Modified REP-CAP plasmid and its use

Abstract

This disclosure provides a modified rep-cap plasmid containing a tetracycline-inducible expression system that can be used to produce a recombinant AAV1 vector.

Description

Technical Field

[0001] This application claims priority to U.S. Patent Application No. 63 / 545,351, filed October 23, 2023, and No. 63 / 456,632, filed April 3, 2023. These applications are incorporated herein by reference.

[0002] The present invention relates to, for example, modified Rep-Cap plasmids for producing recombinant adeno-associated virus (rAAV) and the use of modified Rep-Cap plasmids. rAAV can contain a transgene of interest, which can include any gene that is desired to be expressed and can fit within the AAV capsid.

Background Art

[0003] Research and clinical applications of recombinant adeno-associated virus have increased significantly in recent years, parallel to the regulatory approval of rAAV gene therapy products. For gene therapy to be effective, the rAAV vector must be present at a concentration sufficient to exert a therapeutic effect. The standard approach used to produce rAAV vectors results in concentrations that are too low for patient treatment. Therefore, the rAAV vector must be further concentrated to reach an effective dose in a reasonable volume. Concentration of the vector can also lead to concentration of contaminants or impurities and thus is not without drawbacks. Therefore, there is a need for new approaches to increase the production of AAV vectors.

[0004] Reference to Electronic Sequence Listing This application is electronically filed in.xml format and includes a sequence listing incorporated herein by reference. The.xml copy was created on March 27, 2024, named "11635.xml", and is 68,473 bytes in size. The sequence listing contained in this.xml file is part of this specification and is incorporated herein by reference.

Summary of the Invention

[0005] The present invention provides a modified adeno-associated virus (AAV) rep-cap plasmid and a method for using the same to produce an AAV vector having an AAV capsid (e.g., AAV1 capsid). The modified rep-cap plasmid may contain a tetracycline-inducible expression system (e.g., a Tet-Off system or a Tet-On system) positioned between a polynucleotide encoding the AAV rep protein and a polynucleotide encoding the AAV capsid protein (e.g., AAV1 capsid protein). For example, the rep-cap plasmid may have the configuration shown in Figures 1, 3, 5, 6, or 7 and may contain one or more sequences described herein.

[0006] When a modified rep-cap plasmid is introduced into cells together with a helper plasmid and a transgene plasmid, rAAV (e.g., rAAV1) production is increased compared to rAAV (e.g., rAAV1) production using a conventional rep-cap plasmid (e.g., a rep-cap plasmid lacking a tetracycline-inducible expression system). The tetracycline-inducible expression system can be "off" or "inactivated" (e.g., by exposing a plasmid containing the Tet-Off system to a derivative such as doxycycline, or by maintaining a plasmid containing the Tet-On system in the absence of the derivative) to increase the proportion of complete rAAV vectors produced (i.e., rAAV vectors containing the transgene of interest to be expressed). Therefore, rAAV vectors, such as the rAAV1 vector, can be produced using the compositions and methods described herein for use in gene therapy (e.g., gene therapy approaches that would utilize an AAV1 vector, such as gene therapy approaches targeting the central nervous system, heart, skeletal muscle, retinal pigment epithelium, or inner ear).

[0007] The present invention provides an adeno-associated virus (AAV) rep-cap plasmid comprising, in a 5' to 3' orientation, (a) a polynucleotide encoding an AAV rep protein, (b) a tetracycline-inducible expression system, (c) an AAV promoter such as p41 or TRE-tight, and (d) a polynucleotide encoding an AAV1 capsid protein.

[0008] AAV rep proteins can be AAV2 rep proteins.

[0009] The polynucleotide encoding the AAV rep protein can encode Rep78 and Rep52. Rep78 may have the sequence of SEQ ID NO: 1. Rep52 may have the sequence of SEQ ID NO: 2.

[0010] The polynucleotide encoding the AAV rep protein may have the sequence shown in SEQ ID NO: 3.

[0011] The tetracycline-inducible expression system may also be a tetracycline-off (Tef-Off) system. The Tet-Off system may include, in a 5' to 3' orientation, (a) a first polynucleotide encoding a tetracycline transactivator (tTA) and containing a tetracycline repressor-derived DNA-binding domain and a transcriptional activation domain, and (b) a second polynucleotide containing 1 to 12 tet operator (tetO) sequences. The first polynucleotide may encode a polypeptide having the amino acid sequence of SEQ ID NO: 5. The first polynucleotide may have at least 80% sequence identity with respect to SEQ ID NO: 6 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) (e.g., it may have at least 80% sequence identity with respect to SEQ ID NO: 6 and may encode a polypeptide having the amino acid sequence of SEQ ID NO: 5). The first polynucleotide may have the sequence of SEQ ID NO: 6. Each tetO sequence may have the sequence of SEQ ID NO: 4. Each tetO sequence can be separated from adjacent tetO sequences by 0 to 20 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). A second polynucleotide can contain eight tetO sequences. The second polynucleotide can have at least 80% sequence identity with respect to SEQ ID NO: 7 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity).The second polynucleotide may have at least 80% sequence identity with respect to the sequence of sequence number 7 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity), in addition to eight tetO sequences, each of which may have the sequence of sequence number 4. The Tet-Off system may further include a poly(A) signal sequence located between the first and second polynucleotides.

[0012] A tetracycline-inducible expression system may be a Tet-On system. The Tet-On system may include, in a 5' to 3' orientation, (a) a first polynucleotide encoding a reverse tetracycline trans-activator (rtTA), which may include a tetracycline repressor-derived DNA-binding domain (e.g., a reverse tetracycline repressor) and a transcriptional activation domain, and (b) a second polynucleotide containing 1 to 12 tet operator (tetO) sequences. The first polynucleotide may encode a polypeptide having the amino acid sequence of SEQ ID NO: 20. The first polynucleotide may have at least 80% sequence identity with respect to SEQ ID NO: 21 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) (e.g., it may have at least 80% sequence identity with respect to SEQ ID NO: 21 and may encode a polypeptide having the amino acid sequence of SEQ ID NO: 20). The first polynucleotide may have the sequence of SEQ ID NO: 21. Each tetO sequence may have the sequence of SEQ ID NO: 4. Each tetO sequence can be separated from an adjacent tetO sequence by 0 to 20 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). A second polynucleotide can contain eight tetO sequences. The second polynucleotide can have at least 80% sequence identity with respect to SEQ ID NO: 7 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity).The second polynucleotide may have at least 80% sequence identity with respect to the sequence of sequence number 7 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity), in addition to eight tetO sequences, each of which may have the sequence of sequence number 4. The Tet-On system may further include a poly(A) signal sequence located between the first and second polynucleotides.

[0013] The p41 promoter may have the sequence of sequence number 8.

[0014] A polynucleotide encoding the AAV1 capsid protein can encode a polypeptide having the amino acid sequence of SEQ ID NO: 9. A polynucleotide encoding the AAV capsid protein can have the sequence of SEQ ID NO: 10. A polynucleotide encoding the AAV capsid protein can have the sequence of SEQ ID NO: 11.

[0015] The rep-cap plasmid can contain the polynucleotide sequence of SEQ ID NO: 12. The rep-cap plasmid can contain the polynucleotide sequence of SEQ ID NO: 13. The rep-cap plasmid can contain the polynucleotide sequence of SEQ ID NO: 22.

[0016] The present invention also provides a method for producing recombinant adeno-associated virus by introducing into mammalian cells (a) a rep-cap plasmid of any of the aforementioned inventions, such as the use of a p41 promoter or a TRE tight promoter, (b) a helper plasmid containing one or more helper genes selected from E4, E2a, and VA, and (c) a transgene plasmid containing the transgene of interest flanked by inverse terminal repeats. The introduction step may be performed under conditions that allow for the formation of recombinant adeno-associated virus. These methods may further include a step of collecting recombinant adeno-associated virus. The transgene plasmid may further include a promoter operably ligated to the transgene of interest. The mammalian cells may be HEK-293 cells. The rep-cap plasmid may contain the polynucleotide sequence of SEQ ID NO: 12. The rep-cap plasmid may contain the polynucleotide sequence of SEQ ID NO: 13. The rep-cap plasmid may contain the polynucleotide sequence of SEQ ID NO: 22. The transgene of interest may encode a protein endogenously expressed in the human inner ear. The target transgene can encode, for example, solute carrier family 26, member 4 (pendrin), otoferrin (OTOF), stereocillin (STRC), Atonal BHLH transcription factor 1 (ATOH1), gap junction protein beta 2 (GJB2), or SRY-Box 2 (Sox2).Other transgenes that can be expressed include, for example, GJB6, WFS1, COCH, EYA4, MYO7A, POU4F3, ACTG1, MYO6, REST, NLRP3, COL11A1, TJP2, TBC1D24, SLC26A4, ELMOD3, EPSN, WHRN, IGF1, IGF1R, MYO15A, TMIE, TMC1, TMC2, TMPRSS3, OTOF, CDH23, GIPC3, USH1C, OTOG, TECTA, OTOA, PDCH15, CLDN14, WHRN, ESRRB, MYO3A, HGF, ILDR1, ADCY1, CIB2, MARVELD2, SOX2, SLC26A5, COL4A3, COL4A4, COL4A5, CLPP, PJVK, LRTOMT / COMT2, LOXHD1, TPRM, SYNE4, KCNJ10, PTPRQ, OTOGL, LHFPL5, A1PR2, CABP2, MET, GRXCR2, EPS8, CLIC5, EPS8L2, WBP2, ROR1, CLDN9, USH1G, PKHD1L1, DIAPH3, NDP, USH2A, CLRN1, SANS, HARS1, TRIOBP, and other genes. The transgene of interest can be any gene that can be contained within an AAV capsid and is desired to be expressed. If the total size is such that the entire gene can fit within the AAV capsid, two or more transgenes of interest are possible.

[0017] Viral yield is measured by using ddPCR of the clarified lysate and is at least 1×10 11 vg / ml (e.g., 1×10 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL, or more, e.g., 1×10 11 vg / mL to 5×10 11 vg / mL, 1×10 11 vg / mL to 1×10 12 vg / mL, 1×10 11 vg / mL to 5×10 12 vg / mL, or 1×10 11 vg / mL to 1×1013 It may be vg / mL).

[0018] The present invention also provides a method for producing recombinant adeno-associated virus by introducing into mammalian cells (a) a rep-cap plasmid comprising (i) a polynucleotide encoding the AAV rep protein, (ii) a tetracycline-inducible expression system, (iii) an AAV promoter (such as p41 or TRE-tight), and (iv) a polynucleotide encoding the AAV capsid protein, in a 5' to 3' orientation; (b) a helper plasmid containing one or more helper genes selected from E4, E2a, and VA; and (c) a transgene plasmid containing the desired transgene flanked by reverse-terminal repeats. The introduction step may be performed under conditions that enable the formation of recombinant adeno-associated virus. These methods may further include a step of collecting the recombinant adeno-associated virus. The AAV vector may be a polynucleotide encoding the AAV1, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S capsid protein. The tetracycline-inducible expression system may be a Tet-Off system or a Tet-On system as described herein. The transgene plasmid of the present invention may further comprise a promoter operably ligated to the transgene of interest. The mammalian cell may be HEK-293 cell. The transgene of interest may encode a protein endogenously expressed in the human inner ear. The target transgene can encode, for example, solute carrier family 26, member 4 (pendrin), otoferrin (OTOF), stereocillin (STRC), Atonal BHLH transcription factor 1 (ATOH1), gap junction protein beta 2 (GJB2), or SRY-Box 2 (Sox2).Other transgenes that can be expressed include, for example, GJB6, WFS1, COCH, EYA4, MYO7A, POU4F3, ACTG1, MYO6, REST, NLRP3, COL11A1, TJP2, TBC1D24, SLC26A4, ELMOD3, EPSN, WHRN, IGF1, IGF1R, MYO15A, TMIE, TMC1, TMC2, TMPRSS3, OTOF, CDH23, GIPC3, USH1C, OTOG, TECTA, OTOA, PDCH15, CLDN14, WHRN, ESRRB, MYO3A, HGF, and ILDR1. Examples of target genes include ADCY1, CIB2, MARVELD2, SOX2, SLC26A5, COL4A3, COL4A4, COL4A5, CLPP, PJVK, LRTOMT / COMT2, LOXHD1, TPRM, SYNE4, KCNJ10, PTPRQ, OTOGL, LHFPL5, A1PR2, CABP2, MET, GRXCR2, EPS8, CLIC5, EPS8L2, WBP2, ROR1, CLDN9, USH1G, PKHD1L1, DIAPH3, NDP, USH2A, CLRN1, SANS, HARS1, TRIOBP, and others. The target transgene includes any gene that is desired to be expressed and can fit within the AAV capsid. Two or more target transgenes are possible if the total size of the entire gene can fit within the AAV capsid.

