartitionDegScoreResultMFE (kcal / mol)MFE (kcal / mol)MFE (kcal / mol)AUPSUPDegScoreEstimated mRNA half-life (hr)HEY0025-01 (SEQ ID NO: 9)-463.1-449.9-476.410.398552.914438.7590.690HEY0025-10 (SEQ ID NO: 18)-477.1-472-496.370.396549.793446.8290.678HEY0028-05 (SEQ ID NO: 23)-457.1-457.1-484.350.382530.087430.3460.703HEY0028-06 (SEQ ID NO: 24)-467-467-493.950.385535.385441.8300.685

[0311]

[0312] Comparative Example 1. Production of a triple vector (triple transfection scAAV-FIX) for triple transfection.

[0313] For the existing triple transfection, a triple vector containing a helper gene, a vector containing Rep / Cap genes, and a vector containing GOI (including the portion corresponding to L-ITR to R-ITR) were constructed. Each component was amplified by PCR and inserted into the pMG-Kan vector (Macrogen). The same type of gene as in Example 1 was used for each vector. The genetic sequence of the cloned expression vector was confirmed by request to Macrogen Co., Ltd. (Seoul, Korea). The constructed vector was named “triple transfection scAAV-FIX vector” (also abbreviated as sctFIX).

[0314]

[0315] Comparative Example 2. Production of AIO-SPK9001 Vector

[0316] The portion corresponding to the 5' ITR to the 3' ITR of the pAAV-ApoE_hAAT-FIX39 vector (SPK9001) of US 10799566 B2 was synthesized. Specifically, the portion includes the 5' AAV2 ITR, the ApoE HCR-1 / 2 enhancer, the hAAT promoter, the 5' UTR, the FIX39-Padua CDS, intron A, the 3' UTR, the bGH poly A, and the 3' AAV2 ITR. FIX39-Padua has CpG dinucleotides completely removed from the FIX coding and intron sequences.

[0317] In the All-in-One scAAV-FIX vector of Example 1, the portion corresponding to the 5' ITR to the 3' ITR was replaced with the portion corresponding to the 5' ITR to the 3' ITR of the SPK9001, thereby producing the AIO-SPK9001 vector.

[0318]

[0319] Comparative Example 3. Production of AIO-AMT061 Vector

[0320] The region from the 5' ITR to the 3' ITR of the AMT-061 vector (AAV5-hFIXco-Padua) of Nathwani et al., Blood (2006), 107(7):2653-61 was synthesized. Specifically, the region includes the 5' AAV2 ITR, HCR enhancer, hAAT promoter, modified SV40 intron, hFIX-Padua, SV40 poly A, and the 3' AAV2 ITR.

[0321] In the All-in-One scAAV-FIX vector of Example 1, the portion corresponding to the 5' ITR to the 3' ITR was replaced with the portion corresponding to the 5' ITR to the 3' ITR of the AMT-061, thereby producing the AIO-AMT061 vector.

[0322]

[0323] Comparative Example 4. Production of ttSPK9001 vector

[0324] A triple vector was created in the same manner as in Comparative Example 1, except that the portion corresponding to the 5' ITR to the 3' ITR of Comparative Example 2 was inserted into the vector containing GOI (including the portion corresponding to L-ITR to R-ITR), and this was named ttSPK9001 vector.

[0325]

[0326] Comparative Example 5. Production of AIO-BAX335 Vector

[0327] The portion corresponding to the 5' ITR to the 3' ITR of the BAX335 (TAK-748) vector of BA konkle et al., Blood. 2021;137(6):763-774 was synthesized. Specifically, the portion includes the 5' AAV2 ITR, 3X CRM8, mTTR enhancer, mTTR promoter, MVM intron, hFIX-Padua, bGH poly A, and the 3' AAV2 ITR.

[0328] In the All-in-One scAAV-FIX vector of Example 1, the portion corresponding to the 5' ITR to the 3' ITR was replaced with the portion corresponding to the 5' ITR to the 3' ITR of BAX335, thereby producing an AIO-BAX335 vector.

[0329]

[0330] Comparative Example 6. Production of scAIO-FIX (SPK9001) vector

[0331] An all-in-one vector was constructed in the same manner as in Example 1, except that the transgene (GOI) portion of the All-in-One scAAV-FIX vector of Example 1 was replaced with the FIX39-Padua CDS and intron A used in the AIO-SPK9001 vector of Comparative Example 2. Specifically, the 5' ITR to 3' ITR portion of the vector was composed of the following sequences: AAV2 L-ITR (SEQ ID NO: 3) in which the D region is deleted from the wild-type sequence; a mutant of the mTTR enhancer / promoter (SEQ ID NO: 4); a modified SV40 intron (SEQ ID NO: 5); the FIX39-Padua CDS of Comparative Example 2; the intron A of Comparative Example 2; the rabbit globin polyadenylation signal sequence (rabbit globin pA) (SEQ ID NO: 6); It contains a non-ITR viral DNA sequence corresponding to wtAAV2 nt 4489-4534 derived from the upstream of the 3' ITR in the wild-type AAV2 genome (SEQ ID NO: 7) and the wild-type R-ITR of AAV2 (SEQ ID NO: 8). The constructed vector was named scAIO-FIX (SPK9001).

