Plasmids for improving aav titer and empty full ratio

Abstract

This invention relates to a rep plasmid comprising at least one adeno-associated virus (AAV) replication protein coding sequence encoding at least one functional rep protein, at least one polyadenylation signal sequence downstream of the AAV replication protein coding sequence, and a minimal element comprising one or more rep-binding elements downstream of the polyadenylation signal sequence. Alternatively, this invention provides a transgenic plasmid comprising a CMVie promoter, a transgene, SV40 PolyA, and 5' and 3' terminal inverted repeat sequences derived from J01901.1 AAV2; wherein the transgenic plasmid is selected from one of: conventional single-stranded recombinant AAV virus, or self-complementary recombinant AAV virus.

Description

Technical Field

[0001] This invention relates to a rep or rep cap plasmid comprising at least one adeno-associated virus (AAV) replication protein coding sequence encoding at least one functional rep protein, at least one polyadenylation signal sequence downstream of the AAV replication protein coding sequence, and a minimal element comprising one or more rep binding elements downstream of the polyadenylation signal sequence. The invention also relates to a plasmid system for producing AAV particles, the system comprising a transgenic plasmid and the rep plasmid of the invention. On another aspect, the invention provides a transgenic plasmid comprising a CMVie promoter, a transgene, SV40 PolyA, and 5' and 3' terminal inverted repeat sequences derived from J01901.1 AAV2; wherein the transgenic plasmid is selected from one of: conventional single-stranded genomic recombinant AAV, or self-complementary genomic recombinant AAV. In another aspect, the invention provides a cell comprising the rep plasmid of the invention, the plasmid system of the invention, and / or the transgenic plasmid of the invention. In yet another aspect, the invention provides a kit comprising the expression system or cell. In still another aspect, the invention provides a method for producing a recombinant AAV vector comprising transfecting cells with the plasmid system of the invention. Background Technology

[0002] Several gene delivery technologies have been developed to express target genes in cells, tissues or organisms, including mammalian cells, tissues or organisms, particularly human cells, tissues or organisms.

[0003] Some of these delivery technologies utilize viral vectors derived from lentiviruses, oncogenic retroviruses, adenoviruses, adeno-associated viruses, or similar viruses. Among these vectors, adeno-associated virus (AAV) vectors are considered a promising tool. AAV is a linear single-stranded DNA virus belonging to the parvovirus family. AAV is infectious to a variety of cells, including humans, and can infect non-dividing cells that have stopped differentiating, such as blood cells, muscle cells, or nerve cells. Furthermore, wild-type AAV is non-pathogenic to humans, and AAV particles are physiologically and chemically very stable. These characteristics make it possible to develop recombinant AAV (rAAV) as a vector for gene delivery, particularly gene therapy.

[0004] The single-stranded genome of wild-type AAV contains rep (replication) and cap (cap) genes. These genes produce several rep and cap proteins through alternative translation initiation sites and differential splicing. The coding sequences are flanked by terminal inverted repeats (ITRs), causing the AAV genome to form a T-shaped hairpin structure with ITRs at both ends. The linear single-stranded genome between the hairpin structures encodes the rep and cap proteins. The rep gene encodes four proteins (rep78, rep68, rep52, and rep40) used for viral genome replication and packaging, while cap expression produces three capsid proteins (VP1, VP2, and VP3), which form the protective capsid shell of the AAV genome and participate in cell binding and internalization.

[0005] Recombinant AAVs lacking the wild-type genome (rAAVs) are protein-based nanoparticles designed to cross the cell membrane, where they can transport and deliver their recombinant DNA cargo to the cell nucleus. In the absence of the rep protein, the ITR-encoded transgenes of rAAVs form circular multiplications that persist as episomes in the nucleus of transduced cells. Because the recombinant free DNA does not integrate into the host genome, it eventually becomes diluted over time as the cell undergoes repeated replication cycles. This ultimately leads to the loss of transgenes and transgene expression, with the rate of transgene loss depending on the conversion rate of the transduced cells. These characteristics make rAAVs ideal for gene therapy applications, and AAV vector-mediated gene delivery has recently been approved for the treatment of hereditary blindness and spinal muscular atrophy, and long-term therapeutic effects have been achieved for other rare diseases, including hemophilia and Duchenne muscular dystrophy.

[0006] A typical recombinant adeno-associated virus vector (rAAV vector) has a genomic structure in which the rep and cap genes between the ITRs of the wild-type AAV genome are replaced by one or more transgenes to form a transgenic plasmid for gene delivery in gene therapy applications. An example of a method for producing rAAV vectors involves introducing a transgenic plasmid in which the transgene is inserted between the ITRs into a host cell, and introducing a rep plasmid to provide the rep protein for replication, thereby producing the rAAV vector in the host cell. The rep plasmid may further contain the cap gene, or the cap gene may be located on a separate cap plasmid introduced into the host cell. Furthermore, helper genes from other viruses, such as herpesviruses or adenoviruses, can be introduced into cells to regulate cellular metabolism and AAV gene expression, prevent apoptosis, and perform other functions.

[0007] While wild-type AAVs can provide 100% intact and infectious particles, for rAAVs, the range is typically 1-30% intact particles (the latter is rarely achieved and is only achievable through intensive labor with specific transgene and other element combinations), and only one out of 200-1000 intact particles is infectious. Therefore, producing sufficiently high titers for effective and efficient treatment is challenging when using rAAV vectors for gene therapy applications. High levels of product-related impurities (e.g., empty / partially filled viral capsids) during production further complicate rAAV production. Product-related impurities can include empty AAV capsids, capsid-encapsulated host cell nucleic acids / helper DNA, and non-infectious AAV capsids. Of these impurities, empty capsids have been reported to be the most detrimental to productivity and cause batch-to-batch variability. Purification methods that cannot substantially remove these product-related impurities lead to heightened immune responses and reduced transduction efficiency. This problem is further exacerbated by the fact that, in currently used chromatographic purification methods, product loss increases exponentially with the percentage of empty capsid impurities in the original batch product. Therefore, increasing the concentration of viral genome (i.e., genome titer) and the proportion of intact particles (i.e., intact capsids) in the original batch product is particularly necessary for cost-effective gene therapy with AAV and for obtaining high-purity drug substances for gene therapy applications.

[0008] Recent attempts to improve rAAV production include modifying the AAV ITR to enable transgene expression without the need for second-strand DNA synthesis. Other approaches aim to optimize promoters, AAV transgenes, AAV packaging processes, AAV capsids for enhanced transduction, AAV vectors for enhanced AAV transport or uncoating, plasmid ratios used for AAV production, and subsequent purification methods. Summary of the Invention

[0009] In light of the above background, the object of the present invention is to provide an improved method for the efficient production of rAAV vectors. Specifically, the object of the present invention is to provide rAAV capsids (or rAAV particles) that are properly filled with their intended DNA cargo (referred to as complete capsids), while reducing the proportion of empty capsids (meaning capsids not filled with their intended DNA cargo). Therefore, the object of the present invention is to provide rAAV vectors containing the desired DNA at increased concentrations. Another object of the present invention is to provide rAAV vectors at increased concentrations capable of successfully transducing cells. Another object of the present invention is to provide genomic rAAV vectors containing a higher proportion relative to the total number of viral capsids, wherein the viral capsids may include empty capsids lacking both the genome and the desired gene.

[0010] These objectives are achieved by the rep plasmids of claims 1 to 5, the plasmid systems of claims 6 to 9, the transgenic plasmids of claim 10 or 11, the stable or transient cell expression system of claim 12, the cells of claim 13, the kit of claim 14, and the method for producing recombinant adeno-associated virus vectors of claim 15.

[0011] This invention provides rep plasmids for the efficient production of rAAV vectors and plasmid systems containing rep plasmids. This is achieved by providing a minimal element containing one or more recombinant rep-binding elements located downstream of a polyadenylation signal sequence. The research of this invention unexpectedly found that including at least one recombinant, i.e., non-natural, minimal element containing a rep-binding element downstream of the polyadenylation signal sequence increases rAAV vector yield. Cells used for producing adeno-associated virus particles transfected with the rep plasmids of this invention provide increased concentrations of viral genome and intact capsids. Therefore, the rep plasmids of this invention are particularly suitable for the production of rAAV for gene therapy applications where high titers are required for efficient and / or effective treatment.

[0012] Furthermore, this invention relates to transgenic plasmids, wherein cells transfected with the transgenic plasmids of this invention produce an increased concentration of intact capsids. This is achieved via transgenic plasmids comprising a CMVie promoter, a transgene, SV40PolyA, and 5' and 3' terminal inverted repeat sequences derived from J01901.1 AAV2; wherein the transgenic plasmid is selected from one of the following: (i) Conventional single-stranded recombinant adeno-associated virus, or (ii) Self-complementary genomic recombinant adeno-associated virus.

[0013] Therefore, the transgenic plasmids of the present invention are also particularly suitable for the production of rAAV for gene therapy applications.

[0014] The present invention also relates to stable or transient cellular expression systems comprising the plasmid system of the present invention.

[0015] This invention also relates to a kit comprising the stable or transient cell expression system of this invention or the cells and cell culture medium of this invention. The kit provides an improved method for the efficient production of rAAV vectors using the transgenic plasmids and / or the rep plasmids of this invention.

[0016] The present invention further relates to a method for producing rAAV vectors by transfecting cells with the rep plasmid of the present invention, the plasmid system of the present invention, or the transgenic plasmid of the present invention. Detailed Implementation

[0017] Although some embodiments of the invention are illustrated in detail below, it should be understood that the invention is not limited to the specific embodiments, methodologies, schemes, and reagents described herein, as these may vary within the scope set forth in the claims. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention as defined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0018] In the following description, some elements of the invention will be described. These elements can be discussed with reference to specific embodiments; however, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The examples, features, and specific embodiments described should not be construed as limiting the invention to only the explicitly described embodiments or combinations of explicitly described features. This specification should be understood as disclosing and including embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered as disclosed in this specification unless the context otherwise requires.

[0019] The above objective is achieved by means of the following embodiments according to the present invention: 1. A rep plasmid comprising: (i) at least one adeno-associated virus replication protein coding sequence encoding at least one functional rep protein; (ii) at least one polyadenylation signal sequence located downstream of the adeno-associated virus replication protein coding sequence; and (iii) The smallest element located downstream of the polyadenylation signal sequence, comprising one or more recombinant rep binding elements.

[0020] 2. The rep plasmid of implementation scheme 1, wherein one or more rep binding elements act as binding sites for rep68 and / or rep78.

[0021] 3. The rep plasmid of embodiment 1 or 2, wherein the minimum element comprises at least two rep binding elements.

[0022] 4. A rep plasmid according to any one of embodiments 1 to 3, comprising another minimal element, said other minimal element comprising one or more rep binding elements located downstream of the polyadenylation signal sequence.

[0023] 5. The rep plasmid of any one of embodiments 1 to 4, comprising at least one adeno-associated virus capsid protein coding sequence encoding at least one functional cap protein.

[0024] 6. The rep plasmid according to embodiment 5, wherein the capsid protein coding sequence encodes VP1, VP2 and VP3, or one or more artificial variants thereof.

[0025] 7. The rep plasmid according to embodiment 5 or 6, wherein the capsid protein coding sequence is derived from AAV2.

[0026] 8. The rep plasmid of any one of embodiments 1 to 7, wherein the rep binding element comprises or consists of a sequence selected from or composed of a sequence selected from: SEQ ID NO: 18, 20 and / or 33-41 or a sequence having at least 80% identity with one of the SEQ ID NOs, preferably a sequence having at least 85% identity with one of the SEQ ID NOs, more preferably a sequence having at least 90% identity with one of the SEQ ID NOs, even more preferably a sequence having at least 95% identity with one of the SEQ ID NOs, and most preferably a sequence having at least 99% identity with one of the SEQ ID NOs.

[0027] 9. The rep plasmid of any one of embodiments 1 to 8, wherein the rep binding element is derived from a terminal inverted repeat sequence, preferably from a 3' terminal inverted repeat sequence derived from J01901.1 AAV2.

[0028] 10. The rep plasmid of any one of embodiments 1 to 7, wherein the rep binding element comprises SEQ ID NO: 20 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0029] 11. The rep plasmid of any one of embodiments 1 to 7 or 10, wherein the rep binding element is derived from an AAV promoter sequence, preferably from a p5 promoter sequence derived from J01901.1 AAV2.

[0030] 12. The rep plasmid of any one of embodiments 1 to 11, wherein the minimum element consists of 500 nt or less, 450 nt or less, 300 nt or less, 250 nt or less, 200 nt or less, preferably 150 nt or less or 100 nt or less.

[0031] 13. The rep plasmid of any one of embodiments 1 to 9 or 12, wherein the smallest element comprises SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, or at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0032] 14. A rep plasmid of any one of embodiments 1 to 7 or 10 to 12, wherein the smallest element comprises or is composed of the following sequence: SEQ ID NO: 23 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0033] 15. The rep plasmid of any one of embodiments 1 to 14, wherein the replication protein coding sequence encodes rep78, rep68, rep52 and rep40, or one or more artificial variants thereof.

[0034] 16. The rep plasmid of any one of embodiments 1 to 15, wherein the replication protein coding sequence is derived from AAV2.

[0035] 17. A rep plasmid of any one of embodiments 1 to 16, wherein the rep plasmid further comprises one or more natural or heterologous promoters operatively linked to the coding sequence of the replication protein, preferably wherein the promoters are selected from p5, p19 and p40.

[0036] 18. A plasmid system for producing adeno-associated virus particles, comprising: (i) transgenic plasmids; and (ii) The rep plasmid of any one of Implementation Schemes 1 to 17.

[0037] 19. The plasmid system of embodiment 18, wherein the system further comprises an auxiliary plasmid.