[0019] The virus yield is measured using ddPCR of the clarified lysates, and is at least 1 × 10⁻⁶. 11 vg / ml (for example, 1 × 10) 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL or higher, for example, 1 × 10⁻⁶ 11 vg / mL ~ 5 × 10 11 vg / mL, 1×10 11 vg / mL ~ 1 × 10 12 vg / mL, 1×10 11 vg / mL ~ 5 × 10 12 vg / mL, or 1×10 11vg / mL ~ 1 × 10 13 It may be vg / mL).

[0020] In any of the inventions described above, the tetracycline-inducible expression system may be a Tet-Off system, and these methods may include producing recombinant adeno-associated virus by exposing mammalian cells to a derivative of the Tet-Off system (e.g., production in the presence of doxycycline). These methods result in a higher percentage of complete AAV vectors (i.e., AAV vectors containing the transgene or nucleic acid to be expressed) than producing recombinant adeno-associated virus using the same modified rep-cap plasmid in the absence of a derivative of the Tet-Off system (e.g., exposing the Tet-Off system to a derivative that can increase the percentage of complete AAV vectors by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more percentage points, e.g., 4% complete to 6%, 8%, 10%, 12%, 14% complete, or more).

[0021] In any of the inventions described above, the tetracycline-inducible expression system may be a Tet-On system, and these methods may include producing recombinant adeno-associated virus by maintaining mammalian cells in the absence of a Tet-On system derivative (e.g., producing without exposure to doxycycline). These methods may result in a higher percentage of complete AAV vectors (i.e., AAV vectors containing the transgene or nucleic acid to be expressed) than producing recombinant adeno-associated virus using the same modified rep-cap plasmid in the presence of a Tet-On system derivative (e.g., producing AAV vectors without exposure to the Tet-On system with a derivative that can increase the percentage of complete AAV vectors by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 percent points, e.g., 10% complete to 12%, 14%, 16%, 18%, 20%, 22% complete, or more).

[0022] The present invention also provides methods for increasing the yield of recombinant AAV1-capped adeno-associated virus (AAV1-capped adeno-associated virus) by at least 2.5 times (e.g., 2.5 times, 2.7 times, 3.0 times, 3.1 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 20 times, or more) compared to recombinant AAV1-capped adeno-associated virus produced with pAAV-RC1, which include the step of substituting pAAV-RC1 with any one of the rep-cap plasmids of the aforementioned inventions in the production of adeno-associated virus. The yield of recombinant AAV1-capped adeno-associated virus can be increased by at least 7 times. The yield of recombinant AAV1-capped adeno-associated virus can be increased by at least 8 times. The yield of recombinant AAV1-capped adeno-associated virus can be increased by at least 9 times. The yield of recombinant AAV1-capped adeno-associated virus can be increased by at least 10-fold. The rep-cap plasmid can contain the polynucleotide sequence of SEQ ID NO: 12. The rep-cap plasmid can contain the polynucleotide sequence of SEQ ID NO: 13. The rep-cap plasmid can contain the polynucleotide sequence of SEQ ID NO: 22. The tetracycline-inducible expression system may be a Tet-Off system, and these methods can be carried out in the presence of a Tet-Off system derivative. The tetracycline-inducible expression system may be a Tet-On system, and these methods can be carried out in the absence of a Tet-On system derivative. These methods also increase the percentage of complete vector produced.

[0023] The present invention further provides methods to increase both the yield and percentage of complete recombinant AAV1-capped adeno-associated virus (AAV vector containing the desired transgene or nucleotide sequence to be expressed) compared to recombinant AAV1-capped adeno-associated virus produced with pAAV-RC1, which include the step of replacing pAAV-RC1 with any one of the aforementioned inventions' rep-cap plasmids that may include a Tet-On system in the production of the adeno-associated virus. The formation of recombinant AAV1-capped adeno-associated virus in such a method can be achieved without the addition of derivatives (e.g., without the addition of doxycycline—meaning that the Tet-On system is not induced). The formation of recombinant AAV1-capped adeno-associated virus in such a method can also be achieved with the addition of derivatives (e.g., tetracycline or doxycycline).

[0024] The present invention further provides cells containing the rep-cap plasmid according to the present invention, and a cell culture containing the cells according to the present invention. [Brief explanation of the drawing]

[0025] [Figure 1] A schematic plasmid map of the modified rep-cap plasmid P929 is shown.

[0026] [Figure 2] A schematic plasmid map of the standard rep-cap plasmid P374 is shown.

[0027] [Figure 3] A schematic plasmid map of the modified rep-cap plasmid P2056 is shown.

[0028] [Figure 4] This graph shows the percentage of complete vectors produced using the modified rep-cap plasmid P929 in the presence and absence of doxycycline, as measured by SEC-MALS.

[0029] [Figure 5] A schematic plasmid map of the rep-cap plasmid P2057 is shown.

[0030] [Figure 6] A schematic plasmid map of the rep-cap plasmid P2058 is shown.

[0031] [Figure 7] A schematic plasmid map of the rep-cap plasmid P2059 is shown.

[0032] [Figure 8A-B] Data from the production of AAV1 containing the GJB2 transgene using the RC1 p5AS plasmid or P929 are shown. Figure 8A shows the genome titer, and Figure 8B shows the capsid titer.

[0033] [Figure 9A-C] The production of AAV1 containing a highly sensitive green fluorescent protein (eGFP) transgene using plasmids P374, P929, P2057, P2058, or P2059 is shown. Figure 9A compares the capsid titer for each of the five plasmids. Figure 9B compares the genomic titer for each of the five plasmids using droplet digital PCR (ddPCR). Figure 9C compares the estimated genomic titer for each of the five plasmids, based on the percentage of complete capsid.

[0034] [Figure 10A-B] This document describes the production of AAV1 containing the eGFP transgene using plasmids P374, P929, P2057, P2058, or P2059, and evaluates the completeness of the capsid achieved by each of the five plasmids using mass photometry. Figure 10A shows the percentage of complete capsids. Figure 10B shows the percentage of partially filled capsids. [Modes for carrying out the invention]

[0035] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention belongs.

[0036] As used herein, the terms “AAV” and “adeno-associated virus” refer to dependent parvoviruses within the Parvoviridae family genus of viruses. AAV may refer to AAV derived from naturally occurring “wild-type” viruses, AAV derived from rAAV genomes packaged in capsids derived from capsid proteins encoded by naturally occurring cap genes, and / or rAAV genomes packaged in capsids derived from capsid proteins encoded by non-natural capsid cap genes.

[0037] As used herein, the term "rAAV" refers to recombinant AAV. Recombinant AAV refers to an AAV genome in which some or all of the rep and cap genes are replaced with heterologous sequences.

[0038] As used herein, the term “polynucleotide encoding AAV capsid protein” refers to a nucleic acid sequence encoding a capsid protein that forms or contributes to the formation of the viral capsid or protein shell. In the case of AAV, the polynucleotide encoding the AAV capsid protein encodes the capsid proteins VP1, VP2, and VP3.

[0039] As used herein, “administration” means providing or giving a therapeutic agent (e.g., an rAAV1 vector produced using the modified rep-cap plasmid described herein) to a subject by any valid route. Exemplary routes of administration are described herein below.

[0040] As used herein, the term “cell type” refers to a group of cells that share a statistically separable phenotype based on gene expression data. For example, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation profiles. Cells of a common cell type may include those isolated from common tissues (e.g., epithelial tissue, nerve tissue, connective tissue, or muscle tissue), and / or those isolated from common organs, tissue systems, blood vessels, or other structures and / or regions in an organism.

[0041] As used herein, the terms “conservative mutation,” “conservative substitution,” and “conservative amino acid substitution” refer to the substitution of one or more amino acids with one or more different amino acids that exhibit similar physicochemical properties, such as polarity, static charge, and stereovolume. These properties are summarized in Table 1 below for each of the 20 naturally occurring amino acids. [Table 1]

[0042] From this table, it can be understood that the conserved amino acid families include (i) G, A, V, L, and I, (ii) D and E, (iii) C, S, and T, (iv) H, K, and R, (v) N and Q, and (vi) F, Y, and W. Therefore, a conserved mutation or substitution is the substitution of one amino acid with a member of the same amino acid family (for example, substituting Ser with Thr, or Lys with Arg).

[0043] As used herein, the term “endogenous” refers to a molecule (e.g., polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., human) or in a particular location within an organism (e.g., an organ, tissue, or human cell, such as a human cochlear support cell).

[0044] As used herein, the term “expression” refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. The term “expression product” refers to a protein or RNA molecule produced by any of these events.

[0045] As used herein, the term “exogenous” describes molecules (e.g., polypeptides, nucleic acids, or cofactors) that are not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or human cell, such as a human cochlear support cell). Exogenous substances include those supplied to an organism or to a culture extracted therefrom from an exogenous source.

[0046] As used herein, the term “heterologous” refers to a combination of elements that does not exist in nature. For example, a heterologous transgene refers to a transgene that is not expressed in nature by the promoter to which it is activatably linked.

[0047] As used herein, the term “target host cell” means a cell that is transformed by a nucleic acid sequence, or can be transformed, to express the gene of interest, and which would preferably be derived from a cell line. The term includes offspring of a parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, as long as the gene of interest is present.

[0048] Suitable target host cells for use with the present invention can be easily selected by those skilled in the art. In some embodiments, the cell line is a eukaryotic cell line such as a yeast cell line, an insect cell line (e.g., Sf9 and Sf21 cells), or a mammalian cell line. Preferred mammalian cells include primate cells (including human), canine cells, and rodent cells. The cells may be primary cells or immortalized cells. Suitable cells can be selected from cell lines transfected with viral genes, including but not limited to Vero cells, COS cells, HEK293 cells, HeLa cells, CHO cells, BHK cells, MDCK cells, amniotic fluid cells (human), embryonic cells, immortalized human retinal cells transfected with adenovirus genes, such as PER.C6 cells, or NSO cells, for example, AD5 E1. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell line. Some examples of CHO cells include, but are not limited to, CHO-ori, CHO-K1, CHO-s, CHO-DHB11, CHO-DXB11, CHO-K1SV, and their variants and variants. In other embodiments, the cells are HEK293 cells. Some examples of HEK293 cells include, but are not limited to, HEK293, HEK293A, HEK293E, HEK293F, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T, and their variants and variants.

[0049] As used herein, the term “naturally occurring” refers to a material found in nature or a form of a material found in nature.

[0050] As used herein, the term “operably linked” refers to a first molecule linked to a second molecule in such a way that the first molecule influences the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule, and may or may not be adjacent. For example, if a promoter regulates the transcription of a desired transcribable polynucleotide molecule within a cell, the promoter is operably linked to the transcribable polynucleotide molecule. In addition, two parts of a transcriptional regulatory element are operably linked to each other if they are linked in such a way that the transcriptional activation functionality of one part is not adversely affected by the presence of the other part. The two transcriptional regulatory elements may be operably linked to each other via a linker polynucleotide (e.g., an intervening non-coding polynucleotide) or operably linked to each other without an intervening nucleotide.

[0051] As used herein, the term "pAAV-RC1" refers to a vector containing a polynucleotide encoding the AAV2 rep protein and a polynucleotide encoding the AAV1 capsid protein, but without a tetracycline-inducible expression system. Such vectors are produced by Cell Biolabs, Inc. (cellbiolabs.com / sites / default / files / VPK-421-aav-rc1-vector.pdf).

[0052] As used herein, the term “plasmid” refers to an extrachromosomal circular double-stranded DNA molecule into which additional DNA segments may be ligated. A plasmid is a type of vector, a nucleic acid molecule that can transport another nucleic acid to which it is ligated. Certain plasmids are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial plasmids with bacterial origins of replication, and episomal mammalian plasmids). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Certain plasmids can direct the expression of genes to which they are operably ligated.

[0053] As used herein, the term “polynucleotide” refers to a polymer of nucleosides. Typically, polynucleotides consist of naturally occurring nucleosides in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. The term encompasses molecules containing nucleosides or nucleoside analogs that include chemically or biologically modified bases, modified skeletons, etc., whether or not they are found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. When this application refers to polynucleotides, it is understood that both DNA and RNA, and in each case, both single-stranded and double-stranded forms (as well as complementary strands of each single-stranded molecule) are provided. As used herein, “polynucleotide sequence” may refer to the polynucleotide material itself and / or sequence information (i.e., a series of letters used as abbreviations for bases) that biochemically characterizes a particular nucleic acid. Unless otherwise indicated, the polynucleotide sequences presented herein are presented in the 5′ to 3′ direction.

