Novel dual helper plasmid

Abstract

We provide a dual helper plasmid for recombinant adeno-associated virus production. [Solution] The present invention provides a dual helper plasmid comprising an E2a gene, an E4 gene, a VA RNA gene, and a rep-cap gene, wherein the E2a, E4, and VA RNA genes are sequentially ligated, and the rep-cap gene is located clockwise (from 5' to 3') between the 5' end of the E2a gene and the 3' end of the VA RNA gene. Compared to the triple phenotypic infection method used in traditional adeno-associated virus production, the dual phenotypic infection method of the present invention offers advantages such as 1) increased probability of simultaneous phenotypic infection, 2) increased production rate of recombinant adeno-associated virus, and 3) reduced costs and time for plasmid production and purification, making it useful for the efficient production of gene therapy agents.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to a novel dual helper plasmid for the production of recombinant AAV, which is a gene delivery vector.

[0002] [Background Art] Adeno-associated virus (AAV) is a single-stranded DNA virus and belongs to helper-dependent parvoviruses that require assistance such as adenovirus for growth. It is a non-pathogenic virus with a genome size of about 4.7 kbp and does not induce a cellular immune response. The target of infection varies greatly depending on the serotype, and the virus can transfer genes to non-dividing and dividing cells. In particular, the expression of genes transferred by AAV persists in vivo for a long time [1-3].

[0003] Generally, recombinant adeno-associated virus is produced by triple transfection of host cells (e.g., HEK293 cells) [4]. Recombinant AAV production using triple transfection requires 1) an AAV construct plasmid having a gene expression cassette surrounded by ITRs (inverted terminal repeats), 2) a "Rep-Cap plasmid" that provides the Rep protein necessary for the replication of the adeno-associated virus genome and the capsid protein that constitutes the virus particle, and finally, 3) a "helper plasmid" that provides the adenovirus proteins (E2a, E4) and RNA (VA RNA) useful for the life cycle of the adeno-associated virus. When these three types of plasmids are transfected into a HEK293 cell line that provides the E1 and E3 genes of adenovirus, adeno-associated virus is produced [5]. When these three plasmids are transfected into a HEK293 cell line that provides the E1 and E3 genes of adenovirus, adeno-associated virus is produced [5].

[0004] Recombinant adeno-associated virus vectors are produced only in cells that are simultaneously transfected with the three plasmids mentioned above. If two plasmids are combined into one for double transfection, the probability of co-transfection will increase, and as a result, the production rate of recombinant adeno-associated virus will increase [6,7]. In addition, reducing the number of plasmids required to produce recombinant adeno-associated virus from three to two will save time and costs associated with plasmid production and purification [8].

[0005] Recombinant adeno-associated virus vectors do not possess the rep and cap genes and do not have adenovirus-derived genes, and therefore cannot replicate in vivo.[9]

[0006] However, in the process of creating a dual helper plasmid, the relative positions of the rep, cap, E2, E4, and VA RNA genes within the plasmid change depending on the method used to combine the Rep-Cap plasmid and the helper plasmid. This affects the expression of each gene to different degrees, making the impact on productivity very difficult to predict.

[0007] Therefore, there is a pressing need for a dual-helper plasmid and a method for producing it that is designed to increase the trait infection rate of recombinant adeno-associated virus vectors and the resulting production rate of recombinant adeno-associated virus, while also reducing the time and cost required for production and purification.

[0008] [Prior art document] [Non-patent literature] [Non-Patent Literature 1] BioDrugs. 2017. 31(4):317-334. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. MFNaso, B. Tomkowicz, WLPerry3rd, WRStrohl. [End Page 2]Expert Opin.Drug Saf.2002.1(1):79-91.Safety of Adeno-Associated Viral Gene Therapy Vectors:A Current Evaluation.PEMonahan,K.Jooss,MSSands. [ Abstract ] Gene Ther.1996.3(8):658-68.Safety of single-dose administration of an adeno-associated virus(AAV)-CFTR vector in the primate lung.CKConrad,SSAllen,SAAfione,TCReynolds,SEBeck,MFee-Maki,XBarrazza-Ortiz,RAdams,FBAskin,BJCarter,WBGuggino,TRFlotte. [Reference 4]J.Virology.1998.72(3):2224-2232.Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.X.Xiao,J.Li,RJSamulski. [End Page 5]Viruses.2010.2(8):1681-1703.Adenoviral Producer Cells.I.Kovesdi and SJHedley. [End Page 6]Hum.Gene Ther.1998.9(18):2745-2760.Novel Tools for Production and Purification of Recombinant Adeno-Associated Virus Vectors. [End Page 7]Mol.Ther.2003.7(6):839-850.Helper virus-free,optically controllable,and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6. D. Grimm, MAKay, JAKleinschmidt. [Non-patent document 8] Biores.Open Access.2020.9(1):219-228.Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus.Q.Tang,AMKeeler,S.Zhang,Q.Su,Z.Lyu,Y.Cheng,G.Gao,TRFlotte. [Non-Patent Document 9] Hum.Gene Ther.2017.28(11):1075-1086 Small But Increasingly Mighty:Latest Advances in AAV Vector Research,Design,and Evolution.D.Grimm,H.Buening. [Overview of the prefecture] [Means for solving the problem]

[0009] The present invention provides a dual helper plasmid comprising an E2a gene, an E4 gene, a VA RNA gene, and a rep-cap gene, wherein the E2a, E4, and VA RNA genes are sequentially ligated, and the rep-cap gene is located clockwise (from 5' to 3') between the 5' end of the E2a gene and the 3' end of the VA RNA gene.

[0010] The present invention provides a dual helper plasmid comprising an E2a gene, an E4 gene, a VA RNA gene, and a rep-cap gene, wherein the E2a, E4, and VA RNA genes are sequentially ligated, and the rep-cap gene is located counterclockwise (from 3' to 5') between the 5' end of the E2a gene and the 3' end of the VA RNA gene.

[0011] The present invention provides a dual helper plasmid comprising a regulatory component, the regulatory component comprising (from 5' to 3') the following: (i) The E2a gene containing the sequence presented in Sequence ID No. 34; (ii) The E4 gene containing the sequence presented in Sequence ID No. 35; (iii) VA RNA gene containing the sequence presented in Sequence ID No. 36; (iv) the cap gene containing the sequence presented in SEQ ID NO: 30; and (v) A rep gene containing the sequence presented in Sequence ID No. 29.

[0012] The present invention provides a dual helper plasmid comprising a regulatory element, the regulatory element comprising (from 5' to 3') the following: (i) The E2a gene containing the sequence presented in Sequence ID No. 34; (ii) The E4 gene containing the sequence presented in Sequence ID No. 35; (iii) VA RNA gene containing the sequence presented in Sequence ID No. 36; (iv) the cap gene containing the sequence presented in Sequence ID No. 31; and (v) A rep gene containing the sequence presented in Sequence ID No. 29.

[0013] The present invention provides a dual helper plasmid comprising a regulatory element, the regulatory element comprising (from 5' to 3') the following: (i) The E2a gene containing the sequence presented in Sequence ID No. 34; (ii) The E4 gene containing the sequence presented in Sequence ID No. 35; (iii) VA RNA gene containing the sequence presented in Sequence ID No. 36; (iv) the cap gene containing the sequence presented in Sequence ID No. 32; and (v) A rep gene containing the sequence presented in Sequence ID No. 29.

[0014] The present invention provides a dual helper plasmid comprising a regulatory element, the regulatory element comprising (from 5' to 3') the following: (i) The E2a gene containing the sequence presented in SEQ ID NO: 34; (ii) The E4 gene containing the sequence presented in SEQ ID NO: 35; (iii) The VA RNA gene containing the sequence presented in SEQ ID NO: 36; (iv) The cap gene containing the sequence presented in SEQ ID NO: 33; and (v) The rep gene containing the sequence presented in SEQ ID NO: 29.

[0015] The present invention provides a composition comprising the dual helper plasmid of the present invention.

[0016] The present invention provides a cell comprising the dual helper plasmid or composition of the present invention.

[0017] The present invention provides a method for producing recombinant AAV comprising the step of transforming a cell to contain a first plasmid and a second plasmid, wherein the first plasmid is the dual helper plasmid of the present invention and the second plasmid contains a transgene.

[0018] The present invention provides a method for increasing the production yield of recombinant AAV comprising the step of transforming a cell to contain a first plasmid and a second plasmid, wherein the first plasmid is the dual helper plasmid of the present invention, the second plasmid contains a transgene, and the amount of recombinant AAV produced after the transformation is increased as compared to the corresponding amount produced by a reference method.

[0019] The present invention provides recombinant AAV produced by the method of the present invention.

[0020] The present invention provides a pharmaceutical formulation comprising the recombinant AAV of the present invention and a pharmaceutically acceptable excipient.

[0021] The present invention provides a method for treating a disease or disorder in a subject in need thereof, comprising the step of administering to the subject the recombinant AAV or pharmaceutical formulation of the present invention.

[0022] The present invention provides uses for recombinant AAV or pharmaceutical formulations of the present invention in the manufacture of drugs for treating diseases or disorders in subjects that require them.

[0023] [Brief description of the drawing] [Figure 1] Figures 1A to 1F show the cleavage maps of pHelper-NG and Helper-In-One constructs (pHION8 series) for producing adeno-associated virus vector serotype 8 (AAV8) manufactured by the inventors. Figure 1A schematically shows the pHelper-NG construct for triple phenotypic infection, which contains the E2a, E4 genes and VA RNA gene of adenovirus serotype 5. Figures 1B to 1F schematically show five Helper-In-One-NG plasmid (pHION8) constructs, which are dual helper plasmids manufactured by inserting the rep2-cap8 gene fragment into various regions of the pHelper-NG construct. Figures 1B and 1C schematically show the pHION8-BF construct (Figure 1B) and the pHION8-BR construct (Figure 1C) produced by cloning the rep2-cap8 construct in the forward direction (clockwise, 5'->3') and in the reverse direction (counterclockwise, 3'->5') using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene. Figures 1D and 1E schematically show the pHION8-NF construct (Figure 1D) and the pHION8-NR construct (Figure 1E) produced by cloning the rep2-cap8 construct in the forward direction and in the reverse direction using the NotI site located at the start of the E4 gene. Figure 1F schematically shows the pHION8-AF construct produced by cloning the rep2-cap8 construct in the forward direction using the AsiSI site located between the start of the VA RNA gene and the terminal of the E4 gene.

[0024] [Figure 2] This shows the cleavage map of the pHNG2 construct, a dual helper plasmid for the production of adeno-associated virus vector serotype 2 (AAV2). The pHNG2 construct was produced by inserting the rep2-cap2 gene fragment in the forward direction into the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene within the pHelper-NG plasmid.

[0025] [Figure 3] This shows the cleavage map of the pHNG9 construct, a dual helper plasmid for the production of adeno-associated virus vector serotype 9 (AAV9). For dual phenotypic infection, the rep2-cap9 gene fragment was cloned in the forward direction using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene to produce the pHNG9 construct.

[0026] [Figure 4] This shows the cleavage map of the pHNG5K construct, a dual helper plasmid for the production of adeno-associated virus vector serotype 5 (AAV5). For dual phenotypic infection, the rep2-cap5 gene fragment was cloned in the forward direction using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene to produce the pHNG5K construct.

[0027] [Figure 5] Figures 5A and 5B show the cleavage maps of the pHNG and pHNGR constructs. Figure 5A shows a rep-cap gene fragment inserted in the forward direction between the start portion of the E2a gene and the terminal portion of the VA RNA gene, and Figure 5B shows a rep-cap gene fragment inserted in the reverse direction.

[0028] [Figure 6] This shows the results of observing the effect of double phenotypic infection using the pHION8 series on increasing or decreasing AAV8 production in adherent cell line (HEK293), compared to triple phenotypic infection.

[0029] [Figure 7] This shows the results of observing the effect of double phenotypic infection using the pHION8 series on increasing or decreasing AAV8 production in a suspension cell line (Expi293), compared to triple phenotypic infection.

[0030] [Figure 8] This shows the results of observing the effect of double phenotypic infection using pHNG2 on increasing AAV2 production in adherent cell line (HEK293), compared to triple phenotypic infection.

[0031] [Figure 9] This shows the results of observing the effect of double phenotypic infection using pHNG9 on increasing AAV9 production in adherent cell line (HEK293), compared to triple phenotypic infection.

[0032] [Figure 10] This shows the results of observing the effect of double phenotypic infection using pHNG5K on increasing AAV5 production in adherent cell line (HEK293), compared to triple phenotypic infection.

[0033] [Figure 11] Equivalence results of cell transduction efficiency between AAV8 produced by triple or double transduction with the Helper-In-One construct.

[0034] [Figure 12] Equivalence results of cell transduction efficiency between AAV2 produced by triple transduction or double transduction using pHNG2 constructs.

[0035] [Figure 13] Equivalence results of cell transduction efficiency between AAV9 produced by triple transduction or double transduction using pHNG9 constructs.

[0036] [Modes for carrying out the invention] This invention relates to compositions and methods for producing recombinant AAVs, and to the aforementioned uses of rAAVs for treating diseases or disorders. As described herein, the dual helper plasmids of the present invention comprise multiple genes (e.g., E2a gene, E4 gene, VA RNA gene, rep gene, and cap gene). The applicant has confirmed that by sequencing multiple genes in a specific configuration, yield or productivity can be increased or decreased during recombinant AAV production. Other aspects of the present invention are provided throughout this application.

[0037] I. Definition Throughout this invention, the term "one" or "one" means one or more of the entities. For example, "one polypeptide" is understood to mean one or more polypeptides. Thus, the terms "one" (or "one"), "one or more" and "at least one" may be used interchangeably in this specification.

[0038] Furthermore, as used herein, “and / or” should be considered as specific disclosures of two expressed features or components, each of which may or may not be present. Accordingly, as used herein, the term “and / or” in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone) and “B” (alone). Similarly, as used in phrases such as “A, B and / or C,” the term “and / or” is intended to include each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0039] The term "at least" preceding a number or a sequence of numbers is understood to include all subsequent numbers or integers that are clearly defined in the context in which the term "at least" is adjacent and logically possible. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the stated characteristic. When preceding at least a sequence of numbers or a range, "at least" is understood to modify each number in the sequence or range. "At least" is not limited to integers (for example, "at least 5%" includes 5.0%, 5.1%, and 5.18%, without considering the number of valid digits).