[0038] 20. The plasmid system of embodiment 19, wherein the auxiliary plasmid comprises one or more coding sequences encoding E2A, E4orf6, or E4orf7, or any combination thereof.

[0039] 21. The plasmid system of embodiment 19 or 20, wherein the helper plasmid comprises one or more virus-associated RNAs.

[0040] 22. A plasmid system according to any one of embodiments 18 to 21, wherein the transgenic plasmid comprises a promoter, a transgene, a polyadenylation signal sequence, and 5' and 3' terminal inverted repeat sequences, wherein the transgenic plasmid is selected from one of the following: (i) Conventional single-stranded recombinant adeno-associated virus, or (ii) Self-complementary genomic recombinant adeno-associated virus.

[0041] 23. The plasmid system of embodiment 22, wherein the 3' and 5' terminal inverted repeat sequences are derived from AAV2, preferably J01901.1 AAV2.

[0042] 24. The plasmid system of embodiment 22 or 23, wherein at least one terminal inverted repeat sequence, preferably a 5' terminal inverted repeat sequence, has a deletion compared to the natural terminal inverted repeat sequence.

[0043] 25. The plasmid system of any one of embodiments 22 to 24, wherein the promoter is the CMVie promoter.

[0044] 26. A plasmid system according to any one of embodiments 18 to 25, wherein the transgenic plasmid contains a transgene, the transgene contains a reporter gene, preferably the transgene is a reporter gene.

[0045] 27. The plasmid system of embodiment 26, wherein the reporter gene can be detected by antibody-based assay.

[0046] 28. The plasmid system of embodiment 26 or 27, wherein the reporter gene is a fluorescent molecule.

[0047] 29. The plasmid system of embodiment 26 or 27, wherein the reporter gene is β-galactosidase, luciferase or glutathione S-transferase.

[0048] 30. The plasmid system of any one of embodiments 22 to 29, wherein the polyadenylation signal sequence is an SV40poly(A) signal sequence.

[0049] 31. A plasmid system according to any one of embodiments 18 to 30, wherein the transgenic plasmid comprises the following structure: 5' ITR (inverted)-CMVie promoter-transgenic-SV40 PolyA-ITR (forward and reverse) 3'.

[0050] 32. The plasmid system of embodiment 31, wherein the terminal inverted repeat sequence having a flipped orientation has SEQ ID NO: 1 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0051] 33. The plasmid system of embodiment 31 or 32, wherein the terminal inverted repeat sequences having forward and reverse orientations have SEQ ID NO: 2 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0052] 34. The plasmid system of any one of embodiments 18 to 33, wherein the transgenic plasmid does not contain a post-transcriptional regulatory element of marmot hepatitis virus.

[0053] 35. The plasmid system of any one of embodiments 18 to 34, further comprising a cap plasmid.

[0054] 36. The plasmid system of embodiment 35, wherein the cap plasmid comprises at least one adeno-associated virus capsid protein coding sequence encoding at least one functional cap protein.

[0055] 37. The plasmid system of embodiment 36, wherein the capsid protein coding sequence encodes VP1, VP2, and VP3, or one or more artificial variants thereof.

[0056] 38. A plasmid system according to any one of embodiments 36 or 37, wherein the capsid protein coding sequence is derived from AAV2.

[0057] 39. A transgenic plasmid comprising: CMVie promoter, transgene, SV40 PolyA, 5' and 3' terminal inverted repeat sequences derived from J01901.1 AAV2; The transgenic plasmids mentioned above are selected from one of the following: (i) Conventional single-stranded recombinant adeno-associated virus, or (ii) Self-complementary genomic recombinant adeno-associated virus.

[0058] 40. The transgenic plasmid of embodiment 39, wherein at least one terminal inverted repeat sequence, preferably a 5' terminal inverted repeat sequence, has a deletion compared to the natural terminal inverted repeat sequence, more preferably the terminal inverted repeat sequence containing the deleted sequence has SEQ ID NO: 4 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0059] 41. The transgenic plasmid of embodiment 39 or 40, wherein the transgenic plasmid comprises the following structure: 5' ITR (inverted)-CMVie promoter-transgenic-SV40 PolyA-ITR (forward and reverse) 3'.

[0060] 42. The transgenic plasmid of embodiment 41, wherein the terminal inverted repeat sequence having a flipped orientation has SEQ ID NO: 1 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0061] 43. The transgenic plasmid of embodiment 41 or 42, wherein the terminal inverted repeat sequences having forward and reverse orientations have SEQ ID NO: 2 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0062] 44. A transgenic plasmid according to any one of embodiments 39 to 43, wherein the transgene is a reporter gene, preferably the reporter gene can be detected by an antibody-based assay, more preferably the reporter gene is a fluorescent molecule or the reporter gene is β-galactosidase, luciferase or glutathione S-transferase.

[0063] 45. A transgenic plasmid of any one of embodiments 39 to 44, wherein the plasmid does not contain a post-transcriptional regulatory element of marmot hepatitis virus.

[0064] 46. ​​A rep-cap plasmid comprising: (i) at least one adeno-associated virus replication protein coding sequence encoding at least one functional rep protein; (ii) at least one adeno-associated virus capsid protein coding sequence encoding at least one functional cap protein; (iii) at least one polyadenylation signal sequence located downstream of the adeno-associated virus replication protein coding sequence; and (iv) The smallest element located downstream of the polyadenylation signal sequence, comprising one or more recombinant rep binding elements.

[0065] 47. The rep-cap plasmid of embodiment 46, wherein one or more rep binding elements act as binding sites for rep68 and / or rep78.

[0066] 48. The rep-cap plasmid of embodiment 46 or 47, wherein the minimum element comprises at least two rep-binding elements.

[0067] 49. The rep-cap plasmid of any one of embodiments 46 to 48, comprising an additional minimal element containing one or more rep-binding elements located downstream of the polyadenylation signal sequence.

[0068] 50. The rep-cap plasmid of any one of embodiments 46 to 49, wherein the capsid protein coding sequence encodes VP1, VP2 and VP3, or one or more artificial variants thereof.

[0069] 51. The rep-cap plasmid of any one of embodiments 46 to 50, wherein the capsid protein coding sequence is derived from AAV2.

[0070] 52. The rep-cap plasmid of any one of embodiments 46 to 51, wherein the rep-binding element comprises or consists of a sequence selected from or composed of a sequence selected from: SEQ ID NO: 18, 20 and / or 33-41 or a sequence having at least 80% identity with one of the SEQ ID NOs, preferably a sequence having at least 85% identity with one of the SEQ ID NOs, more preferably a sequence having at least 90% identity with one of the SEQ ID NOs, even more preferably a sequence having at least 95% identity with one of the SEQ ID NOs, and most preferably a sequence having at least 99% identity with one of the SEQ ID NOs.

[0071] 53. The rep-cap plasmid of any one of embodiments 46 to 52, wherein the rep-binding element is derived from a terminal inverted repeat sequence, preferably from a 3' terminal inverted repeat sequence derived from J01901.1 AAV2.

[0072] 54. The rep plasmid of any one of embodiments 46 to 51, wherein the rep binding element comprises SEQ ID NO:20 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0073] 55. A rep plasmid of any one of embodiments 46 to 51 or 54, wherein the rep binding element is derived from an AAV promoter sequence, preferably from a p5 promoter sequence derived from J01901.1 AAV2.

[0074] 56. The rep-cap plasmid of any one of embodiments 46 to 55, wherein the minimum element consists of 500 nt or less, 450 nt or less, 300 nt or less, 250 nt or less, 200 nt or less, preferably 150 nt or less, or 100 nt or less.

[0075] 57. A rep-cap plasmid of any one of embodiments 46 to 53 or 56, wherein the smallest element comprises SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, or at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0076] 58. A rep-cap plasmid according to any one of embodiments 46 to 51 or 54 to 56, wherein the smallest element comprises SEQ ID NO: 23 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, most preferably a sequence having at least 99% identity with said SEQ ID NO, or composed of the above sequences.

[0077] 59. The rep-cap plasmid of any one of embodiments 46 to 58, wherein the replication protein coding sequence encodes rep78, rep68, rep52 and rep40, or one or more artificial variants thereof.

[0078] 60. The rep-cap plasmid of any one of embodiments 46 to 59, wherein the replication protein coding sequence is derived from AAV2.

[0079] 61. The rep-cap plasmid of any one of embodiments 46 to 60, wherein the rep plasmid further comprises one or more natural or heterologous promoters operatively linked to the coding sequence of the replication protein, preferably wherein the promoters are selected from p5, p19 and p40.

[0080] 62. A stable or transient cell expression system comprising a plasmid system and cell line according to any one of embodiments 18 to 38.

[0081] 63. A cell comprising a rep plasmid of any one of embodiments 1 to 17, a plasmid system of any one of embodiments 18 to 38, a transgenic plasmid of any one of embodiments 39 to 45, or a rep-cap plasmid of any one of embodiments 46 to 61.

[0082] 64. The cell of embodiment 63, wherein the cell is a mammalian cell.

[0083] 65. A kit comprising a stable or transient cell expression system of embodiment 62, or cells of any one of embodiments 63 or 64, and cell culture medium.

[0084] 66. The kit according to embodiment 65, further comprising culture medium feed, culture medium additive or transfection agent or any combination thereof.

[0085] 67. The kit according to implementation plan 65 or 66, which also includes a manual.

[0086] 68. A method for producing a recombinant adeno-associated virus vector, the method comprising: (i) Transfect cells with the rep plasmid of any one of embodiments 1 to 17, the plasmid system of any one of embodiments 18 to 38, the transgenic plasmid of any one of embodiments 39 to 45, or the rep-cap plasmid of any one of embodiments 46 to 61; (ii) Culturing transfected cells to produce the adeno-associated virus vector; and (iii) Isolate the recombinant adeno-associated virus vector.

[0087] 69. The method for producing a recombinant adeno-associated virus vector according to embodiment 68, wherein the cell is HEK293 cell.

[0088] definition

[0089] Unless otherwise specified in the specification or claims, the terms used to explain the invention have the following meanings. Further definitions are set forth throughout the detailed description.

[0090] The 3' UTR sequence is the 3' untranslated region, known to regulate various mRNA-based processes, such as mRNA localization, mRNA stability, and translation. Furthermore, the 3' UTR can establish 3' UTR-mediated protein-protein interactions (PPIs), thus transferring the genetic information encoded in the 3' UTR to proteins. This function has been shown to regulate a variety of protein characteristics, including protein complex formation and post-translational modifications, but is also expected to alter protein conformation.

[0091] The 5'UTR sequence, or 5' untranslated region, is located in the non-coding genome upstream of the coding sequence and plays a crucial role in regulating gene expression. Numerous cis-regulatory elements can reside within the 5'UTR sequence, interacting with transcriptional mechanisms to regulate mRNA abundance. The 5' UTR can contain various RNA-based regulatory elements, including secondary structures, RNA-binding protein motifs, upstream open reading frames (uORFs), internal ribosome entry sites, terminal oligopyrimidine (TOP) sequences, and G-quadruplexes. These elements can alter the efficiency of mRNA translation; some elements can also affect mRNA transcription levels through changes in stability or degradation.

[0092] The terms “a,” “an,” and “the,” as well as similar designations, used in the context of describing this invention (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0093] As used herein, the terms “about” or “approximately” indicate a range of ±10% of a reference value. For example, “about 10” is defined as a range of 9 to 11. Generally, those skilled in the art will understand the extent of variation covered by “about” or “approximately” in that context.

[0094] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, type 1 AAV (e.g., AAV of serotype 1, also known as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), type 11 AAV (e.g., AAV11), and type 12 AAV. Type AAV (e.g., AAV12), Type 13 AAV (e.g., AAV13), Type rh32.33 AAV (e.g., AAVrh32.33), Type rh8 AAV (e.g., AAVrh8), Type rhlO AAV (e.g., AAVrhlO), Type rh74 AAV (e.g., AAVrh74), Type hu.68 AAV (e.g., AAVhu.68), Bird AAV (e.g., AAAV), Cattle AAV (e.g., BAAV), Dog AAV, Horse AAV, Sheep AAV, Snake AAV, Bearded Dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV currently known or subsequently discovered.

[0095] As used herein, “and / or” means and covers any and all possible combinations of one or more of the related listed items, as well as combinations lacking when interpreted in the alternative form (“or”). The use of an alternative (e.g., “or”) should be understood to mean one, both, or any combination of the alternatives.

[0096] A "cap plasmid" is a plasmid that contains a sequence encoding a capsid protein. The terms "cap plasmid" and "capsid plasmid" are used interchangeably in this document.

[0097] Unless otherwise expressly stated, the term "comprising" is used in the context of this document to indicate that, in addition to the members of the list introduced by "comprising," other members may optionally exist. However, as a particular embodiment of the invention, each use of the term "comprising" should also include the possibility that no other members exist; that is, for the purposes of this embodiment, "comprising" can be understood to have the meaning of "consisting of."

[0098] As used herein, in the context of nucleic acid or amino acid sequences, "derived from" means that the sequence is identical to the sequence from which it originates, or has a specified percentage of identical amino acid residues or nucleotides to the sequence from which it originates. When comparing and aligning maximum correspondence within a comparison window or specified region using the BLAST or BLAST2.0 sequence comparison algorithm with default parameters or by manual alignment and visual inspection, the specified percentage may be at least about 60% of the specified sequence, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. For example, the 3' and 5' inverted repeat sequences may be derived from AAV2, preferably J01901.1 AAV2, meaning that the 3' and 5' inverted repeat sequences may be identical to, or have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with, the 3' and 5' inverted repeat sequences contained in the AAV2 genome, preferably J01901.1 AAV2 genome. In the context of this disclosure, “J01901.1” should be understood to refer to the GenBank accession number “J01901.1” of the complete wild-type AAV2 genome listed in SEQ ID NO: 19.