[0054] As used herein, the term “promoter” refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives the transcription of the transgene. Preferred AAV promoters include, but are not limited to, the p41 and TRE-tight promoters. Other AAV promoters that can be used include the p40 promoter.

[0055] The “sequence identity percentage (%)” with respect to a reference polynucleotide or reference polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or reference polypeptide sequence, after the sequences have been aligned to achieve the maximum possible sequence identity percentage and gaps have been introduced as necessary. Alignment for the purpose of determining nucleic acid or amino acid sequence identity percentage can be achieved in various ways within the capabilities of a person skilled in the art, for example, using readily available computer software such as BLAST, BLAST-2, or Megalign software. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. For example, a sequence identity percentage value can be generated using the sequence comparison computer program BLAST. As an example, the sequence identity percentage of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (alternatively, it can be expressed as a given nucleic acid or amino acid sequence A having a specific sequence identity percentage to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows:

[0056] 100×(fraction X / Y)

[0057] In the formula, X is the number of nucleotides or amino acids scored as identical in the alignments of A and B by a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the sequence identity percentage of A to B will not be equal to the sequence identity percentage of B to A.

[0058] As used herein, the term “pharmaceutical composition” means a mixture containing a therapeutic agent, optionally one or more pharmaceutically acceptable excipients, diluents, and / or carriers, administered to a subject such as a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition affecting or potentially affecting the subject.

[0059] As used herein, the term “pharmaceutically acceptable” means a compound, material, composition and / or dosage form suitable for contact with the tissues of a subject, such as those of a mammal (e.g., human), without transient toxicity, irritation, allergic reactions and other problematic complications, and with a reasonable benefit-to-risk ratio.

[0060] As used herein, the term “polynucleotide encoding AAV rep protein” refers to nucleic acid sequences encoding non-structural proteins (i.e., rep78, rep68, rep52, and rep40) necessary for viral replication and production.

[0061] As used herein, the term "rep-cap plasmid" refers to a plasmid that provides viral rep and cap gene function. This plasmid may be useful for AAV production from rAAV genomes lacking functional rep and / or capsid gene sequences.

[0062] As used herein, the term “subject” refers to any organism to which a composition according to the present invention may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). Subjects may be humans and animals that seek, require, are receiving, or may receive treatment in the future, and are being cared for by specialists trained in specific diseases and conditions. Preferably, the subject is human.

[0063] As used herein, the terms “transcriptional regulatory element” and “regulatory sequence” refer to polynucleotides that, at least in part, regulate the transcription of a gene of interest. Transcriptional regulatory elements may include promoters, enhancers, and other polynucleotides (e.g., polyadenylation signals) that regulate or assist in the regulation of gene transcription. Examples of transcriptional regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0064] As used herein, the term “transfection” refers to any of the various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, nucleofection, squeeze-poration, acoustic perforation, optical transfection, magnetofection, and imparefection.

[0065] As used herein, the terms “transduction” and “transduction” refer to a method of introducing a vector construct or a portion thereof into a cell. When the vector construct is contained in a viral vector, such as an AAV vector, transduction refers to the viral infection of the cell, and the subsequent transfer and integration of the vector construct or a portion thereof into the cell genome.

[0066] As used herein, the term “transgene” refers to recombinant nucleic acid (e.g., DNA) that encodes a gene product such as a peptide, protein, or RNA (e.g., mRNA encoding a protein, or inhibitory RNA such as miRNA or shRNA). In addition to the coding region of the gene product, a transgene may include, or may be operably linked to, one or more elements for promoting or enhancing expression, such as promoters, enhancers, destabilization domains, response elements, reporter elements, insulating elements, polyadenylation signals, and / or other functional elements. According to the present invention, any known and suitable promoters, enhancers, destabilization domains, response elements, reporter elements, insulating elements, polyadenylation signals, and / or other functional elements can be utilized. Examples of transgenes are specified herein.

[0067] As used herein, “treatment” and “to treat” in relation to a disease or condition mean means to obtain a beneficial or desired outcome, such as a clinical outcome. Beneficial or desired outcomes may include, but are not limited to, the reduction or recovery of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the severity of the disease or condition; a stable (i.e., non-exacerbating) state of the disease, disorder, or condition; prevention of the spread of the disease or condition; delay or slowing of the progression of the disease or condition; recovery or mitigation of the disease or condition; and remission (whether partial or complete), whether detectable or undetectable. To “recover” or “mitigate” a disease or condition means that the severity and / or undesirable clinical symptoms of the disease, disorder, or condition are reduced and / or the progression is slowed or prolonged compared to the degree or course of time in the absence of treatment. “Treatment” may also mean extending survival compared to the expected survival time in the absence of treatment. Those requiring treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those for whom the condition or disorder should be prevented.

[0068] As used herein, the term “vector” refers to nucleic acid vectors, such as DNA vectors including plasmids, cosmids, or artificial chromosomes, RNA vectors, viruses, or any other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering polynucleotides encoding exogenous proteins to prokaryotic or eukaryotic cells. Examples of such expression vectors are described, for example, in Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the compositions and methods described herein contain polynucleotide sequences and additional sequence elements used for protein expression, for example. Certain vectors that may be used for the expression of viral capsid proteins, as described herein, include vectors containing regulatory sequences such as promoter and enhancer regions that direct gene transcription. Other useful vectors for transgene expression contain polynucleotide sequences that increase the translation rate of the transgene or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signaling sites to direct the efficient transcription of the gene supported on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may contain polynucleotides encoding markers for the selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or norseoslysin.

[0069] As used herein, “viral capsid protein” refers to a capsid protein that constitutes a proteinaceous shell. Such a proteinaceous shell is generally composed of one or more viral capsid proteins and, when assembled, can be filled with one or more polynucleotide molecules. The viral capsid proteins described herein may be, for example, viral proteins (VP)1, VP2, and VP3. Furthermore, the viral capsid proteins described herein may refer to viral capsid proteins from AAV1.

[0070] As used herein, the term "VP1" refers to a capsid protein that is a component of an AAV capsid, such as AAV1. The AAV1 VP1 protein has the sequence of Sequence ID No. 9. As used herein, VP1 may have a surface binding site that interacts with one or more molecules on the surface of a cell to initiate the process of cell entry (e.g., endocytosis and receptor-mediated fusion). As used herein, VP1 may self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. VP1 exhibits self-binding properties and may self-assemble around the outside of each VP1-containing capsid.

[0071] As used herein, the term "VP2" refers to a capsid protein that is a component of an AAV capsid, such as AAV1. The AAV1 VP2 protein has the sequence of Sequence ID No. 14. As used herein, VP2 can facilitate the entry of a capsid into a host cell, for example, by mediating association with and exit from the host cell's endoplasmic nettle, and by facilitating the entry of nucleic acid molecules into the host cell nucleus. As used herein, VP2 can self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. VP2 can self-assemble within each VP2-containing capsid.

[0072] As used herein, the term "VP3" refers to a capsid protein that is a component of an AAV capsid, such as AAV1. The AAV1 VP3 protein has the sequence of Sequence ID No. 15. As used herein, VP3 can facilitate the entry of a capsid into a host cell, for example, by mediating association with and exit from the host cell's endoplasmic nettle, and by facilitating the entry of nucleic acid molecules into the host cell nucleus. As used herein, VP3 can self-assemble into a structure consisting of VP1, VP2, and / or VP3 molecules. VP3 can self-assemble within each VP3-containing capsid.

[0073] As used herein, the term “wild type” refers to the most frequently occurring genotype for a particular gene in a given organism.

[0074] explanation This specification describes compositions and methods for producing AAV vectors (e.g., AAV1 vector). The present invention provides a modified rep-cap plasmid containing a tetracycline-inducible expression system (e.g., a Tet-Off or Tet-On system) positioned between a polynucleotide encoding an AAV rep protein and a polynucleotide encoding an AAV capsid protein (e.g., AAV1 capsid protein). The polynucleotide encoding the AAV capsid protein (e.g., AAV1 capsid protein) can encode AAV capsid proteins VP1, VP2, and VP3. In addition, the present invention provides a method for producing AAV vectors (e.g., AAV1 vector) using the modified rep-cap plasmid (e.g., by transducing cells using the modified rep-cap plasmid, a helper plasmid, and a transgene plasmid). These methods may include turning off the tetracycline-inducible expression system (e.g., producing an AAV vector using a modified rep-cap plasmid containing the Tet-Off system in the presence of a Tet-Off system derivative, or producing an AAV vector using a modified rep-cap plasmid containing the Tet-On system in the absence of a Tet-On system derivative). Using the compositions and methods described herein, AAV production (e.g., AAV1 production) can be increased compared to approaches using unmodified rep-cap plasmids (e.g., rep-cap plasmids containing the same polynucleotides encoding the rep and capsid proteins but lacking the tetracycline-inducible expression system). In addition, using the compositions and methods described herein can increase the proportion of complete AAV vectors produced (e.g., AAV vectors containing the transgene or polynucleotide to be expressed) compared to approaches using the same modified rep-cap plasmid with the tetracycline-inducible expression system turned on. AAV vectors produced using the methods described herein (e.g., AAV1 vectors) may contain the transgene of interest and can be used for gene therapy applications.

[0075] Modified rep-cap plasmid Three plasmids are required for the production of an AAV vector: 1) a rep-cap plasmid containing the AAV structural and packaging genes, 2) a helper plasmid containing genes encoding proteins necessary for viral replication, and 3) a transgene plasmid (also called a transfer plasmid) containing the transgene to be expressed by the AAV vector. The rep-cap plasmid contains two genes: a replication (rep) gene and a capsid (cap) gene. The rep gene encodes four proteins involved in viral genome replication and packaging: Rep78, Rep68, Rep52, and Rep40. The cap gene encodes three AAV viral capsid proteins, VP1, VP2, and VP3, which together form the outer capsid shell protecting the viral genome. Alternative splicing and different translation initiation sites produce the three VP proteins. The cap gene also encodes an alternative open reading frame assembly activation protein (AAP) and a membrane-associated accessory protein (MAAP). AAP is thought to provide a scaffold function for capsid assembly.

[0076] The present invention is partly based on the discovery of modifications to rep-cap plasmids that can result in increased AAV production, such as AAV1 (for example, when the modified rep-cap plasmid is introduced into cells along with a helper plasmid and a transgene plasmid). These modifications may include introducing a tetracycline-inducible expression system between the polynucleotide encoding the AAV rep protein and the polynucleotide encoding the AAV1 capsid protein. Producing larger quantities of AAV vectors, such as AAV1, is desirable because it facilitates production and reduces the concentration required to achieve effective doses for gene therapy applications in reasonable volumes. The inventors have also made the surprising discovery that a higher percentage of complete vectors can be produced using modified rep-cap plasmids by producing AAV vectors under conditions that "turn off" the tetracycline-inducible expression system (for example, producing an AAV vector using a modified rep-cap plasmid containing a Tet-Off system in the presence of a Tet-Off system derivative, or producing an AAV vector using a modified rep-cap plasmid containing a Tet-On system in the absence of a Tet-On system derivative). Producing a higher percentage of complete vectors is desirable because it means that more of the resulting product can be used for therapeutic purposes. Therefore, AAV1 vectors can be produced using the compositions and methods described herein for use in gene therapy (e.g., therapy involving the expression of transgenes in cells that can be transduced by the AAV1 vector).

[0077] The modified rep-cap plasmids described herein may include a polynucleotide encoding an AAV rep protein and a polynucleotide encoding an AAV cap protein, such as an AAV1 cap protein. The plasmid may contain any of the polynucleotide sequences disclosed or specified herein, as well as other existing polynucleotides including regulatory sequences that are useful for constructing the rep-cap plasmids described herein.

[0078] A polynucleotide encoding an AAV rep protein may be, for example, a polynucleotide encoding an AAV2 rep protein. A polynucleotide encoding an AAV rep protein can encode both Rep78 and Rep52. The Rep78 protein encoded by the polynucleotide encoding an AAV rep protein (e.g., an AAV2 rep protein) may have the amino acid sequence of Sequence ID No. 1 provided below.

[0079] MPGFYEIVIKVPSDLDEHLPGISDSFVNWVAEKEWELPPDSDMDLNLIEQAPLTVAEKLQRDFLTEWRRVSKAPEALFFVQFEKGESYFHMHVLVETTGVKSMVLGRFLSQIREKLIQRIYRGIEPTLPNWFAVTKTRNGAGGGNKVVDECYIPN YLLPKTQPELQWAWTNMEQYLSACLNLTERKRLVAQHLTHVSQTQEQNKENQNPNSDAPVIRSKTSARYMELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQ YAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRYQNKCSRHVGMNLMLFPCRQCERLNQNSNICFTHGVKDCLECFPVSESQPVSVVKKAYQKLCYIHHIMGKVPDACTACDLVNVDLDDCVSEQ (Sequence ID 1)

[0080] The Rep52 protein encoded by the polynucleotide encoding the AAV rep protein (e.g., the AAV2 rep protein) may have the amino acid sequence of Sequence ID No. 2 provided below.