[0332]

[0333] Experimental Example 1. GOI Functional Evaluation of All-in-One scAAV-FIX Vector (FIX Protein Expression / Activity)

[0334] To verify the GOI functionality of the All-in-One scAAV-FIX vector, the vector was injected into target cells via Lipofectamine (Invitrogen), and then the expression of FIX protein and blood coagulation ability were confirmed.

[0335] The experimental vectors are four types of All-in-One scAAV-FIX vectors in which codon-optimized GOIs of SEQ ID NO: 9, 18, 23, or 24 of Example 2 are inserted into the vector of Example 1. Each was named scAIO-FIX-1 (including SEQ ID NO: 9 as GOI), scAIO-FIX-2 (including SEQ ID NO: 18 as GOI), scAIO-FIX-3 (including SEQ ID NO: 23 as GOI), and scAIO-FIX-4 (including SEQ ID NO: 24 as GOI).

[0336] As a control group, AIO-SPK9001 of Comparative Example 2 and AIO-AMT061 of Comparative Example 3 were used.

[0337] Target cells were Huh7 cells, cultured in DMEM medium supplemented with 10% FBS at 37°C and 5% CO2. One day before treatment with the all-in-one vector, Huh7 cells were seeded in a 24-well plate at 1.0E05 cells / well. The all-in-one vector pDNA was mixed with a cationic lipid transfection reagent (Lipofectamine) at a 1:1 ratio, incubated at room temperature for 25 minutes, and then slowly added dropwise to the target cells. At this time, the pDNA concentration in the cells was 1 μg / well. After transfection, the cells were incubated for 4 hours at 37°C and 5% CO2. Afterwards, all the culture medium was removed, and DMEM medium containing 0.005 μg / mL of vitamin K3 was added. After 72 hours of culture, the Huh7 culture medium was harvested, and the FIX expression level was measured using the Human Factor IX ELISA Kit (ELABSCIENCE). Additionally, FIX coagulation ability was analyzed using the Factor IX chromogenic assay kit (BIOPHEN).

[0338] Figure 2 is a graph showing FIX protein expression and activity (blood coagulation ability) to evaluate the GOI functionality of the All-in-One scAAV-FIX vector.

[0339] As a result, as shown in Fig. 2, compared to AIO-SPK9001 or AIO-AMT061, the FIX expression level of the All-in-One scAAV-FIX vector was improved by 1.9 to 2.6 times, and the FIX blood coagulation ability was confirmed to be increased by 1.3 to 1.4 times.

[0340]

[0341] Experimental Example 2. Productivity Verification of the All-in-One scAAV-FIX Vector

[0342] Feasibility studies for AAV production using the All-in-One scAAV-FIX vector were conducted at flask scale.

[0343] The cells used in this experiment were suspension HEK293F cells, and the experimental conditions were 37°C, 8% CO2, and 80% humidity. The medium used to evaluate AAV production was serum-free media (SFM).

[0344] The experimental vector is an All-in-One scAAV-FIX vector (scAIO-FIX-3) in which the codon-optimized GOI of sequence number 23 of Example 2 is inserted into the vector of Example 1.

[0345] As a control, a triple transfection scAAV-FIX vector (named scttFIX-3, including SEQ ID NO: 23 as GOI) was used, in which the codon-optimized GOI of SEQ ID NO: 23 of Example 2 was inserted into the vector of Comparative Example 1.

[0346] Since the All-in-One scAAV-FIX vector has 1 copy of each of the GOI, Rep / Cap, and Helper genes inserted and each gene is processed with the same copy number, the productivity of the GOI, Rep / Cap, and Helper vectors of the triple transfection condition control group (Triple) was compared with the same amount of pDNA.

[0347] Both the control and experimental groups were transiently transfected with 3 μg / mL of pDNA (plasmid DNA) using PEI (Polyethylenimine) in 3E06 cells / mL (30 mL, 125 flask). Seventy-two hours after transfection, cells were lysed to confirm AAV productivity, and the produced AAV viral genome (VG) was measured by qPCR using ITR-specific primers using the AAVpro® Titration Kit (TAKARA). In addition, AAV viral protein (VP) was measured using the AAV Titration ELISA (PROGEN). The quality (%Full / empty) of the produced AAV particles was confirmed through the measured VG / VP ratio value.

[0348] Figure 3 shows the results showing the AAV productivity (viral genome / L) and quality of produced AAV particles (%Full / empty) of the All-in-One scAAV-FIX vector and the triple vector at the flask scale.

[0349] As a result, as shown in Fig. 3, when the same amount of pDNA was used, it was confirmed that the all-in-one vector could produce AAV with a 1.1-fold improvement in quality compared to triple transfection and a 2.4-fold higher productivity (VG / L).

[0350]

[0351] Experimental Example 3. Verification of the transduction ability and GOI functionality of recombinant scAAV produced by the All-in-One scAAV-FIX vector.