[0099] As used herein, the terms “wild-type” and “natural” are synonymous and are well known in the art. In this document, these terms specifically refer to polynucleotide sequences or genes present in the genome of AAV or adenovirus strains / serotypes, or proteins encoded by genes present in the genome of AAV or adenovirus strains / serotypes. Preferably, a wild-type AAV strain / serotype may refer to the AAV2 genome shown in GenBank accession number J01901.1.

[0100] In this document, the term "DNA" refers to a nucleic acid molecule that is entirely or at least substantially composed of deoxyribonucleotide residues. In a preferred embodiment, the DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2'-position of the β-D-furanose group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, substantially pure DNA, synthetic DNA, recombinant DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alteration may refer to the addition of a non-nucleotide substance to the internal DNA nucleotides or the ends of the DNA. This document also considers that the nucleotides in the DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the purposes of this disclosure, such altered DNA is considered an analogue of naturally occurring DNA. A molecule is considered to contain "major deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is based on the total number of nucleotide residues in the molecule exceeding 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%). The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogues).

[0101] DNA can be recombinant DNA and can be obtained by cloning nucleic acids, especially cDNA. cDNA can be obtained through reverse transcription of RNA.

[0102] As used herein, "feed" or "supplement" refers to a composition that, when added to cells in a standard culture, may benefit cell maintenance, expansion, growth, or survival, or affect cell performance, or increase culture lifespan, or maintain cells in a pseudostationary phase of continued product expression, or lead to an increase in the final product titer. The terms "feed" or "supplement" are used interchangeably in this disclosure and refer to solid and liquid forms (including aggregated forms) of a culture medium component containing one or more amino acids, sugars, vitamins, buffers, and sometimes peptides, hydrolysates, components, growth factors, hormones, etc., required for the rebalancing or supplementation or regulation of the growth or performance of cells or a cell culture system. The difference between a feed or supplement and cell culture medium is that it is added to a cell culture medium in which cells can be cultured. As will be understood by those skilled in the art, sometimes a feed / supplement may primarily contain those amino acids, sugars, vitamins, buffers, etc., required for the rebalancing or supplementation or regulation of the growth or performance of cells or a cell culture system. A feed or supplement may be concentrated or non-concentrated, or may be a partial concentration of only certain components.

[0103] As used herein, the term "gene" refers to a segment (e.g., a coding region) of a DNA molecule that encodes a polypeptide chain. In some embodiments, a gene is located by a region immediately before, after, and / or inserted into the coding region that is involved in generating the polypeptide chain (e.g., regulatory elements such as promoters, enhancers, polyadenylated sequences, 5'-untranslated regions, 3'-untranslated regions, or introns).

[0104] As used herein, the term "encoding" refers to the sequence information of a first molecule that directs the production of a second molecule having a defined nucleotide sequence (e.g., mRNA) or a defined amino acid sequence. For example, a DNA molecule can encode an RNA molecule (e.g., through transcription involving a DNA-dependent RNA polymerase). A coding sequence that encodes a protein is a sequence that directs the production of said protein. Thus, if transcription and translation of mRNA corresponding to a gene produces a polypeptide in a cell or other biological system, the coding sequence, gene, cDNA, or single-stranded RNA (e.g., mRNA) encodes the polypeptide. In some embodiments, a coding sequence that encodes a target polypeptide refers to a coding strand whose nucleotide sequence can be identical to the mRNA sequence of such a target polypeptide. In some embodiments, a coding sequence that encodes a target polypeptide refers to a non-coding strand of such a target polypeptide reagent that can be used as a template for gene or cDNA transcription. As understood in the art, the phrase "coding sequence that encodes a peptide or protein" refers to a plasmid containing that coding sequence, which, if present in a suitable environment, such as within a cell and / or in a cell-free translation system, can direct the assembly of amino acids to produce a peptide or protein through a translation process.

[0105] In the context of this invention, a functional rep protein is a replication (rep) protein involved in AAV replication and / or AAV assembly. In some embodiments, the functional rep protein is involved in AAV replication and viral assembly. In some embodiments, the functional rep protein is involved in AAV replication. In some embodiments, the functional rep protein is involved in the assembly of AAV virions. In some embodiments, the functional rep protein binds to DNA. In some embodiments, the functional rep protein does not bind to DNA.

[0106] In the context of this invention, a functional cap protein is a cap protein capable of forming a capsid. A functional cap protein can be any of VP1, VP2, VP3, or an artificial variant thereof capable of forming a capsid.

[0107] In the context of two or more nucleic acid or peptide sequences, the term "identical" or "identity" percentage (%) refers to the fact that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides when measured using the BLAST or BLAST2.0 sequence comparison algorithm with default parameters or by manual alignment and visual inspection (i.e., having approximately 60% identity in a specified region when comparing and aligning the maximum correspondence in a comparison window or specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity).

[0108] Terminal inverted repeats (ITRs) are guanine-cytosine-rich structures involved in the replication and capsiding of the AAV genome, and they are integrated into and excised from the host genome. ITRs are conserved native AAV-derived DNA sequences in recombinant AAVs (rAAVs) because they allow for replication, capsiding, and long-term maintenance and expression in target cells. ITRs can be incomplete, truncated, and / or modified. A truncated ITR means that one or more nucleotides of the native sequence may be missing. A modified ITR means that the ITR may contain one or more nucleotides of a non-ITR sequence. Non-ITR sequences can be inserted into or added to one or both ends of an ITR sequence. Modified ITR sequences may contain substitutions involving one or more nucleotide exchanges. It has been shown that the rAAV genome can be replicated even with incomplete, truncated, or modified ITR sequences. It should be understood that the invention is not limited to specific ITR sequences, and any ITR sequence now known or discovered thereafter can be used.

[0109] A “minimum element” is a recombinant polynucleotide sequence containing one or more rep-binding elements. The minimum element is preferably a short polynucleotide sequence, preferably having a length of 500 nt or less, more preferably 450 nt or less, more preferably 400 nt or less, more preferably 350 nt or less, more preferably 300 nt or less, more preferably 250 nt or less, more preferably 200 nt or less, most preferably 150 nt or less, or 100 nt or less. The minimum element may contain a nucleotide sequence that is not naturally present in AAV. Such a non-natural nucleotide sequence may be upstream and / or downstream of one or more rep-binding elements. In some embodiments, the minimum element contains one rep-binding element, preferably one rep-binding element and a non-natural nucleic acid. In some embodiments, the minimum element contains two or more rep-binding elements or is composed of two or more rep-binding elements, preferably two or more rep-binding elements. In some embodiments, the minimum element is derived from an ITR sequence. In some embodiments, the minimum element is derived from a truncated and / or modified ITR sequence.

[0110] "Rep-binding elements" are well known in the art and are understood by those skilled in the art to be polynucleotide sequences present in adeno-associated viruses, but also in other viruses and organisms containing AAV replication protein (rep protein) binding sites. Rep-binding elements are known in the art and are involved in AAV transcriptional regulation, DNA replication, and genome integration. In some preferred embodiments, one or more rep-binding elements act as binding sites for rep68 and / or rep78. In some preferred embodiments, one or more rep-binding elements act as binding sites for either rep68 or rep78. In some embodiments, one or more rep-binding elements act as binding sites for both rep68 and rep78. In some embodiments, one or more rep-binding elements act as binding sites for rep68. In some embodiments, one or more rep-binding elements act as binding sites for rep78. Not wishing to be bound by theory, this document anticipates that rep-binding elements enable genome replication to be initiated by allowing rep68 and / or rep78 to bind, thereby increasing rAAV vector yield, for example, by providing increased concentrations of viral genome and intact capsids.

[0111] As used herein, when nucleic acid sequences (e.g., coding sequences) and regulatory sequences are covalently linked in a manner that brings the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequence, they are said to be "operably linked." Two DNA sequences are considered to be operably linked if the nucleic acid sequence is desired to be translated into a functional protein, if a promoter is induced in the 5' regulatory sequence to lead to transcription of the coding sequence, and if the nature of the link between the two DNA sequences does not (1) lead to the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.

[0112] The “polyadenylation signal sequence” (PAS) is a sequence known to those skilled in the art. A polyadenylation signal sequence typically comprises a conserved hexameric motif required for mRNA polyadenylation, a U-rich and / or GU-rich sequence downstream of the hexameric motif, and a dinucleotide sequence preceding the polyadenylation cleavage site and located between the U-rich and / or GU-rich sequence and the hexameric motif. This sequence can be recognized by a cleavage and polyadenylation specificity factor (CPSF) within the RNA cleavage complex. The hexameric motif varies among eukaryotes but can be AATAAA or a modification thereof. The CPSF is a central component of the 3' processing mechanism of polyadenylated mRNA and recognizes the PAS, thereby providing sequence specificity for pre-mRNA cleavage and polyadenylation, and catalyzing pre-mRNA cleavage. In some embodiments, the polyadenylation signal sequence may be a sequence containing AATAAA or a modified sequence thereof. The modified AATAAA sequence may be a sequence in which one or two nucleic acids are deleted, substituted, inserted, and / or added. Other PAS are known, such as ATTAAA, AGTAAA, TATAAA, CATAAA, GATAAA, AATTA, AATCA, AATGA, AAAAAG, and ACTAAA, and may be used in the context of this disclosure.

[0113] A "poly(A) tail" (or "poly(A) sequence") is an adenine nucleotide chain that is typically added to mRNA molecules during RNA processing to increase molecular stability and make translation possible. This process is called polyadenylation and usually adds 100 to 250 adenine nucleotides.

[0114] The terms “nucleic acid sequence,” “nucleotide sequence,” “polynucleotide,” and “nucleic acid” are used interchangeably herein to refer to one or more nucleotides, preferably deoxyribonucleic acid (DNA). These terms include DNA, ribonucleic acid (RNA), combinations thereof, and their modified forms. The term includes genomic DNA, cDNA, mRNA, recombinant-derived molecules, and chemically synthesized molecules. In some preferred embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is a mixture of DNA and RNA. Polynucleotides can exist as single-stranded or double-stranded molecules and as linear or covalently circularly closed molecules. Polynucleotides can be isolated. According to this disclosure, the term “isolated polynucleotide” means that the polynucleotide is (i) obtained, for example, by in vitro amplification of DNA using polymerase chain reaction (PCR), or by in vitro transcription of RNA (using, for example, RNA polymerase), (ii) obtained by clonal recombination, (iii) purified, for example, by enzyme digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.

[0115] The term "plasmid" refers to extrachromosomal circular DNA that can be expressed in a given cell. Plasmids can also be engineered using standard molecular biology techniques (Sambrook et al., Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), New York).

[0116] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this document to refer to polymers of amino acids.

[0117] The term "recombination," when used in the context of polynucleotides, refers to a polynucleotide having a non-naturally linked nucleotide sequence. The term recombination is well known in the art, and those skilled in the art understand that "non-naturally linked" means the removal of a nucleotide sequence from its natural sequence background. Thus, for example, the recombinant minimal element used herein may be a naturally occurring nucleotide sequence or a fragment thereof, however, linked with another nucleotide sequence such that the combination is not naturally occurring. For example, the minimal element is linked with one or more other nucleic acid sequences, for example, in the rep plasmid disclosed herein, to produce a polynucleotide having a non-naturally linked nucleotide sequence. In this embodiment, the point where the minimal element sequence of the rep plasmid and the adjacent nucleic acid sequence are linked together is non-natural, i.e., not naturally linked together. In this embodiment, the minimal element is considered recombinant because it is placed adjacent to a sequence that is not adjacent to it in its natural sequence background. In another embodiment, the recombinant polynucleotide can be produced by artificially combining two additionally separate sequence segments. Such artificial combination is typically accomplished by chemical synthesis, or more generally by artificially manipulating isolated nucleic acid fragments, such as through genetic engineering techniques. Recombinant polynucleotides (RPNs) include vectors containing amplified or assembled polynucleotides that can be used to transform or transfect suitable host cells. The host cell containing the RPN is called a "recombinant host cell." The polynucleotide is then expressed in the recombinant host cell to produce a "recombinant polypeptide." The RPN may also contain non-coding functions.

[0118] A "rep plasmid" is a plasmid that contains the coding sequence for a replicating protein. The terms "rep plasmid" and "replication plasmid" are used interchangeably in this document. As described herein, a rep plasmid is a recombinant polynucleotide.

[0119] A "rep cap plasmid" or "rep-cap plasmid" is a plasmid containing both the replication protein-coding sequence and the capsid protein-coding sequence. The terms "rep cap plasmid" and "replication capsid plasmid" are used interchangeably herein. As described herein, a rep-cap plasmid is a recombinant polynucleotide.

[0120] "Recombinant AAV (rAAV)" and "AAV" are used interchangeably in this application.

[0121] As used herein, a “transgenic” is a nucleic acid introduced into a cell, including but not limited to genes or nucleic acids having sequences not normally present in the AAV, genes present in the AAV genome but not normally transcribed and translated (“expressed”), or any other gene or nucleic acid desired to be located between ITR sequences. Transgenics may include one or more transcriptional regulatory sequences and any other nucleic acids, such as introns, which may be necessary for optimal expression of the selected nucleic acid. Transgenics can be as short as a few nucleotides, but preferably can be at least about 50, 100, 150, 200, 250, 300, 350, 400, or 500 nucleotides (nt) long. Transgenics may contain coding or non-coding sequences.