[0081] MELVGWLVDKGITSEKQWIQEDQASYISFNAASNSRSQIKAALDNAGKIMSLTKTAPDYLVGQQPVEDISSNRIYKILELNGYDPQYAASVFLGWATKKFGKRNTIWLFGPATTGKTNIAEAIAHTVPFYGCVNWTNENFPFNDCVDKMVIWWEEGKMTAKVVESAKAILGGSKVRVDQKCKSSAQIDPTPVIVTSNTNMCAVIDGNSTTFEHQQPLQDRMFKFELTRRLDHDFGKVTKQEVKDFFRWAKDHVVEVEHEFYVKKGGAKKRPAPSDADISEPKRVRESVAQPSTSDAEASINYADRYQNKCSRHVGMNLMLFPCRQCERLNQNSNICFTHGVKDCLECFPVSESQPVSVVKKAYQKLCYIHHIMGKVPDACTACDLVNVDLDDCVSEQ (Sequence ID 2)

[0082] The polynucleotide encoding the AAV rep protein contained in the rep-cap plasmid described herein may be a polynucleotide having the sequence of SEQ ID NO: 3 provided below.

[0083]

[0084] The modified rep-cap plasmids described herein may also include a tetracycline-inducible expression system. The tetracycline-inducible expression system may be located at the 3' of the polynucleotide encoding the AAV rep protein within the modified rep-cap plasmid.

[0085] A tetracycline-inducible expression system may be a tetracycline-off (Tet-Off) system. A Tet-Off system may contain a tetracycline-regulated transactivator (tTA) and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) tetracycline operator (tetO) sequences. The tTA may contain a tetracycline repressor (tetR)-derived DNA-binding domain and a transcriptional activation domain. The tTA may be a fusion of tetR and the C-terminal domain of VP16 (virion protein 16), an essential transcriptional activation domain from HSV (herpes simplex virus). A Tet-Off system may contain seven tetO sequences. A Tet-Off system may contain eight tetO sequences. One or more tetO sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) can be positioned directly adjacent to each other (e.g., linked without any intervening sequences between the tetO sequences, e.g., the 3' end of a first tetO sequence is positioned immediately before the 5' end of a second tetO sequence), or they can be linked by a nucleic acid linker (e.g., a nucleic acid linker can be positioned between each tetO sequence in the Tet-Off system, or between at least two of the tetO sequences in the Tet-Off system). According to the present invention, in which one or more tetO sequences can be linked by a linker, each linker can contain 1 to 20 or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides) without disrupting the function of the tetO sequences. Each tetO sequence in the Tet-Off system can be separated from the next tetO sequence by a linker containing 20 or more nucleotides. Each tetO sequence in the Tet-Off system can be separated from the next tetO sequence by a linker containing 18 nucleotides. Within the Tet-Off system, the polynucleotide sequence encoding tTA can be located at 5' of one or more tetO sequences.The tetracycline-inducible expression system may include polyadenylation (poly-A) signal sequences positioned at 3' of the polynucleotide sequence encoding tTA and 5' of the polynucleotide sequence encoding tetO (i.e., the poly-A signal sequence may be positioned between the tTA sequence and the tetO sequence in the modified rep-cap plasmid).

[0086] tTA may include proteins that have at least 85% sequence identity to SEQ ID NO: 5 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity) and retain function as tTA (i.e., proteins that have sequences that function as rtTA, excluding proteins that have sequences that function as rtTA). tTA may include proteins having amino acid sequences containing one or more conserved amino acid substitutions to SEQ ID NO: 5 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more conserved amino acid substitutions), provided that the encoded tTA analogue retains the ability to bind to the tetO sequence and promote expression in the absence of an effector. The present invention provides that 10% or less of the amino acids in a tTA protein may be replaced with conserved amino acid substitutions. The polynucleotide sequence encoding the tTA protein can be any polynucleotide sequence encoding SEQ ID NO: 5 due to the redundancy of the gene code. tTA may have the amino acid sequence of SEQ ID NO: 5 provided below.

[0087] MSRLDKSKVINGALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (Sequence ID 5)

[0088] The tTA protein of SEQ ID NO: 5 can be encoded by a polynucleotide having at least 80% sequence identity with respect to the sequence of SEQ ID NO: 6 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). The tTA protein of SEQ ID NO: 5 can be encoded by the polynucleotide sequence of SEQ ID NO: 6 provided below.

[0089] ATGAGTCGGCTGGATAAATCTAAAGTCATAAACGGCGCTCTGGAATTACTCAATGAAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGGCCATCGAGATGCTGGACAGG CATCATACCCACTTCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCT TCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGGAAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGGAGACAAGCAATTGAGCTGTTTCGACCGGCAGGGAGCCGAACCTGCCTTTCGGCCTGGAACTAATCATATGTGGCCTTGGAGAAACAGCTAAAAGTGCGAAAGCGCGGGCCGGCCGACGCCCTTGACGATTTTGACGACTTTGACCTTGATATGCTGCTGCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCGGGTAA (sequence number 6)

[0090] A tetracycline-inducible expression system may be a tetracycline-on (Tet-On) system. A Tet-On system may comprise a reverse tetracycline-regulated transactivator (rtTA) and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) tetracycline operator (tetO) sequences. The rtTA may include variants of a tetracycline repressor (tetR)-derived DNA-binding domain that can bind to tetO only in the presence of an effector (called a reverse tetR). The present invention also provides that the rtTA may be a fusion of a reverse tetR and the C-terminal domain of VP16 (virion protein 16), an essential transcriptional activation domain from HSV (herpes simplex virus). A Tet-On system may contain seven tetO sequences. A Tet-On system may contain eight tetO sequences. One or more tetO sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) can be positioned directly adjacent to each other (e.g., linked without any intervening sequences between the tetO sequences, e.g., the 3' end of a first tetO sequence is positioned immediately before the 5' end of a second tetO sequence), or they can be linked by a nucleic acid linker (e.g., a nucleic acid linker can be positioned between each tetO sequence in the Tet-On system, or between at least two of the tetO sequences in the Tet-On system). According to the present invention, in which one or more tetO sequences can be linked by a linker, each linker can contain 1 to 20 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides) without disrupting the function of the tetO sequences. Each tetO sequence in the Tet-On system can be separated from the next tetO sequence by a linker containing 20 nucleotides. Each tetO sequence in the Tet-On system can be separated from the next tetO sequence by a linker containing 18 nucleotides. Within the Tet-On system, the polynucleotide sequence encoding rtTA can be located at 5' of one or more tetO sequences.The tetracycline-inducible expression system may include a polyadenylation (poly-A) signal sequence located at 3' of the polynucleotide sequence encoding rtTA and 5' of the polynucleotide sequence encoding the tetO sequence (i.e., the poly-A signal sequence may be located between the rtTA sequence and the tetO sequence in the modified rep-cap plasmid). The rtTA may include proteins that have at least 85% sequence identity to SEQ ID NO: 20 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) and can retain function as rtTA (i.e., proteins that do not have a sequence that functions as tTA). rtTA comprises a protein having an amino acid sequence that can contain one or more conserved amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more conserved amino acid substitutions) relative to SEQ ID NO: 20, provided that the encoded rtTA analog retains the ability to bind to the tetO sequence and promote expression in the presence of an effector. The present invention provides that 10% or less of the amino acids in the rtTA protein can be replaced with conserved amino acid substitutions. The polynucleotide sequence encoding the rtTA protein can be any polynucleotide sequence capable of encoding SEQ ID NO: 20 due to gene coding redundancy. rtTA may have the amino acid sequence of SEQ ID NO: 20 provided below.

[0091] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDFDLDMLPADALDDFDLDMLPG (Sequence ID 20)

[0092] The rtTA protein of SEQ ID NO: 20 may be encoded by a polynucleotide having at least 80% sequence identity with respect to the sequence of SEQ ID NO: 21 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). The rtTA protein of SEQ ID NO: 20 may be encoded by the polynucleotide sequence of SEQ ID NO: 21 provided below.

[0093] ATGAGTCGGCTGGATAAATCTAAAGTCATAAACTCTGCTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGG CATCATACCCACTCCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTCCTTCCACATCGCGACGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCT TCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAACAAGCAATTGAGCTGTTTCGACCGGGAGCCGAACCTGCCTTTCGGCCTGGAACTAATCATATGTGGCCTTGGAGAAACAGCTAAAAGTGCGAAAGCGCGGGCCGACCGACGCCCTTGACGATTTTGACGATTATTGACATGCTCCCCGGTTA (sequence number 21)

[0094] Each of the one or more tetO sequences in a tetracycline-inducible expression system (e.g., Tet-Off or Tet-On) in the modified rep-cap plasmid described herein may have the following sequence: TCCCTATCAGTGATAGAGA (SEQ ID NO: 4). A tetracycline-inducible expression system (e.g., Tet-Off or Tet-On) may contain eight tetO sequences, and a polynucleotide containing eight tetO sequences may have at least 80% sequence identity with respect to the sequence of SEQ ID NO: 7 (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity). A polynucleotide containing eight tetO sequences may have at least 80% sequence identity with respect to the sequence of Sequence ID No. 7, separate from the eight tetO sequences (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity), and each of these sequences may contain the sequence of Sequence ID No. 4. The portion of the tetracycline-inducible expression system containing eight tetO sequences may contain the sequence of Sequence ID No. 7 provided below.

[0095] TCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTC TAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGA (Sequence number 7)

[0096] The modified rep-cap plasmids described herein may include an AAV promoter positioned between a tetracycline-inducible expression system and a polynucleotide encoding the AAV1 capsid protein. The AAV promoter may be an AAV p41 promoter. The p41 promoter may have the sequence of SEQ ID NO: 8 provided below.

[0097] TGTTCAAATTTGAACTGACTAAGCGGCTCCCGCCAGATTTTGGCAAGATTACTAAGCAGGAAGTCAAGGACTTTTTTGCTTGGGCAAAGGTCAATCAGGTGCCGTGACTCACGAGTTTAAAGTTCCCAGGGAATTGGCGGGAACTA AAGGGGCGGAGAAATCTCTAAAACGCCCACTGGGTGACGTCACCAATACTAGCTATAAAAGTCTGGAGAAGCGGGCCAGGCTCTCATTTGTTCCCGAGACGCCTCGCAGTTCAGACGTGACTGTTGATCCCGCTCCTCTGCGACCGCT (Sequence number 8)

[0098] Using the modified rep-cap plasmid described herein, an AAV vector containing the AAV1 capsid can be produced.

[0099] The AAV1 capsid protein VP1 may have the amino acid sequence of Sequence ID No. 9.

[0100] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPD PQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPA DVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL (Sequence ID 9)

[0101] The AAV1 capsid protein VP2 may have the amino acid sequence of SEQ ID NO: 14.

[0102] TAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMN PLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL (Sequence ID 14)

[0103] The AAV1 capsid protein VP3 may have the amino acid sequence of SEQ ID NO: 15.

[0104] MASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIAN NLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPA GMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRPL (Sequence ID 15)

[0105] The polynucleotide encoding the AAV1 capsid protein may have the sequence of Sequence ID No. 10 provided below.

[0106]

[0107] The polynucleotide encoding the AAV1 capsid protein may have the sequence of Sequence ID No. 11 provided below.

[0108]

[0109] The modified rep-cap plasmid may contain, in 5' to 3' order, a polynucleotide encoding an AAV rep protein (e.g., the AAV2 rep protein, such as the polynucleotide of SEQ ID NO: 3), a tetracycline-inducible expression system (e.g., a Tet-Off system containing tTA encoded by SEQ ID NO: 6, or a Tet-On system containing rtTA encoded by a series of tetO sequences of SEQ ID NO: 4, such as a series of tetO sequences contained in SEQ ID NO: 21 and SEQ ID NO: 7), an AAV promoter (e.g., the AAV p41 promoter of SEQ ID NO: 8), and a polynucleotide encoding an AAV1 capsid protein (e.g., the AAV1 VP1, VP2, and VP3 proteins, such as the polynucleotide of SEQ ID NO: 10 or SEQ ID NO: 11). The modified rep-cap plasmid may contain the sequence of SEQ ID NO: 12 provided below.

[0110]

[0111] The modified rep-cap plasmid may contain the sequence of SEQ ID NO: 13 provided below.

[0112]

[0113] The modified rep-cap plasmid may contain the sequence of SEQ ID NO: 22 provided below.