[0040] Where the term “includes” is used herein, similar forms are also provided, which are described using the terms “constituted” and / or “essentially constituted.”

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by a person of ordinary skill in the art of the present invention. For example, the following references provide the general meanings of many of the terms used herein: The Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press.

[0042] Units, prefixes, and symbols are based on the SI (Systems International de Nobel Memorial). The units are displayed in an acceptable format. Numerical ranges include a number that limits the range. Unless otherwise specified, amino acid sequences are recorded from left to right, from amino to carboxyl. The titles provided herein are not limitations on the various aspects of the invention that may have by referring to the entire specification. Therefore, the terms defined below are more fully defined by referring to the entire specification.

[0043] The term "approximately" is used herein to mean roughly, approximately, around, or in a range. When the term "approximately" is used with a numerical range, it modifies that range by extending the boundary above or below the specified numerical value. Generally, the term "approximately" can modify a numerical value above or below (higher or lower than) the specified value by, for example, a 10% variation.

[0044] As used herein, the term "adeno-associated virus" (AAV) refers to AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh.74, snake AAV, bird AAV, cattle AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and their AAV serotypes and lineages (Gao et al. (J.Virol.78:6381 (2004)) and Moris et al. This includes, but is not limited to, the work of al. (Virol. 33:375 (2004)) and other AAVs currently known or to be discovered later (e.g., reference (FIELDS). See et al. Virology 2, Chapter 69 (4th ed., Lippincott-Raven Publishers). In some respects, “AAV” includes derivatives of known AAVs. In some respects, “AAV” includes modified or artificial AAVs.

[0045] The terms “administer,” “to administer,” and their synonyms mean introducing a composition (e.g., a recombinant AAV signaling vector produced using the dual helper plasmid of the present invention) into a target organism via a pharmaceutically acceptable route. Introducing a composition into a target organism is by any suitable route, including intratumorally, orally, pulmonarily, intratranasally, parenterally (intavenously, intra-arterially, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intrathecally, periocularly, or topically. Administration includes self-administration and administration by another person. A suitable route of administration ensures that the composition or agonist performs its intended function. For example, if the appropriate route is intravenous, the composition or agonist is administered by introducing it into the vein of the target body.

[0046] In the present invention, “antibiotic resistance gene” means a gene inserted into a plasmid for the purpose of conferring drug resistance to a microorganism so that it can survive exposure to antibiotics. As described in this application, in some respects, an antibiotic resistance gene can be introduced into a plasmid (e.g., a dual helper plasmid) to screen for cells having a desired plasmid using cloning. Examples of antibiotic resistance genes useful in the present invention include, but are not limited to, resistance genes for ampicillin, kanamycin, chloramphenicol, gentamicin, streptomycin, tetracycline, erythromycin, vancomycin, penicillin, spectinomycin, chloramphenicol, sulfadiazine, and trimethoprim.

[0047] Furthermore, as described herein, the dual helper plasmid of the present invention includes rep and cap genes present with the dual helper plasmid in a specific orientation / configuration. In some aspects, the rep and cap genes are in a "clockwise direction," "forward direction," or "5'→3'" (see, for example, Figure 5A), meaning that the rep-cap genes are inserted or included in a clockwise direction, or from the replication initiation site (5') to the replication termination site (3'). In some aspects, the rep and cap genes are in a "counterclockwise direction," "reverse direction," or "3'→5'" (see, for example, Figure 5B), meaning that the rep-cap genes are inserted or included in a counterclockwise direction, or from the replication termination site (3') to the replication initiation site (5').

[0048] In this specification, the term “rep gene” means a gene that encodes one or more open reading frames (ORFs), the ORFs encoding AAV Rep proteins or their variants or derivatives. Such AAV Rep proteins (or their variants or derivatives) are involved in AAV genome replication and / or AAV genome packaging, and the wild-type rep gene encodes four Rep proteins: Rep78, Rep68, Rep52, and Rep40. Unless otherwise specified, “rep gene” includes wild-type rep genes, their derivatives, and artificial rep genes having equivalent function. In some aspects, the rep gene may be the rep2 gene derived from adeno-associated virus serotype 2 (AAV2). In some aspects, the rep2 gene includes the nucleic acid sequence presented in Sequence ID No. 29.

[0049] In this specification, the term “cap gene” means a gene that encodes one or more open reading frames (ORFs), the ORFs encoding the AAV Cap structural protein, or its variants or derivatives. Four proteins are translated from the cap gene. Of these, the VP1, VP2, and VP3 proteins are structural proteins that make up AAV particles, and the AAP (assembly-activating protein) promotes the formation (assembly) of AAV particles by the structural proteins. Unless otherwise specified, “cap gene” includes the wild-type cap gene, its derivatives, and artificial cap genes having equivalent function. In some aspects, the cap gene is a cap gene derived from adeno-associated virus serotype 2 (AAV2; cap2), serotype 5 (AAV5; cap5), serotype 8 (AAV8; cap8), or serotype 9 (AAV9; cap9). In some aspects, the cap gene contains the nucleic acid sequences presented in SEQ ID NO: 30 (cap2), SEQ ID NO: 31 (cap5), SEQ ID NO: 32 (cap8), or SEQ ID NO: 33 (cap9).

[0050] In this specification, the term “E2a gene” means the gene encoding the adenovirus protein E2a, which regulates the AAV promoter, assists in AAV genome replication, and is involved in increased productivity of the capsid protein through splicing of Rep mRNA and increased stability of capsid mRNA. Unless otherwise specified, “E2a gene” includes the wild-type E2a gene, its derivatives, and artificial E2a genes having equivalent function. In specific embodiments of the present invention, the E2a gene is the E2a gene derived from adenovirus serotype 5 (Ad5). In some aspects, the E2a gene comprises the nucleic acid sequence presented in Sequence ID No. 34.

[0051] In this specification, the term "E4 gene" refers to the gene encoding the adenovirus protein E4, which is involved in the synthesis of the second-strand of the AAV genome during the AAV life cycle and assists in AAV genome replication by suppressing the formation of the MRN (Mre11-Rad50-Nbs1) complex, an intracellular mechanism that represses AAV genome replication. Unless otherwise specified, "E4 gene" includes the wild-type E4 gene, its derivatives, and artificial E4 genes with equivalent function. In some aspects, the E4 gene is the E4 gene derived from adenovirus serotype 5 (Ad5). In some aspects, the E4 gene includes the nucleic acid sequence presented in Sequence ID No. 35.

[0052] In this specification, the terms “VA RNA gene” or “VA RNA region” mean a VA region that produces VA RNA, which helps increase the stability and translation efficiency of AAV capsid mRNA and prevent the degradation of the Rep52 protein. Unless otherwise specified, “VA RNA gene” includes wild-type VA RNA genes, their derivatives, and artificial VA RNA genes having equivalent function. In some aspects, VA RNA genes are derived from adenovirus serotype 5 (Ad5) VA It is an RNA gene. In some respects, the VA RNA gene contains the nucleic acid sequence presented in SEQ ID NO: 36.

[0053] In this specification, the term "conserved" means a nucleotide or amino acid residue of a polynucleotide or polypeptide sequence that occurs unchanged at the same position in two or more sequences being compared. Relatively conserved nucleotides or amino acids are more conserved between related sequences than nucleotides or amino acids that appear elsewhere in the sequence.

[0054] In this specification, the term “control element” means a nucleic acid sequence that modulates (e.g., increases or decreases) the expression of an operablely linked nucleic acid (e.g., an exogenous gene). Non-limiting examples of suitable control elements are provided elsewhere in the invention.

[0055] "Completely conserved" or "identical" is used when two or more sequences are 100% identical to each other in some aspects. "Highly conserved" is used when two or more sequences are at least 70%, 80%, 90%, or 95% identical to each other in some aspects. "Highly conserved" is used when two or more sequences are approximately 70%, 80%, 90%, 95%, 98%, or 99% identical to each other in some aspects. "Conserved" is used when two or more sequences are at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% identical to each other in some aspects. A sequence is described as "conserved" if, in some aspect, two or more sequences are identical to each other by approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%. Sequence conservation may apply to the entire length of a polynucleotide or polypeptide, or to a portion, region, or feature thereof.

[0056] In this specification, the term “enhancer” means a segment of DNA that contains a sequence capable of providing improved transcription and, in some cases, can act independently of their orientation toward other regulatory sequences. Enhancers may act cooperatively or additively with promoters and / or other enhancing elements.

[0057] The terms “excipient” and “carrier” are used interchangeably and refer to inert substances added to a pharmaceutical composition to facilitate the administration of recombinant AAV signaling vectors produced using the dual helper plasmids provided herein, including compounds, such as exogenous genes.

[0058] The term "exon" refers to a limited portion of a protein-encoding nucleic acid, or a nucleic acid sequence represented in the mature form of an RNA molecule after a portion of pre-processed (or precursor) RNA has been removed by splicing. The mature RNA molecule may be a messenger RNA (mRNA) or a functional form of unencoded RNA such as rRNA or tRNA.

[0059] The term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or polypeptide. This includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA) and the translation of mRNA into a polypeptide. Expression produces a “gene product” or “encoded protein.” As used herein, a gene product may be a nucleic acid, such as RNA produced by the transcription of a gene. In this specification, a gene product may be a nucleic acid or polypeptide translated from a transcript. Gene products described herein include nucleic acids having post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides having post-translational modifications, such as phosphorylation, methylation, glycosylation, lipid addition, binding to other protein subunits, or proteolytic cleavage.

[0060] In this specification, the term "identity" refers to the overall monomer conservation between polymer molecules, for example, between polynucleotide molecules. In relation to the term "identical" without additional modifiers, for example, polynucleotide A being identical to polynucleotide B means that the polynucleotide sequences are 100% identical (100% sequence identity). For example, describing two sequences as "70% identical" is equivalent to describing something that has "70% sequence identity".

[0061] For example, the calculation of percentage identity between two polypeptide or polynucleotide sequences may be performed by aligning the two sequences for optimal comparison purposes (for example, gaps may be introduced in either or both of the first and second polypeptide or polynucleotide sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain aspects, the length of the aligned sequences for comparison purposes is a reference sequence length of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the reference sequence length. For amino acids or polynucleotides at the relevant amino acid positions, the bases are compared.

[0062] Molecules are identical at a given position if that position in the first sequence is occupied by the same amino acid or nucleotide in the second sequence. Percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap; this must be introduced for optimal alignment of the two sequences. Sequence comparison and measurement of percentage identity between two sequences can be achieved using mathematical algorithms.

[0063] Suitable software programs for aligning various sequences (e.g., polynucleotide sequences) are available from various sources. One suitable program for measuring percent sequence identity is bl2seq, part of the BLAST product suite available on the U.S. government's National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). bl2seq compares the two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics program suite and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0064] Sequence sorting may be performed using methods known in the industry, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), or MUSCLE.

[0065] Various regions within a single polynucleotide or polypeptide target sequence aligned with a polynucleotide or polypeptide reference sequence may each possess their own percentage sequence identity. It should be noted that the percentage sequence identity value is rounded to the nearest tenth of the value. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Also, the length value is always an integer.

[0066] In some aspects, the percentage identity (%ID) of the first amino acid sequence (or nucleic acid sequence) to the second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100x(Y / Z), where Y is the number of amino acid residues (or nuclear bases) recorded as being identical in the alignment of the first and second sequences (aligned by visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percentage identity between the first and second sequences will be higher than the percentage identity of the second sequence to the first sequence.

[0067] Those skilled in the art will understand that the generation of sequence sorting for the calculation of percent sequence identity is not limited to binary sequence-to-sequence comparison driven exclusively by primary sequence data. They will also understand that sequence sorting can be obtained by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structure), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to obtain multiple sequence sorting is T-Coffee, available at www.tcoffee.org, and alternatively, available from EBI, for example. Furthermore, they will understand that the final sorting used to calculate percent sequence identity can be selected automatically or manually.

[0068] In this specification, the terms “isolated,” “purified,” “extracted,” and their synonyms are used interchangeably and refer to a state of a preparation of a specific composition of the present invention, such as a polynucleotide preparation containing foreign genes and off-label nucleic acid sequences, that has undergone one or more purification processes. In some aspects, isolation or purification as used herein refers to the process of removing, partially removing (e.g., fractionating) the polynucleotides described herein from a composition of the present invention, such as a sample containing contaminants.

[0069] In this specification, the term “linked” means a first amino acid sequence or polynucleotide sequence covalently or noncovalently linked to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid sequence or polynucleotide sequence may be directly linked to or juxtaposed with the second amino acid sequence or polynucleotide sequence, or an intervening sequence may covalently link the first sequence to the second sequence. The term “linked” means that the first and second polynucleotide sequences are fused at the 5' or 3' end, and also includes insertions into any two nucleotides of the second polynucleotide sequence (or the first polynucleotide sequence) of the entire first polynucleotide sequence (or the second polynucleotide sequence). The first polynucleotide sequence may be linked to the second polynucleotide sequence by a phosphodiester bond or a linker, which may be, for example, a polynucleotide.

[0070] The terms “miRNA,” “miR,” and “microRNA” are interchangeable and refer to microRNA molecules found in eukaryotes involved in RNA-based gene regulation. This term is used to describe single-stranded RNA molecules processed from precursors. In some aspects, the term “antisense oligomer” may also be used to describe the microRNA molecules of the present invention. The names and sequences of miRNAs related to the present invention are provided herein. MicroRNAs recognize and bind to target mRNAs through incomplete base pairing, inducing destabilization or translational repression of the target mRNA and downregulating target gene expression. Conversely, targeting miRNAs with molecules containing miRNA-binding sites (generally molecules containing sequences complementary to the seed region of the miRNA) can reduce or suppress miRNA-induced translational repression, which causes upregulation of target genes.