[0122] As used herein, the term "variant" refers to a molecule, such as a gene or protein, that shares one or more specific structural features, elements, components, or portions with a reference molecule. Thus, a variant is a molecule, such as a gene or protein, that shares one or more specific structural features, elements, components, or portions with a reference molecule. A variant can be a gene that shares one or more specific structural features, elements, components, or portions with a reference gene. A variant can be a protein that shares one or more specific structural features, elements, components, or portions with a reference protein. Typically, a "variant" has significant structural similarity to a reference molecule, such as sharing a core or common structure, but also differs in some discrete manner. In some embodiments, a variant is a molecule that can be produced from a reference molecule, for example, through chemical manipulation of the reference molecule. In some embodiments, a variant is a molecule that can be produced by a synthetic method that is substantially similar to (e.g., shares multiple steps) the synthetic method used to produce the reference molecule. In some embodiments, a variant is produced by, or can be produced by, a synthetic method different from the synthetic method used to produce the reference molecule.

[0123] As used herein, the terms “viral vector,” “viral vector,” and “gene delivery vector” refer to a viral particle that functions as a nucleic acid delivery vector and contains nucleic acid molecules packaged within a viral capsid. Exemplary viral vectors include adeno-associated virus vectors (AAV).

[0124] The description of numerical ranges herein is intended only to refer to each individual value falling within that range in a concise manner. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were listed separately herein. Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all embodiments or exemplary language (e.g., “such”) provided herein is intended only to better illustrate the invention and not to limit the scope of the invention as defined by the claims. The language in the specification should not be construed as indicating that any unclaimed element is necessary for the practice of the invention.

[0125] References to “an embodiment,” “implementation,” “exemplary implementation,” “some implementations,” “certain implementations,” “various implementations,” etc., indicate that one or more implementations of the disclosed technology as described herein may include a particular feature, structure, or characteristic, but not every implementation is required to include that particular feature, structure, or characteristic.

[0126] All patents, patent applications and other publications cited in this application are incorporated herein by reference in their entirety for all purposes.

[0127] The rep plasmid of the present invention

[0128] This invention provides a rep plasmid comprising: (i) at least one adeno-associated virus replication protein coding sequence encoding at least one functional rep protein; (ii) at least one polyadenylation signal sequence located downstream of the adeno-associated virus replication protein coding sequence; and (iii) The smallest element located downstream of the polyadenylation signal sequence, comprising one or more recombinant rep binding elements.

[0129] Not wanting to be bound by theory, it is anticipated that adding a recombinant rep binding element downstream of the polyadenylation signal sequence will improve replication and / or rAAV assembly. As demonstrated in the examples (see...). Figures 1 to 6 The recombinant rep binding element downstream of the polyadenylation signal sequence (10 to 12) leads to increased concentrations of the viral genome and intact capsid, resulting in higher titers. Therefore, the rep plasmids of this invention are particularly suitable for rAAV production for gene therapy applications, resulting in fewer empty capsid impurities, which have been reported to be most detrimental to productivity and cause batch-to-batch variability in the product. Furthermore, improved transduction efficiency can be obtained due to the high titers.

[0130] Even more surprisingly, the combination of the rep plasmid of the present invention with the self-complementary AAV (scAAV) transgenic plasmid provides a synergistic effect in terms of viral genome and intact capsid concentration.

[0131] In one alternative aspect, the recombinant rep binding element is located downstream of the stop codon in the replication protein coding sequence and the polyadenylation signal sequence, meaning that the recombinant rep binding element is located downstream of the stop codon in the replication protein coding sequence and upstream of the polyadenylation signal sequence. In another aspect, the recombinant rep binding element is located downstream of the stop codon in the capsid protein coding sequence and the polyadenylation signal sequence, meaning that the recombinant rep binding element is located downstream of the stop codon in the capsid protein coding sequence and upstream of the polyadenylation signal sequence.

[0132] In some implementations, the rep plasmid is a rep-cap plasmid that also contains a rep-cap sequence encoding at least one adeno-associated virus capsid protein encoding at least one functional cap protein.

[0133] Analytical techniques capable of providing information about the concentration of viral genome and intact capsid are well known. Exemplary methods for detecting capsid titers of adeno-associated virus (AAV) and for determining the ratio of intact to empty capsids of AAV are described in WO2023 / 139224, which is incorporated herein by reference in its entirety. For example, the percentage of viral capsids containing the genome is typically quantified by electron microscopy of viral vector solutions. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can be used to characterize capsid content and other quality properties. Density-based separation by analytical ultracentrifugation (AUC) can also be used to resolve empty, partial, and intact capsids and to quantify the levels of each population. Charge-based chromatographic separation based on the difference between empty and intact capsids can also be used to quantify the ratio of empty to intact capsids. Preferred methods for determining and quantifying genome titers are quantitative polymerase chain reaction (qPCR) and digital droplet PCR (ddPCR), with DNA content quantified by fluorescence during or after amplification in a thermal cycler. The preferred method for capsid quantification is conventional enzyme-linked immunosorbent assay (ELISA).

[0134] The minimum element is a recombinant polynucleotide sequence containing one or more rep-binding elements. In some embodiments, the minimum element is a polynucleotide sequence containing one or more rep-binding elements downstream of a polyadenylation signal sequence.

[0135] In some embodiments, the minimum element is a short polynucleotide sequence, preferably 500 nt or less, more preferably 450 nt or less, more preferably 400 nt or less, more preferably 350 nt or less, more preferably 300 nt or less, more preferably 250 nt or less, more preferably 200 nt or less, and most preferably 150 nt or less or 100 nt or less.

[0136] In some embodiments, the minimum element comprises or consists of one or more rep-binding elements. In some embodiments, the length of the minimum element is preferably 5-500 nt, preferably 10-400 nt, preferably 20-300 nt, preferably 30-200 nt, and preferably 40-150 nt. In other embodiments, the length of the minimum element is preferably 5-300 nt, preferably 10-250 nt, preferably 20-200 nt, or preferably 20-150 nt. In some embodiments, the length of the minimum element corresponds to the length of the rep-binding element. In other embodiments, the length of the minimum element corresponds to the length of the rep-binding element and other nucleic acids, which are not considered rep-binding element nucleic acids and may be referred to as non-natural nucleotide sequences as disclosed herein.

[0137] In some particularly preferred embodiments, the smallest element is in a reverse orientation.

[0138] In some preferred embodiments, the smallest element is derived from an ITR sequence, preferably a 3' ITR sequence, more preferably a 3' ITR sequence from AAV2, and even more preferably a 3' ITR from J01901.1 AAV2. As demonstrated in the embodiments (see, for example) Figures 1 to 5 In AAV production, the smallest element unexpectedly leads to significantly higher titers and intact capsid concentrations. This is particularly advantageous when used in gene therapy, where high concentrations of AAV are required for treatment. Furthermore, this simplifies the AAV production process and reduces the need for purification processes that reduce AAV yield.

[0139] In some preferred embodiments, the minimum element is derived from a truncated and / or modified ITR sequence, preferably a truncated and / or modified 3' ITR sequence, more preferably a truncated and / or modified 3' ITR sequence from AAV2, and even more preferably a truncated and / or modified 3' ITR from J01901.1 AAV2.

[0140] The smallest element may include rep-binding elements derived from the ITR sequence, preferably the 3' ITR sequence, and non-natural nucleotide sequences. In this context, it should be understood that the non-natural sequence may not be derived from a sequence immediately adjacent to the rep-binding element sequence of the ITR. For example, the non-natural sequence may be a sequence located further upstream or downstream of the rep-binding element in the ITR.

[0141] In some implementations, the minimum element does not contain or is not composed of a naturally occurring or wild-type AAV ITR sequence. A naturally occurring or wild-type AAV ITR sequence refers to the full-length ITR sequence contained in the wild-type AAV genome. Specifically, a naturally occurring or wild-type AAV ITR sequence refers to any naturally occurring or wild-type full-length and / or self-complementary ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also known as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), and type 11 AAV (e.g., AAV10). Such as AAV11), type 12 AAV (e.g., AAV12), type 13 AAV (e.g., AAV13), type rh32.33 AAV (e.g., AAVrh32.33), type rh8 AAV (e.g., AAVrh8), type rhlO AAV (e.g., AAVrhlO), type rh74 AAV (e.g., AAVrh74), type hu.68 AAV (e.g., AAVhu.68), bird AAV (e.g., AAAV), cow AAV (e.g., BAAV), dog AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV currently known or subsequently discovered.In some embodiments, the minimum element does not contain or is not composed of any naturally occurring or wild-type full-length or self-complementary ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also referred to as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), and type 10 AAV (e.g., AAV10). AAVs of type 11 (e.g., AAV11), type 12 (e.g., AAV12), type 13 (e.g., AAV13), type rh32.33 (e.g., AAVrh32.33), type rh8 (e.g., AAVrh8), type rhlO (e.g., AAVrhlO), type rh74 (e.g., AAVrh74), type hu.68 (e.g., AAVhu.68), bird AAVs (e.g., AAAV), cattle AAVs (e.g., BAAV), dog AAVs, horse AAVs, sheep AAVs, snake AAVs, bearded dragon AAVs, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or subsequently discovered.In some embodiments, the minimum element does not contain or is not composed of any naturally occurring or wild-type full-length and self-complementary ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also referred to as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), and type 10 AAV (e.g., AAV10). AAVs of types 11 (e.g., AAV11), 12 (e.g., AAV12), 13 (e.g., AAV13), rh32.33 (e.g., AAVrh32.33), rh8 (e.g., AAVrh8), rhlO (e.g., AAVrhlO), rh74 (e.g., AAVrh74), hu.68 (e.g., AAVhu.68), avian AAVs (e.g., AAAV), bovine AAVs (e.g., BAAV), canine AAVs, horse AAVs, sheep AAVs, snake AAVs, bearded dragon AAVs, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other currently known or later discovered AAVs. In some embodiments, the minimum element is not composed of naturally occurring or wild-type AAV ITR sequences. In this context, naturally occurring or wild-type AAV ITR sequences refer to the full-length ITR sequences contained in the wild-type AAV genome.

[0142] In specific embodiments, the minimum element is not present in the rep plasmid of the present invention as part of a naturally occurring or wild-type AAV ITR sequence. Typically, the rep plasmid of the present invention does not contain a naturally occurring or wild-type AAV ITR sequence. A naturally occurring or wild-type AAV ITR sequence refers to the full-length ITR sequence contained in the wild-type AAV genome. Specifically, naturally occurring or wild-type AAV... An ITR sequence refers to any naturally occurring or wild-type full-length and / or self-complementary ITR sequence of any AAV serotype, including but not limited to AAV type 1 (e.g., AAV of serotype 1, also known as AAV1), AAV type 2 (e.g., AAV2), AAV type 3 (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), AAV type 4 (e.g., AAV4), AAV type 5 (e.g., AAV5), AAV type 6 (e.g., AAV6), AAV type 7 (e.g., AAV7), AAV type 8 (e.g., AAV8), AAV type 9 (e.g., AAV9), AAV type 10 (e.g., AAV10), and AAV type 11. AV (e.g., AAV11), type 12 AAV (e.g., AAV12), type 13 AAV (e.g., AAV13), type rh32.33 AAV (e.g., AAVrh32.33), type rh8 AAV (e.g., AAVrh8), type rhlO AAV (e.g., AAVrhlO), type rh74 AAV (e.g., AAVrh74), type hu.68 AAV (e.g., AAVhu.68), bird AAV (e.g., AAAV), cow AAV (e.g., BAAV), dog AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV currently known or subsequently discovered.

[0143] In some embodiments, the minimum element is not present in the rep plasmid of the present invention as part of any naturally occurring or wild-type full-length or self-complementary AAV ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also referred to as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), and type 11 AAV (e.g., AAV1). 1) Type 12 AAV (e.g., AAV12), Type 13 AAV (e.g., AAV13), rh32.33 AAV (e.g., AAVrh32.33), rh8 AAV (e.g., AAVrh8), rhlO AAV (e.g., AAVrhlO), rh74 AAV (e.g., AAVrh74), hu.68 AAV (e.g., AAVhu.68), bird AAV (e.g., AAAV), cattle AAV (e.g., BAAV), canine AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other currently known or later discovered AAV.In some embodiments, the minimum element is not present in the rep plasmid of the present invention as part of the naturally occurring or wild-type full-length and self-complementary AAV ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also referred to as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), and type 11 AAV (e.g., AAV1). 1) Type 12 AAV (e.g., AAV12), Type 13 AAV (e.g., AAV13), rh32.33 AAV (e.g., AAVrh32.33), rh8 AAV (e.g., AAVrh8), rhlO AAV (e.g., AAVrhlO), rh74 AAV (e.g., AAVrh74), hu.68 AAV (e.g., AAVhu.68), bird AAV (e.g., AAAV), cattle AAV (e.g., BAAV), canine AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other currently known or later discovered AAV.