[0352] To verify the transducing capability and GOI functionality of the recombinant self-complementary AAV (scAAV) virus produced by the All-in-One scAAV-FIX vector, scAAV was injected into target cells to confirm the expression of FIX protein and blood coagulation ability.

[0353] The target cells were Huh7 cells, and they were cultured in DMEM medium supplemented with 10% FBS at 37°C and 5% CO2 conditions. One day before treatment with the recombinant virus, Huh7 cells were plated in a 48-well plate at 2.5E04 cells / well. Both the recombinant virus produced by the all-in-one FIX vector of Example 1 and the recombinant viruses produced by the control vectors of Comparative Examples 2 to 5 were obtained as highly pure recombinant AAV viruses using CsCl ultracentrifugation. The obtained viruses were diluted in DMEM medium without FBS to be treated at 1E05, 3E05, or 1E06 vg / cell. The viruses were added to the 48-well plate on which Huh7 cells, from which the culture medium had been completely removed, were plated in DMEM medium supplemented with 350 ng / mL of vitamin K3 and without FBS. Four days after virus treatment, Huh7 cultures were harvested, and FIX expression levels were measured using a Human Factor IX ELISA Kit (ELABSCIENCE). In addition, FIX coagulation activity was analyzed using a Factor IX chromogenic assay kit (BIOPHEN).

[0354] Figure 4 is a graph showing the FIX expression level of recombinant AAV viruses produced by All-in-One scAAV-FIX vector or triple transfection.

[0355] Figure 5 is a graph showing the FIX activity of recombinant AAV viruses produced by the All-in-One scAAV-FIX vector or triple transfection.

[0356] Additionally, the FIX expression levels and FIX activity levels of FIGS. 4 and 5 are shown in Table 4 below.

[0357] MOI (vg / cell) FIX protein expression (ng / mL) FIX activity (mIU / mL) 1.1E053.3E051.0E061.1E053.3E051.0E06ttSPK9001335.2785.71716.61079.32242.43471.7AIO-SPK9001545.71133.32157.31767.52578.53418.8AIO-AMT061527.31207.52508.32720.13521.73820.8scA IO-FIX-1939.11581.82515.13324.53916.53992.9scAIO-FIX-2798.71429.62263.03050.03963.64293.1s cAIO-FIX-3874.11664.72614.22885.44145.64248.3scAIO-FIX-4833.81611.02508.13010.64126.04339.7

[0358] As a result, as shown in FIGS. 4, 5, and Table 4, when the MOI was 1.1E05 vg / cell, a 1.6-fold increase in FIX expression was confirmed in AIO-SPK9001 (545.7 ng / mL) produced from the all-in-one vector compared to SPK9001 (ttSPK9001, 335.2 ng / mL) produced by the triple transfection method (FIG. 4), and a 1.6-fold (ttSPK9001: 1079.3 mIU / mL, AIO-SPK9001: 1767.5 mIU / mL) improvement in FIX blood coagulation ability was confirmed (FIG. 5). In addition, compared to ttSPK9001, the four All-in-One scAAV-FIX vectors showed 2.4 to 2.8 times higher FIX expression (scAIO-FIX-1: 939.1 ng / mL, scAIO-FIX-2: 798.7 ng / mL, scAIO-FIX-3: 874.1 ng / mL, scAIO-FIX-4: 833.8 ng / mL) (Fig. 4) and 2.7 to 3.1 times higher FIX coagulation activity (scAIO-FIX-1: 3324.5 mIU / mL, scAIO-FIX-2: 3050.0 mIU / mL, scAIO-FIX-3: 2885.4 mIU / mL, scAIO-FIX-4: 3010.6 mIU / mL) (Fig. 5).

[0359] Additionally, in the all-in-one vector system, compared to the control group (AIO-SPK9001), the four All-in-One scAAV-FIX vectors showed a 1.5 to 1.7-fold increase in FIX protein expression (Fig. 4) and a 1.6 to 1.9-fold increase in FIX blood coagulation ability (Fig. 5).

[0360] Figure 6 is a graph showing the FIX activity of recombinant AAV viruses produced in the All-in-One vector system according to the type of GOI.

[0361] Additionally, the FIX activity levels of Fig. 6 are shown in Table 5 below.

[0362] MOI(vg / cell)FIX active (mIU / mL)1.1E053.3E051.0E06AIO-BAX3353396.5453190.453218.79scAIO-FIX-13324.4893916.5383992.9scAI O-FIX-23049.953963.6224293.11scAIO-FIX-32885.4134145.554248.29scAIO-FIX-43010.6364125.994339.67

[0363] As shown in Figure 6 and Table 5, in the all-in-one vector system, an increase in FIX blood coagulation activity of 1.2 to 1.3 times was confirmed in the four types of All-in-One scAAV-FIX vectors compared to the control group (AIO-BAX335).

[0364]

[0365] Experimental Example 4. Evaluation of GOI Functionality According to Codon-Optimized Sequence and Regulatory Elements (FIX Protein Expression / Activity)

[0366] To evaluate the influence of the components between the 5'-ITR and 3'ITR of the All-in-One scAAV-FIX vector of Example 1 and the factor IX codon-optimized sequence of Example 2 on GOI functionality, the vector was injected into target cells via Lipofectamine (Invitrogen), and then the expression of the FIX protein and blood coagulation ability were confirmed.