[0071] The terms "nucleic acid," "nucleic acid molecule," "nucleotide sequence," "polynucleotide," and their synonyms are mutually interchangeable and refer to a nucleotide sequence linked by phosphodiester bonds. Polynucleotides are presented herein in the 5' to 3' direction. The polynucleotides of the present invention may be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are represented herein by the following single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0072] In this specification, the terms “operatively linked” or “operably linked” mean that linked DNA sequences are adjacent to each other and perform a desired function. For example, a promoter may be operatively linked to a coding region when it is used to initiate transcription of a coding sequence (e.g., an exogenous gene). The promoter and coding region do not necessarily have to be adjacent to each other as long as such a functional relationship is maintained.

[0073] "Pharmacochemically acceptable carrier," "pharmacochemically acceptable excipient," and their synonyms include any formulation and any carrier or diluent approved by a U.S. federal regulatory agency or described in the U.S. Pharmacopoeia for use in animals, including humans, that prohibits the administration of the composition to a subject and does not induce the production of undesirable physiological effects to the extent that it does not interfere with the biological activity and properties of the administered compound. This includes preferred excipients and carriers that are useful in the manufacture of pharmaceutical compositions, are generally safe and non-toxic.

[0074] In this specification, the term “pharmaceutical composition” means one or more compositions described herein (e.g., polynucleotides, vectors, cells and / or recombinant viruses) mixed with or combined with one or more other chemical components, such as pharmaceutically acceptable carriers and excipients, or suspended therein.

[0075] As used herein, the terms “promoter” and “promoter sequence” may be used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located at 3' of the promoter sequence. Promoters may be derived entirely from a native gene, or they may consist of various elements derived from various promoters found in nature, or they may include synthetic DNA segments. It will be understood by those skilled in the art that various promoters can direct gene expression in various tissues or cell types, at various developmental stages, or in response to various environmental or physiological conditions. In general, promoters that cause gene expression in most cell types are usually called “constitutive promoters.” Promoters that cause gene expression in specific cell types are usually called “cell-specific promoters” or “tissue-specific promoters.” Promoters that express genes at specific stages of development or cell differentiation are typically called "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that induce gene expression after exposure or treatment of cells with promoter-inducing agents, biological molecules, chemicals, ligands, or light are typically called "inducible promoters" or "regulatory promoters." Furthermore, it is generally recognized that DNA fragments of different lengths can possess the same promoter activity because the precise boundaries of regulatory sequences are not fully defined.

[0076] The promoter sequence is typically bound to its 3' end by a transcription start site and extended upward (5' direction) to contain the minimum number of bases or elements necessary to initiate transcription at a level detectable from the background. Within the promoter sequence, in addition to the protein-binding domain (consensus sequence) responsible for RNA polymerase binding, a transcription start site (conveniently defined, for example, by mapping to nuclease S1) will be identified. In some aspects, promoters usable in this invention include tissue-specific promoters.

[0077] In this specification, the terms “gene regulatory region” or “regulatory region” mean a nucleotide sequence located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding region that affects the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. When a coding region is intended to be expressed in eukaryotic cells, the polyadenylation signal and transcription termination sequence will generally be located at 3' of the coding sequence.

[0078] In some respects, nucleic acid compositions (e.g., expression constructs) provided herein may include promoters and / or other expression (e.g., transcription) regulators operably associated with one or more coding regions. In an operably associated relationship, a coding region to a gene product is associated with one or more regulatory regions in such a way that it places the expression of the gene product under the influence or control of the regulatory regions. For example, a coding region and a promoter are "operably associated" if the induction of promoter function induces the transcription of mRNA encoding the gene product encoded by the coding region, and the linkage characteristics between the promoter and the coding region should not interfere with the promoter's ability to direct the expression of the gene product or the ability of the DNA template to be transcribed. In addition to promoters, other expression regulators, such as enhancers, operators, repressors, and transcription termination signals, may also be operably associated with coding regions that direct the expression of a gene product.

[0079] In this specification, the term "transgene" refers to one or more polynucleotides or polynucleotide regions encoded within a recombinant expression construct, or polynucleotides or regulatory nucleic acids that encode expression products, polypeptides, or multiple polypeptides of such polynucleotides or polynucleotide regions. This means regulatory nucleic acid. In some respects, the exogenous gene may be a nucleotide sequence encoding a therapeutic peptide for a specific disease, intended for sustained expression within the body of a subject or patient. In some respects, "operatically linked" or "operatably linked" means that the linked DNA sequences are located adjacent to each other so as to perform a desired function. For example, if a particular promoter is used to help initiate transcription of a coding sequence (e.g., an exogenous gene), such a promoter may be operationally linked to the coding region. The promoter and coding region do not necessarily have to be located adjacent to each other as long as such a functional relationship is maintained.

[0080] In this specification, the terms “vector” or “construct” mean a construct into which a nucleic acid or gene can be inserted, preferably including a carrier into which a nucleic acid sequence can be inserted for introduction into cells capable of replicating the nucleic acid sequence. The nucleic acid sequence may be exogenous or heterologous. The nucleic acid sequence may be a foreign gene. Examples of constructs include, but are not limited to, plasmids, cosmids, and viruses (e.g., AAV). Those skilled in the art can construct such vectors or constructs by standard recombination techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Co. Sons, Inc., NY, 1994.

[0081] In this specification, the terms “expression vector” or “expression construct” mean a vector or construct containing a nucleotide sequence that encodes at least a portion of a transcribed gene product. In some cases, the transcribed RNA molecule is translated into a protein, polypeptide, or peptide. Expression constructs may contain various control elements. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleotide sequences that provide other functions.

[0082] In this specification, the term “subject” means an AAV (produced using the methods provided herein) or a composition containing such AAV to which an AAV is administered. Non-limiting examples include humans, livestock (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.) and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.) requiring diagnosis, treatment, or therapy, and especially humans. The methods described herein are applicable to both human therapeutic and veterinary applications.

[0083] In this specification, “subject in need thereof” includes subjects that benefit from the administration of the compositions described herein, such as mammals.

[0084] In this specification, the term “therapeutic effective amount” refers to the amount of a reagent or pharmaceutical compound containing a sufficient amount of the composition of the present invention (e.g., a polynucleotide comprising an exogenous gene and an off-nucleic acid sequence) to exhibit the desired therapeutic effect. The therapeutic effective amount may also be a “prophylactically effective amount,” since prevention can be considered therapy.

[0085] In this specification, the term “transgene” means a polynucleotide or modulatory or regulatory nucleic acid that encodes a recombinant expression construct or an expression product of a polynucleotide or polynucleotide region, a polypeptide or multiple polypeptide. In some aspects, the transgene may be heterologous to the cell into which it is inserted (i.e., not spontaneously expressed from the cell).

[0086] The terms “treat,” “treatment,” or “treating” mean, for example, reducing the severity of a disease or condition; shortening the duration of disease progression; improving or eliminating one or more symptoms associated with a disease or condition; or providing a beneficial effect to a subject with a disease or condition without necessarily curing the disease or condition. The terms also include the prevention or mitigation of a disease or condition or its symptoms.

[0087] The term "upstream" refers to the nucleotide sequence located 5' relative to the reference nucleotide sequence.

[0088] In this specification, the terms “vector” or “construct” mean any carrier into which a nucleic acid or gene can be inserted, such as a signaling carrier into which a nucleic acid sequence can be inserted for introduction into a replicable cell. The nucleic acid sequence may be exogenous or heterologous. The nucleic acid sequence may be a foreign gene. Examples of constructs include, but are not limited to, plasmids, cosmids, and viruses (e.g., AAV). Those skilled in the art can construct such vectors or constructs by standard recombination techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc., NY, 1994). In this specification, the terms “expression vector” or “expression construct” mean a vector or construct comprising a nucleotide sequence encoding at least a portion of a transcribed gene product. In some cases, the transcribed RNA molecule is translated into a protein, polypeptide, or peptide. Expression constructs may contain various control elements. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleotide sequences that provide other functions.

[0089] A vector may be engineered to encode a selectable marker or reporter that provides for the selection or identification of cells contaminated with the vector. Expression of a selectable marker or reporter enables the identification and / or selection of host cells that contain and express other coding regions included in the vector. Examples of selectable marker genes known and used in the art include genes that provide resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, etc., and genes used as phenotypic markers, namely anthocyanin regulatory genes, isopentanyltransferase genes, etc. Examples of reporters known and used in this field include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), and β-glucuronidase (Gus). Selectable markers may also be considered as reporters.

[0090] II. Dual Helper Plasmids II.A.E2a gene, E4 gene, VA RNA gene, rep gene, cap gene Some aspects of the present invention relate to dual helper plasmids. As demonstrated herein, such dual helper plasmids may be particularly useful for producing recombinant adeno-associated viruses (AAVs) (e.g., containing an exogenous gene encoding a protein of interest).

[0091] Adeno-associated virus (AAV) is a single-stranded DNA virus and a helper-dependent human parvovirus. Its genome size is approximately 4.7 kbp. The N-terminus of the genome encodes the rep gene, which is involved in viral replication and viral gene expression, while the C-terminus encodes the cap gene, which encodes the viral capsid protein. Approximately 145-base reverse-terminal repeat sequences (ITRs) are inserted at both ends. These 145-bp ITRs, with their T-shaped structure, function as the replication origin during viral gene replication and act as a primary packaging signal. The ITR is the only cis-acting base sequence required to construct recombinant AAV constructs. While it exhibits enhancer activity in the presence of the Rep protein, it has very weak activity in the absence of the Rep protein. Therefore, when cloning foreign genes into recombinant AAV constructs, this must be taken into consideration when appropriately configuring enhancers, promoters, pA, etc., to create the expression construct (RJ). Samulski and N. Muzyczka, Annu. Rev. Virolo. 2014. 1:427-451. Four proteins are translated from the rep gene, which are classified into rep78, rep68, rep52, and rep40 according to their molecular weight, and play an important role in AAV DNA replication. Four proteins are translated from the cap gene, of which VP1, VP2, and VP3 are structural proteins that make up AAV particles, and the assembly-activating protein (AAP) promotes the formation of AAV particles by the aforementioned structural proteins. For the adeno-associated virus to replicate efficiently, certain proteins and RNA derived from a helper virus such as adenovirus or herpes simplex virus are required (Muzyczka N. Curr Top Microbiol Immunol 158,97-129,1992).

[0092] In this specification, the term “dual-helper plasmid” means a plasmid capable of providing two or more of the requirements for generating AAV in a cell. To produce a recombinant AAV vector containing a foreign gene, the following components must be provided to the host cell: (1) the foreign gene, (2) rep and cap proteins, and (3) E2a, E4, and VA RNA proteins. By conventional methods, as described elsewhere in this invention, the different requirements are provided to the cell by three different plasmids: (1) an AAV construct plasmid containing the foreign gene on the ITR side, (2) a Rep-Cap plasmid, and (3) a helper plasmid.

[0093] As is evident from the present invention, in some aspects, the dual helper plasmids described herein provide the requirements (2) and (3) described above. For example, in some aspects, the dual helper plasmid comprises a rep gene, a cap gene, an E2a gene, an E4 gene, and a VA RNA gene.

[0094] As demonstrated herein, the dual helper plasmids described herein include, in addition to the genes mentioned above, the genes are arranged in a specific configuration within the plasmid. For example, in some aspects, the E2a gene, the E4 gene, and the VA RNA gene are sequentially ligated within the dual helper plasmid, and the rep gene and the cap gene (referred to herein as "rep-cap gene") sequentially ligate the 5' end of the E2a gene and the 3' end of the VA RNA gene in a clockwise direction (from 5' to 3'). More specifically, in some aspects, the 5' end of the rep-cap gene is ligated to the 5' end of the E2a gene, and the 3' end of the rep-cap gene is ligated to the 3' end of the VA RNA gene. An unrestrictive example of such a dual helper plasmid is shown in Figure 5A.

[0095] In some aspects, the E2a gene, E4 gene, and VA RNA gene are sequentially ligated, and the rep-cap gene is located counterclockwise (from 3' to 5') between the 5' end of the E2a gene and the 3' end of the VA RNA gene. More specifically, in some aspects, the 3' end of the rep-cap gene is ligated to the 5' end of the E2a gene, and the 5' end of the rep-cap gene is ligated to the 3' end of the VA RNA gene. A non-restrictive example of such a dual helper plasmid is shown in Figure 5B.

[0096] As is evident from the present invention, in some aspects, the respective E2a, E4, and VA RNA genes described above are derived from adenoviruses. In some aspects, the cap gene and rep gene are derived from the same AAV serotype. For example, in some aspects, both the cap gene and rep gene are derived from AAV2 (see, for example, the pUC-R2C2 construct described in Example 1-4). In some aspects, the cap gene and rep gene are derived from AAVs with various serotypes. For example, as demonstrated herein, in some aspects, the cap gene is derived from AAV8 (cap8 or a fragment thereof) and the rep gene is derived from AAV2 (rep2 or a fragment thereof) (see, for example, the pUC-R2C8 construct described in Example 1-2). In some aspects, the cap gene is derived from AAV9 (cap9 or a fragment thereof) and the rep gene is derived from AAV2 (rep2 or a fragment thereof) (see, for example, the pUC-R2C9 construct described in Example 1-6). In some aspects, the cap gene is derived from AAV5 (cap5 or a fragment thereof), and the rep gene is derived from AAV2 (rep2 or a fragment thereof) (see, for example, the pUC-R2C5 constructs described in Examples 1-7).