[0144] In some embodiments, the rep plasmid of the present invention does not contain a naturally occurring or wild-type AAV ITR sequence. A naturally occurring or wild-type AAV ITR sequence refers to the full-length ITR sequence contained in the wild-type AAV genome. Specifically, a naturally occurring or wild-type AAV ITR sequence refers to any naturally occurring or wild-type full-length and / or self-complementary ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also called AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), and type 11 AAV. AV (e.g., AAV11), type 12 AAV (e.g., AAV12), type 13 AAV (e.g., AAV13), type rh32.33 AAV (e.g., AAVrh32.33), type rh8 AAV (e.g., AAVrh8), type rhlO AAV (e.g., AAVrhlO), type rh74 AAV (e.g., AAVrh74), type hu.68 AAV (e.g., AAVhu.68), bird AAV (e.g., AAAV), cow AAV (e.g., BAAV), dog AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV currently known or subsequently discovered.In some embodiments, the rep plasmid of the present invention does not contain any naturally occurring or wild-type full-length or self-complementary ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also referred to as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), and type 10 AAV (e.g., AAV10). AAVs of type 11 (e.g., AAV11), type 12 (e.g., AAV12), type 13 (e.g., AAV13), type rh32.33 (e.g., AAVrh32.33), type rh8 (e.g., AAVrh8), type rhlO (e.g., AAVrhlO), type rh74 (e.g., AAVrh74), type hu.68 (e.g., AAVhu.68), bird AAVs (e.g., AAAV), cattle AAVs (e.g., BAAV), canine AAVs, horse AAVs, sheep AAVs, snake AAVs, bearded dragon AAVs, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or subsequently discovered.In some embodiments, the rep plasmid of the present invention does not contain any naturally occurring or wild-type full-length and self-complementary ITR sequences of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also referred to as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), and type 10 AAV (e.g., AAV10). AAVs of type 11 (e.g., AAV11), type 12 (e.g., AAV12), type 13 (e.g., AAV13), type rh32.33 (e.g., AAVrh32.33), type rh8 (e.g., AAVrh8), type rhlO (e.g., AAVrhlO), type rh74 (e.g., AAVrh74), type hu.68 (e.g., AAVhu.68), bird AAVs (e.g., AAAV), cattle AAVs (e.g., BAAV), canine AAVs, horse AAVs, sheep AAVs, snake AAVs, bearded dragon AAVs, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or subsequently discovered.

[0145] In some implementations, the minimum element is derived from the ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also known as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), and type 11 AAV (e.g., A... AAV11), AAVs of type 12 (e.g., AAV12), AAVs of type 13 (e.g., AAV13), AAVs of type rh32.33 (e.g., AAVrh32.33), AAVs of type rh8 (e.g., AAVrh8), AAVs of type rhlO (e.g., AAVrhlO), AAVs of type rh74 (e.g., AAVrh74), AAVs of type hu.68 (e.g., AAVhu.68), AAVs of birds (e.g., AAAV), AAVs of cattle (e.g., BAAV), AAVs of dogs, AAVs of horses, AAVs of sheep, AAVs of snakes, AAVs of bearded dragons, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or to be discovered thereafter.

[0146] In some embodiments, the minimal element allows one or more rep proteins to bind to the polynucleotide sequence. In some embodiments, one or more rep-binding elements contained within the minimal element allow one or more rep proteins to bind to the polynucleotide sequence. The minimal element preferably allows for the initiation of genome replication. The replicated sequence can further initiate subsequent rounds of genome replication.

[0147] The minimum element may contain a nucleotide sequence that is not naturally present in AAV or a sequence from which it is derived. The non-natural nucleotide sequence may be upstream and / or downstream of the rep-binding element. In some embodiments, the non-natural nucleotide sequence may be upstream, downstream, and / or within the rep-binding element.

[0148] In some embodiments, the minimum element comprises one rep-binding element. In some embodiments, the minimum element comprises two or more rep-binding elements. In some embodiments, the minimum element consists of one rep-binding element. In some embodiments, the minimum element consists of two or more rep-binding elements. If the minimum element comprises two or more rep-binding elements, these rep-binding elements may be derived from different AAV serotypes. For example, one rep-binding element may be derived from AAV2, and one rep-binding element may be derived from AAV3. In some embodiments, the minimum element includes a first rep-binding element derived from an ITR sequence and a second rep-binding element derived from a non-ITR sequence. In some implementations, the minimum element includes a first rep binding element and a second rep binding element, wherein the first rep binding element is derived from a 3' ITR sequence, preferably from a 3' ITR sequence of AAV2, more preferably from a 3' ITR of J01901.1 AAV2, and the second rep binding element is derived from an AAV promoter sequence, preferably a p5 promoter sequence, more preferably from a p5 promoter sequence of AAV2, and even more preferably from a p5 promoter sequence of J01901.1 AAV2.

[0149] In some embodiments, the minimum element is derived from a non-ITR sequence, such as an AAV promoter sequence. In some embodiments, the minimum element is derived from an AAV promoter sequence, preferably a p5 promoter sequence, more preferably a p5 promoter sequence from AAV2, and even more preferably a p5 promoter sequence from J01901.1 AAV2. In some embodiments, the minimum element comprises or is composed of a non-ITR sequence (e.g., an AAV promoter sequence). In some embodiments, the minimum element comprises or is composed of an AAV promoter sequence, preferably a p5 promoter sequence, more preferably a p5 promoter sequence from AAV2, and even more preferably a p5 promoter sequence from J01901.1 AAV2, or is derived from the aforementioned sequences. In some embodiments, the minimum element includes SEQ ID NO:23 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, most preferably a sequence having at least 99% identity with the SEQ ID NO, or is composed of the above sequences.

[0150] In some embodiments, the minimum element is derived from a truncated and / or modified AAV promoter sequence, preferably a truncated and / or modified p5 promoter sequence, more preferably a truncated and / or modified p5 promoter sequence from AAV2, and even more preferably a truncated and / or modified p5 promoter sequence from J01901.1 AAV2. In some embodiments, the rep-binding element is derived from the p5 promoter. The minimum element derived from a non-ITR may comprise a rep-binding element derived from an AAV promoter sequence (preferably a p5 promoter sequence) and a non-natural nucleotide sequence. In this context, it should be understood that the non-natural sequence may not be derived from a sequence immediately adjacent to the rep-binding element sequence of the p5 promoter. For example, the non-natural sequence may be a sequence located further upstream or downstream of the rep-binding element in the p5 promoter.

[0151] In some implementations, the minimum element is derived from the promoter sequence of any AAV serotype, preferably the p5 promoter sequence, wherein the AAV serotypes include, but are not limited to, type 1 AAV (e.g., AAV of serotype 1, also known as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), and type 11 AA. V (e.g., AAV11), AAV type 12 (e.g., AAV12), AAV type 13 (e.g., AAV13), AAV type rh32.33 (e.g., AAVrh32.33), AAV type rh8 (e.g., AAVrh8), AAV type rhlO (e.g., AAVrhlO), AAV type rh74 (e.g., AAVrh74), AAV type hu.68 (e.g., AAVhu.68), AAV of birds (e.g., AAAV), AAV of cattle (e.g., BAAV), AAV of dogs, AAV of horses, AAV of sheep, AAV of snakes, AAV of bearded dragons, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV currently known or subsequently discovered.

[0152] In some embodiments, the minimum element is derived from an ITR sequence and includes a rep-binding element derived from a non-ITR sequence. For example, the minimum element includes a non-natural nucleotide sequence derived from an ITR sequence and a rep-binding element derived from an AAV promoter sequence. In some embodiments, the minimum element comprises a non-natural nucleotide sequence derived from an ITR sequence, preferably from a 3' ITR sequence, more preferably from a 3' ITR sequence from AAV2, and even more preferably from a 3' ITR sequence from J01901.1 AAV2, and the rep-binding element is derived from a p5 promoter, preferably from a p5 promoter from AAV2, and more preferably from a p5 promoter from J01901.1 AAV2.

[0153] In some embodiments, the minimum element comprises nucleotide sequences from different AAV serotypes, such as rep-binding elements and non-natural nucleotide sequences, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also known as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), and type 10 AAV (e.g., AAV10). Types of AAVs include: AAV11 (e.g., AAV11), AAV12 (e.g., AAV12), AAV13 (e.g., AAV13), AAVs of rh32.33 (e.g., AAVrh32.33), AAVs of rh8 (e.g., AAVrh8), AAVs of rhlO (e.g., AAVrhlO), AAVs of rh74 (e.g., AAVrh74), AAVs of hu.68 (e.g., AAVhu.68), avian AAVs (e.g., AAAV), bovine AAVs (e.g., BAAV), canine AAVs, equine AAVs, sheep AAVs, snake AAVs, bearded dragon AAVs, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other currently known or hereafter discovered AAVs. For example, the minimum element may include a rep-binding element derived from the AAV2 serotype and a non-natural nucleotide sequence from any AAV serotype other than AAV2. In some embodiments, the minimum element comprises a rep-binding element derived from an ITR sequence (preferably derived from a 3' ITR sequence) and a non-natural nucleotide sequence derived from any serotype other than AAV2 (e.g., AAV3). In some embodiments, the minimum element comprises a rep-binding element derived from a non-ITR sequence (preferably derived from an AAV promoter sequence, more preferably derived from a p5 promoter sequence) and a non-natural nucleotide sequence derived from any serotype other than AAV2 (e.g., AAV3).

[0154] In some preferred embodiments, one or more rep binding elements act as binding sites for rep68 and / or rep78. In some preferred embodiments, one or more rep binding elements act as binding sites for either rep68 or rep78. In some embodiments, one or more rep binding elements act as binding sites for both rep68 and rep78. In some embodiments, one or more rep binding elements act as binding sites for rep68. In some embodiments, one or more rep binding elements act as binding sites for rep78.

[0155] In some embodiments, the minimum element comprises at least two rep binding elements. In some embodiments, the minimum element comprises at least three rep binding elements. In some embodiments, the minimum element comprises at least four rep binding elements. In some embodiments, the minimum element comprises at least five rep binding elements. In some embodiments, the minimum element comprises at least six rep binding elements. In some embodiments, the minimum element comprises at most two rep binding elements. In some embodiments, the minimum element comprises at most three rep binding elements. In some embodiments, the minimum element comprises at most four rep binding elements. In some embodiments, the minimum element comprises at most five rep binding elements. In some embodiments, the minimum element comprises at most six rep binding elements.

[0156] In some implementations, the rep plasmid includes an additional minimal element containing one or more rep-binding elements. This additional minimal element may be located downstream or upstream of the polyadenylation signal.

[0157] In some embodiments, at least one polyadenylation signal sequence contained in the rep plasmid of the present invention and located downstream of the adeno-associated virus (AAV) replication protein coding sequence is a natural polyadenylation signal sequence, i.e., a polyadenylation sequence contained in the wild-type AAV genome. In some embodiments, at least one polyadenylation signal sequence downstream of the AAV replication protein coding sequence is a heterologous polyadenylation signal sequence, i.e., a polyadenylation signal sequence not contained in the wild-type AAV genome. In some embodiments, the rep plasmid contains a natural polyadenylation signal sequence downstream of the AAV replication protein coding sequence. In some embodiments, the rep plasmid contains a heterologous polyadenylation signal sequence downstream of the AAV replication protein coding sequence.

[0158] In some embodiments, the native or heterologous polyadenylation signal sequence may be a sequence comprising AATAAA or a modified sequence thereof. The modified AATAAA sequence may be a sequence in which one or two nucleic acids are deleted, substituted, inserted, and / or added. Other polyadenylation signal sequences are known, such as ATTAAA, AGTAAA, TATAAA, CATAAA, GATAAA, AATTA, AATCA, AATGA, AAAAAG, and ACTAAA, and may be used in the context of this disclosure. For clarity, the plasmids disclosed herein may contain sequences (or elements) that can control or influence polyadenylation, such as U / GU-rich sequences, which may be located upstream or downstream of the AATAAA or modified sequence, for example, separated by several (random) nucleotides (5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 2000, or more nucleotides).

[0159] In some embodiments, the natural or heterologous polyadenylation signal sequence used in the context of this disclosure is a strong polyadenylation signal sequence. In some preferred embodiments, the heterologous polyadenylation signal sequence used in the context of this disclosure is a strong polyadenylation signal sequence. In some embodiments, the natural polyadenylation signal sequence used in the context of this disclosure is a strong polyadenylation signal sequence. Without being bound by theory, it is anticipated that a strong polyadenylation signal improves nuclear output of rep protein mRNA, stabilizes mRNA by protecting it from enzymatic degradation, and / or increases rep protein mRNA translation, thereby improving rAAV vector production, for example, by providing increased concentrations of viral genome and an intact capsid. The strength of a given polyadenylation signal sequence as described herein refers to the ability of the polyadenylation signal sequence to terminate transcription and initiate polyadenylation at the 3' end of the mRNA. The strength of a polyadenylation signal sequence can be determined based on the degree of polyadenylation of a given mRNA. Therefore, those skilled in the art can readily determine the intensity of polyadenylation signal sequences by using methods such as PCR-based methods (e.g., digital PCR, real-time quantitative PCR, RNA-seq), Northern blotting, and microarray methods to determine the level of mRNA polyadenylation. For example, the intensity of polyadenylation signal sequences can be determined by methods described in the following literature: Hoque et al., “Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing,” Nature Methods, Vol. 10, pp. 133–139 (2013), doi: 10.1038 / nmeth.2288 and Proudfoot, “Ending the message: poly(A) signals then and now,” Genes Dev. 2011 Sep 1; 25(17): 1770–1782, doi:10.1101 / gad.17268411.

[0160] Non-limiting examples of heterologous strong polyadenylation signal sequences are SV40 and rabbit β-globin poly(A) signal sequences.

[0161] In some embodiments, the heteropolyadenylation signal sequence is capable of forming a suitable poly(A) sequence at the 3' end of the RNA. Examples of heteropolyadenylation signal sequences are, but are not limited to, the adenovirus L3 poly(A) signal sequence, the HSV TK poly(A) signal sequence, the hGH poly(A) signal sequence, the spA poly(A) signal sequence, the rabbit gbpA poly(A) signal sequence, the sNRP1 poly(A) signal sequence, the bGH poly(A) signal sequence, synthetic poly(A) signal sequences, mouse β-globin poly(A) signal sequences, rabbit β-globin poly(A) signal sequences, H4-based poly(A) signal sequences, and the SV40 poly(A) signal sequence. In some preferred embodiments, the strong heteropolyadenylation signal sequence is the rabbit β-globin poly(A) signal sequence. In some preferred embodiments, the strong heteropolyadenylation signal sequence comprises SEQ ID NO:21 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with said SEQ ID NO, or is composed of the above sequences.