[0114]

[0115] Method for delivering exogenous polynucleotides to target cells The modified rep-cap plasmids described herein can be introduced into cells together with helper plasmids and transgene plasmids using methods known in the art to produce rAAV vectors (e.g., rAAV1 vectors). Techniques that can be used to introduce polynucleotides such as DNA and RNA (e.g., encoding the rep proteins, tetracycline-inducible expression systems, and AAV1 capsid proteins described herein) into target host cells (e.g., mammalian cells) are well known in the art. For example, mammalian cells (e.g., human target cells) can be made permeable by applying an electrostatic position to the cells of interest using electroporation. Mammalian cells, such as human cells, exposed to an external electric field in this manner are then more readily able to take up exogenous nucleic acids (e.g., nucleic acids that can be expressed in the cell). Electroporation of mammalian cells is described in detail, for example, Chu et al., Nucleic Acids Research 15:1311 (1987). A similar technique, NUCLEOFECTION®, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. NUCLEOFECTION® and useful protocols for implementing this technique are detailed, for example, in Distler et al., Experimental Dermatology 14:315 (2005), and US2010 / 0317114.

[0116] An additional technique useful for transfection of target host cells is squeeze-poration. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to applied stress. This technique is advantageous in that it does not require a vector for the delivery of nucleic acids into cells, such as human target host cells. Squeeze-poration is described in detail, for example, Sharei et al., Journal of Visualized Experiments 81:e50980 (2013).

[0117] Lipofection is another technique useful for transfection of target host cells. This method involves filling liposomes with polynucleotides that present cationic functional groups, often quaternary amines or protonated amines, toward the outer surface of the liposomes. This facilitates electrostatic interactions between the liposomes and cells due to the anionic nature of the cell membrane, ultimately leading to the uptake of exogenous nucleic acids, for example, by direct fusion of the liposomes with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, US7,442,386. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acids is to bring cells into contact with cationic polymer-polynucleotide complexes. Exemplary cationic molecules that associate with polynucleotides to confer a positive charge favorable to interaction with the cell membrane include activated dendrimers (e.g., described in Dennig, Topics in Current Chemistry 228:227 (2003)), polyethyleneimines, and DEAE-dextran, the use of which as transfection agents is detailed, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997).

[0118] Another useful tool for inducing the uptake of exogenous nucleic acids by target host cells is laser transmission, also known as optical transfection. This technique involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeate the cells, allowing polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to, and in some cases superior to, electroporation.

[0119] Imparefection is another technique that can be used to deliver genetic material to target host cells. It relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene, intended for intracellular delivery, is bound to the surface of the nanostructure. A chip with an array of these needles is then pressed into cells or tissue. Cells impaled by the nanostructure can then express the delivered gene. An example of this technique is described in Shalek et al., PNAS 107:25 1870 (2010).

[0120] MAGNETOFECTION® can also be used to deliver nucleic acids to target host cells. The principle of MAGNETOFECTION® is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made of fully biodegradable iron oxide and are coated with specific cationic intrinsic molecules that vary depending on the application. Their association with gene vectors (e.g., plasmids or viral vectors) is achieved by salt-inducible colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated into the target host cells by the influence of an external magnetic field generated by a magnet. This technique is detailed in Scherer et al., Gene Therapy 9:102 (2002). Magnetic beads are another tool that can be used to transfect target host cells in a gentle and efficient manner, as this method utilizes the applied magnetic field to guide nucleic acid uptake. This technique is detailed, for example, in US2010 / 0227406.

[0121] Another useful tool for inducing the uptake of exogenous nucleic acids by target host cells is sonoporation, a technique that involves using sound (typically ultrasonic frequencies) to modify the permeability of the cell plasma membrane in order to make the cell permeable and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, Rhodes et al., Methods in Cell Biology 82:309 (2007).

[0122] Microvesicles represent another potential vehicle that can be used to modify the genome of a target host cell according to the methods described herein. For example, microvesicles induced by the co-overexpression of the glycoprotein VSV-G with a genome-modifying protein, such as a nuclease, can be used to efficiently deliver the protein to the cell, which then catalyzes site-specific cleavage of endogenous polynucleotide sequences so as to prepare the cell's genome for the covalent incorporation of the desired polynucleotide, such as a gene or regulatory sequence. The use of such vesicles, also called gesicles, for genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [Abstract], in Methylation changes in early embryonic genes in cancer [Abstract], and in Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0123] AAV vectors for polynucleotide delivery Using the modified rep-cap plasmid described herein, polynucleotides (e.g., polynucleotides contained in the transgene plasmid) can be incorporated into an rAAV (e.g., rAAV1) vector and / or virion to facilitate introduction into cells. According to the present invention, an rAAV vector (e.g., rAAV1) produced using the modified rep-cap plasmid described herein is a recombinant polynucleotide construct comprising (1) a promoter, (2) a sequence to be expressed (e.g., a polynucleotide encoding a protein or RNA molecule), and (3) a viral sequence to facilitate the incorporation and expression of the sequence to be expressed. The viral sequence may include the AAV sequence required in cis for DNA replication and packaging into the virion (e.g., functional ITR). The expressed sequence may encode an RNA molecule or protein endogenously expressed in cells transducible by the AAV1 vector (e.g., RNA molecules or proteins endogenously expressed in cells of the central nervous system, heart, skeletal muscle, retinal pigment epithelium, or inner ear), or a sequence encoding a target RNA molecule or protein suitable for expression in cells transducible by the AAV1 vector (e.g., an RNA molecule or protein intended to have a therapeutic effect in the target cell or to investigate the biology of the target cell). The expressed sequence may encode a protein endogenously expressed in the human ear.

[0124] The transgene sequence to be expressed can encode one of the following: solute carrier family 26, member 4 (pendrin), otoferlin (OTOF), stereocilin (STRC), atonal BHLH transcription factor 1 (ATOH1), gap junction protein beta 2 (GJB2), and SRY-Box 2 (Sox2). The expressed sequence can encode, for example, pendrin. The expressed sequence can encode, for example, GJB2. The expressed sequence can encode, for example, OTOF. The expressed sequence can encode, for example, STRC. The expressed sequence can encode, for example, atonal BHLH transcription factor 1 (ATOH1). The expressed sequence can encode, for example, SRY-Box 2 (Sox2).

[0125] Other transgenes that can be expressed include, for example, GJB6, WFS1, COCH, EYA4, MYO7A, POU4F3, ACTG1, MYO6, REST, NLRP3, COL11A1, TJP2, TBC1D24, SLC26A4, ELMOD3, EPSN, WHRN, IGF1, IGF1R, MYO15A, TMIE, TMC1, TMC2, TMPRSS3, CDH23, GIPC3, STRC, USH1C, OTOG, TECTA, OTOA, PDCH15, CLDN14, WHRN, ESRRB, MYO3A, HGF, I Examples include LDR1, ADCY1, CIB2, MARVELD2, SOX2, SLC26A5, COL4A3, COL4A4, COL4A5, CLPP, PJVK, LRTOMT / COMT2, LOXHD1, TPRM, SYNE4, KCNJ10, PTPRQ, OTOGL, LHFPL5, A1PR2, CABP2, MET, GRXCR2, EPS8, CLIC5, EPS8L2, WBP2, ROR1, CLDN9, USH1G, PKHD1L1, DIAPH3, NDP, USH2A, CLRN1, SANS, HARS1, and TRIOBP. The target transgene includes any gene that is desired to be expressed and can fit within the AAV capsid. Two or more target transgenes are possible if the total size of the entire gene can fit within the AAV capsid.

[0126] Such rAAV vectors may contain a marker gene or a reporter gene. Useful rAAV vectors have one or more AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. The AAV ITR may be of any serotype suitable for a particular application. The ITR may be an AAV2 ITR. Methods for using rAAV vectors are described, for example, in Pupo et al., Mol Ther. 7:3515-3541 (2022) and Wang et al., Nat Rev Drug Discov. 18:358-378 (2019).

[0127] Polynucleotides (e.g., polynucleotides contained in transgene plasmids cotransfected into cells together with helper plasmids and modified rep-cap plasmids described herein) can be incorporated into rAAV virions to facilitate the introduction of polynucleotides or vectors into cells. The AAV capsid protein constitutes the external non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, necessary for virion assembly. Construction of rAAV virions is described, for example, in US5,173,414, US5,139,941, US5,863,541, US5,869,305, US6,057,152, and US6,376,237, as well as in Rabinowitz et al., J.Virol.76:791 (2002) and Bowles et al., J.Virol.77:423 (2003).

[0128] manufacturing The production of rAAV vectors for gene therapy can be carried out in vitro using a suitable producing cell line. The producing cells can be any cell type possessing the genes necessary to facilitate AAV genome replication, capsid assembly, and packaging. Exemplary producing cells include human embryonic kidney 293 (HEK-293) cells or derivatives thereof, HeLa cells, human amniotic fluid cells, CHO cells, BHK cells, and insect cells. One strategy for delivering all the elements necessary for rAAV production can utilize two plasmids (e.g., the modified rep-cap plasmid and transgene plasmid described herein) and a helper virus. This method relies on transfection of the producing cells with a plasmid containing a gene cassette encoding the required gene product, and infection of the cells with an adenovirus to provide helper function. This system uses plasmids having two different gene cassettes. The first may be a proviral plasmid (i.e., a transgene plasmid) encoding recombinant DNA to be packaged as rAAV. The second may be a plasmid containing polynucleotides encoding the rep protein and capsid protein (e.g., the modified rep-cap plasmid described herein). To introduce these various elements into cells, cells can be transfected with two plasmids in addition to being infected with adenovirus. Alternatively, the adenovirus infection step can be replaced by transfection with a helper plasmid containing the VA, E2A, and E4 genes. Another alternative approach may involve incorporating the helper genes (e.g., VA, E2A, and / or E4) into a rep-cap plasmid (e.g., the modified rep-cap plasmid described herein), so that only two plasmids are needed to produce the rAAV vector.

[0129] Adenoviruses have traditionally been used as helper viruses for rAAV production, but other DNA viruses, such as herpes simplex virus type 1 (HSV-1), are also known to be usable. The minimum set of HSV-1 genes required for AAV2 replication and packaging has been identified, including the initial genes UL5, UL8, UL52, and UL29. These genes encode components of the HSV-1 core replication mechanism, namely helicase, primase, primase accessory proteins, and single-strand DNA binding proteins. This rAAV helper property of HSV-1 has been utilized in the design and construction of recombinant herpesvirus vectors capable of providing the helper virus gene products necessary for rAAV production.

[0130] The production of rAAV1 vectors using the modified rep-cap plasmid described herein may result in an increased yield of rAAV1 virus compared to the production of rAAV1 virus using an unmodified rep-cap plasmid that contains a polynucleotide encoding the AAV2 rep protein and a polynucleotide encoding the AAV1 capsid protein and lacks a tetracycline-inducible expression system (e.g., the pAAV-RC1 vector produced by Cell Biolabs, Inc.). For example, the production of rAAV1 vectors using the modified rep-cap plasmid described herein may result in a yield of rAAV1 virus that can be increased by at least 2.5 times (e.g., 2.5, 2.7, 3.0, 3.1, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more) compared to the production of rAAV1 vectors using an unmodified AAV2 rep-AAV1 cap plasmid (e.g., pAAV-RC1). The yield can be increased by at least 7 times (e.g., 7, 8, 9, 10, 15, 20, or more). Production of rAAV1 virus using the modified rep-cap plasmid described herein (e.g., by introducing the modified rep-cap plasmid into cells together with the transgene plasmid and helper plasmid) yields at least 1 × 10⁶ of rAAV1 virus, as determined by ddPCR of the clarified lysate. 11 vg / mL (e.g., 1 × 10) 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL or higher, for example, 1 × 10⁻⁶ 11 vg / mL ~ 5 × 10 11 vg / mL, 1×10 11 vg / mL ~ 1 × 10 12 vg / mL, 1×10 11 vg / mL ~ 5 × 10 12 vg / mL, or 1×10 11 vg / mL ~ 1 × 10 13(vg / mL), or determined by ddPCR of the purified vector, at least 1 × 10 13 vg / mL (e.g., 1 × 10) 13 vg / mL, 2×10 13 vg / mL, 3×10 13 vg / mL, 4×10 13 vg / mL, 5×10 13 vg / mL or higher, for example, 1 × 10⁻⁶ 13 vg / mL ~ 5 × 10 13 vg / mL, 1×10 13 vg / mL ~ 1 × 10 14 vg / mL, 1×10 13 vg / mL ~ 5 × 10 14 vg / mL, or 1×10 13 vg / mL ~ 1 × 10 15 This can result in a viral yield of vg / mL. Increased rAAV1 production can be achieved when the tetracycline-inducible expression system is active (e.g., by producing rAAV1 using a modified rep-cap plasmid containing the Tet-Off system in the absence of a Tet-Off system derivative, or by producing rAAV1 using a modified rep-cap plasmid containing the Tet-On system in the presence of a Tet-On system derivative). Increased rAAV1 production can also be achieved when the tetracycline-inducible expression system is "inactive" (e.g., by producing rAAV1 using a modified rep-cap plasmid containing the Tet-Off system in the presence of a Tet-Off system derivative, or by producing rAAV1 using a modified rep-cap plasmid containing the Tet-On system in the absence of a Tet-On system derivative). The tetracycline-inducible expression system contained in the modified rep-cap plasmids described herein may leak, meaning that some rAAV1 production may still occur even when the tetracycline-inducible expression system is "inactive".