[0367] The experimental vectors are four types of All-in-One scAAV-FIX vectors (scAIO-FIX-1 (including SEQ ID NO. 9 as GOI), scAIO-FIX-2 (including SEQ ID NO. 18 as GOI), scAIO-FIX-3 (including SEQ ID NO. 23 as GOI), scAIO-FIX-4 (including SEQ ID NO. 24 as GOI)) in which codon-optimized GOIs of SEQ ID NO. 9, 18, 23, or 24 of Example 2 are inserted into the vector of Example 1.

[0368] As a control group, the AIO-SPK9001 vector of Comparative Example 2 and the scAIO-FIX (SPK9001) vector of Comparative Example 6 were used.

[0369] Target cells were Huh7 cells cultured in DMEM medium supplemented with 10% FBS at 37°C and 5% CO2. One day before treatment with the all-in-one vector, Huh7 cells were seeded in a 24-well plate at 1.0E05 cells / well. The all-in-one vector pDNA was mixed with a cationic lipid transfection reagent (Lipofectamine) at a 1:3 ratio and incubated at room temperature for 25 minutes before being added to the target cells. At this time, the pDNA concentration in the cells was 1 μg / well. After transfection, the cells were incubated for 4 hours at 37°C and 5% CO2. Afterwards, all the culture medium was removed, and DMEM medium containing 0.005 μg / mL of vitamin K3 was added. After 85 hours of culture, the Huh7 culture medium was harvested and the FIX expression level was measured using the Human Factor IX ELISA Kit (ELABSCIENCE). Additionally, FIX coagulation ability was analyzed using the Factor IX chromogenic assay kit (BIOPHEN).

[0370] Figure 7 is a graph showing FIX protein expression and activity (blood coagulation ability) to evaluate GOI functionality according to the components and codon-optimized sequences between 5'-ITR and 3'ITR of the All-in-One scAAV-FIX vector.

[0371] As a result, as shown in Fig. 7, compared to the AIO-SPK9001 vector, the All-in-One scAAV-FIX 4 vectors were found to have significantly improved FIX expression levels and FIX coagulability, and even when the GOI portion in the All-in-One scAAV-FIX 4 vectors was changed to be the same as that in the AIO-SPK9001 vector (scAIO-FIX(SPK9001)), it was confirmed that the improved FIX expression levels and FIX coagulability were maintained compared to the AIO-SPK9001 vector. This means that the excellent FIX expression levels and FIX coagulability in the All-in-One scAAV-FIX 4 vectors are much more affected by other components between 5'-ITR and 3'ITR than by the GOI portion.

[0372]

[0373] Experimental Example 5. Verification of transduction ability and GOI functionality of recombinant scAAV produced by the All-in-One scAAV-FIX vector (in vivo, wild-type mouse)

[0374] To verify the transducing capability and GOI functionality of the recombinant self-complementary AAV (scAAV) virus produced by the All-in-One scAAV-FIX vector, scAAV was injected into a wild-type mouse model to confirm the expression of FIX protein and blood coagulation ability.

[0375] A highly pure recombinant AAV virus was obtained using the anion exchange chromatography (AEX) purification method of the recombinant virus produced by the all-in-one FIX vector of Example 1. The obtained recombinant AAV was administered to male C57Bl / 6 mice at doses of 5.0E11, 6.0E12, and 5.0E13 vg / kg. After a two-week observation period, mouse plasma was isolated and the protein expression of hFIX and blood coagulation ability in the plasma were confirmed.

[0376] Figure 8 is a graph showing the expression and activity of FIX protein (blood coagulation ability) in plasma after intravenous administration of a recombinant AAV virus produced by the All-in-One scAAV-FIX (scAIO-FIX-3) vector to a mouse model.

[0377] As a result, as shown in Fig. 8, hFIX protein was detected in mouse plasma in a dose-dependent amount during the second week after administration (Week 2: 5.0E11 vg / kg 7.5 ug / mL, 6.0E12 vg / kg 69.3 ug / mL, 5.0E13 vg / kg 255 ug / mL). In addition, the blood coagulation ability of hFIX was confirmed in a dose-dependent manner during the second week (5.0E11 vg / kg 1,840, 6.0E12 vg / kg 16,040, 5.0E13 vg / kg 38,587).

[0378]

[0379] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A codon-optimized polynucleotide encoding Factor IX (FIX), comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9 to 41.

2. A codon-optimized polynucleotide encoding FIX, wherein the polynucleotide comprises a nucleotide sequence of any one of SEQ ID NOs: 9 to 41, according to claim 1.

3. A codon-optimized polynucleotide encoding FIX, wherein the FIX of claim 1 is a FIX variant comprising an R338L mutation (FIX-R338L); a FIX variant comprising T148A and R338L mutations (FIX-T148A / R338L); or a wild-type FIX.

4. A codon-optimized polynucleotide encoding FIX according to claim 3, wherein the codon-optimized polynucleotide encoding FIX-R338L comprises any one nucleotide sequence selected from SEQ ID NOs: 9 to 18 and 39.