[0162] In some embodiments, the natural polyadenylation signal sequence comprises SEQ ID NO: 26 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with said SEQ ID NO, or is composed of the above sequences.

[0163] In some embodiments, the rep plasmid includes more than one polyadenylation signal sequence downstream of the adenosine-associated virus (AAV) replication protein coding sequence. In some embodiments, the rep plasmid includes at least one, at least two, at least three, at least four, or at least five polyadenylation signal sequences downstream of the AAV replication protein coding sequence. In some embodiments, the rep plasmid includes two, three, four, or five polyadenylation signal sequences downstream of the AAV replication protein coding sequence. In some embodiments, the rep plasmid includes two polyadenylation signal sequences downstream of the AAV replication protein coding sequence.

[0164] In some embodiments, at least one, at least two, at least three, at least four, or at least five polyadenylation signal sequences downstream of the adeno-associated virus replication protein coding sequence may be selected from natural or heterologous polyadenylation signal sequences. In some embodiments, at least one, at least two, at least three, at least four, or at least five polyadenylation signal sequences downstream of the adeno-associated virus replication protein coding sequence may be selected from strong natural or strong heterologous polyadenylation signal sequences.

[0165] In some embodiments, the rep plasmid includes a first polyadenylation signal sequence downstream of the adenosine-associated virus (AAV) replication protein coding sequence and a second polyadenylation signal sequence downstream of the first polyadenylation signal sequence. In some embodiments, the rep plasmid includes a first native polyadenylation signal sequence downstream of the AAV replication protein coding sequence and a second heterologous polyadenylation signal sequence downstream of the first native polyadenylation signal sequence.

[0166] In some embodiments, the rep plasmid comprises a first polyadenylation signal sequence downstream of the adenosine-associated virus (AAV) replication protein coding sequence, a minimal element containing a rep-binding element downstream of the first polyadenylation signal sequence, and a second polyadenylation signal sequence downstream of the minimal element. In some embodiments, the rep plasmid comprises a first native polyadenylation signal sequence downstream of the AAV replication protein coding sequence, a minimal element containing a rep-binding element downstream of the first native polyadenylation signal sequence, and a second heterologous polyadenylation signal sequence downstream of the minimal element. In some embodiments, the rep plasmid comprises a first native polyadenylation signal sequence downstream of the AAV replication protein coding sequence, a minimal element containing a rep-binding element downstream of the first native polyadenylation signal sequence, and a second strong heterologous polyadenylation signal sequence downstream of the minimal element.

[0167] In some implementations, the rep plasmid includes a first natural polyadenylation signal sequence downstream of the adeno-associated virus replication protein coding sequence, a minimum element containing a rep binding element downstream of the first natural polyadenylation signal sequence, and a second rabbit β-globin poly(A) signal sequence downstream of the minimum element.

[0168] In some embodiments, the rep plasmid comprises: a first natural polyadenylation signal sequence downstream of an adeno-associated virus replication protein coding sequence, the first natural polyadenylation signal sequence comprising or consisting of the sequence SEQ ID NO: 26 or a sequence having at least 60%, 70%, 80%, 90%, 95%, or 99% identity with the SEQ ID NO; a minimal element comprising a rep-binding element downstream of the first natural polyadenylation signal sequence; and a second strong heteropolyadenylation signal sequence downstream of the minimal element, the second strong heteropolyadenylation signal sequence comprising or consisting of the sequence SEQ ID NO: 21 or a sequence having at least 60%, 70%, 80%, 90%, 95%, or 99% identity with the SEQ ID NO.

[0169] In some embodiments, the rep plasmid comprises: a first natural polyadenylation signal sequence downstream of an adeno-associated virus replication protein coding sequence, the first natural polyadenylation signal sequence comprising or consisting of a sequence SEQ ID NO: 21 or a sequence having at least 60%, 70%, 80%, 90%, 95%, or 99% identity with said SEQ ID NO; a minimum element downstream of the first natural polyadenylation signal sequence comprising a rep-binding element, wherein the rep-binding element comprises or consists of a sequence SEQ ID NO: 20 or a sequence having at least 60%, 70%, 80%, 90%, 95%, or 99% identity with said SEQ ID NO; and a second strong heteropolyadenylation signal sequence downstream of the minimum element, the second strong heteropolyadenylation signal sequence comprising or consisting of a sequence SEQ ID NO: 21 or a sequence having at least 60%, 70%, 80%, 90%, 95%, or 99% identity with said SEQ ID NO.

[0170] In some embodiments, the rep plasmid comprises: a first natural polyadenylation signal sequence downstream of an adeno-associated virus replication protein coding sequence, the first natural polyadenylation signal sequence comprising SEQ ID NO: 21 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with said SEQ ID NO, or composed of the above sequences; a minimum element downstream of the first natural polyadenylation signal sequence, wherein the minimum element comprises SEQ ID NO: 23 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with said SEQ ID NO; and a second strong heteropolyadenylation signal sequence downstream of the minimum element, the second strong heteropolyadenylation signal sequence comprising SEQ ID NO: 21 or a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with said SEQ ID NO, or composed of the above sequences.

[0171] In some embodiments, the rep plasmid comprises at least one adeno-associated virus capsid protein coding sequence encoding at least one functional cap protein. In some embodiments, the capsid protein coding sequence encodes VP1, VP2, and VP3, or one or more artificial variants thereof. In some embodiments, the rep plasmid does not contain a capsid protein coding sequence. In such embodiments, the rep plasmid may therefore omit the p40 promoter, as this promoter is typically used for cap transcription.

[0172] In a preferred embodiment, the VP1 protein comprises SEQ ID NO: 12 or a sequence having at least 80% identity with SEQ ID NO, preferably a sequence having at least 85% identity with SEQ ID NO, more preferably a sequence having at least 90% identity with SEQ ID NO, even more preferably a sequence having at least 95% identity with SEQ ID NO, most preferably a sequence having at least 99% identity with SEQ ID NO, or is composed of the above sequences.

[0173] In a preferred embodiment, the VP2 protein comprises SEQ ID NO: 13 or a sequence having at least 80% identity with SEQ ID NO, preferably a sequence having at least 85% identity with SEQ ID NO, more preferably a sequence having at least 90% identity with SEQ ID NO, even more preferably a sequence having at least 95% identity with SEQ ID NO, most preferably a sequence having at least 99% identity with SEQ ID NO, or is composed of the above sequences.

[0174] In a preferred embodiment, the VP3 protein comprises SEQ ID NO: 14 or a sequence having at least 80% identity with SEQ ID NO, preferably a sequence having at least 85% identity with SEQ ID NO, more preferably a sequence having at least 90% identity with SEQ ID NO, even more preferably a sequence having at least 95% identity with SEQ ID NO, most preferably a sequence having at least 99% identity with SEQ ID NO, or is composed of the above sequences.

[0175] In another preferred embodiment, the capsid protein (cap) coding sequence is derived from AAV2. In some embodiments, the capsid protein coding sequence is derived from an AAV serotype selected from the group consisting of: AAV type 1 (e.g., AAV of serotype 1, also known as AAV1), AAV type 2 (e.g., AAV2), AAV type 3 (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), AAV type 4 (e.g., AAV4), AAV type 5 (e.g., AAV5), AAV type 6 (e.g., AAV6), AAV type 7 (e.g., AAV7), AAV type 8 (e.g., AAV8), AAV type 9 (e.g., AAV9), AAV type 10 (e.g., AAV10), and AAV type 11 (e.g., AAV10). AAV11), AAVs of type 12 (e.g., AAV12), AAVs of type 13 (e.g., AAV13), AAVs of type rh32.33 (e.g., AAVrh32.33), AAVs of type rh8 (e.g., AAVrh8), AAVs of type rhlO (e.g., AAVrhlO), AAVs of type rh74 (e.g., AAVrh74), AAVs of type hu.68 (e.g., AAVhu.68), AAVs of birds (e.g., AAAV), AAVs of cattle (e.g., BAAV), AAVs of dogs, AAVs of horses, AAVs of sheep, AAVs of snakes, AAVs of bearded dragons, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or to be discovered thereafter.

[0176] In some implementations, the rep and / or cap coding sequences may be derived from AAV2, AAV9, and AAV5 serotypes.

[0177] In some implementations, the rep-binding element is derived from any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also known as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), and type 11 AAV (e.g., AAV11). Type 12 AAV (e.g., AAV12), Type 13 AAV (e.g., AAV13), rh32.33 AAV (e.g., AAVrh32.33), rh8 AAV (e.g., AAVrh8), rhlO AAV (e.g., AAVrhlO), rh74 AAV (e.g., AAVrh74), hu.68 AAV (e.g., AAVhu.68), bird AAV (e.g., AAAV), cattle AAV (e.g., BAAV), canine AAV, horse AAV, sheep AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAV currently known or subsequently discovered.

[0178] In some embodiments, the minimum element comprises one or more rep-binding elements. In some embodiments, the rep-binding element comprises or consists of sequences selected from or composed of sequences of: SEQ ID NO: 18, 20 and / or 33-41 or sequences having at least 80% identity with one of the SEQ ID NOs, preferably sequences having at least 85% identity with one of the SEQ ID NOs, more preferably sequences having at least 90% identity with one of the SEQ ID NOs, even more preferably sequences having at least 95% identity with one of the SEQ ID NOs, and most preferably sequences having at least 99% identity with one of the SEQ ID NOs.

[0179] In a preferred embodiment, the rep binding element comprises SEQ ID NO: 18 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In a preferred embodiment, the rep binding element consists of the following sequence: SEQ ID NO: 18 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0180] In some embodiments, the rep binding element comprises SEQ ID NO: 33 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 33 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0181] In some embodiments, the rep binding element comprises SEQ ID NO: 35 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 35 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0182] In some embodiments, the rep binding element comprises SEQ ID NO: 36 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 36 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0183] In some embodiments, the rep binding element comprises SEQ ID NO: 37 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 37 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0184] In some embodiments, the rep binding element comprises SEQ ID NO: 40 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 40 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0185] In a preferred embodiment, the rep binding element is derived from a terminal inverted repeat sequence, preferably from a 3' terminal inverted repeat sequence derived from J01901.1 AAV2.

[0186] In some embodiments, the rep binding element comprises SEQ ID NO: 20 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 20 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0187] In some embodiments, the rep binding element comprises SEQ ID NO: 34 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 34 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0188] In some embodiments, the rep binding element comprises SEQ ID NO: 38 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 38 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0189] In some embodiments, the rep binding element comprises SEQ ID NO: 39 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 39 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0190] In some embodiments, the rep binding element comprises SEQ ID NO: 41 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In some embodiments, the rep binding element consists of the following sequence: SEQ ID NO: 41 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0191] In some implementations, the rep binding element is derived from the AAV promoter sequence, preferably from the p5 promoter sequence derived from J01901.1AAV2.

[0192] In a preferred embodiment, the replication protein (rep) coding sequence is derived from AAV2. In some embodiments, the replication protein coding sequence is derived from an AAV serotype selected from the group consisting of: AAV type 1 (e.g., AAV of serotype 1, also known as AAV1), AAV type 2 (e.g., AAV2), AAV type 3 (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), AAV type 4 (e.g., AAV4), AAV type 5 (e.g., AAV5), AAV type 6 (e.g., AAV6), AAV type 7 (e.g., AAV7), AAV type 8 (e.g., AAV8), AAV type 9 (e.g., AAV9), AAV type 10 (e.g., AAV10), and AAV type 11 (e.g., AAV10). AAV11), AAVs of type 12 (e.g., AAV12), AAVs of type 13 (e.g., AAV13), AAVs of type rh32.33 (e.g., AAVrh32.33), AAVs of type rh8 (e.g., AAVrh8), AAVs of type rhlO (e.g., AAVrhlO), AAVs of type rh74 (e.g., AAVrh74), AAVs of type hu.68 (e.g., AAVhu.68), AAVs of birds (e.g., AAAV), AAVs of cattle (e.g., BAAV), AAVs of dogs, AAVs of horses, AAVs of sheep, AAVs of snakes, AAVs of bearded dragons, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other AAVs currently known or to be discovered thereafter.

[0193] In a preferred embodiment, the sequence length of the smallest element is 500 nt or less, 450 nt or less, 300 nt or less, 250 nt or less, 200 nt or less, preferably 150 nt or less or 100 nt or less.

[0194] In a preferred embodiment, the minimum element comprises SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, or at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO. In a preferred embodiment, the minimum element consists of the following sequence: SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, or at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0195] In a preferred embodiment, the replication protein coding sequence encodes rep78, rep68, rep52, and rep40, or one or more of their artificial variants. The coding sequences for rep78, rep68, rep52, and / or rep40 may be located within the same open reading frame. The rep protein is typically translated from transcripts produced by the p5 and p19 promoters.

[0196] In a preferred embodiment, the rep78 protein comprises or consists of the following sequence: SEQ ID NO: 8 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0197] In a preferred embodiment, the rep68 protein comprises or consists of the following sequence: SEQ ID NO: 9 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0198] In a preferred embodiment, the rep52 protein comprises or consists of the following sequence: SEQ ID NO:10 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0199] In a preferred embodiment, the rep40 protein comprises or consists of the following sequence: SEQ ID NO:11 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0200] In a preferred embodiment, the rep plasmid further comprises one or more natural or heterologous promoters operatively linked to the coding sequence of the replication protein, preferably wherein the promoters are selected from p5, p19 and p40.

[0201] In some embodiments, the rep plasmid may contain an AAV promoter selected from p5, p19, and / or p40. The promoter may be derived from a serotype different from the replication protein coding sequence. The promoter may be derived from a serotype different from the capsid protein coding sequence. In some embodiments, the rep plasmid may contain a non-natural promoter, meaning a promoter not derived from AAV. In some embodiments, the rep plasmid contains a mouse mammary tumor virus (MMTV) promoter. In some embodiments, the MMTV promoter is operatively linked to the replication protein coding sequence. In some embodiments, the MMTV promoter is operatively linked to the capsid protein coding sequence.