[0131] Under conditions where the tetracycline-inducible expression system is "inactive," the production of rAAV1 vectors using the modified rep-cap plasmids described herein (e.g., producing rAAV1 using a modified rep-cap plasmid containing the Tet-Off system in the presence of a Tet-Off system derivative, or producing rAAV1 using a modified rep-cap plasmid containing the Tet-On system in the absence of a Tet-On system derivative) may result in an increased proportion of complete AAV1 vectors compared to the production of complete AAV1 vectors using the same rep-cap plasmids under conditions where the tetracycline-inducible expression system is "active" (e.g., producing rAAV1 using a modified rep-cap plasmid containing the Tet-Off system in the absence of a Tet-Off system derivative, or producing rAAV1 using a modified rep-cap plasmid containing the Tet-On system in the presence of a Tet-On system derivative). Under "inactivation" conditions, rAAV1 production using the rep-cap plasmid described herein can increase the production of complete rAAV1 vector by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more percentage points (e.g., 4% complete to 6%, 8%, 10%, 12%, 14% complete or more, or 10% complete to 12%, 14%, 16%, 18%, 20%, 22% complete or more).

[0132] The production of rAAV1 virus using the non-inducible (i.e., "inactive," e.g., without the addition of doxycycline or any other derivative) Tet-On modified rep-cap plasmid described herein (e.g., by introducing the modified rep-cap plasmid described herein into cells together with the transgene plasmid and helper plasmid) is determined by ddPCR of the clarified lysate to be at least 1 × 10⁻⁶. 11 vg / mL (e.g., 1 × 10) 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL or higher, for example, 1 × 10⁻⁶ 11vg / mL ~ 5 × 10 11 vg / mL, 1×10 11 vg / mL ~ 1 × 10 12 vg / mL, 1×10 11 vg / mL ~ 5 × 10 12 vg / mL, or 1×10 11 vg / mL ~ 1 × 10 13 When determined by ddPCR of vg / mL or purified vector, at least 1 × 10 13 vg / mL (e.g., 1 × 10) 13 vg / mL, 2×10 13 vg / mL, 3×10 13 vg / mL, 4×10 13 vg / mL, 5×10 13 vg / mL or higher, for example, 1 × 10⁻⁶ 13 vg / mL ~ 5 × 10 13 vg / mL, 1×10 13 vg / mL ~ 1 × 10 14 vg / mL, 1×10 13 vg / mL ~ 5 × 10 14 vg / mL, or 1×10 13 vg / mL ~ 1 × 10 15 A viral yield of vg / mL, and as determined using SEC-MALS, can yield at least 20% (e.g., 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or more) of complete AAV1 vector (AAV1 vector also containing the transgene).

[0133] The rep-cap plasmids described herein may contain a polynucleotide encoding the AAV1 capsid protein and are intended for use in the production of rAAV1 vectors. However, if the polynucleotide encoding the AAV1 capsid protein is replaced with a polynucleotide encoding a different AAV capsid protein, modified rep-cap plasmids can be used to produce rAAV vectors with different capsids. For example, the polynucleotide encoding the AAV1 capsid protein may be replaced in any of the rep-cap plasmids described herein with a polynucleotide encoding the AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S capsid protein. Using such rep-cap plasmids, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vectors can be produced using the methods described herein (for example, by introducing the rep-cap plasmid together with the transgene plasmid and helper plasmid into producing cells).According to the present invention, AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vectors can be produced using these modified rep-cap plasmids by producing rAAV vectors under conditions that would "inactivate" the tetracycline-inducible expression system (for example, producing an AAV vector using a modified rep-cap plasmid containing the Tet-Off system in the presence of a Tet-Off system derivative, or producing an AAV vector using a modified rep-cap plasmid containing the Tet-On system in the absence of a Tet-On system derivative). Under "inactivation" conditions, modified rep-cap plasmids containing polynucleotides encoding AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S capsid proteins were used to obtain AAV2, AAV2quad(YF), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh The production of 43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S vectors may yield a higher percentage of complete vectors than the production of rAAV vectors using the same rep-cap plasmids under "activated" conditions (e.g., conditions under which the tetracycline-inducible expression system would be "on," such as producing an AAV vector using a modified rep-cap plasmid containing the Tet-Off system in the absence of a Tet-Off system derivative, or producing an AAV vector using a modified rep-cap plasmid containing the Tet-On system in the presence of a Tet-On system derivative).

[0134] Recombinant AAV vectors (e.g., AAV1 virus) produced using the modified rep-cap plasmids described herein can be purified by any suitable purification method known in the art, such as chromatography, ultracentrifugation, centrifugation, soft agglutination, filtration, and / or ultrafiltration / dialysis filtration. For example, rAAV1 virus can be purified by chromatography. rAAV1 virus can be purified by ultracentrifugation. rAAV1 virus can be purified by centrifugation. rAAV1 virus can be purified by soft agglutination. rAAV1 virus can be purified by filtration. rAAV1 virus can also be purified by ultrafiltration / dialysis filtration.

[0135] kit The modified rep-cap plasmids described herein may be provided in a kit. The kit may include a modified rep-cap plasmid and a helper plasmid, or a modified rep-cap plasmid, a helper plasmid, and a transgene plasmid. The kit may also include instructions for users of the kit, such as scientists in the art, to carry out any one of the methods described herein. For example, the kit may include instructions for users of the kit to produce an rAAV vector (e.g., an rAAV1 vector) using the modified rep-cap plasmid described herein. [Examples]

[0136] The following examples are provided to those skilled in the art to explain how the compositions and methods described herein can be used, prepared, and evaluated, and are intended to be merely illustrative of the present invention and not intended to limit the scope of what the inventors consider to be their own invention.

[0137] Example 1. Comparison of AAV1 production using standard rep-cap plasmid and modified rep-cap plasmid.

[0138] AAV production To enable AAV production, plasmids encoding AAV2 rep / AAV1 cap in a 2:1:2 concentration ratio, a pHelper plasmid, and a transfer plasmid were combined with FectoVIR (Polypus) in a 1:1 FectoVIR:DNA (deoxyribonucleic acid) ratio and added to a suspension VPC (virus-producing cell, originating from 293) culture. The plasmid encoding AAV2 rep / AAV1 cap used for AAV production was either the modified rep-cap plasmid P929 (Figure 1) or the standard rep-cap plasmid P374 (Figure 2). P929 was developed according to the present invention to provide AAV1 rep / cap in the PHP.B tet-off backbone. Compared to P374, P929 is thought to contain two silent single-point mutations (which may not be silent in the AAP ORF) in the VP1:2:3 CAP open reading frame and possess a wild-type AAV1 CAP sequence.

[0139] The transfer plasmids used for AAV production were transfer plasmid P1381, containing a CMV promoter operably linked to a polynucleotide encoding enhanced GFP; transfer plasmid P1900, containing a pendrin promoter operably linked to a polynucleotide encoding pendrin; and transfer plasmid P707, containing a CMV promoter operably linked to a polynucleotide encoding GFP. The same pHelper plasmid (P376) was used with all combinations of rep-cap plasmids and transfer plasmids. Cell culture medium and cells were harvested after incubation at 37°C, 8% CO2, and 125 rpm for 72 hours. Cell lysates were then treated with benzonase nuclease for degradation of non-viral DNA and RNA (ribonucleic acid), and Triton X-100 (10% Triton X-100 w / v), and lysed during incubation at 37°C, 8% CO2, and 125 rpm. The cell lysates were then filtered through a 0.22 μm filter before purification.

[0140] AAV particles were concentrated from cell culture lysates via gravity affinity chromatography using AAVX resin, followed by buffer exchange using a centrifugal filtration unit. The AAV was formulated in 10 mM sodium phosphate, 180 mM NaCl, 5% sucrose (w / v), and 0.001% poloxamer 188 (w / v) at pH 7.3 and stored at -80°C. The resulting AAV and their transfer plasmid sources are shown in Table 2. [Table 2]

[0141] Vector genome titer obtained by droplet digital polymerase chain reaction

[0142] The total number of vector genomes within AAV was quantified using droplet digital polymerase chain reaction (ddPCR). The ddPCR method used a set of primers and probes specific to the target gene. The primer / probe sets used for samples AAV1439, AAV1440, AAV1442, and AAV1443 targeted the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a sequence present in all four viruses. The primer and probe sequences included the forward primer: TTGTGAAAGATTGACTGGTATTCT (SEQ ID NO: 16), the reverse primer: AGGCATTAAAGCAGCGTA (SEQ ID NO: 17), and the probe: TAACTATGTTGCTCCTTTTAC (SEQ ID NO: 18). For the remaining two samples (AAV1441 and AAV1444), the primer / probe included in the pre-designed TaqMan gene expression assay Hs01070627_m1 from ThermoFisher was used. This unique set specifically targets sequences in the SLC26A4 gene encoding the pendrin protein, found in two viruses. The sequence is AAGGGATGGCATATGCCCTACTAGCTGCAGTTCCTGTCGGATATGGTCTCTACTCTGCTTTTTTCCCTATCC (Sequence ID 19).

[0143] To perform ddPCR testing, DNAse digestion (30 minutes at 37°C) was performed to remove all non-capsidized DNA, ensuring that only endonuclease-resistant (capsidized) vector genomes were quantified. This step was carried out using recombinant DNase I from Sigma-Aldrich (10 units / μL, catalog no. 4716728001). Next, the DNAse was neutralized using proteinase K (ProK, >600 mAU / mL, Qiagen catalog no. 19133) to digest the AAV capsid and release any capsidized DNA, followed by heat treatment to inactivate the enzyme and open the capsid. Incubation parameters were 30 minutes at 56°C, 5 minutes at 95°C, and 5 minutes to 1 hour at 4°C. Subsequently, the samples were diluted and polymerase chain reaction (PCR) master mixture containing Bio-Rad PCR Supermix (1863023) and primer and probe sets was added.

[0144] In the ddPCR assay, the PCR mixture (test sample, master mixture with primers, and probe) was distributed into nanoliter-scale droplets using a water-oil emulsion system with a Bio-Rad Automatic Droplet Generator. The test sample was randomly distributed into the droplets and amplified by PCR using a Bio-Rad C1000 Touch Thermal Cycler. At the end of the PCR reaction, the fluorescence in each droplet was measured individually using a Bio-Rad QX200 Droplet Reader. The internal probe was labeled with a 5' fluorescent reporter dye and a non-fluorescent quencher at the 3' end. In each PCR cycle, Taq polymerase 5' exonuclease activity hydrolyzed the internal probe, releasing fluorophores from the probe and quencher. Each droplet was assigned as negative (no measurable DNA) or positive (one or more copies of DNA) based on the fluorescence intensity threshold determined from a non-template control. The absolute concentration of DNA in each sample was determined according to a Poisson distribution based on the ratio of positive to negative droplets for different dilutions of the test sample. The dilution-corrected concentration of the target gene (GOI) is reported in units of vg / mL.

[0145] As shown in Table 3, AAV1 production using the modified rep-cap plasmid P929 resulted in increased titer compared to AAV1 production using the standard rep-cap plasmid P374. For example, a comparison of clarified lysate ddPCR titers (vg / mL) showed a 13.9-fold difference between the vector genome titer of AAV1 produced with the modified rep-cap plasmid P929 and transfer plasmid P1381 and that of the standard rep-cap plasmid P374 and transfer plasmid P1381, a 13.2-fold difference between the vector genome titer of AAV1 produced with the modified rep-cap plasmid P929 and transfer plasmid P1900 and that of the standard rep-cap plasmid P374 and transfer plasmid P1900, and a 9.1-fold difference between the vector genome titer of AAV1 produced with the modified rep-cap plasmid P929 and transfer plasmid P707 and that of the standard rep-cap plasmid P374 and transfer plasmid P707. Comparison of purified ddPCR titers (vg / mL) also showed increased titers for AAV1 produced using modified rep-cap plasmid P929 compared to AAV1 produced using standard rep-cap plasmid P374, with a 16.5-fold difference for AAV1 produced with transfer plasmid P1381, a 15.2-fold difference for AAV1 produced with transfer plasmid P1900, and a 68.89-fold difference for AAV1 produced with transfer plasmid P707.