5. A codon-optimized polynucleotide encoding FIX according to claim 3, wherein the codon-optimized polynucleotide encoding FIX-T148A / R338L comprises any one nucleotide sequence selected from SEQ ID NOs: 19 to 28 and 40.

6. A codon-optimized polynucleotide encoding FIX according to claim 3, wherein the codon-optimized polynucleotide encoding wild-type FIX comprises any one nucleotide sequence selected from SEQ ID NOs: 29 to 38 and 41.

7. A codon-optimized polynucleotide encoding FIX, comprising a nucleotide sequence of any one of SEQ ID NOs: 9, 18, 23, and 24, according to claim 1.

8. A codon-optimized polynucleotide encoding FIX according to any one of claims 1 to 7, wherein the polynucleotide has a reduced number of CpG dinucleotides compared to the non-codon-optimized wild-type sequence.

9. A codon-optimized polynucleotide encoding FIX according to any one of claims 1 to 8, wherein no CpG dinucleotide is present in the polynucleotide.

10. A codon-optimized polynucleotide encoding FIX, according to any one of claims 1 to 9, wherein mRNA transcribed by the polynucleotide has an increased half-life compared to mRNA transcribed by a non-codon-optimized wild-type sequence.

11. A codon-optimized polynucleotide encoding FIX according to any one of claims 1 to 10, wherein mRNA transcribed by the polynucleotide has a half-life of 0.6 hours or longer.

12. A codon-optimized polynucleotide encoding FIX according to any one of claims 1 to 11, wherein the polynucleotide has a cryptic splicing site removed.

13. A codon-optimized polynucleotide encoding FIX, according to any one of claims 1 to 12, wherein the polynucleotide exhibits reduced immunotoxicity compared to a non-codon-optimized wild-type sequence.

14. A codon-optimized polynucleotide encoding FIX, wherein the polynucleotide is expressed at a higher level than a wild-type sequence that is not codon-optimized, according to any one of claims 1 to 13.

15. A codon-optimized polynucleotide encoding FIX, wherein the FIX protein expressed by the polynucleotide exhibits a higher level of activity than the FIX protein expressed by the non-codon-optimized wild-type sequence according to any one of claims 1 to 14.

16. A plasmid for producing a recombinant adeno-associated virus (AAV) for treating hemophilia B, comprising the nucleotide sequence of the following genes arranged in a single nucleic acid molecule: (a) Helper virus genes required for AAV production; (b) Rep gene of AAV; (c) Cap gene of AAV; and (d) A transgene comprising a codon-optimized polynucleotide encoding a FIX of any one of claims 1 to 15.

17. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the transgene is arranged between ITRs (Inverted Terminal Repeats) according to claim 16.

18. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the transgene is arranged between L-ITR and R-ITR according to claim 16.

19. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the nucleic acid molecule is circular according to claim 16.

20. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the nucleotide sequence of the helper virus gene of claim 16 is derived from at least one selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus.

21. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the nucleotide sequence of the helper virus gene according to claim 16 is derived from adenovirus.

22. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the nucleotide sequence of the helper virus gene according to claim 16 is derived from adenovirus 2.

23. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the helper virus gene comprises at least one selected from E1, E2, E2a, E4, E4orf1, E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DBP (DNA-binding protein), and mutants thereof, according to claim 16.

24. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the helper virus genes comprise E2a, E4, and VA, according to claim 16.

25. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the nucleotide sequence of the Rep gene according to claim 16 is derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.

26. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the nucleotide sequence of the Rep gene according to claim 16 is derived from AAV2.

27. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the Rep gene according to claim 16 comprises at least one selected from Rep78, Rep68, Rep52, Rep40, and mutants thereof.

28. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the Rep gene comprises Rep78, Rep68, Rep52, and Rep40, according to claim 16.

29. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the nucleotide sequence of the Cap gene according to claim 16 is derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.

30. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the nucleotide sequence of the Cap gene according to claim 16 is derived from AAV8.

31. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the Cap gene encodes at least one selected from a capsid protein, a VP1 protein, a VP2 protein, a VP3 protein, and variants thereof, according to claim 16.

32. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the Cap gene according to claim 16 is a wild-type Cap gene of AAV8.

33. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the ITR in claim 17 or 18 is derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.

34. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the ITR according to claim 17 or 18 is derived from AAV2.

35. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the ITR according to claim 17 or 18 is a wild-type ITR or a mutant thereof.

36. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the ITR comprises all or part of a wild-type ITR sequence according to claim 17 or 18.

37. A plasmid for producing recombinant AAV for treating hemophilia B, wherein either the L-ITR or the R-ITR does not contain trs (terminal resolution site) according to claim 18.

38. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein one of the L-ITR and the R-ITR does not contain trs and the other is a wild-type ITR according to claim 18.

39. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein one of the L-ITR and the R-ITR is an ITR mutant having a D region deleted, and the other is a wild-type ITR according to claim 18.

40. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the L-ITR is composed of sequence number 3 and the R-ITR is composed of sequence number 8, according to claim 18.