[0097] Non-limiting examples of AAV that can be used in the present invention are AAVrh.10(AAVrh10), AAV-DJ(AAVDJ), AAV-DJ8(AAVDJ8), AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV. AV4-4、AAV5、AAV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、 AAV9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27.3、AAV42.12、AAV42-lb、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5 b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25 、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAVl-7 / rh.48、AAVl-8 / rh.49、AAV2-15 / rh.6 2、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / rl1.64、AAV4-9 / rh.54、AAV4-1 9 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.l7、AAV33.4 / hu.l5、AAV33.8 / hu.l6、AAV52 / hu.l9、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAV A3.4、AAVA3.5、AAV A3.7、AAVC1、AAVC2、AAVC5、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60 、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-l / hu.1、AAVH-5 / hu. .3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVc y.5、AAVCy.5Rl、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu. AVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23、AAVhu.2 AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39 、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44Rl、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AA Vhu.48、AAVhu.48Rl、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.12、AAVrh.13、AAVrh. h.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh. rh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh. AVrh.52、AAVrh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64Rl、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.8R、AAVrh.8R A586R mutant、AAVrh8R R533A mutant、AAAV、BAAV、caprine AAV、bovine AAV、AAVhEl.1、AAVhErl.5、AAVhER1.14、AAVhErl.8、AAVhErl.16、AAVhErl.18、AAVhErl.35、AAVhErl.7、AAVhErl .36、AAVhEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhER1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK04、AAV-LK05、AAV- LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18、AAV-LK18 LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAV- PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV- b、AAV SM10-2 Shuffle100-1、AAV Shuffle100-3、AAV Shuffle100-7、AAV Shuffle10-2、AAV Shuffle10-6、AAV Shuffle10-8、AAV Shuffle100-2、AAV SM10-1、AAV SM10-8、AAV SM100-3、AAV SM100-10、B P61AAV、B P62AAV Including P63AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV10, Japanese AAV10 serotypes, and combinations thereof.

[0098] In some respects, the adeno-associated virus serotypes useful for the present invention include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or combinations thereof. Therefore, in some respects, one or more of the multiple genes in the dual helper plasmid originate from AAV1. For example, in some respects, the rep gene originates from AAV1. In some respects, the cap gene originates from AAV1. In some respects, both the rep gene and the cap gene originate from AAV1. In some respects, one or more of the multiple genes in the dual helper plasmid originate from AAV2. For example, in some respects, the rep gene originates from AAV2. In some respects, the cap gene originates from AAV2. In some respects, both the rep gene and the cap gene originate from AAV2. In some respects, one or more of the multiple genes in the dual helper plasmid originate from AAV3. For example, in some respects, the rep gene originates from AAV3. In some respects, the cap gene originates from AAV3. In some aspects, both the rep gene and the cap gene originate from AAV3. In some aspects, one or more of the multiple genes in the dual helper plasmid originate from AAV4. For example, in some aspects, the rep gene originates from AAV4. In some aspects, the cap gene originates from AAV4. In some aspects, both the rep gene and the cap gene originate from AAV4. In some aspects, one or more of the multiple genes in the dual helper plasmid originate from AAV5. For example, in some aspects, the rep gene originates from AAV5. In some aspects, the cap gene originates from AAV5. In some aspects, both the rep gene and the cap gene originate from AAV5. In some aspects, one or more of the multiple genes in the dual helper plasmid originate from AAV6. For example, in some aspects, the rep gene originates from AAV6. In some aspects, the cap gene originates from AAV6. In some aspects, both the rep gene and the cap gene originate from AAV6.In some aspects, one or more of the multiple genes in a dual helper plasmid originate from AAV7. For example, in some aspects, the rep gene originates from AAV7. In some aspects, the cap gene originates from AAV7. In some aspects, both the rep gene and the cap gene originate from AAV7. In some aspects, one or more of the multiple genes in a dual helper plasmid originate from AAV8. For example, in some aspects, the rep gene originates from AAV8. In some aspects, the cap gene originates from AAV8. In some aspects, both the rep gene and the cap gene originate from AAV8. In some aspects, one or more of the multiple genes in a dual helper plasmid originate from AAV9. For example, in some aspects, the rep gene originates from AAV9. In some aspects, the cap gene originates from AAV9. In some aspects, both the rep gene and the cap gene originate from AAV9. In some aspects, one or more of the multiple genes in a dual helper plasmid originate from AAVrh10. For example, in some aspects, the rep gene originates from AAVrh10. In some aspects, the cap gene originates from AAVrh10. In some aspects, both the rep gene and the cap gene originate from AAVrh10.

[0099] In some aspects, the dual helper plasmid of the present invention comprises an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the rep gene is derived from AAV2. The nucleic acid sequence for the AAV2 rep gene is presented in SEQ ID NO: 29. In some aspects, the dual helper plasmid comprises an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the rep gene comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the sequence presented in SEQ ID NO: 29. In some aspects, the dual helper plasmid comprises an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the rep gene comprises the nucleic acid sequence described in SEQ ID NO: 29.

[0100] In some respects, the dual helper plasmids described herein include an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the cap gene is derived from AAV2, AAV5, AAV8, or AAV9.

[0101] In some respects, the cap gene is derived from AAV2. Therefore, in some respects, the dual helper plasmid described herein comprises a rep gene (rep2) derived from AAV2 and a cap gene (cap2) derived from AAV2. The nucleic acid sequence for the AAV2 cap gene is presented in SEQ ID NO: 30. In some respects, the dual helper plasmid comprises an E2a gene, an E4 gene, a VA RNA gene, a rep gene and a cap gene, wherein the cap gene comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the sequence presented in SEQ ID NO: 30. In some respects, the dual helper plasmid comprises an E2a gene, an E4 gene, a VA RNA gene, a rep gene and a cap gene, wherein the cap gene comprises the nucleic acid sequence presented in SEQ ID NO: 30.

[0102] In some aspects, the cap gene is derived from AAV5. For example, in some aspects, the dual helper plasmid described herein includes a rep gene (rep2) derived from AAV2 and a cap gene (cap5) derived from AAV5. The nucleic acid sequence for the AAV5 cap gene is presented in SEQ ID NO: 31. In some aspects, the dual helper plasmid includes an E2a gene, an E4 gene, a VA RNA gene, a rep gene and a cap gene, wherein the cap gene contains a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the sequence presented in SEQ ID NO: 31. In some aspects, the dual helper plasmid includes an E2a gene, an E4 gene, a VA RNA gene, a rep gene and a cap gene, wherein the cap gene contains the nucleic acid sequence presented in SEQ ID NO: 31.

[0103] In some aspects, the cap gene is derived from AAV8. For example, in some aspects, the dual helper plasmid described herein includes a rep gene (rep2) derived from AAV2 and a cap gene (cap8) derived from AAV8. The nucleic acid sequence for the AAV8 cap gene is presented in SEQ ID NO: 32. In some aspects, the dual helper plasmid includes an E2a gene, an E4 gene, a VA RNA gene, a rep gene and a cap gene, wherein the cap gene contains a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the sequence presented in SEQ ID NO: 32. In some aspects, the dual helper plasmid includes an E2a gene, an E4 gene, a VA RNA gene, a rep gene and a cap gene, wherein the cap gene contains the nucleic acid sequence presented in SEQ ID NO: 32.

[0104] In some aspects, the cap gene is derived from AAV9. For example, in some aspects, the dual helper plasmid described herein includes a rep gene (rep2) derived from AAV2 and a cap gene (cap9) derived from AAV9. The nucleic acid sequence for the AAV9 cap gene is presented in SEQ ID NO: 33. In some aspects, the dual helper plasmid includes an E2a gene, an E4 gene, a VA RNA gene, a rep gene and a cap gene, wherein the cap gene contains a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the sequence presented in SEQ ID NO: 33. In some aspects, the dual helper plasmid includes an E2a gene, an E4 gene, a VA RNA gene, a rep gene and a cap gene, wherein the cap gene contains the nucleic acid sequence presented in SEQ ID NO: 33.

[0105] In some respects, the dual helper plasmids provided herein comprise an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the E2a gene is derived from adenovirus serotype 5 (Ad5). The nucleic acid sequence for the Ad5E2a gene is presented in Sequence ID No. 34. Therefore, in some respects, dual helper plasmids useful for the present invention comprise an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the E2a gene comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the sequence presented in Sequence ID No. 34. In some respects, the dual helper plasmid includes the E2a gene, the E4 gene, the VA RNA gene, the rep gene, and the cap gene, wherein the E2a gene includes the nucleic acid sequence presented in Sequence ID No. 34.

[0106] In some respects, the dual helper plasmids provided herein comprise an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the E4 gene is derived from adenovirus serotype 5 (Ad5). The nucleic acid sequence for the Ad5 E4 gene is presented in Sequence ID No. 35. In some respects, dual helper plasmids useful for the present invention comprise an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the E4 gene comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the sequence presented in Sequence ID No. 35. In some respects, the dual helper plasmid includes the E2a gene, the E4 gene, the VA RNA gene, the rep gene, and the cap gene, wherein the E4 gene includes the nucleic acid sequence presented in Sequence ID No. 35.

[0107] In some respects, the dual helper plasmids provided herein comprise an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the VA RNA gene is derived from adenovirus serotype 5 (Ad5). The nucleic acid sequence for the Ad5 VA RNA gene is presented in SEQ ID NO: 36. In some respects, dual helper plasmids useful for the present invention comprise an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene, wherein the VA RNA gene comprises a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with the sequence presented in SEQ ID NO: 36. In some respects, the dual helper plasmid includes the E2a gene, the E4 gene, the VA RNA gene, the rep gene, and the cap gene, wherein the VA RNA gene includes the nucleic acid sequence presented in Sequence ID No. 36.

[0108] As is evident from the present invention, in some aspects, the dual helper plasmids described herein include genes derived from the same or multiple viral sources. In some aspects, the described dual helper plasmids include (i) an E2a gene derived from Ad5 (e.g., SEQ ID NO: 34); (ii) an E4 gene derived from Ad5 (e.g., SEQ ID NO: 35); (iii) a VA RNA gene derived from Ad5 (e.g., SEQ ID NO: 36); (iv) a cap gene derived from AAV2 (e.g., SEQ ID NO: 30); and (v) a rep gene derived from AAV2 (e.g., SEQ ID NO: 29). In some aspects, the described dual helper plasmids include (i) an E2a gene derived from Ad5 (e.g., SEQ ID NO: 34); (ii) an E4 gene derived from Ad5 (e.g., SEQ ID NO: 35); (iii) a VA RNA gene derived from Ad5 (e.g., SEQ ID NO: 36); (iv) a cap gene derived from AAV5 (e.g., SEQ ID NO: 31); and (v) a rep gene derived from AAV2 (SEQ ID NO: 29). In some respects, the described dual helper plasmid includes (i) an E2a gene derived from Ad5 (e.g., SEQ ID NO: 34); (ii) an E4 gene derived from Ad5 (e.g., SEQ ID NO: 35); (iii) a VA RNA gene derived from Ad5 (e.g., SEQ ID NO: 36); (iv) a cap gene derived from AAV8 (e.g., SEQ ID NO: 32); and (v) a rep gene derived from AAV2 (e.g., SEQ ID NO: 29). In some respects, the described dual helper plasmid includes (i) an E2a gene derived from Ad5 (e.g., SEQ ID NO: 34); (ii) an E4 gene derived from Ad5 (e.g., SEQ ID NO: 35); (iii) a VA RNA gene derived from Ad5 (e.g., SEQ ID NO: 36); (iv) a cap gene derived from AAV9 (e.g., SEQ ID NO: 33); and (v) a rep gene derived from AAV2 (e.g., SEQ ID NO: 29).

[0109] As described and demonstrated herein, one or more sequences of the aforementioned genes (e.g., E2a gene, E4 gene, VA RNA gene, rep gene, and cap gene) may help improve certain properties of the dual helper plasmid of the present invention. For example, in some aspects, the described dual helper plasmid comprises (i) an E2a gene derived from Ad5 (e.g., SEQ ID NO: 34); (ii) an E4 gene derived from Ad5 (e.g., SEQ ID NO: 35); (iii) a VA RNA gene derived from Ad5 (e.g., SEQ ID NO: 36); (iv) a cap gene derived from AAV2, AAV5, AAV8, or AAV9 (e.g., SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, respectively); and (v) a rep gene derived from AAV2 (e.g., SEQ ID NO: 29), wherein the rep gene and cap gene are oriented clockwise. For example, in some aspects, the 5' end of the rep gene is ligated to the 5' end of the E2a gene, the 3' end of the rep gene is ligated to the 5' end of the cap gene, and the 3' end of the cap gene is ligated to the 3' end of the VA RNA gene (see Figure 5A). In some aspects, the described dual helper plasmid includes (i) an E2a gene derived from Ad5 (e.g., SEQ ID NO: 34); (ii) an E4 gene derived from Ad5 (e.g., SEQ ID NO: 35); (iii) a VA RNA gene derived from Ad5 (e.g., SEQ ID NO: 36); (iv) a cap gene derived from AAV2, AAV5, AAV8, or AAV9 (e.g., SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, respectively); and (v) a rep gene derived from AAV2 (e.g., SEQ ID NO: 29), wherein the rep gene and cap gene are oriented counterclockwise. In some aspects, the 5' end of the rep gene is ligated to the 3' end of the VA RNA gene, the 3' end of the rep gene is ligated to the 5' end of the cap gene, and the 3' end of the cap gene is ligated to the 5' end of the E2a gene (see Figure 5B).

[0110] Based on the features described herein (e.g., specific sequences of the E2a gene, E4 gene, VA RNA gene, rep gene, and cap gene), the dual helper plasmids of the present invention exhibit one or more of the following improved properties: 1) increased probability of co-phenotypic infection so that the host cell has all the components necessary to produce recombinant AAV; 2) increased productivity of recombinant adeno-associated virus; and 3) reduced costs and time for plasmid production and purification. Additional disclosures relating to such properties are provided elsewhere in the present invention.

[0111] II.B. Additional Features As is evident from the present invention, in some aspects, the dual helper plasmids described herein include one or more additional features useful for recombinant AAV production. For example, in some aspects, the dual helper plasmids described herein further include an antibiotic resistance gene. For example, in some aspects, the dual helper plasmids further include a selection marker. In this specification, the term “selection marker” means any gene that can be used to identify cells expressing a nucleic acid sequence. Thus, such selection markers may be used to identify and enrich transformed cells after phenotypic infection with the dual helper plasmids described herein. Selection markers usable with the present invention include any suitable selection markers known in the art. Non-restrictive examples of suitable screening markers include (i) enzymes encoding resistance to antibiotics (i.e., "antibiotic resistance genes"), such as kanamycin, neomycin, puromycin, hygromycin, blasticidin, or zeocin; or (ii) fluorescent proteins, such as green fluorescent protein (GFP), red fluorescent protein (RFP), or blue fluorescent protein (BFP).