[0146] Vector capsid titer determined by enzyme-linked immunosorbent assay

[0147] The total capsid content in all samples was quantified using a sandwich enzyme-linked immunosorbent assay (ELISA).

[0148] The AAV1 capsid assay was performed as a two-step colorimetric ELISA using commercially available capture and detection reagents, calibration standards, and assay diluents. Two different versions of the same single-chain camelid antibody that binds to the AAV capsid with high affinity were used. The capture antibody (ThermoFisher 7103522100, 1 mg / mL) was an anti-AAV antibody conjugated to biotin, and the detection antibody (ThermoFisher 7303522100, 0.5 mg / mL) was the same anti-AAV antibody conjugated to horseradish peroxidase (HRP).

[0149] Each well of a streptavidin-coated plate (ThermoFisher No. 15124) was incubated with 100 ng of captured biotin conjugate antibody at room temperature for 1 hour. After washing, diluted test samples containing calibration standards and AAV1 capsids were added to the plate wells and incubated at room temperature for 1 hour. The standard curve was 1.00 × 10⁻⁶. 11 ~1.64 × 10 8 The sample consisted of 2.5 serial dilutions of empty AAV1 capsids (Vigene RS-AAV1-ET) in the range of capsid / mL. The plate was washed, and 100 μL of detection HRP conjugate antibody diluted 1:10,000 was added to the wells and incubated at room temperature for 1 hour. After washing, 3,3',5,5'-tetramethylbenzidine (TMB) substrate (ThermoFisher 34028) was added to the plate wells. After 10 minutes, the reaction was stopped by adding 25% sulfuric acid (Sigma-Aldrich 84736-1L), yielding a colorimetric reaction proportional to the concentration of AAV1 capsid in the calibration standard and the diluted test sample. Absorbance was measured at 450 nm on a PerkinElmer EnVision Multimode Plate Reader. Concentrations were determined by interpolation from a standard curve fitted to a 4-parameter logistic (4PL) model using GraphPad Prism. Three measurements were performed for each standard, assay control, and test sample. The dilution-corrected concentration of AAV1 capsid was reported in capsids / mL.

[0150] As shown in Table 3, AAV1 production using the modified rep-cap plasmid P929 resulted in increased capsid titer (cp / mL) and total capsid compared to AAV1 production using the standard rep-cap plasmid P374. For example, a comparison of clarified lysate capsid titers (cp / mL) showed an 18.8-fold difference between the capsid titers of AAV1 produced with modified rep-cap plasmid P929 and transfer plasmid P1381 and those of standard rep-cap plasmid P374 and transfer plasmid P1381, a 15.8-fold difference between the capsid titers of AAV1 produced with modified rep-cap plasmid P929 and transfer plasmid P1900 and those of standard rep-cap plasmid P374 and transfer plasmid P1900, and a 27.2-fold difference between the capsid titers of AAV1 produced with modified rep-cap plasmid P929 and transfer plasmid P707 and those of standard rep-cap plasmid P374 and transfer plasmid P707. [Table 3]

[0151] Example 2. Comparison of AAV1 production using standard rep-cap plasmid and modified rep-cap plasmid.

[0152] AAV production To enable AAV production, plasmids encoding AAV2 rep / AAV1 cap in a 2:1:2 concentration ratio, a pHelper plasmid, and a transfer plasmid were combined with FectoVIR (Polypus) in a 1:1 FectoVIR:DNA (deoxyribonucleic acid) ratio and added to a suspension VPC (virus-producing cell, origin 293) culture. The plasmid encoding AAV2 rep / AAV1 cap used for AAV production was either the modified rep-cap plasmid P2056 (Figure 3) or the standard rep-cap plasmid P374 (Figure 2). The transfer plasmid used for AAV production was transfer plasmid P1381, which contains a CMV promoter operably ligated to a polynucleotide encoding GFP. The same pHelper plasmid (P376) was used with each rep-cap plasmid and transfer plasmid combination. The P2056 rep / cap plasmid contains a doxycycline-induced rtTA induction system. The inventors tested the P2056 / P1381 / P374 system and three concentrations of doxycycline (1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml) added simultaneously during transfection of an untreated control. Furthermore, the P376 / P1381 / P374 system without tetracycline-inducible rep / cap was tested by simultaneously adding 1 μg / ml of doxycycline during transfection to determine the effect of doxycycline on transfection. Cell culture media and cells were collected after incubation at 37°C, 8% CO2, and 125 rpm for 72 hours. Next, the cell lysates were treated with benzonase nuclease to degrade non-viral DNA and RNA (ribonucleic acid) as well as Triton X-100 (10% Triton X-100 w / v), and lysed during incubation at 37°C, 8% CO2, and 125 rpm. The cell lysates were then filtered through a 0.22 μm filter before purification.

[0153] AAV particles (AAV1556 for P2056 / P1381 / P374 cotransfection, and AAV1442 for P376 / P1381 / P374 cotransfection) were concentrated from cell culture lysates via gravity affinity chromatography using AAVX resin, followed by buffer exchange using a centrifugal filtration unit. The AAV was formulated in 10 mM sodium phosphate, 180 mM NaCl, 5% sucrose (w / v), and 0.001% poloxamer 188 (w / v) at pH 7.3 and stored at -80°C.

[0154] Vector genome titer obtained by droplet digital polymerase chain reaction

[0155] The total number of vector genomes in the AAV was quantified using droplet digital polymerase chain reaction (ddPCR), as described in Example 1, using the same forward and reverse primers (SEQ ID NOs. 16 and 17) and probe (SEQ ID NOs. 18).

[0156] As shown in Table 5, AAV1 production using the modified rep-cap plasmid P2056 resulted in increased titer compared to AAV1 production using the standard rep-cap plasmid P374, as determined by ddPCR. For example, a comparison of clarified lysate ddPCR titers (vg / mL) showed a 2.7-fold difference between the vector genome titer of AAV1 produced with the modified rep-cap plasmid P2056 and that of the standard rep-cap plasmid P374 in the absence of added doxycycline, and a 3.1-fold difference in the presence of 1.0 μg / ml doxycycline. A comparison of purified ddPCR titers (vg / mL) also showed increased titer of AAV1 produced with the modified rep-cap plasmid P2056 compared to AAV1 produced with the standard rep-cap plasmid P374, with a 5.0-fold difference for AAV1 produced in the absence of doxycycline and a 3.0-fold difference for AAV1 produced in the presence of 1 μg / ml doxycycline. Interestingly, in both the clarified lysate and subsequent affinity purification, the titer of AAV1 produced by the modified rep-cap plasmid P2056 was minimally affected by the presence or absence of added doxycycline, as determined by ddPCR. We believe this is due to the fact that the Tet-On system used in P2056 leaked, enabling capsid protein production even in the absence of added doxycycline.

[0157] Vector capsid and genome titer analysis, as well as empty / complete analysis by size exclusion chromatography-multi-angle light scattering.

[0158] Size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis was performed using an Agilent 1260 Infinity II HPLC instrument equipped with a multi-wavelength UV-Vis detector and a Wyatt Dawn Mals detector. Separation was achieved at a flow rate of 1.0 mL / min using a 7.8 × 300 mm Agilent Bio SEC-5 column filled with 5 μm particles having a 1000 Å pore, and a mobile phase containing 300 mM NaCl, 20 mM Na phosphate, pH 7.4, and 0.001% (w / v) Pluronic® F68. The volume of each injection was 50 μL. Capsid titer (cp / mL) and genome titer (vg / mL) were determined using a viral vector procedure based on conjugate analysis within Astra software (version 8.1.2.1). In comparison with other techniques used to determine the empty / complete ratio of AAV, the procedure described in Wyatt Technology Application Note 1617 ("Quantification of Quality Attributes of AAV Gene Therapy Vectors by SEC-UV-MALS-dRI") and discussed in Werle et al., Mol Ther Methods Clin Dev. 23:254-262, 2021, involves the use of signals from multi-angle light scattering along with two concentration sources, such as UV absorbance at 260 nm and 280 nm. These signals are used to calculate total protein and DNA mass, as well as capsid and transgene molar mass (Mw), which are then converted to capsid and genome titers. Finally, the percentage of complete capsid is determined by the following formula: (capsid titer) / (genome titer) × 100. The processing parameters used for the SEC-MALS analysis are listed in Table 4. [Table 4]

[0159] The results of the SEC-MALS analysis are shown in Table 5. SEC-MALS measurements showed that AAV1 production using the modified rep-cap plasmid P2056 resulted in a 4.7-fold increase in purified viral titer compared to AAV1 production using the standard rep-cap plasmid P374 in the absence of doxycycline, and a 3.5-fold increase in the presence of 1.0 μg / ml doxycycline. Capsid titers using P2056 also increased 4.1-fold in the absence of doxycycline and 6.7-fold in the presence of 1.0 μg / ml doxycycline compared to P374. The SEC-MALS results also confirmed that doxycycline has minimal effect on purified viral titer when using the modified rep-cap plasmid P2056. Surprisingly, the production of the complete AAV1 vector using P2056 was higher in the absence of added doxycycline (23.1%) than in its presence (10.1–18.5%). [Table 5]

[0160] In another experiment, AAV1 was produced using the modified rep-cap plasmid P929 in the presence of 1.0 μg / ml doxycycline, 0.1 μg / ml doxycycline, or 0.01 μg / ml doxycycline, or in the absence of doxycycline. The percentage of complete AAV1 vector was assessed using SEC-MALS in a manner similar to that described above. Surprisingly, the production of complete AAV1 vector using P929 was higher in the presence of added doxycycline than in its absence (Figure 4).

[0161] Example 3. Comparison of AAV1 production encoding the GJB2 transgene using standard Rep-Cap plasmid and modified Rep-Cap plasmid. The GJB2 transgene encodes the connexin 26 protein. Mutations in this gene are associated with hearing loss.

[0162] A comparison was made between the AAV-pRC1 variant and P929 in the production of AAV1 encoding the GJB2 transgene (see Figure 1). The AAV-pRC1 variant, designated RC1 p5AS, was used as a control. Production was carried out in the presence or absence of 1.5 μg / ml doxycycline. The same pHelper plasmid (P376) was used.

[0163] Production was carried out in a 125 mL shaking flask with a working volume of 30 mL. After incubation at 37 °C, 8% CO2, and 120 RPM for 72 hours, the cell cultures were lysed using 1% Tween 20 and 100 U / mL denalase nuclease. Cell lysates were clarified by centrifugation before quantification of genome and capsid titers.

[0164] Figures 8A and 8B show data from the production of AAV1 containing the GJB2 transgene using RC51 p5AS or P929. Figure 8A shows genomic titers, indicating that P929 was superior to RC1 p5AS in the presence of doxycycline (more than twice as superior) and in the absence of doxycycline (more than three times superior). Figure 8B shows capsid titers, indicating that P929 was superior to RC1 p5AS in the presence of doxycycline (approximately three times superior) and in the absence of doxycycline (more than ten times superior). Table 6 below shows the aggregated data. [Table 6]

[0165] Example 4. AAV1 expressing a highly sensitive green fluorescent protein transgene This example compares plasmids P374 (Figure 2), P929 (Figure 1), P2057 (Figure 5), P2058 (Figure 6), and P2059 (Figure 7).

[0166] Plasmid 2057 is based on P929, and its promoter has been modified to a TRE tight promoter. Plasmid 2058 contains tTA (tetracycline transactivator) linked to the Rep gene by the 2A peptide. The 2A peptide is an 18-22 amino acid peptide that mediates the autocleavage of polypeptides during translation in eukaryotic cells. Plasmid 2059 contains tTA regulated under its own promoter and not regulated by Rep.

[0167] Using each of the five plasmids, an AAV1 vector carrying the eGFP transgene was produced. The vector was produced in biological replicas in the absence of doxycycline. A plasmid ratio of 2:1:2 for cap-encoding plasmid:helper plasmid:eGFP plasmid was used for production. Figures 9A–9C show the production titer of AAV1 containing the highly sensitive green fluorescent protein (eGFP) transgene using plasmids P374, P929, P2057, P2058, or P2059. The same pHelper plasmid (p376) was used. Production was carried out in a 125 mL shaking flask using the method described in Example 3.

[0168] Figure 9A compares the capsid titers measured by ELISA in clarified lysate samples for each of the five plasmids. P2059 achieved the highest capsid titer (Cp / ml), followed by P2058 and P929. P2057 achieved a slightly lower capsid titer compared to P374.

[0169] Figure 9B compares the genomic titers of each of the five plasmids using droplet digital PCR (ddPCR) in clarified lysate samples. The results are similar to those in Figure 9A. All tetracycline-modulated constructs (P929, P2057, P2058, and P2059), which can be modulated by derivatives such as tetracycline and doxycycline, achieved higher genomic titers (vg / ml) than the P374 control. P2059 showed the best performance in terms of genomic titer, followed by P2058, P929, and P2057.