41. A plasmid for producing a recombinant AAV for treating hemophilia B, further comprising a nucleotide sequence consisting of sequence number 7 upstream of the R-ITR according to claim 18.

42. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the recombinant AAV produced by the plasmid according to any one of claims 37 to 41 is self-complementary AAV (scAAV).

43. A plasmid for producing recombinant AAV for treating hemophilia B, according to any one of claims 16 to 42, wherein the nucleic acid molecule further comprises an expression regulatory element operably linked to (d).

44. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the expression regulatory element of claim 43 is a tissue-specific expression regulatory element.

45. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the expression regulatory element according to claim 43 or 44 comprises at least one selected from an enhancer and a promoter.

46. ​​In claim 45, The above enhancer is a liver tissue-specific enhancer, The above promoter is a liver tissue-specific promoter, Plasmids for producing recombinant AAV for treating hemophilia B.

47. In claim 45, The above enhancer is mTTR (mouse transthyretin) enhancer or a variant thereof, The above promoter is an mTTR promoter or a variant thereof, Plasmids for producing recombinant AAV for treating hemophilia B.

48. In claim 45, The nucleic acid molecule comprises an enhancer and a promoter operably linked to (d), The above enhancer and promoter are mutants of the mTTR enhancer and promoter in which nucleotides at positions 1 to 22 and 124 to 138 are deleted from the nucleotide sequence of the wild-type mTTR enhancer and promoter, and ACAGGA is inserted after position 361. Plasmids for producing recombinant AAV for treating hemophilia B.

49. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the variant of the mTTR enhancer and promoter comprises sequence number 4 in claim 48.

50. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the nucleic acid molecule further comprises an intron according to any one of claims 16 to 49.

51. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the intron of claim 50 is a modified SV40 intron.

52. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the modified SV40 intron comprises sequence number 5 in claim 51.

53. A plasmid for producing recombinant AAV for treating hemophilia B, according to any one of claims 16 to 52, wherein the nucleic acid molecule further comprises a polyadenylation (poly A) signal sequence.

54. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the polyadenylation signal sequence of claim 53 is a rabbit globin poly A signal sequence.

55. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the rabbit globin poly A signal sequence of claim 54 comprises SEQ ID NO:

6.

56. A plasmid for producing recombinant AAV for treating hemophilia B, according to any one of claims 16 to 55, wherein the nucleic acid molecule further comprises a replication origin.

57. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the replication origin is a pUC origin, a pBR322 origin, a pMB1 origin, a pSC101 origin, a p15A origin, or a synthetic origin, according to claim 56.

58. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the replication origin is the pBR322 origin according to claim 56.

59. A plasmid for producing recombinant AAV for treating hemophilia B, according to any one of claims 16 to 58, wherein the nucleic acid molecule further comprises a selectable marker.

60. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the selection marker is at least one selected from ampicillin, streptavidin, kanamycin, hygromycin, neomycin, puromycin, blasticidin, and zeocin, according to claim 59.

61. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the selection marker of claim 59 is kanamycin.

62. A plasmid for producing recombinant AAV for treating hemophilia B, wherein in the nucleic acid molecule, (a), (b), (c), and (d) are each independently arranged in any order in the forward orientation or the reverse orientation according to any one of claims 16 to 61.

63. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein in the nucleic acid molecule of claim 62, (a), (b), (c), and (d) are arranged in the following order, wherein (a), (b), (c), and (d) are each independently in the normal or reverse orientation: 1) (d) - (b) - (c) - (a); 2) (d) - (c) - (b) - (a); 3) (d) - (a) - (b) - (c); 4) (d) - (a) - (c) - (b); 5) (b) - (c) - (d) - (a); 6) (c) - (b) - (d) - (a); 7) (b) - (c) - (a) - (d); 8) (c) - (b) - (a) - (d); 9) (a) - (b) - (c) - (d); 10) (a) - (c) - (b) - (d); 11) (a) - (d) - (b) - (c); or 12) (a) - (d) - (c) - (b).

64. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the plasmids of claim 62 are arranged in the order of (a) - (b) - (c) - (d), wherein (a), (b), (c), and (d) are each independently in the normal or reverse orientation.

65. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the plasmids of claim 62 are arranged in the order of (a) - (b) - (c) - (d), wherein (a), (b), (c), and (d) are all oriented in the correct direction.

66. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein in claim 65, E2a is in the reverse orientation, E4 is in the normal orientation, and VA is in the reverse orientation.

67. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the ITRs are arranged on both sides of (d) according to any one of claims 62 to 66.

68. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the plasmid backbone is arranged between (a) and (b) according to any one of claims 62 to 67.

69. A plasmid for producing recombinant AAV for treating hemophilia B, wherein the plasmid backbone comprises at least one selected from an origin of replication and a selection marker according to claim 68.

70. A plasmid for producing a recombinant AAV for treating hemophilia B, wherein the nucleic acid molecule according to claim 69 is arranged in the following order: (a) - plasmid backbone - (b) - (c) - L-ITR - enhancer and promoter - intron - (d) - polyadenylation signal sequence - R-ITR.

71. A plasmid for producing a recombinant AAV for treating hemophilia B, further comprising a nucleotide sequence consisting of SEQ ID NO: 7 between the polyadenylation signal sequence and R-ITR according to claim 70.