[0202] In a preferred embodiment, the p5 promoter comprises or consists of the following sequence: SEQ ID NO: 23 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0203] In a preferred embodiment, the p19 promoter comprises or consists of the following sequence: SEQ ID NO:24 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0204] In a preferred embodiment, the p40 promoter comprises or consists of the following sequence: SEQ ID NO:25 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0205] In some embodiments, the MMTV promoter comprises or consists of the following sequence: SEQ ID NO: 32 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0206] In some embodiments, the rep plasmid contains at least one accessory protein coding sequence encoding at least one accessory protein. Accessory proteins of adeno-associated viruses are known in the art and may be selected from AAP (assembly-activation protein), MAAP (membrane-associated accessory protein), and X, or variants thereof, or any combination thereof.

[0207] In a preferred embodiment, the AAP protein comprises or is composed of the following sequence: SEQ ID NO: 15 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0208] In a preferred embodiment, the X protein comprises or is composed of the following sequence: SEQ ID NO: 16 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0209] In a preferred embodiment, the MAAP protein comprises or consists of the following sequence: SEQ ID NO:22 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0210] In some embodiments, the replication protein-coding sequence is capable of encoding one or more heterozygous rep proteins from different AAV serotypes. In a preferred embodiment, the replication protein-coding sequence is derived from AAV2. In a preferred embodiment, the replication protein-coding sequence is derived from AAV2, and the promoter operatively linked to the replication protein-coding sequence is the MMTV promoter.

[0211] In some implementations, the rep plasmid may include helper genes. These helper genes may be selected from Ela, Elb, E2A, E4orf6, E4orf7, or VA RNA, or any combination thereof.

[0212] In some implementations, the rep plasmid is used for rAAV production, where rAAV can be used for gene therapy applications. In some implementations, rAAV production is transient.

[0213] The rep plasmid may contain one or more 5' and / or 3' UTRs located upstream and / or downstream of the replication protein coding sequence. In some embodiments, the rep plasmid further includes regulatory sequences, which include promoters, binding sites, and / or non-coding RNAs. These sequences may be independently derived from AAV, including different AAV serotypes.

[0214] In a preferred embodiment, the rep plasmid comprises SEQ ID NO: 7 or a sequence having at least 80% identity with SEQ ID NO, preferably a sequence having at least 85% identity with SEQ ID NO, more preferably a sequence having at least 90% identity with SEQ ID NO, even more preferably a sequence having at least 95% identity with SEQ ID NO, and most preferably a sequence having at least 99% identity with SEQ ID NO. In a preferred embodiment, the rep plasmid consists of the following sequence: SEQ ID NO: 7 or a sequence having at least 80% identity with SEQ ID NO, preferably a sequence having at least 85% identity with SEQ ID NO, more preferably a sequence having at least 90% identity with SEQ ID NO, even more preferably a sequence having at least 95% identity with SEQ ID NO, and most preferably a sequence having at least 99% identity with SEQ ID NO.

[0215] In a preferred embodiment, the rep plasmid comprises a sequence of SEQ ID NO: 30 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In a preferred embodiment, the rep plasmid consists of the following sequence: SEQ ID NO: 30 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0216] In a preferred embodiment, the rep plasmid comprises a sequence of SEQ ID NO: 31 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO. In a preferred embodiment, the rep plasmid consists of the following sequence: SEQ ID NO: 31 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0217] The plasmid system of the present invention

[0218] This invention provides a plasmid system for producing adeno-associated virus particles, comprising: (i) transgenic plasmids; and (ii) The rep plasmid of the present invention.

[0219] like Figures 1 to 6 As shown in Figures 10 to 12, the plasmid system of the present invention unexpectedly increases the concentration of viral genome and intact capsid, due to the fact that the rep plasmid includes a minimal element containing a recombinant rep binding element located downstream of the polyadenylation signal sequence.

[0220] In a preferred embodiment, the plasmid system further comprises a helper plasmid. Typically, AAV may be replication-defective and may require co-infection with adenovirus or herpesvirus for efficient replication. Single adenovirus genes that contribute to AAV helper function are well known and have been identified, in particular, by testing the ability of adenovirus mutants to mediate AAV replication. In a further preferred embodiment, the helper plasmid comprises one or more coding sequences encoding Ela, Elb, E2A, E4orf6, E4orf7, or VARNA, or any combination thereof. These genes (i.e., helper genes) are known to support or participate in AAV replication.

[0221] In a preferred embodiment, the helper plasmid contains one or more virus-associated RNAs. Adenovirus 'virus-associated' RNA (VA RNA) is considered to be an abundant, heterologous, non-coding RNA transcript, typically containing 150-200 nucleotides. For example, VA RNAI is recognized for its function in alleviating the blockade of protein synthesis by cellular antiviral activity through inhibition of double-stranded RNA-activated protein kinase (PKR). Recent evidence shows that VA RNA interferes with several other host cell processes.

[0222] In a preferred embodiment, the transgenic plasmid comprises a promoter, a transgene, a polyadenylation signal sequence, and 5' and 3' terminal inverted repeat sequences, wherein the transgenic plasmid is selected from one of the following: (i) Conventional single-stranded recombinant adeno-associated virus, or (ii) Self-complementary genomic recombinant adeno-associated virus.

[0223] In some embodiments, the transgenic plasmid is a self-complementary AAV (scAAV) plasmid. Because conventional ssAAV viruses rely on DNA replication mechanisms to synthesize complementary DNA strands, transgenic expression can be delayed. To overcome this rate-limiting step, scAAV contains a complementary sequence that spontaneously anneals after infection, thus eliminating the need for host cell DNA synthesis. Methods for generating scAAV plasmids are well known to those skilled in the art. In some embodiments, the scAAV plasmid contains approximately 2.4 kb or less of transgene.

[0224] In some embodiments, the plasmid system may contain two or more transgenic plasmids. The first transgenic plasmid may contain a 3' splice donor, while the second transgenic plasmid may contain a 5' splice acceptor. When the two plasmids are expressed in cells, they can form a tandem and be spliced ​​together, thereby expressing the full-length transgene. In other embodiments, the transgene is dissociated into two transgenic plasmids, but with substantial sequence overlap. Co-expression can induce homologous recombination and expression of the full-length transgene.

[0225] In some implementations, the 3' and 5' terminal inverted repeat sequences are independently derived from the ITR sequence of any AAV serotype, including but not limited to type 1 AAV (e.g., AAV of serotype 1, also known as AAV1), type 2 AAV (e.g., AAV2), type 3 AAV (e.g., AAV3, including types 3A and 3B, i.e., AAV3A and AAV3B), type 4 AAV (e.g., AAV4), type 5 AAV (e.g., AAV5), type 6 AAV (e.g., AAV6), type 7 AAV (e.g., AAV7), type 8 AAV (e.g., AAV8), type 9 AAV (e.g., AAV9), type 10 AAV (e.g., AAV10), and type 11 AAV. AVs (e.g., AAV11), type 12 AAVs (e.g., AAV12), type 13 AAVs (e.g., AAV13), rh32.33 type AAVs (e.g., AAVrh32.33), rh8 type AAVs (e.g., AAVrh8), rhlO type AAVs (e.g., AAVrhlO), rh74 type AAVs (e.g., AAVrh74), hu.68 type AAVs (e.g., AAVhu.68), avian AAVs (e.g., AAAV), bovine AAVs (e.g., BAAV), canine AAVs, horse AAVs, sheep AAVs, snake AAVs, bearded dragon AAVs, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B, and any other currently known or subsequently discovered AAVs. In a preferred embodiment, the 3' and 5' terminal inverted repeat sequences are derived from J01901.1 AAV2.

[0226] In some embodiments, the terminal inverted repeat sequence, preferably the 5' terminal inverted repeat sequence, is deleted compared to the natural terminal inverted repeat sequence. In some embodiments, the transgenic plasmid containing the 5' ITR deletion is the scAAV plasmid.

[0227] In some implementations, the transgenic plasmid contains a cap protein coding sequence.

[0228] The transgenic plasmid can contain any suitable promoter. Exemplary suitable promoters may be the chicken β-actin (CBA) promoter, the short CMV early enhancer / chicken β-actin (sCAG) promoter, the human cytomegalovirus (hCMV) promoter, the mouse phosphoglycerate kinase (mPGK) promoter, and the human synaptic protein (hSYN) promoter. In a preferred embodiment, the promoter is the CMVie promoter.

[0229] In a preferred embodiment, the transgene is a reporter gene. In a preferred embodiment, the reporter gene can be detected by an antibody-based assay. In a further preferred embodiment, the reporter gene is a fluorescent molecule. Exemplary fluorescent molecules suitable as reporter genes are GFP, eGFP, mGFP, eYFP, citrine, eCFP, mCFP, Cerulean, dtTomato, and any variants thereof. In some embodiments, the reporter gene is β-galactosidase, luciferase, or glutathione S-transferase, or any variant thereof.

[0230] In some implementations, the genetic material is up to 4.4 kb. In other implementations, the genetic material is up to 2.4 kb.

[0231] In some embodiments, the polyadenylation signal sequence is capable of forming a suitable poly(A) sequence at the 3' end of the RNA. Exemplary polyadenylation signal sequences are adenovirus L3 poly(A) signal sequences, HSV TK poly(A) signal sequences, hGH poly(A) signal sequences, spA poly(A) signal sequences, rabbit gbpA poly(A) signal sequences, sNRP1 poly(A) signal sequences, bGH poly(A) signal sequences, synthetic poly(A) signal sequences, mouse β-globin poly(A) signal sequences, rabbit β-globin poly(A) signal sequences, H4-based poly(A) signal sequences, and SV40 poly(A) signal sequences. In a preferred embodiment, the polyadenylation signal sequence is the SV40 poly(A) signal sequence.

[0232] In a preferred embodiment, the transgenic plasmid comprises the following structure: 5' ITR (inverted)-CMVie promoter-transgenic-SV40 PolyA-ITR (forward and reverse) 3'.

[0233] In a preferred embodiment, the terminal inverted repeat sequence with a flipped orientation has SEQ ID NO: 1 or a sequence that is at least 80% identical to the SEQ ID NO, preferably a sequence that is at least 85% identical to the SEQ ID NO, more preferably a sequence that is at least 90% identical to the SEQ ID NO, even more preferably a sequence that is at least 95% identical to the SEQ ID NO, and most preferably a sequence that is at least 99% identical to the SEQ ID NO.

[0234] In a preferred embodiment, the terminal inverted repeat sequence with forward and reverse orientation has SEQ ID NO: 2 or a sequence that is at least 80% identical to the SEQ ID NO, preferably a sequence that is at least 85% identical to the SEQ ID NO, more preferably a sequence that is at least 90% identical to the SEQ ID NO, even more preferably a sequence that is at least 95% identical to the SEQ ID NO, and most preferably a sequence that is at least 99% identical to the SEQ ID NO.

[0235] In some implementations, the transgenic plasmid does not contain post-transcriptional regulatory elements of marmot hepatitis virus.

[0236] In some embodiments, the plasmid system comprises the rep plasmid of the present invention, which does not contain a cap protein coding sequence. In some such embodiments, the plasmid system comprises a cap plasmid. The cap plasmid may contain at least one adeno-associated virus capsid protein coding sequence encoding at least one functional cap protein. The capsid protein coding sequence may encode one or more of VP1, VP2, and VP3 or their artificial variants. In a preferred embodiment, the capsid protein coding sequence is derived from AAV2.

[0237] In a preferred embodiment, the VP1 protein comprises or consists of the following sequence: SEQ ID NO: 12 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0238] In a preferred embodiment, the VP2 protein comprises or is composed of the following sequence: SEQ ID NO: 13 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0239] In a preferred embodiment, the VP3 protein comprises or consists of the following sequence: SEQ ID NO: 14 or a sequence having at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

[0240] In a particularly preferred embodiment, the plasmid system comprises the transgenic plasmids of the present invention. In some embodiments, the transgenic plasmid is an scAAV transgenic plasmid. In some embodiments, the transgenic plasmid is an ssAAV transgenic plasmid. In some embodiments, the plasmid system may comprise two or more transgenic plasmids of the present invention.

[0241] In some implementations, the plasmid system is used for rAAV production, preferably transient rAAV production, where rAAV can be used for gene therapy applications.

[0242] The transgenic plasmid of the present invention

[0243] The present invention also provides a transgenic plasmid comprising: a CMVie promoter, a transgene, SV40 PolyA, and 5' and 3' terminal inverted repeat sequences derived from J01901.1 AAV2; wherein the transgenic plasmid is selected from one of the following: (i) Conventional single-stranded recombinant adeno-associated virus, or (ii) Self-complementary genomic recombinant adeno-associated virus.

[0244] like Figure 3 As shown, the transgenic plasmids of the present invention unexpectedly lead to an increase in viral genome concentration, thereby enhancing the replication of ssAAV and scAAV transgenic plasmids. During AAV production, transgenic molecules flanked by ITRs (i.e., nucleic acid sequences encoding the target protein) are packaged into AAV particles, which can be used for gene therapy.