[0170] Figure 9C compares the estimated genomic titers for each of the five plasmids, based on the percentage of complete capsid. The results are similar to those in Figure 9B.

[0171] Figures 10A–10B show the production of AAV1 containing the eGFP transgene using plasmids P374, P929, P2057, P2058, or P2059, and the completeness of the capsid achieved by each of the five plasmids was evaluated using mass photometry. The clarified lysate samples were purified before quantifying the completeness of the capsid using mass photometry.

[0172] Figure 10A shows the percentage of complete capsids. P2057 had the highest percentage of complete capsids at 64%, followed by P2058 at 35%, and P929 at 34%. The percentage of complete capsids produced by P2059 was slightly lower than that of the P374 control.

[0173] Figure 10B shows the percentage of partially filled capsids. P2057 showed the highest percentage. All constructs produced vectors showed minimal partially filled capsids. The aggregated data is shown in Table 7 below. [Table 7]

[0174] The data from Example 7 are compelling. P2059 and 2058 showed the best performance in terms of overall capsid and genome titer. However, P2057 showed the best performance in terms of the percentage of complete capsid. Therefore, if complete capsid purification is easily achieved, P2059 and P2058 are logical choices. However, if complete capsid purification is more difficult, P2057 may be the best choice despite its lower performance in terms of overall capsid and genome titer.

[0175] Other inventions Although the present invention has been described in relation to its specific examples, further modifications are possible, and it will be understood that this application is intended to cover any variations, uses, or adaptations of the present invention, and to be subject to the claims, including any deviations from the invention that may be applied to known or customary practices in the relevant art, generally in accordance with the principles of the invention, and which may be applied to essential features described previously.

Claims

1. From 5' to 3', (a) A polynucleotide encoding the AAV rep protein, (b) Tetracycline-off (Tef-Off) system or tetracycline-on (Tet-On) system, (c) AAV promoter and, (d) Polynucleotides encoding the AAV1 capsid protein and The adeno-associated virus (AAV) rep-cap plasmid contains this plasmid.

2. The rep-cap plasmid according to claim 1, wherein the AAV promoter is the AAV p41 promoter.

3. The rep-cap plasmid according to claim 2, comprising a tetracycline-off (Tef-Off) system.

4. The rep-cap plasmid according to claim 2, comprising a tetracycline (Tef-On) system.

5. The rep-cap plasmid according to claim 2, wherein the AAV rep protein is the AAV2 rep protein.

6. The rep-cap plasmid according to claim 2, wherein the polynucleotide encoding the AAV rep protein encodes Rep78 and Rep52.

7. The rep-cap plasmid according to claim 6, wherein Rep78 has the sequence of Sequence ID No.

1.

8. The rep-cap plasmid according to claim 6, wherein Rep52 has the sequence of Sequence ID No.

2.

9. The rep-cap plasmid according to claim 2, wherein the polynucleotide encoding the AAV rep protein has the sequence of Sequence ID No.

3.

10. The Tet-Off system is oriented from 5' to 3'. (a) A first polynucleotide encoding a tetracycline trans-activator (tTA), wherein the tTA comprises a tetracycline repressor-derived DNA-binding domain and a transcription-activating domain, (b) A second polynucleotide containing 1 to 12 tet operator (tetO) sequences and The rep-cap plasmid according to claim 2, comprising:

11. The rep-cap plasmid according to claim 10, wherein the first polynucleotide encodes a polypeptide having the amino acid sequence of SEQ ID NO:

5.

12. The rep-cap plasmid according to claim 11, wherein the first polynucleotide has at least 80% sequence identity with respect to SEQ ID NO:

6.

13. The rep-cap plasmid according to claim 12, wherein the first polynucleotide has the sequence of SEQ ID NO:

6.

14. The rep-cap plasmid according to claim 10, wherein each tetO sequence has the sequence of sequence number 4.

15. The rep-cap plasmid according to claim 10, wherein each tetO sequence is separated from adjacent tetO sequences by 0 to 20 nucleotides.

16. The rep-cap plasmid according to claim 10, wherein the second polynucleotide comprises eight tetO sequences.

17. The rep-cap plasmid according to claim 16, wherein the second polynucleotide has at least 80% sequence identity with respect to SEQ ID NO:

7.

18. The rep-cap plasmid according to claim 17, wherein the second polynucleotide has the sequence of Sequence ID No.

7.

19. The aforementioned Tet-On system is oriented from 5' to 3', (a) A first polynucleotide encoding a reverse tetracycline trans-activator (rtTA), wherein the rtTA comprises a tetracycline repressor-derived DNA-binding domain and a transcription-activating domain, (b) A second polynucleotide containing 1 to 12 tet operator (tetO) sequences and The rep-cap plasmid according to claim 2, comprising:

20. The rep-cap plasmid according to claim 19, wherein the first polynucleotide encodes a polypeptide having the amino acid sequence of SEQ ID NO:

20.

21. The rep-cap plasmid according to claim 20, wherein the first polynucleotide has at least 80% sequence identity with respect to SEQ ID NO:

21.

22. The rep-cap plasmid according to claim 21, wherein the first polynucleotide has the sequence of Sequence ID No.

21.

23. The rep-cap plasmid according to claim 19, wherein each tetO sequence has the sequence of sequence number 4.

24. The rep-cap plasmid according to claim 19, wherein each tetO sequence is separated from adjacent tetO sequences by 0 to 20 nucleotides.

25. The rep-cap plasmid according to claim 19, wherein the second polynucleotide comprises eight tetO sequences.

26. The rep-cap plasmid according to claim 25, wherein the second polynucleotide has at least 80% sequence identity with respect to SEQ ID NO:

7.

27. The rep-cap plasmid according to claim 26, wherein the second polynucleotide has the sequence of Sequence ID No.

7.

28. The rep-cap plasmid according to claim 2, wherein the p41 promoter has the sequence of Sequence ID No.

8.

29. The rep-cap plasmid according to claim 2, wherein the polynucleotide encoding the AAV1 capsid protein encodes a polypeptide having the amino acid sequence of SEQ ID NO:

9.

30. The rep-cap plasmid according to claim 29, wherein the polynucleotide encoding the AAV capsid protein has the sequence of Sequence ID No.

10.

31. The rep-cap plasmid according to claim 29, wherein the polynucleotide encoding the AAV capsid protein has the sequence of Sequence ID No.

11.

32. The rep-cap plasmid according to claim 2, wherein the Tet-Off system or Tet-On system further comprises a polyA signal sequence located between the first polynucleotide and the second polynucleotide.

33. The rep-cap plasmid according to claim 2, comprising the polynucleotide sequence of sequence number 12.

34. The rep-cap plasmid according to claim 2, comprising the polynucleotide sequence of sequence number 13.

35. The rep-cap plasmid according to claim 2, comprising the polynucleotide sequence of sequence number 22.

36. A method for producing recombinant adeno-associated virus, (I) (a) The rep-cap plasmid according to claim 1, (b) A helper plasmid containing one or more helper genes selected from E4, E2a, and VA, and (c) Transgene plasmid containing the target transgene flanked by reverse terminal repeats A step of introducing into mammalian cells, wherein the introduction step is performed under conditions that enable the formation of the recombinant adeno-associated virus, (II) A step of collecting the recombinant adeno-associated virus and Methods that include...

37. The method according to claim 36, wherein the AAV promoter is the AAV p41 promoter.

38. The method according to claim 37, wherein the mammalian cells are HEK-293 cells.

39. The method according to claim 37, wherein the rep-cap plasmid contains the polynucleotide sequence of SEQ ID NO:

12.

40. The method according to claim 37, wherein the rep-cap plasmid comprises the polynucleotide sequence of SEQ ID NO:

13.

41. The method according to claim 37, wherein the rep-cap plasmid contains the polynucleotide sequence of SEQ ID NO:

22.

42. The method according to claim 37, wherein the introduced gene for the purpose encodes a protein that is normally expressed in the human ear.

43. The method according to claim 42, wherein the target transgene encodes solute carrier family 26, member 4 (pendrin), otoferrin (Otof), stereocillin (STRC), Atonal BHLH transcription factor 1 (ATOH1), gap junction protein beta 2 (GJB2), or SRY-Box 2 (Sox2).

44. The method according to claim 42, wherein the introduced gene for the purpose encodes pendrin, GJB2, or OTOF.

45. The virus yield, as measured using ddPCR of the clarified lysate, is at least 1 × 10⁻⁶. １１ The method according to claim 37, wherein the concentration is vg / ml.

46. (a) The rep-cap plasmid contains a Tet-On system, (b) The method according to claim 37, wherein the production of recombinant AAV1 virus is achieved without inducing the Tet-On system.

47. A method for increasing the yield of recombinant AAV1-capped adeno-associated virus by at least 2.5 times compared to recombinant AAV1-capped adeno-associated virus produced by pAAV-RC1, the method comprising the step of substituting pAAV-RC1 with the rep-cap plasmid described in claim 2 in the production of the adeno-associated virus.

48. The method according to claim 47, wherein the yield is increased by at least seven times.

49. The method according to claim 47, wherein the rep-cap plasmid comprises the polynucleotide sequence of SEQ ID NO:

12.

50. The method according to claim 47, wherein the rep-cap plasmid comprises the polynucleotide sequence of SEQ ID NO:

13.

51. The method according to claim 47, wherein the rep-cap plasmid contains the polynucleotide sequence of SEQ ID NO:

22.

52. The rep-cap plasmid according to claim 2, wherein the rep-cap plasmid has the arrangement shown in Figure 1.

53. The rep-cap plasmid according to claim 2, wherein the rep-cap plasmid has the arrangement shown in Figure 6.

54. The rep-cap plasmid according to claim 2, wherein the rep-cap plasmid has the arrangement shown in Figure 7.

55. A cell comprising the rep-cap plasmid described in claims 1 to 54.

56. The cell according to claim 55, further comprising a target transgene encoding a protein normally expressed in the human ear.

57. The cell according to claim 56, wherein the target transgene encodes solute carrier family 26, member 4 (pendrin), otoferrin (Otof), stereocillin (STRC), atonal BHLH transcription factor 1 (ATOH1), gap junction protein beta 2 (GJB2), or SRY-Box 2 (Sox2).

58. The cell according to any one of claims 55 to 57, wherein the cell is a mammalian cell.

59. The cell according to claim 58, wherein the mammalian cell is a HEK-293 cell.

60. A cell culture comprising the cells described in any one of claims 55 to 59.

61. From 5' to 3', (a) A polynucleotide encoding the AAV rep protein, (b) Tetracycline-off (Tef-Off) system or tetracycline-on (Tet-On) system, (c) TRE Tight Promoter and (d) Polynucleotides encoding the AAV1 capsid protein and The adeno-associated virus (AAV) rep-cap plasmid contains this plasmid.

62. The rep-cap plasmid according to claim 51, wherein the rep-cap plasmid has the arrangement shown in Figure 5.

63. A cell comprising the rep-cap plasmid according to claim 61 or 62.

64. The cell according to claim 63, further comprising a target transgene encoding a protein normally expressed in the human ear.

65. The cell according to claim 64, wherein the target transgene encodes solute carrier family 26, member 4 (pendrin), otoferrin (Otof), stereocillin (STRC), Atonal BHLH transcription factor 1 (ATOH1), gap junction protein beta 2 (GJB2), or SRY-Box 2 (Sox2).

66. The cell according to any one of claims 63 to 65, wherein the cell is a mammalian cell.

67. The cell according to claim 66, wherein the mammalian cell is a HEK-293 cell.

68. A cell culture comprising the cells described in any one of claims 63 to 67.

69. A method for producing recombinant adeno-associated virus, (I) (a) The rep-cap plasmid according to claim 61 or 62, (b) A helper plasmid containing one or more helper genes selected from E4, E2a, and VA, and (c) Transgene plasmid containing the target transgene flanked by reverse terminal repeats A step of introducing into mammalian cells, wherein the introduction step is performed under conditions that enable the formation of the recombinant adeno-associated virus, (II) A step of collecting the recombinant adeno-associated virus and Methods that include...

70. The method according to claim 69, wherein the mammalian cells are HEK-293 cells.

71. The method according to claim 69 or 70, wherein the cells further include a target transgene encoding a protein normally expressed in the human ear.

72. The method according to claim 71, wherein the target transgene encodes solute carrier family 26, member 4 (pendrin), otoferrin (Otof), stereocillin (STRC), Atonal BHLH transcription factor 1 (ATOH1), gap junction protein beta 2 (GJB2), or SRY-Box 2 (Sox2).

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