72. A step of introducing a plasmid of any one of claims 16 to 71 into a host cell; and Comprising the step of isolating recombinant AAV from said host cell, Method for producing a recombinant adeno-associated virus (AAV) for treating hemophilia B.

73. In claim 72, 0.25×10 6 Dog or 3×10 6 A method for producing a recombinant AAV for treating hemophilia B, wherein the amount of plasmid DNA (pDNA) introduced per host cell is about 0.5 to about 4 μg, about 0.5 to about 3 μg, about 1 to about 4 μg, or about 1 to about 3 μg.

74. A method for producing a recombinant AAV for treating hemophilia B, according to claim 72, wherein the host cell is a mammalian cell or an insect cell.

75. A method for producing a recombinant AAV for treating hemophilia B according to claim 74, wherein the mammalian cell is selected from HEK293 cells, HEK293F cells, HEK293T cells, and cells derived therefrom.

76. A method for producing a recombinant AAV for treating hemophilia B, wherein the insect cell is a Sf9 cell, a Sf21 cell, a TN-5B1-4 cell, or a High Five cell, according to claim 74.

77. A method for producing a recombinant AAV for treating hemophilia B according to claim 72, wherein the introduction is selected from physical methods including electroporation, cell compression, sonication, optical transfection, protoplast fusion, impalfection, magnetofection, gene gunning, and particle bombardment; methods using a cationic polymer such as polyethyleneimine (PEI) and a chemical reagent including calcium phosphate; and methods using a cationic lipid such as lipofection.

78. A method for producing a recombinant AAV for treating hemophilia B, wherein the separation is performed using centrifugation or chromatography according to claim 72.

79. A method for producing a recombinant AAV for treating hemophilia B, wherein the centrifugation method according to claim 78 is a cesium chloride-based ultra-high-speed centrifugation method.

80. A method for producing a recombinant AAV for treating hemophilia B, wherein the chromatography according to claim 78 is at least one selected from affinity chromatography, ion exchange chromatography, column chromatography, gel filtration chromatography, thin layer chromatography, radial flow chromatography, interference chromatography, and reversed phase chromatography.

81. A method for producing a recombinant AAV for treating hemophilia B, wherein the recombinant AAV produced by the method according to any one of claims 72 to 80 has an increased Full capsid ratio (%Full capsid) compared to the recombinant AAV produced by triple transfection.

82. A method for producing a recombinant AAV for treating hemophilia B according to any one of claims 72 to 81, wherein the recombinant AAV produced by the method exhibits an increased FIX expression level compared to the recombinant AAV produced by triple transfection.

83. A method for producing a recombinant AAV for treating hemophilia B, according to any one of claims 72 to 82, wherein the recombinant AAV produced by the method exhibits increased FIX coagulation ability compared to the recombinant AAV produced by triple transfection.

84. A recombinant AAV produced by the plasmid of any one of claims 16 to 71.

85. In claim 84, the recombinant AAV is a self-complementary AAV (scAAV).

86. Containing AAV capsid and genome, The above genome contains a transgene, The transgene comprises a codon-optimized polynucleotide encoding a FIX of any one of claims 1 to 15. Recombinant adeno-associated virus (AAV) for treating hemophilia B.

87. A recombinant AAV for treating hemophilia B according to claim 86, wherein the AAV capsid is any one AAV capsid selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.

88. A recombinant AAV for treating hemophilia B, wherein the AAV capsid of claim 86 is a wild-type AAV8 capsid.

89. A recombinant AAV for treating hemophilia B according to claim 86, wherein the genome is a linear single-stranded DNA.

90. A recombinant AAV for treating hemophilia B, wherein the transgene is arranged between ITRs (Inverted Terminal Repeats) according to claim 86.

91. A recombinant AAV for treating hemophilia B, wherein the transgene is arranged between L-ITR and R-ITR, according to claim 86.

92. A recombinant AAV for treating hemophilia B according to claim 90, wherein the ITR is derived from at least one selected from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV11, AAV12, and AAV13.

93. A recombinant AAV for treating hemophilia B, wherein the ITR of claim 90 is derived from AAV2.

94. A recombinant AAV for treating hemophilia B, wherein the ITR of claim 90 is a wild-type ITR or a mutant thereof.

95. A recombinant AAV for treating hemophilia B according to claim 90, wherein the ITR comprises all or part of a wild-type ITR sequence.

96. A recombinant AAV for treating hemophilia B according to claim 91, wherein either the L-ITR or the R-ITR does not contain trs (terminal resolution site).

97. In claim 91, Either of the above L-ITR and R-ITR does not contain trs, The other one is the wild type ITR, Recombinant AAV for treating hemophilia B.

98. In claim 91, Either of the above L-ITR and R-ITR is an ITR mutant with a deletion of the D region, The other one is the wild type ITR, Recombinant AAV for treating hemophilia B.

99. In claim 91, The above L-ITR consists of sequence number 3, The above R-ITR is composed of sequence number 8, Recombinant AAV for treating hemophilia B.