[0112] In some aspects, the screening markers include antibiotic resistance genes. In some aspects, the antibiotic resistance genes include ampicillin resistance genes, kanamycin resistance genes, or both. The nucleic acid sequence for the ampicillin resistance gene is presented in SEQ ID NO: 37. The nucleic acid sequence for the kanamycin resistance gene is presented in SEQ ID NO: 38.

[0113] III. Expression constructs As is evident from the present invention, in some aspects, the dual helper plasmids provided herein may be used in combination with one or more additional plasmids, for example, when generating recombinant AAV. In some aspects, the additional plasmids include recombinant expression constructs, such as AAV construct plasmids (hereinafter referred to as “expression constructs”). Exemplary aspects of expression constructs are described below.

[0114] III.A. Foreign genes (e.g., located on the side of the ITR) As further described elsewhere in the present invention, in some aspects, the dual helper plasmids provided herein include an E2a gene, an E4 gene, a VA RNA gene, a rep gene, and a cap gene. In such aspects, the exogenous gene (e.g., introduced into the recombinant AAV produced) may be provided by a separate expression construct (e.g., an AAV construct plasmid). Thus, in some aspects, the expression construct includes the exogenous gene. In some aspects, the exogenous gene is located on the side of the reverse terminal repeat (ITR). The exogenous gene useful in the present invention is not particularly limited, as long as it can be translated into a polypeptide when introduced into the cell. Thus, any suitable exogenous gene of interest may be used in conjunction with the present invention. In some aspects, the exogenous gene encodes a polypeptide (or any variant thereof), a fusion protein, an antibody or its antigen-binding fragment, an RNA-based molecule (e.g., miRNA, shRNA, ribozyme, siRNA), or any combination thereof.

[0115] In some aspects, exogenous genes encode proteins useful for treating diseases or disorders as described herein. In some aspects, exogenous genes encode therapeutic peptides for specific diseases intended for sustained expression in the subject body or patient's body.

[0116] III.B. Adjustment elements In some respects, the expression construct usable with the dual helper plasmid of the present invention further comprises a regulatory element. In some respects, the regulatory element is operably ligated to an exogenous gene.

[0117] Therefore, in some respects, the expression constructs described herein (e.g., AAV construct plasmids) include the following: (a) ITR (Inverse Terminal Repeat Sequence); (b) foreign genes; and (c) Regulatory elements operably linked to foreign genes.

[0118] Modulatory elements useful in the present invention include enhancers (e.g., CMV enhancers), promoters (e.g., CMV promoter, EF-1α promoter, β-actin promoter), exons (e.g., exon 1, exon 2), introns (e.g., intron A), splicing donor or receptor sequences, or combinations thereof. In some aspects, the modulatory elements may include sequences for transcription termination (e.g., poly-A), sequences for stable expression of foreign genes (e.g., WPRE sequences), sequences for reducing foreign gene-specific immunity (e.g., miRNA target sequences), or combinations thereof. Details regarding foreign genes and modulatory elements (ITR sequences, enhancer sequences, promoter sequences, E1 sequences, splicing donor sequences, fragment sequences including EF-1α introns or splicing receptors, EF-1α E2 sequences, WPRE sequences, miR sequences, poly sequences, etc.) can be found in US2022 / 0010332A1, the full text of which is incorporated herein by reference.

[0119] As described above, in some respects, an expression construct useful for the present invention comprises (i) an exogenous gene and (ii) a regulatory element operably linked to the exogenous gene, the regulatory element comprising the following components: 1) CMV enhancer sequence; 2) CMV promoter sequence, EF-1α promoter sequence, or chicken β-actin promoter sequence; 3) CMV E1 sequence, EF-1α E1 sequence, or chicken β-actin E1 sequence; 4) Splicing donor sequence; 5) EF-1α intron fragment sequence; and 6) EF-1α E2 sequence.

[0120] IV. Vectors In some respects, vectors comprising any plasmid provided herein (e.g., dual helper plasmids and / or expression constructs containing exogenous genes) are provided herein. Recombinant AAVs produced using plasmids provided herein are also provided herein. Such vectors are useful for recombinant expression in host cells and target cells for therapeutic interventions.

[0121] As is evident from the present invention, in some aspects, the vectors useful in the present invention are derived from AAV. AAV has a unique and attractive function as a vector system for delivering foreign DNA to cells. AAV infection of cells in culture is generally non-cellular, and natural infections in humans and other animals are silent and asymptomatic. Moreover, AAV can infect many different types of mammalian cells and target many different tissues in vivo. Furthermore, AAV has the additional advantage of promoting a weaker immune response compared to other forms of gene carriers, and is persistently expressed in both dividing and quiescent cells based on non-integrating, episomal vector DNA, making it a particularly attractive viral system for gene carriers. In addition, AAV can withstand the conditions used to inactivate adenoviruses (56-65°C for several hours), so the importance of low-temperature storage of rAAV-based vaccines is relatively low.

[0122] Non-limiting examples of adeno-associated virus types or serotypes usable in the present invention are provided elsewhere in the invention.

[0123] V. Cell In some aspects, the present invention provides cells (hereinafter referred to as “modified cells”) containing plasmids described herein (e.g., dual helper plasmids and expression constructs containing foreign genes). In some aspects, cells (e.g., host cells) are transfected with dual helper plasmids and AAV constituent plasmids to produce recombinant AAV. In some aspects of the present invention, the modified cells of the present invention (e.g., containing dual helper plasmids and expression constructs containing foreign genes) can improve one or more aspects of recombinant AAV production. For example, in some aspects, the modified cells provided herein can produce recombinant AAV in a higher yield compared to control cells. In some aspects, the control cells include the corresponding cells modified to contain the following three distinct plasmids: (1) a first plasmid containing the rep gene and cap gene (“rep-cap plasmid”); (2) the E2a gene, E4 gene and VA gene. (3) A second plasmid containing an RNA gene ("helper plasmid"); and (4) a third plasmid containing an exogenous gene.

[0124] In some aspects, compared to control cells, cells of the present invention (including, for example, expression vectors containing a dual helper plasmid and an exogenous gene) can increase the amount of recombinant AAV produced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least 100% or more. In some aspects, compared to control cells, cells of the present invention can increase the amount of recombinant AAV produced by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, or at least about 100 times or more. In some respects, the cells provided herein (modified to include, for example, expression constructs containing dual helper plasmids and exogenous genes) can reduce production and / or purification times compared to control cells. In some respects, production and / or purification times are reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% or more compared to control cells.

[0125] As is evident from the present invention, cells are also provided that have been modified (e.g., transfected) to contain recombinant AAV produced using the plasmids of this specification. Such cells may be particularly useful, for example, for producing proteins encoded by the exogenous genes described herein. Without being bound by any one theory, in some respects, when cells (e.g., host cells) are transfected with the plasmids described herein (e.g., an expression construct comprising a dual helper plasmid and an exogenous gene operably linked to a regulatory element), the produced recombinant AAV contains one or more features of the individual plasmids. For example, in some respects, the produced recombinant AAV contains an exogenous gene operably linked to a regulatory element.

[0126] As described herein, in some respects, one or more of the regulatory elements described herein can enhance the expression of a protein encoded by an exogenous gene ("encoded protein") in a cell. Therefore, in some respects, cells described herein (e.g., those transfected with a recombinant AAV signaling vector containing an exogenous gene operably linked to one or more of the regulatory elements described herein) exhibit a greater increase in the expression of encoded proteins compared to control cells. In some respects, control cells are transfected with the signaling vector but lack one or more of the regulatory elements described herein.

[0127] In some aspects, the cells described herein are modified (e.g., transfected) in vitro to produce a composition of interest. For example, in some aspects, cells are transfected in vitro with a dual helper plasmid and expression construct (e.g., those described herein) to produce recombinant AAV. In some aspects, cells are transfected in vitro with a recombinant gene carrier vector to produce a protein encoded by a foreign gene. In some aspects, the cells described herein can produce the encoded protein in vivo (e.g., in a subject administered with recombinant AAV containing a foreign gene). In some aspects, the cells described herein can produce the composition of interest (e.g., the encoded protein) both in vitro and in vivo.

[0128] As described herein, in certain aspects, the present invention provides host cells transformed, transduced, or transfected with, for example, recombinant expression constructs for exogenous gene expression. Hereinafter, the term “host cell” includes eukaryotic and prokaryotic cells and means cells of an organism transducible so that a gene encoded by a plasmid or expression construct (e.g., AAV construct plasmid, Rep-Cap plasmid, helper plasmid, dual helper plasmid, etc.) can be expressed or replicated. In certain aspects, this means isolated (eukaryotic) host cells. Hereinafter, the term “transfection” is intended to include transduction and transformation. Host cells may be transfected, transduced, or transformed with any plasmid, construct, and / or vector described herein. These terms refer to the process by which an exogenous nucleic acid molecule is transmitted or introduced into a host cell.

[0129] In some respects, the host cell is a eukaryotic cell. In some respects, the host cell is selected from the group consisting of mammalian cells, insect cells, yeast cells, transgenic mammalian cells, and plant cells. In some respects, the host cell is a prokaryotic cell. In some respects, the prokaryotic cell is a bacterial cell.

[0130] In some aspects, the cells useful for the present invention (e.g., host cells) include insect cells, mammalian cells, or both. In some aspects, the insect cells may be Sf9 cells. In some aspects, the mammalian cells may include HEK293 cells, HeLa cells, ARPE-19 cells, RPE-1 cells, HepG2 cells, Hep3B cells, Huh-7 cells, C8D1a cells, Neuro2A cells, CHO cells, MES13 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, MCF-7 cells, HC70 cells, HCC1428 cells, BT-549 cells, PC3 cells, LNCaP cells, Capan-1 cells, Panc-1 cells, and MIA cells. This includes PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells, PC-12 cells, NIH3T3 cells, or combinations thereof.

[0131] In some aspects, cells useful to the present invention include human cells. In some aspects, human cells are cells of a subject to which the recombinant gene transfer vector described herein is administered. In some aspects, human cells are derived from a donor (e.g., a healthy human subject).

[0132] VI. Composition The present invention also relates to compositions comprising dual helper plasmids as described herein. As is evident from the present invention, in some aspects such compositions further comprise additional plasmids, for example, expression constructs as described herein (e.g., AAV construct plasmids for exogenous gene expression). As described elsewhere in the present invention, such compositions may be useful for producing recombinant AAVs.

[0133] Various plasmids (e.g., dual helper plasmids), constructs (e.g., expression constructs), vectors (e.g., recombinant AAV carrier vectors), and cells disclosed herein (hereinafter referred to as “active compounds”) may be included in a pharmaceutically appropriate composition for administration. Therefore, in some aspects, the present invention relates to such pharmaceutically appropriate compositions. For example, in some aspects, the present invention provides a pharmaceutically appropriate composition comprising recombinant AAV produced using a plasmid (e.g., dual helper plasmid) described herein.

[0134] In some respects, the pharmaceutical compositions described herein further include pharmaceutically acceptable carriers.

[0135] Pharmaceutically acceptable carriers usable in the present invention include those commonly used in dosage forms. Non-limiting examples of such pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and combinations thereof. The pharmaceutical compositions of the present invention may further contain one or more additives such as lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and combinations thereof. Additional details regarding suitable pharmaceutically acceptable carriers and formulations are described in the literature (Remington's Pharmaceutical Sciences (19th ed., 1995)).

[0136] The pharmaceutical compositions of the present invention are formulated in dosage forms to be compatible with the intended route of administration. Non-limiting examples of such routes of administration are provided elsewhere in the present invention.

[0137] The pharmaceutical compositions described herein may be provided in single-dose or multiple-dose forms. In some aspects, the pharmaceutical compositions provided herein are available in the form of solutions (e.g., in oily or aqueous media), suspensions, emulsions, extracts, powders, granules, tablets, or capsules. In some aspects, dosage forms containing the pharmaceutical compositions described herein may further include dispersants, stabilizers, or both.

[0138] VII. Kit This specification discloses kits comprising one or more of the various plasmids (e.g., dual helper plasmids), constructs (e.g., expression constructs), vectors (e.g., recombinant AAV carrier vectors), cells, and compositions (e.g., pharmaceutical compositions) that have been disclosed. In some aspects, the kits include instructions for use (e.g., instructions for administering any or a combination thereof to an object requiring it).

[0139] In this specification, the terms “kit” and “system” are intended to represent at least one of various plasmids (e.g., dual helper plasmids), constructs (e.g., expression constructs), vectors (e.g., recombinant AAV carrier vectors), cells, and compositions (e.g., pharmaceutical compositions), or any combination thereof, provided in some aspects together with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packaging for sale, instructions for use, etc.).

[0140] VIII. Uses and Methods VIII.A.AAV Production Method A particular aspect of the present invention relates to a method for producing AAV. More specifically, in some aspects, the present invention provides a method for producing recombinant AAV containing a foreign gene. In some aspects, such a method involves transfecting cells (e.g., host cells) with a dual helper plasmid (e.g., one described herein) and an expression construct (e.g., an AAV construct plasmid) containing a foreign gene. In some aspects, the cells are transfected simultaneously with the dual helper plasmid and the expression construct. In some aspects, the cells are transfected sequentially with the dual helper plasmid and the expression construct. Unless otherwise noted, any suitable transfecting method known in the art may be used in conjunction with the present invention.

[0141] In some aspects, a method for producing recombinant AAV further includes the step of culturing cells that have been plasma-infected under conditions suitable for the production of recombinant AAV. In some aspects, the method further includes the step of recovering the produced recombinant AAV.

[0142] The method for producing recombinant AAV described herein offers clear advantages. As described elsewhere in the invention, more conventional methods for producing AAV require triple phenotypic infection, in which host cells are phenotypic infected with three distinct plasmids: i) a Rep-Cap plasmid containing genes encoding the Rep and Cap proteins; ii) a helper plasmid containing genes encoding adenovirus proteins (E2a, E4) and VA RNA; and iii) an AAV construct plasmid for exogenous gene expression. The recombinant AAV is produced only if the cells are successfully phenotypic infected with all three plasmids described above (i.e., each gene is absorbed and transmitted to the cell nucleus, and subsequently transcribed, replicated in the cell, and / or translated). Due to the associated complexity, such methods requiring triple phenotypic infection may result in low yields, high costs, and / or more labor-intensive outcomes.