100. A recombinant AAV for treating hemophilia B, according to any one of claims 96 to 99, wherein the recombinant AAV is self-complementary AAV (scAAV).

101. A recombinant AAV for treating hemophilia B, further comprising a nucleotide sequence consisting of SEQ ID NO: 7 upstream of the R-ITR according to claim 91.

102. A recombinant AAV for treating hemophilia B according to claim 86, wherein the genome further comprises an expression regulatory element operably linked to the transgene.

103. A recombinant AAV for treating hemophilia B according to claim 102, wherein the expression regulatory element is a tissue-specific expression regulatory element.

104. A recombinant AAV for treating hemophilia B according to claim 102, wherein the expression regulatory element comprises at least one selected from an enhancer and a promoter.

105. In claim 104, The above enhancer is a liver tissue-specific enhancer, The above promoter is a liver tissue-specific promoter, Recombinant AAV for treating hemophilia B.

106. In claim 104, The above enhancer is mTTR (mouse transthyretin) enhancer or a variant thereof, The above promoter is an mTTR promoter or a variant thereof, Recombinant AAV for treating hemophilia B.

107. A recombinant AAV for treating hemophilia B according to claim 104, wherein the enhancer and promoter are mutants of an mTTR enhancer and promoter in which nucleotides at positions 1 to 22 and 124 to 138 are deleted from the nucleotide sequence of a wild-type mTTR enhancer and promoter, and ACAGGA is inserted after position 361.

108. A recombinant AAV for treating hemophilia B according to claim 107, wherein the variant of the mTTR enhancer and promoter comprises SEQ ID NO:

4.

109. A recombinant AAV for treating hemophilia B, according to claim 86, wherein the genome additionally comprises an intron.

110. A recombinant AAV for treating hemophilia B, wherein the intron is a modified SV40 intron, according to claim 109.

111. A recombinant AAV for treating hemophilia B, wherein the modified SV40 intron comprises SEQ ID NO: 5, according to claim 110.

112. A recombinant AAV for treating hemophilia B according to claim 86, wherein the genome further comprises a polyadenylation (poly A) signal sequence.

113. A recombinant AAV for treating hemophilia B according to claim 112, wherein the polyadenylation signal sequence is a rabbit globin poly A signal sequence.

114. A recombinant AAV for treating hemophilia B according to claim 113, wherein the rabbit globin poly A signal sequence comprises SEQ ID NO:

6.

115. A recombinant AAV for treating hemophilia B according to any one of claims 86 to 114, wherein the genome comprises, in 5' to 3' order, an L-ITR, an enhancer, a promoter, an intron, a codon-optimized polynucleotide encoding the FIX of any one of claims 1 to 15, a polyadenylation signal sequence, and an R-ITR.

116. In claim 115, The above L-ITR is an ITR variant of AAV2, wherein the variant does not contain trs; The above enhancer and promoter are mutants of the mTTR enhancer and promoter, wherein the mutant has nucleotides at positions 1 to 22 and 124 to 138 deleted and ACAGGA inserted after position 361 in the nucleotide sequence of the wild-type mTTR enhancer and promoter; The above intron is a modified SV40 intron; The above polyadenylation signal sequence is a rabbit globin polyadenylation (rabbit globin poly A) signal sequence; and The above R-ITR is the wild-type ITR of AAV2. Recombinant AAV for treating hemophilia B.

117. In claim 116, The above L-ITR consists of sequence number 3; The above enhancer and promoter are composed of sequence number 4; The above intron consists of sequence number 5; The above polyadenylation signal sequence is composed of sequence number 6; and The above R-ITR is composed of sequence number 8, Recombinant AAV for treating hemophilia B.

118. A recombinant AAV for treating hemophilia B according to any one of claims 86 to 117, wherein the genome further comprises a nucleotide sequence consisting of SEQ ID NO: 7 between the polyadenylation signal sequence and the R-ITR.

119. A recombinant AAV produced by a plasmid according to any one of claims 16 to 71 or a recombinant AAV according to any one of claims 86 to 118; and Comprising a pharmaceutically acceptable carrier, A pharmaceutical composition for preventing or treating hemophilia B.

120. A method for delivering Factor IX (FIX) to a subject in need thereof, comprising administering to the subject an effective amount of a recombinant AAV produced by the plasmid of any one of claims 16 to 71, a recombinant AAV of any one of claims 86 to 118, or a pharmaceutical composition of claim 119.

121. A method of delivering FIX to a subject in need thereof, wherein the subject is a hemophilia B patient, according to claim 120.

122. A method for treating hemophilia B, comprising administering to a subject an effective amount of a recombinant AAV produced by the plasmid of any one of claims 16 to 71, a recombinant AAV of any one of claims 86 to 118, or a pharmaceutical composition of claim 119.

123. A method for treating hemophilia B according to claim 122, wherein the subject is a mammal.

124. A method for treating hemophilia B according to claim 123, wherein the mammal is a human.

125. Use of a plasmid according to any one of claims 16 to 71, a recombinant AAV produced by said plasmid, or a recombinant AAV according to any one of claims 86 to 118, for the manufacture of a medicament for the treatment of hemophilia B.

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