[0143] Compared to such approach methods, the method for producing recombinant AAV provided herein can yield higher yields of recombinant AAV. In some aspects, compared to methods requiring triple trait infection (i.e., the “control method”), the amount of recombinant AAV produced by the present invention increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or more. In some respects, compared to control cells, the cells of the present invention can increase the amount of recombinant AAV produced by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 75 times, or at least about 100 times or more.

[0144] As described elsewhere in the present invention, in some aspects, the method for producing recombinant AAV provided herein can reduce production and / or purification time. Further advantages are described elsewhere in the present invention.

[0145] VIII.B. Protein Production Methods Furthermore, methods for producing a protein (or polypeptide) encoded by a foreign gene are disclosed herein. In some aspects, such methods include the steps of culturing cells of the present invention under appropriate conditions (e.g., transfected with a recombinant AAV signaling vector containing the foreign gene) and recovering the encoded protein. In certain aspects, the method for producing a protein encoded by a foreign gene includes the step of administering recombinant AAV to a target organism requiring it.

[0146] VIII.C. Therapeutic uses In some respects, the present invention provides applications for recombinant AAV produced using the dual helper plasmid of the present invention for various therapeutic applications.

[0147] For example, in some aspects, the present invention provides a method for treating a disease in a subject in need, comprising the step of administering a recombinant AAV, which contains an exogenous gene and is produced as described herein, to the subject. In some aspects, the method for treating a disease provided herein comprises the step of administering any modified cells as described herein (e.g., transfected with a recombinant AAV gene carrier vector containing an exogenous gene) to a subject in need. In some aspects, the method for treating a disease provided herein comprises the step of administering any pharmaceutical composition as described herein (e.g., which contains recombinant AAV provided herein) to a subject in need. As is evident from the present invention, the methods may be used, for example, to treat and / or prevent any disease of interest by modifying an exogenous gene.

[0148] The diseases to be prevented, mitigated, or treated by the present invention are not limited and include all diseases for which the number of drug administrations should be reduced. Non-limiting examples of such diseases include ophthalmic and neurological diseases. In some aspects, said ophthalmic diseases include diabetic retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal edema, and mascular tumentia, or combinations thereof. In some aspects, said neurological diseases include diseases affecting the central nervous system, the peripheral nervous system, or both. Non-limiting examples of neurological diseases include anxiety, depression, post-traumatic stress disorder (PTSD), bipolar disorder, and attention deficit hyperactivity disorder. Deficiency hyperactivity disorder (ADHD), autism, schizophrenia, neuropathic pain, glaucoma, toxicosis, arachnoid cyst, catatonia, encephalitis, epilepsy / seizure, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Batten disease, Tourette's syndrome This includes syndrome, traumatic brain injury, cerebrospinal injury, stroke, tremor (essential or Parkinson's disease), dystonia, intellectual disability, brain tumor, or a combination thereof.

[0149] Some aspects of the present invention relate to gene therapy agents capable of achieving the continuous expression of exogenous genes, or to methods of using such formulations to treat diseases.

[0150] The use of gene transmission systems provided herein (e.g., recombinant AAVs containing foreign genes) allows for the administration of therapeutic agents (e.g., recombinant AAVs containing foreign genes) at intervals that can significantly reduce the number of drug administrations for the convenience of the physician, patient, or subject. For example, in some aspects, therapeutic agents may be administered at intervals of about 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or more than 1 year. In some aspects, the interval is about 2 to 3 months. In some aspects, the interval is about 6 months. In some aspects, the interval is about 1 year. In some aspects, the interval is at least about 1 year. In some aspects, depending on the patient's symptoms, the drug may be administered 2 to 3 times at intervals of approximately 1 to 2 weeks initially, and thereafter at intervals of approximately 2 to 3 months, approximately 6 months, or more than 1 year if necessary.

[0151] In any method provided herein, including an administration step (e.g., administering any AAV, cells, and / or pharmaceutical composition described herein to a subject requiring it), the therapeutic agent may be administered to the subject orally or parenterally. In some aspects, the therapeutic agent (e.g., any AAV, cells, and / or pharmaceutical composition described herein) is administered to the subject parenterally. Unrestricted examples of parenteral administration include: intravenous infusion, percutaneous administration, subcutaneous infusion, intramuscular infusion, intravitreal injection, subretinal injection, suprachoroidal injection, eye drop administration, intracerebral infusion, intrathecal injection, intraamniotic injection, intraarterial injection, intraarticular injection, intracardiac injection, intracavernous injection, intracerebral infusion, intracisional injection, intracoronary injection, intracranial injection, intradural injection, epidural injection, and intrahippocampal injection. injection), intranasal injection, intraosseous injection, intraperitoneal injection, intrapleural injection, intraspinal injection, intrathoracic injection, intrathymic injectionThis includes injections such as intrauterine injection, intravaginal injection, intraventricular injection, intravesical injection, subconjunctival injection, intratumoral injection, local injection, intraperitoneal injection, and combinations thereof.

[0152] As will be apparent to those skilled in the art, the appropriate dosage of a therapeutic agent (e.g., the pharmaceutical composition of the present invention) may vary depending on factors such as dosage form, method of administration, age, body weight, patient's sex, pathological condition and diet, administration time, route of administration, excretion rate and response sensitivity. In some aspects, the daily dosage of a therapeutic agent (e.g., the pharmaceutical composition of the present invention) is approximately 0.00001 to 100 mg / kg.

[0153] The present invention will be described in more detail below using examples. The following examples are provided to illustrate the present invention more concretely, and it will be obvious to those with ordinary skill in the art that the scope of the present invention is not limited by these examples.

[0154] Examples Materials and methods Example 1. Production of pUC-R2C2, 5, 8, and 9 constructs for triple phenotypic infection. Example 1-1. Manufacturing of pRC8 structure By performing a polymerase chain reaction (PCR) using pIDT-Cap8-Kan (Integrated DNA Technologies, USA) as a template and oligo #001 / 002 combination, the cap8 gene fragment was obtained. By performing a PCR using pRC6 (Takara Bio, Japan) as a template and oligo #003 / 004 combination, the plasmid backbone DNA fragment containing the rep2 gene was obtained. Gibson Assembly (registered trademark) (NEB, USA, Cat The pRC8 construct was prepared by ligating both DNA fragments using No. E2611).

[0155] Examples 1-2. Fabrication of pUC-R2C8 structure A plasmid backbone was secured by performing PCR using the oligo #005 / 006 combination with pUC57-WPRE (GenScript, USA) as a template. The cap8 gene fragment was secured by performing PCR using oligo #007 / 008 with the pRC8 construct from Example 1-1 as a template, and the rep2 gene fragment was secured using oligo #009 / 004. The pUC-R2C8 construct was then produced by ligating the three DNA fragments obtained above using Gibson Assembly®.

[0156] Examples 1-3. Manufacturing of pRC2 structures Using pRC2-mi342 (Takara Bio, Japan) as a template, oligo The cap2 gene fragment was secured by performing PCR using the #007 / 010 combination, and the plasmid backbone fragment containing the rep2 gene was secured by performing PCR using the oligo #003 / 004 combination with pRC6 (Takara Bio, Japan) as a template. The pRC2 construct was then manufactured by ligating both NA fragments using Gibson Assembly®.

[0157] Examples 1-4. Fabrication of pUC-R2C2 structures A plasmid backbone fragment containing the rep2 gene was obtained by performing PCR using the oligo #011 / 012 combination with the pUC-R2C8 construct from Example 1-2 as a template, and a cap2 gene fragment was obtained by performing PCR using the oligo #013 / 014 combination with the pRC2 construct from Example 1-3 as a template. The pUC-R2C2 construct was then produced by ligating the two DNA fragments using Gibson Assembly®.

[0158] Examples 1-5. Manufacturing of pRC9 structures The cap9 gene fragment was obtained by performing PCR using the oligo #001 / 015 combination with pIDT-Cap9-Kan (Integrated DNA Technologies, USA) as a template, and the plasmid backbone fragment containing the rep2 gene was obtained by performing PCR using the oligo #003 / 004 combination with pRC6 (Takara Bio, Japan) as a template. The pRC9 construct was then manufactured by ligating both DNA fragments using Gibson Assembly® (NEB, USA, Cat No. E2611).

[0159] Examples 1-6. Fabrication of pUC-R2C9 structures A plasmid backbone was secured by performing PCR using the oligo #005 / 006 combination with pUC57-WPRE (GenScript, USA) as a template. The cap9 gene fragment was secured by performing PCR using oligo #007 / 008 with the pRC9 construct from Example 1-5 as a template, and the rep2 gene fragment was secured using oligo #009 / 004 with pRC8 from Example 1-1 as a template. The pUCR2C9 construct was then manufactured by ligating the three DNA fragments obtained above using Gibson Assembly®.

[0160] Examples 1-7. Manufacturing of pUC-R2C5 structures A plasmid backbone was secured by performing PCR using the oligo #005 / 006 combination with pUC57-WPRE (GenScript, USA) as a template, and a rep2-cap5 gene fragment was secured by performing PCR using oligo #008 / 009 with the pRC5 construct (TakaraBio, Japan) as a template. The pUC-R2C5 construct was then manufactured by ligating the two DNA fragments obtained above using Gibson Assembly®.

[0161] Example 2. Manufacturing of pHION8 series constructs Example 2-1. pHelper-NG construction manufacturing pHelper-NG was produced by cloning both constructs, VA-E4-pUC (GeneScript, USA) and E2a-pUC57 (GeneScript, USA), by binding them to a common SalI / BamHI site (Figure 1A). pHelper-NG contains the E2a, E4, and VA RNA genes of adenovirus serotype 5.

[0162] Example 2-2. Insertion of the rep2-cap8 gene at various positions in the pHelper-NG construct. Example 2-2-1. pHION8-BamHI-Forward or Reverse (pHION8-BF or -BR) Cloning Using the pUC-R2C8 construct from Example 1-2 as a template, the rep2-cap8 gene fragment was obtained by PCR using the oligo #016 / 017 combination and inserted into the BamHI region of pHelper-NG produced in Example 2-1 (Figures 1B and 1C). Figure 1B is a schematic diagram of the pHION8-BF construct, in which the rep2-cap8 gene fragment was inserted in the forward clockwise direction (clockwise, 5'->3') using the BamHI region located between the start of the E2a gene and the end of the VA RNA gene to produce the construct. Figure 1C is a schematic diagram of the pHION8-BR construct, in which the rep2-cap8 construct was inserted in the reverse counterclockwise direction (counterclockwise, 3'->5') using the BamHI region located between the start of the E2a gene and the end of the VA RNA gene to produce the construct. The aforementioned pHION8-BF was named pHNG8, and pHION8-BR was named pHNGR8.

[0163] Example 2-2-2. pHION8-NotI-forward or -reverse (pHION8-NF or -NR) cloning Using the pUC-R2C8 construct from Example 1-2 as a template, the rep2-cap8 gene fragment was obtained by PCR using the oligo #018 / 019 combination and inserted into the NotI site of pHelper-NG produced in Example 2-1 (Figures 1D and 1E). Figure 1D is a schematic diagram of the pHION8-NF construct, in which the rep2-cap8 gene fragment was inserted in the positive direction (clockwise) using the NotI site located between the start of the E4 gene and the start of the AmpR gene to produce the construct. Figure 1E is a schematic diagram of the pHION8-NR construct, in which the rep2-cap8 gene fragment was inserted in the reverse direction (counterclockwise) using the NotI site located between the start of the E4 gene and the start of the AmpR gene to produce the construct.

[0164] Example 2-2-3. pHION8-AsiSI-forward cloning (pHION8-AF) Using the pUC-R2C8 construct from Example 1-2 as a template, the rep2-cap8 gene fragment was obtained by PCR using the oligo #020 / 021 combination. This fragment was then inserted in the positive direction (clockwise) into the AsiSI site between the VA RNA gene and the E4 gene of pHelper-NG produced in Example 2-1 to produce the pHION8-AF construct (Figure 1F).

[0165] Example 3. Production of pHNG2, pHNG5K, and pHNG9 structures Example 3-1. Preparation of pHNG2 structure The pUC-R2C2 constructs from Examples 1-4 were used as templates, and the rep2-cap2 gene fragment was obtained by PCR using oligo #016 / 017. This fragment was then inserted into the BamHI region of pHelper-NG produced in Example 2-1, and the resulting construct was named pHNG2.

[0166] Figure 2 is a schematic diagram of the pHNG2 construct. The pHNG2 construct was prepared by inserting the rep2-cap2 gene fragment in the positive direction (clockwise) using the BamHI site located between the E2a gene and the VA RNA gene.

[0167] Example 3-2. pHNG9 Construct Production Using the pUC-R2C9 constructs from Examples 1-6 as templates, the rep2-cap9 gene fragment was obtained by PCR using oligo #016 / #017. The construct produced by inserting this fragment into the BamHI region of pHelper-NG manufactured in Example 2-1 was named pHNG9. Figure 3 is a schematic diagram of the pHNG9 construct, in which the rep2-cap9 gene fragment was inserted in the positive direction (clockwise) using the BamHI region located between the E2a gene and the VA RNA gene to produce the pHNG9 construct.

[0168] Example 3-3. pHNG5K Structure Manufacturing Example 3-3-1. pHelper-NG-Kan construction Kanamycin resistance gene fragments were secured by PCR using the pMK-RQ-1-PM construct (Geneart, Thermo Fisher Scientific, USA) as a template and the oligo #018 / 019 combination. E4 gene fragments were secured by PCR using the pHelper-NG construct from Example 2-1 as a template and the oligo #020 / 021 combination. The N-terminal fragment of the E2a gene in the VA RNA gene was secured by PCR using the oligo #022 / 023 combination, and the origin fragment at the C-terminus of the E2a gene was secured by PCR using the oligo #024 / 025 combination. The above four DNA fragments were ligated using Gibson Assembly® to produce pHelper-NG-Kan constructs.

[0169] Example 3-3-2. Insertion of the rep2-cap5 gene into the pHelper-NG-Kan construct. Using the pUC-R2C5 constructs from Examples 1-7 as templates, the rep2-cap5 gene fragment was obtained by PCR using oligos #016 and #017. This fragment was then inserted into the BamHI region of pHelper-NG-Kan prepared in Example 3-3-1, and the resulting construct was named pHNG5K (K stands for antibiotic resistance gene, indicating the use of a kanamycin resistance gene). Figure 4 is a schematic diagram of the pHNG5K construct, in which the rep2-cap5 gene fragment was inserted in the positive direction (clockwise) using the BamHI region located between the E2a gene and the VA RNA gene to produce the pHNG5K construct.

[0170] Table 1 shows the sequence of oligos used to manufacture the aforementioned structure:

[0171] [Table 1] Furthermore, constructs produced by inserting the rep-cap gene fragment in the forward direction (clockwise, 5'->3') between the E2a gene and the VA RNA gene were collectively named pHNG (pHNG2, pHNG5 (including pHNG5K), pHNG8, pHNG9, etc.), and constructs produced by inserting it in the reverse direction (counterclockwise, 3'->5') were collectively named pHNGR (pHNGR8, etc.). The cleavage maps of these structures are shown in Figures 5A and 5B.

[0172] Example 4. Production of AAV construct plasmids containing foreign genes The AAV construct plasmid containing the foreign gene was manufactured by the method described in Korean Patent Application No. 10-2020-0084038. Specifically, it was manufactured based on the pUC57 plasmid, with the AAV2 ITR nucleotide sequence, an element necessary for AAV capsid packaging, present at both ends, and containing a CMV enhancer, a chicken β-actin promoter, a hybrid intron, and the foreign gene eGFP gene, WPRE sequence, four repeat miR142-3p target sequence, and bovine growth hormone pA sequence.

[0173] Example 5. Confirmation of base sequence The base sequences of all the constructs produced above were confirmed by DNA sequencing (Bionics, Korea).

[0174] Example 6. Cell Culture HEK293 cell lines were cultured in moist conditions at 5% CO2 and 37°C using MEM medium (Gibco, USA, Cat No. 42360-032) containing 10% FBS (Fetal Bovine Serum, Gibco, USA, Cat No. 16000-044) and 1% benicillin-streptomycin (Gibco, USA, Cat No. 15140-163). Expi293 cell lines were cultured in moist conditions at 8% CO2 and 37°C with shaking at 250 rpm using Expi293 medium (Gibco, USA, Cat No. A14351-01) supplemented with 1% benicillin-streptomycin.

[0175] Example 7. Plasma Infection Example 7-1. Plasma infection of adherent cells for AAV production For phenotypic infection, HEK293 cell lines were washed twice with DPBS (Gibco, USA, Cat No. 14190-250), then treated with Trypsin-EDTA (Gibco, USA, Cat No. 25200-114) to detach from the culture dish and secure. They were then placed in 150mm culture dishes in 2×10⁶ cells. 7Cells were inoculated into a cell / dish. After 24 hours of incubation, in the case of triple transfection, 3.73 pmole of pHelper-NG plasmid, pUC-R2C2, pUC-R2C8, or pUC-R2C9 plasmid, and an AAV construct plasmid containing an exogenous gene were dissolved in 500 μl of Opti-MEM (Gibco, USA, Cat No. 51985-034). In the case of double transfection, 3.73 pmole of pHION8 plasmid and an AAV construct plasmid containing an exogenous gene were dissolved in 500 μl of Opti-MEM (Gibco, USA, Cat No. 51985-034). Then, polyethyleneimine (PEI, Polyscience, USA, Cat No. 23966-1) at twice the total amount of DNA was diluted in 500 µl of Opti-MEM, and the two solutions were immediately mixed to prepare a phenotypic infection solution. After reacting at room temperature for 30 minutes, a total of 1 ml of the phenotypic infection solution was added to the culture dish in which the HEK293 cells had been cultured.

[0176] Example 7-2. Plasma infection of suspension cells for AAV production For AAV production, 6 × 10 in a 1 L Erlenmeyer culture flask 8Cells were inoculated into 220 ml of Expi293 medium. After culturing for approximately 3-4 hours for stabilization, for triple transfection, 3.73 pmole of pHelper-NG plasmid, pUC-R2C2, pUC-R2C5, pUC-R2C8, or pUC-R2C9 plasmid, and an AAV construct plasmid containing an exogenous gene were dissolved in 10 ml of Opti-MEM (Gibco, USA, Cat No. 51985-034). For double transfection, 3.73 pmole of pHION8 plasmid, pHNG2 plasmid, or pHNG9 plasmid, and an AAV construct plasmid containing an exogenous gene were dissolved in 10 ml of Opti-MEM (Gibco, USA, Cat No. 51985-034). Then, polyethyleneimine (PEI, Polyscience, USA, Cat No. 23966-1) was diluted in 10 ml of Opti-MEM at twice the amount of total DNA, and the two solutions were immediately mixed to prepare a phenotypic infection solution. After reacting at room temperature for 30 minutes, a total of 20 ml of the phenotypic infection solution was added to the culture flask.

[0177] Example 8. Purification of AAV 72 hours after phenotypic infection in Example 7-1 or Example 7-2, NaCl was added to the culture dish and culture flask to a concentration of 500 mM and the mixture was allowed to react for 3 hours. All culture medium was collected and debris was removed by centrifugation. The mixture was then centrifuged at 4°C at 4000 rpm using a centricon (VIVASPIN 20, 100,000 MWCO PES, Sartorius, Germany) with a molecular weight cut-off pore size of 100 kDa until approximately 200 μl of supernatant remained. The supernatant containing the recombinant adeno-associated virus vector was then collected and used for experimentation or stored at -80°C.

[0178] Example 9. Determination of AAV titer qPCR (Bio-Rad, USA, CFX96) was performed to determine the titer of AAV purified in Example 8. AAV was treated with DNase I reaction buffer (New England Biolab, USA, M0303S) at 37°C for 1 hour. Subsequently, the DNase I-treated sample was treated with protease K (Invitrogen, USA, Cat No. AM2548) at 55°C for 30 minutes, followed by reaction at 95°C for 15 minutes to inactivate protease K. The prepared sample was used as a template for qPCR, and the AAV construct plasmid (7.4 × 10⁶) was obtained. 8 ~7.4×10 4 Standard curves were determined using a 10-fold dilution, and recombinant adeno-associated virus 2 Reference Standard Stock (rAAV2-RSS, ATCC, USA, Cat No. VR-1616) or recombinant adeno-associated virus 8 Reference Standard Stock (rAAV8-RSS, ATCC, USA, Cat No. VR-1816) was used as the positive control group. For qPCR to determine titer, 2xSsoAdvanced Universal Probe Supermix (Bio-Rad, USA, Cat No. VR-1616) was used. No. 172-5282) and bGH poly A sequence-specific primers (#026, #027) and probe (#028) were used. qPCR was performed by denaturing at 95°C for 10 minutes, followed by 40 repetitions of 30 seconds at 95°C and 1 minute at 60°C. Standard curves and quantification were analyzed using Bio-Rad CFXMaestro 1.1 software (Bio-Rad, USA).

[0179] Example 10. In vitro adeno-associated virus vector-based phenotypic infection For trait infection with adeno-associated virus vectors, use 4 × 10⁶ per well in a 24-well culture plate. 5HEK293 cells were inoculated. 24 hours later, each well was treated with MG132 (Sigma-Aldrich, USA, Cat No. M7449) at a concentration of 5 μM for 8 hours. AAV2 cells were treated with 2,500 MOI (Multiplicity of Infection), and AAV8 and AAV9 cells were treated with 10,000 MOI, followed by 72-hour incubation.

[0180] Example 11. Measurement of eGFP gene expression intensity using fluid cell analysis. eGFP expression was measured by fluid cell analysis (Beckman Coulter, USA, CytoFlex). 72 hours after phenotypic infection, cells were washed with DPBS and detached with trypsin. Cells were secured by centrifugation at 1500 rpm for 5 minutes, and resuspended in 500 μl of DPBS supplemented with 2% FBS. In fluid cell analysis, FSC Single-cell regions were identified from the vs SSCplot, and the expression level of FL1-A (green) was measured. All fluid cell analysis results were analyzed using FlowJo software 10.5.3 (Becton Dickinson & Company, USA).

[0181] Example 12. Statistical Analysis All experiments were repeated at least three times. Comparisons between the two groups were performed using Prism software. Comparisons were performed using Student's t-test with 8.1.1 (GraphPad Software, Inc., USA), and for comparisons of three or more groups, one-way ANOVA and Tukey's multiple comparisons test were used. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0182] Experimental results 1. Selection of Helper-In-One plasmid constructs demonstrating increased AAV8 production. 1-1. Observation of the effect of triple phenotypic infection and double phenotypic infection using the pHION8 series on increasing or decreasing AAV8 production in adherent cell line (HEK293). The effect of various rep2-cap8 gene positions within five Helper-In-One plasmid constructs (pHION8 series; pHION8-BF, pHION8-BR, pHION8-NF, pHION8-NR, and pHION8-AF) used for double phenotypic infection on AAV8 production was tested in adherent cell line (HEK293). When AAV8 production using the triple phenotypic infection method was set to 100%, various results were obtained in AAV production during double phenotypic infection depending on the rep2-cap8 gene position. AAV8 production increased by 244.3% when using the pHION8-BF construct (pHNG8) and by 170.2% when using the pHION8-BR construct (pHNGR8). An increase of 126.0% was observed when using the pHION8-NF construct, but this was not statistically significant (p=0.82). On the other hand, when pHION8-NR and pHION8-AF constructs were used, the production of AAV8 actually decreased (62.4% (p=0.50), 71.7% (p=0.77)) (Figure 6).

[0183] 1-2. Effect of triple phenotypic infection and double phenotypic infection using the pHION8 series on increasing or decreasing AAV8 production in suspension cell line (Expi293). The same experiment as described in Experiment 1-1 above was tested on suspension cells (Expi293F). Similar to the previously observed adherent cell lines, when the pHION8-BF construct was used during double phenotypic infection, AAV8 production increased statistically significantly by 177.1% (p<0.001) compared to the triple phenotypic infection method, and when the pHION8-BR construct was used, it increased by 173.1% (p<0.001). However, when constructs such as pHION8-NF, pHION8-NR, and pHION8-AF were used, a statistically significant decrease in production was observed, at levels of 40.9%, 66.7%, and 43.1%, respectively (Figure 7).

[0184] These results suggest that the production of AAV can be determined by the relative position and orientation of the rep2-cap8 gene within the dual helper plasmid. Subsequently, constructs in which the rep2-cap gene is located in the forward direction (BF) at the BamHI site within the pHION plasmid for dual trait infection were named pHNG, and constructs in which it is located in the reverse direction (BR) were named pHNGR. The serotype of the adeno-associated virus from which the cap gene originated was then represented numerically (pHION8-BF=pHNG8, pHION8-BR=pHNGR8).

[0185] 2. Increased AAV2 production in adherent cell line (HEK293) through triple phenotypic infection and double phenotypic infection using pHNG2. A pHNG2 construct was prepared by inserting a rep2-cap2 gene fragment in the forward direction into the BamHI site located between the start region of the E2a gene and the terminal region of the VA RNA gene within the pHelper-NG plasmid, and this construct was used in a double transfection method (Figure 2).

[0186] A comparison of AAV2 production using the triple-phenotypic infection method and the double-phenotypic infection method using pHNG2 revealed that AAV2 production was significantly increased by 279.6% (p<0.001) in the double-phenotypic infection method compared to the triple-phenotypic infection method (Figure 8). These results suggest that, similar to the increase in AAV8 production due to double-phenotypic infection with pHNG8 described above, positioning the rep2-cap2 gene between the start portion of the E2a gene and the terminal portion of the VA RNA gene can increase AAV2 production through double-phenotypic infection.

[0187] 3. Increased AAV9 production in adherent cell line (HEK293) through dual phenotypic infection using pHNG9 For double transfection, the rep2-cap9 gene fragment was cloned in the forward direction using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene to produce the pHNG9 construct (Figure 3).

[0188] A comparison of AAV9 production by triple phenotypic infection and double phenotypic infection using pHNG9 revealed that AAV9 production was significantly increased by 214.0% (p<0.001) in double phenotypic infection compared to triple phenotypic infection (Figure 9). This result was similar to the increase in AAV8 or AAV2 production observed in double phenotypic infection using pHNG8 and pHNG2, as described above. In conclusion, this result suggests that in adeno-associated virus serotype 9 (AAV9), positioning the rep2-cap9 gene between the start of the E2a gene and the terminal of the VA RNA gene can increase AAV production by the double phenotypic infection method.

[0189] 4. Increased AAV5 production in adherent cell line (HEK293) through dual phenotypic infection using pHNG5K. For double transfection, the Rep2-Cap5 gene fragment was cloned in the forward direction using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene to produce the pHNG5K construct (Figure 4).

[0190] A comparison of AAV5 production by triple phenotypic infection and double phenotypic infection using pHNG5K revealed that AAV5 production was significantly increased by 194.7% (p=0.002) in double phenotypic infection compared to triple phenotypic infection (Figure 10). This result was similar to the increase in AAV8, AAV2, or AAV9 production observed in double phenotypic infection using pHNG8, pHNG2, and pHNG9, as described above.

[0191] In conclusion, these results suggest that in adeno-associated virus serotype 5 (AAV5), positioning the rep2-cap5 gene between the start portion of the E2a gene and the terminal portion of the VA RNA gene can increase AAV production via the dual-phenotype infection method.

[0192] 5. Equivalence of cell transduction efficiency between AAV8s produced by triple or double phenotypic infection (pHNG8 or pHNGR8 constructs). We compared the cell infectivity and gene expression capacity of AAV8 produced by triple phenotypic infection with that produced by double phenotypic infection using pHNG8 and pHNGR8. These AAV8 strains were designed to express the eGFP protein. HEK293 cell lines were infected with AAV8 under conditions of 10,000 MOI, and after 72 hours, the level of intracellular eGFP expression was measured using fluid cell analysis. The results showed that, compared to AAV8 produced by triple phenotypic infection, AAV8 produced by double phenotypic infection using pHNG8 and pHNGR8 exhibited similar cell infectivity (Triple: 6.0%, pHNG8: 5.4%, pHNGR8: 5.7%), and the eGFP expression levels in infected cells were also similar (pHNG8: 93.4%, pHNGR8: 94.6%). The above results indicate that there is no difference in the cellular infectivity and post-infection gene expression ability between AAV8 produced by triple phenotypic infection and AAV8 produced by double phenotypic infection using pHNG8 and pHNGR8 (Figure 11).

[0193] 6. Equivalence of AAV2 transduction efficiency produced by triple or double transduction (pHNG2) In a study on gene transfer using AAV2, the cell infectivity and gene expression capacity of AAV2 produced by triple phenotypic infection and AAV2 produced by double phenotypic infection using pHNG2 were compared. These AAV2 molecules were designed to express the eGFP protein. HEK293 cell lines were infected with AAV2 under conditions of 2,500 MOI, and after 72 hours, the percentage of cells expressing eGFP and the level of intracellular eGFP expression were measured by fluid cell analysis. The results showed that, compared to AAV2 produced by triple phenotypic infection, AAV2 produced by double phenotypic infection using pHNG2 had at least equivalent cell infectivity (Triple: 32.0%, BF: 44.7%; p=0.38), and the eGFP expression level per infected cell was equivalent or slightly higher (129.6%, p=0.02). However, no statistically significant differences were observed in either case. These results suggest that the cellular infectivity and post-infection gene expression capacity of AAV2 produced by dual phenotypic infection using pHNG2 are at least equivalent to those of AAV2 produced by triple phenotypic infection (Figure 12).

[0194] 7. Equivalence of AAV9 transduction efficiency produced by triple or double transduction (pHNG9) When transferring genes using AAV9, we compared the cell infectivity and gene expression capacity of AAV9 produced by triple phenotypic infection and AAV9 produced by double phenotypic infection using pHNG9. These AAV9 molecules were designed to express the eGFP protein. HEK293 cell lines were infected with AAV9 under conditions of 10,000 MOI, and the level of intracellular eGFP expression was measured by fluid cell analysis after 72 hours. The results showed that AAV9 produced by double phenotypic infection using pHNG9 exhibited similar eGFP expression levels to AAV9 produced by triple phenotypic infection (90.9%, p=0.35). These results indicate that there is no difference in cell infectivity and gene expression capacity between AAV9 produced by triple phenotypic infection and AAV9 produced by double phenotypic infection using pHNG9 (Figure 13).

[0195] Although embodiments of the present invention have been described above, any person with ordinary skill in the relevant art can modify and change the present invention in various ways by adding, changing, deleting, or adding components, without departing from the spirit of the invention as described in the claims, and this too can be said to be within the scope of the rights of the present invention. [Brief explanation of the drawing]

[0196] [Figure 1A] Figures 1A to 1F show the cleavage maps of the pHelper-NG and Helper-In-One constructs (pHION8 series) for producing adeno-associated virus vector serotype 8 (AAV8) manufactured by the present inventors. Figure 1A schematically shows the pHelper-NG construct for triple phenotypic infection, which includes the E2a, E4 genes and VA RNA gene of adenovirus serotype 5. [Figure 1B] Figures 1A to 1F show the cleavage maps of pHelper-NG and Helper-In-One constructs (pHION8 series) for producing adeno-associated virus vector serotype 8 (AAV8) manufactured by the inventors. Figures 1B to 1F schematically show five Helper-In-One-NG plasmid (pHION8) constructs, which are dual helper plasmids manufactured by inserting rep2-cap8 gene fragments into various regions of the pHelper-NG construct. Figures 1B and 1C schematically show the pHION8-BF construct (Figure 1B), manufactured by cloning the rep2-cap8 construct in the forward direction (clockwise, 5'->3') and the pHION8-BR construct (Figure 1C), manufactured by cloning in the reverse direction (counterclockwise, 3'->5') using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene. [Figure 1C]Figures 1A to 1F show the cleavage maps of pHelper-NG and Helper-In-One constructs (pHION8 series) for producing adeno-associated virus vector serotype 8 (AAV8) manufactured by the inventors. Figures 1B to 1F schematically show five Helper-In-One-NG plasmid (pHION8) constructs, which are dual helper plasmids manufactured by inserting rep2-cap8 gene fragments into various regions of the pHelper-NG construct. Figures 1B and 1C schematically show the pHION8-BF construct (Figure 1B), manufactured by cloning the rep2-cap8 construct in the forward direction (clockwise, 5'->3') and the pHION8-BR construct (Figure 1C), manufactured by cloning in the reverse direction (counterclockwise, 3'->5') using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene. [Figure 1D] Figures 1A to 1F show the cleavage maps of pHelper-NG and Helper-In-One constructs (pHION8 series) for producing adeno-associated virus vector serotype 8 (AAV8) manufactured by the inventors. Figures 1B to 1F schematically show five Helper-In-One-NG plasmid (pHION8) constructs, which are dual helper plasmids manufactured by inserting rep2-cap8 gene fragments into various regions of the pHelper-NG construct. Figures 1D and 1E schematically show the pHION8-NF construct (Figure 1D), manufactured by cloning the rep2-cap8 construct in the forward direction using the NotI site located at the start of the E4 gene, and the pHION8-NR construct (Figure 1E), manufactured by cloning it in the reverse direction. [Figure 1E]Figures 1A to 1F show the cleavage maps of pHelper-NG and Helper-In-One constructs (pHION8 series) for producing adeno-associated virus vector serotype 8 (AAV8) manufactured by the inventors. Figures 1B to 1F schematically show five Helper-In-One-NG plasmid (pHION8) constructs, which are dual helper plasmids manufactured by inserting rep2-cap8 gene fragments into various regions of the pHelper-NG construct. Figures 1D and 1E schematically show the pHION8-NF construct (Figure 1D), manufactured by cloning the rep2-cap8 construct in the forward direction using the NotI site located at the start of the E4 gene, and the pHION8-NR construct (Figure 1E), manufactured by cloning it in the reverse direction. [Figure 1F] Figures 1A to 1F show the cleavage maps of pHelper-NG and Helper-In-One constructs (pHION8 series) for producing adeno-associated virus vector serotype 8 (AAV8) manufactured by the inventors. Figures 1B to 1F schematically show five Helper-In-One-NG plasmid (pHION8) constructs, which are dual helper plasmids manufactured by inserting rep2-cap8 gene fragments into various regions of the pHelper-NG construct. Figure 1F schematically shows the pHION8-AF construct manufactured by cloning the rep2-cap8 construct in the forward direction using the AsiSI site located between the start of the VA RNA gene and the terminal of the E4 gene. [Figure 2] This shows the cleavage map of the pHNG2 construct, a dual helper plasmid for the production of adeno-associated virus vector serotype 2 (AAV2). The pHNG2 construct was produced by inserting the rep2-cap2 gene fragment in the forward direction into the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene within the pHelper-NG plasmid. [Figure 3]This shows the cleavage map of the pHNG9 construct, a dual helper plasmid for the production of adeno-associated virus vector serotype 9 (AAV9). For dual phenotypic infection, the rep2-cap9 gene fragment was cloned in the forward direction using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene to produce the pHNG9 construct. [Figure 4] This shows the cleavage map of the pHNG5K construct, a dual helper plasmid for the production of adeno-associated virus vector serotype 5 (AAV5). For dual phenotypic infection, the rep2-cap5 gene fragment was cloned in the forward direction using the BamHI site located between the start of the E2a gene and the terminal of the VA RNA gene to produce the pHNG5K construct. [Figure 5A] Figures 5A and 5B show the cleavage maps of the pHNG and pHNGR constructs. Figure 5A shows the rep-cap gene fragment inserted in the positive direction between the start portion of the E2a gene and the terminal portion of the VA RNA gene. [Figure 5B] Figures 5A and 5B show the cleavage maps of the pHNG and pHNGR constructs. Figure 5B shows the rep-cap gene fragment inserted in the reverse direction. [Figure 6] This study observed the effect of double phenotypic infection using the pHION8 series on increasing or decreasing AAV8 production in adherent cell line (HEK293), compared to triple phenotypic infection. [Figure 7] This study observed the effect of double phenotypic infection using the pHION8 series on increasing or decreasing AAV8 production in a suspension cell line (Expi293), compared to triple phenotypic infection. [Figure 8] This study observed the effect of double phenotypic infection using pHNG2 on increasing AAV2 production in adherent cell line (HEK293), compared to triple phenotypic infection. [Figure 9]This study observed the effect of double phenotypic infection using pHNG9 on increasing AAV9 production in adherent cell line (HEK293), compared to triple phenotypic infection. [Figure 10] This study observed the effect of double phenotypic infection using pHNG5K on increasing AAV5 production in adherent cell line (HEK293), compared to triple phenotypic infection. [Figure 11] This is the result of the equivalence of cell transduction efficiency between AAV8 produced by triple or double transduction with the Helper-In-One construct. [Figure 12] This shows the equivalence of cell transduction efficiency between AAV2s produced by triple transduction (Triple) or double transduction (Double) using the pHNG2 construct. [Figure 13] This shows the equivalence of cell transduction efficiency between AAV9 produced by triple transduction or double transduction using the pHNG9 construct.

Claims

1. A composition for producing recombinant AAV, The composition comprises a first plasmid and a second plasmid, (a) The first plasmid is a dual helper plasmid containing the E2a gene, the E4 gene, the VA RNA gene and the rep-cap gene, wherein the E2a gene, the E4 gene and the VA RNA gene are sequentially linked, (i) The rep-cap gene is located clockwise (from 5' to 3') between the 5' end of the E2a gene and the 3' end of the VA RNA gene, or (ii) The rep-cap gene is located counterclockwise (from 3' to 5') between the 5' end of the E2a gene and the 3' end of the VA RNA gene; and (b) The second plasmid is (i) Inverted terminal repeat (ITR); (ii) foreign genes; and (iii) Regulatory elements operably linked to the foreign gene Includes, The first plasmid and the second plasmid are configured to produce recombinant AAV by in vitro double-phenotypic infection of cells providing the adenovirus E1 gene. A composition characterized by the following features.

2. The composition according to claim 1, characterized in that the 5' end of the rep-cap gene is ligated to the 5' end of the E2a gene, and the 3' end of the rep-cap gene is ligated to the 3' end of the VA RNA gene.

3. The composition according to claim 1, characterized in that the 3' end of the rep-cap gene is ligated to the 5' end of the E2a gene, and the 5' end of the rep-cap gene is ligated to the 3' end of the VA RNA gene.

4. The composition according to claim 1, characterized in that the rep-cap gene comprises a rep gene and a cap gene, and the 3' end of the rep gene is ligated to the 5' end of the cap gene.

5. The composition according to claim 4, characterized in that the rep gene includes the rep2 gene derived from adeno-associated virus serotype 2 (AAV2).

6. The composition according to claim 4, characterized in that the cap gene comprises a cap gene derived from adeno-associated virus serotype 2 (AAV2; cap2), serotype 5 (AAV5; cap5), serotype 8 (AAV8; cap8), or serotype 9 (AAV9; cap9).

7. The composition according to claim 1, characterized in that the E2a gene comprises an E2a gene derived from adenovirus serotype 5 (Ad5).

8. The composition according to claim 1, characterized in that the E4 gene comprises an E4 gene derived from adenovirus serotype 5 (Ad5).

9. The composition according to claim 1, characterized in that the VA RNA gene comprises a VA RNA gene derived from adenovirus serotype 5 (Ad5).

10. The aforementioned dual helper plasmid is (i) The E2a gene containing the sequence presented in Sequence ID No. 34; (ii) The E4 gene containing the sequence presented in Sequence ID No. 35; (iii) VA RNA gene containing the sequence presented in Sequence ID No. 36; (iv) The cap gene containing the sequence presented in SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33; and (v) rep gene containing the sequence presented in Sequence ID No. 29 The composition according to claim 4, characterized by containing the following:

11. The aforementioned regulatory elements include promoters, enhancers, exon sequences, intron sequences, splicing donor or receptor sequences, miRNA target sequences, and woodchuck hepatitis virus posttranscriptional regulatory elements. The composition according to claim 1, characterized by comprising a regular element (WPRE) sequence, a polyadenylation (pA) sequence, or any combination thereof.

12. The composition according to claim 1, characterized in that the foreign gene encodes a wild-type polypeptide or any variant thereof, a fusion protein, an antibody or its antigen-binding fragment, an RNA-based molecule or any combination thereof.

13. In vitro cells for producing recombinant AAV, The in vitro cells described above include a first plasmid and a second plasmid. (a) The first plasmid is a dual helper plasmid containing the E2a gene, the E4 gene, the VA RNA gene and the rep-cap gene, wherein the E2a gene, the E4 gene and the VA RNA gene are sequentially linked, (i) The rep-cap gene is located clockwise (from 5' to 3') between the 5' end of the E2a gene and the 3' end of the VA RNA gene, or (ii) The rep-cap gene is located counterclockwise (from 3' to 5') between the 5' end of the E2a gene and the 3' end of the VA RNA gene; and (b) The second plasmid is (i) Inverted terminal repeat (ITR); (ii) foreign genes; and (iii) Regulatory elements operably linked to the foreign gene including In vitro cells characterized by the following features.

14. This is a kit for producing a modified AAV. The kit includes a first plasmid, a second plasmid, and instructions for use. (a) The first plasmid is a dual helper plasmid containing the E2a gene, the E4 gene, the VA RNA gene and the rep-cap gene, wherein the E2a gene, the E4 gene and the VA RNA gene are sequentially linked, (i) The rep-cap gene is located clockwise (from 5' to 3') between the 5' end of the E2a gene and the 3' end of the VA RNA gene, or (ii) The rep-cap gene is located counterclockwise (from 3' to 5') between the 5' end of the E2a gene and the 3' end of the VA RNA gene; and (b) The second plasmid is (i) Inverted terminal repeat (ITR); (ii) foreign genes; and (iii) Regulatory elements operably linked to the foreign gene Includes, The first plasmid and the second plasmid are configured to produce recombinant AAV by in vitro double-phenotypic infection of cells providing the adenovirus E1 gene. A kit characterized by the following features.

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