[0245] In some implementations, the transgenic plasmid is a self-complementary AAV (scAAV) plasmid. A potential disadvantage of AAV is its single-stranded DNA (ssDNA) genome. Because the virus relies on DNA replication mechanisms to synthesize the complementary strand, transgenic expression can be delayed. Any ssAAV genome that reaches the nucleus still requires the synthesis or recruitment of the complementary strand for gene expression. To overcome this rate-limiting step, scAAV contains a complementary sequence that can spontaneously anneal (SA) after infection, thus eliminating the need for the host cell to synthesize DNA. This interstrand base pairing or strand annealing is possible because, unlike many autonomous parvoviruses, AAV packages both positive and negative DNA strands with equal efficiency. By packaging the two strands as a single molecule, the need for dsDNA conversion (whether achieved via SA or DNA synthesis) can be circumvented. This can be achieved by taking advantage of the tendency to generate dimeric inverted repeat sequences during the AAV replication cycle. These dimers can be packaged in the same manner as the conventional AAV genome, and the two halves of the ssDNA molecule can fold and pair bases to form a half-length dsDNA molecule. While this further limits the transgene-carrying capacity of the already small viral vector, it offers considerable benefits in terms of transgene expression efficiency and initiation speed, as dsDNA transformation is independent of host cell DNA synthesis and vector concentration. The yield of scAAV genomes can be increased by inhibiting the unwinding of a terminal repeat sequence. This is typically achieved by deleting the terminal unwinding site sequence from one ITR, preventing the rep protein from generating the necessary ssDNA nick. The replication complex, initiated on another ITR, then replicates via a hairpin and returns to the initiation end. Replication proceeds to the ends of the template molecule, leaving a mutated ITR in the middle. This dimer inverted repeat sequence can then undergo normal replication cycling. Each substituted daughter strand contains an ssDNA inverted repeat sequence with an ITR at each end and a mutated ITR in the middle. Production and purification of scAAV vectors from mutated ITR constructs are identical to those for conventional ssAAV.

[0246] In a preferred embodiment, the terminal inverted repeat sequence, preferably the 5' terminal inverted repeat sequence, has a deletion compared to the natural terminal inverted repeat sequence. In some embodiments, the transgenic plasmid containing the deletion is the scAAV transgenic plasmid.

[0247] Transgenic plasmids are constructed using known techniques to provide at least the operatively linked components in the transcriptional direction, a control element including a transcription initiation region, target DNA, and a transcription termination region. Control elements that are functional in mammalian cells are selected. The resulting construct containing the operatively linked components has functional AAV ITR sequences on its flanks (5' and 3'). Termination signals (e.g., polyadenylation sites) may also be included in the plasmid.

[0248] To avoid being bound by theory, it is considered that the ITR is the only cis-acting element required for packaging to allow the production of rAAV. Even though the rolling circle DNA replication mechanism primarily amplifies (i.e., replicates) transgenic expression cassette DNA sequences flanked by the ITR due to the presence of the D sequence within the ITR, plasmid DNA backbones (e.g., origin of replication, antibiotic resistance gene expression cassettes, etc.) can also be packaged into the vector capsid, although less frequently due to the absence of flanking D sequence domains. AAVs can efficiently package genomes similar in size to or smaller than the wild-type viral genome (~4.7 kb). Packaging of the plasmid backbone can be hindered by increasing its size to a degree that is unfavorable for its packaging into the capsid. Backbone amplification can be achieved through additional "filler" sequences (i.e., filler components) that make the plasmid backbone larger than the wild-type AAV genome. It is believed that the presence of an enlarged plasmid backbone reduces the likelihood of rAAV packaging the plasmid backbone into the vector capsid. In some embodiments, the plasmid backbone is enlarged by using filler sequences. In some embodiments, the filler sequence is biologically silent, lacking at least one of an enhancer, promoter, splicing regulator, non-coding RNA, antisense sequence, and / or coding sequence. In some embodiments, each of the enhancer, promoter, splicing regulator, non-coding RNA, antisense sequence, and coding sequence is absent from the filler sequence. In some embodiments, the filler sequence comprises an inert intron DNA sequence found in the human genome. By utilizing DNA sequences from the human genome, it is believed that the filler sequence is also less likely to elicit an immune response when the plasmid is packaged into a capsid.

[0249] In a preferred embodiment, the transgenic plasmid comprises the following structure: 5' ITR (inverted)-CMVie promoter-transgenic-SV40 PolyA-ITR (forward and reverse) 3'.

[0250] In a preferred embodiment, the terminal inverted repeat sequence with a flipped orientation has SEQ ID NO: 1 or a sequence that is at least 80% identical to the SEQ ID NO, preferably a sequence that is at least 85% identical to the SEQ ID NO, more preferably a sequence that is at least 90% identical to the SEQ ID NO, even more preferably a sequence that is at least 95% identical to the SEQ ID NO, and most preferably a sequence that is at least 99% identical to the SEQ ID NO.

[0251] In a preferred embodiment, the terminal inverted repeat sequence with forward and reverse orientation has SEQ ID NO: 2 or a sequence that is at least 80% identical to the SEQ ID NO, preferably a sequence that is at least 85% identical to the SEQ ID NO, more preferably a sequence that is at least 90% identical to the SEQ ID NO, even more preferably a sequence that is at least 95% identical to the SEQ ID NO, and most preferably a sequence that is at least 99% identical to the SEQ ID NO.

[0252] In a preferred embodiment, the terminal inverted repeat sequence contains a deletion and has SEQ ID NO: 4 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0253] In a preferred embodiment, the CMVie promoter comprises or consists of the following sequence: sequence SEQ ID NO: 26 or a sequence having at least 80% identity with said SEQ ID NO, preferably a sequence having at least 85% identity with said SEQ ID NO, more preferably a sequence having at least 90% identity with said SEQ ID NO, even more preferably a sequence having at least 95% identity with said SEQ ID NO, and most preferably a sequence having at least 99% identity with said SEQ ID NO.

[0254] In a preferred embodiment, the transgene is a reporter gene. In a preferred embodiment, the reporter gene can be detected by an antibody-based assay. In a further preferred embodiment, the reporter gene is a fluorescent molecule. Exemplary fluorescent molecules suitable as reporter genes are GFP, eGFP, mGFP, eYFP, citrine, eCFP, mCFP, Cerulean, dtTomato, and any variant thereof. In some embodiments, the reporter gene is β-galactosidase, luciferase, or glutathione S-transferase, or any variant thereof. In some embodiments, the transgene is at most 4.4 kb. In some embodiments, the transgene is at most 2.4 kb. In some preferred embodiments, the reporter gene has the sequence of SEQ ID NO: 17, or preferably a sequence at least 85% identical to said SEQ ID NO, more preferably a sequence at least 90% identical to said SEQ ID NO, even more preferably a sequence at least 95% identical to said SEQ ID NO, and most preferably a sequence at least 99% identical to said SEQ ID NO.

[0255] In a preferred embodiment, the transgenic plasmid does not contain post-transcriptional regulatory elements of marmot hepatitis virus.

[0256] In a preferred embodiment, the transgenic plasmid has the sequence of SEQ ID NO: 3, or preferably a sequence that is at least 85% identical to the SEQ ID NO, more preferably a sequence that is at least 90% identical to the SEQ ID NO, even more preferably a sequence that is at least 95% identical to the SEQ ID NO, and most preferably a sequence that is at least 99% identical to the SEQ ID NO.

[0257] In a preferred embodiment, the transgenic plasmid has the sequence of SEQ ID NO: 5, or preferably a sequence that is at least 85% identical to the SEQ ID NO, more preferably a sequence that is at least 90% identical to the SEQ ID NO, even more preferably a sequence that is at least 95% identical to the SEQ ID NO, and most preferably a sequence that is at least 99% identical to the SEQ ID NO.

[0258] In some implementations, the transgenic plasmid is used for rAAV production, preferably transient rAAV production, wherein the rAAV can be used for gene therapy applications.

[0259] In some particularly preferred embodiments, the rep plasmid comprises a reverse-oriented minimal element, wherein said minimal element comprises the sequence shown in SEQ ID NO: 18, preferably the amino acid sequence shown in SEQ ID NO: 6, wherein said sequence is derived from a 3' inverted repeat sequence from J01901.1 AAV2. In some of these embodiments, the rep plasmid is a repcap plasmid, which also comprises a capsid protein coding sequence.

[0260] In some particularly preferred embodiments, the transgenic plasmid contains the following structure: 5' ITR (inverted)-CMVie promoter-transgenic-SV40 PolyA-ITR (positive / negative) 3' The transgene is a reporter gene, preferably a fluorescent reporter gene having the sequence shown in SEQ ID NO: 17, wherein the terminal inverted repeat sequence with the flipped orientation has the sequence shown in SEQ ID NO: 1, and the terminal inverted repeat sequence with the forward and reverse orientation has the sequence shown in SEQ ID NO: 2.

[0261] The present invention provides a stable or transient cell expression system and cells.

[0262] This invention provides a stable or transient cell expression system comprising the plasmid system and cell line of this invention.

[0263] According to a preferred embodiment, a stable cell expression system comprises genetic elements of the plasmid system, rep plasmid, and / or transgenic plasmid disclosed herein. For example, genome editing can be used to stably integrate one or more sequences present on a plasmid or plasmid system disclosed herein into the chromosome of a cell to obtain cells capable of stably producing rAAV particles. Stable integration is advantageous because it allows for the repeated use of the same genome-edited cell stock without requiring genetic modification of the cells prior to culturing them to produce rAAV particles. The integration can be performed by providing at least one site-directed endonuclease, preferably selected from a wide range of nucleases, ZFN, TALEN, CRISPR-nucleases, nickases, or inactivated variants thereof. Integration can also be performed by providing at least one nucleic acid molecule encoding a site-directed endonuclease, preferably selected from a wide range of nucleases, ZFN, TALEN, CRISPR-nucleases, nickases, or inactivated variants thereof. CRISPR gene editing is well known to those skilled in the art. For example, CRISPR-guided gene integration can be performed by providing at least one suitable functional guide RNA molecule or nucleic acid molecule encoding it, and the genetic element required to generate rAAV in the form of a template to be integrated into the genome. The template can be cleaved by at least one site-directed endonuclease, such as a CRISPR nuclease. The manner in which the genetic element is integrated into the host cell genome is not limited, and various different techniques are known and can be used by those skilled in the art. However, depending on the specific implementation, a CRISPR nuclease, such as Cas12a or Cas9, can be used to integrate the viral vector to generate one or more of the required genetic elements.

[0264] In some embodiments, the stable or transient cell expression system comprises the rep plasmid and the transgenic plasmid of the present invention. In some embodiments, the stable or transient cell expression system comprises the rep plasmid of the present invention. In some embodiments, the stable or transient cell expression system comprises the transgenic plasmid of the present invention. In some embodiments, the stable or transient cell expression system comprises the plasmid system of the present invention. In some embodiments, the stable or transient cell expression system comprises the rep plasmid and cap plasmid of the present invention. In some embodiments, the stable or transient cell expression system comprises the rep plasmid, cap plasmid, and helper plasmid of the present invention. In some embodiments, the stable or transient cell expression system comprises the rep plasmid and helper plasmid of the present invention. In some embodiments, the stable or transient cell expression system comprises the rep plasmid and helper plasmid of the present invention.

[0265] The present invention further provides cells comprising the rep plasmid of the present invention, the plasmid system of the present invention, or the transgenic plasmid of the present invention. In some embodiments, the cells comprise the rep plasmid and the transgenic plasmid of the present invention. In some embodiments, the cells comprise the rep plasmid of the present invention. In some embodiments, the cells comprise the transgenic plasmid of the present invention. In some embodiments, the cells comprise the plasmid system of the present invention. In some embodiments, the cells comprise the rep plasmid and the cap plasmid of the present invention. In some embodiments, the cells comprise the rep plasmid, the cap plasmid, and the helper plasmid of the present invention. In some embodiments, the cells comprise the rep plasmid and the helper plasmid of the present invention. In some embodiments, the cells comprise the rep plasmid and the helper plasmid of the present invention.

[0266] In a preferred embodiment, the cell is a mammalian cell. The cell may be HEK293 cell, HELA cell, or insect cell, or derivatives thereof. Most preferably, the cell is a HEK293 cell.

[0267] The reagent kit of the present invention

[0268] The present invention provides a kit comprising the stable or transient cell expression system of the present invention, or the cells of the present invention, and cell culture medium.

[0269] In some embodiments, the kit includes culture medium feed, culture medium additives, or transfection reagents, or any combination thereof. In some embodiments, the kit includes a manual.

[0270] The method for producing the recombinant adeno-associated virus vector of the present invention

[0271] This invention provides a method for producing a recombinant adeno-associated virus vector, comprising: (i) Transfecting cells with the rep plasmid of the present invention, the plasmid system of the present invention, or the transgenic plasmid of the present invention; (ii) Culturing transfected cells to produce the adeno-associated virus vector; and (iii) Isolate the recombinant adeno-associated virus vector.

[0272] The cells may be HEK293 cells, HELA cells, or insect cells, or derivatives thereof. In a preferred embodiment, the cells are mammalian cells, preferably HEK293 cells.

[0273] sequence list

[0274] This application includes a sequence list, which has been electronically submitted and is incorporated herein by reference in its entirety. The sequence list file is named 230267WO_Sequence Listing.XML and is 82.090 bytes in size.

[0275] SEQ ID NO: 1 is an exemplary polynucleotide sequence of the 5' ITR sequence applicable to the transgenic plasmid of this disclosure.

[0276] SEQ ID NO: 2 is an exemplary polynucleotide sequence of the 3' ITR sequence applicable to the transgenic plasmid of this disclosure.

[0277] SEQ ID NO: 3 is an exemplary polynucleotide sequence of the ssAAV transgenic plasmid disclosed herein.

[0278] SEQ ID NO: 4 is an exemplary polynucleotide sequence of a 5' ITR sequence that contains a deletion of a transgenic plasmid suitable for use in this invention, particularly the scAAV transgenic plasmid of this disclosure.

[0279] SEQ ID NO: 5 is an exemplary polynucleotide sequence of the scAAV transgenic plasmid disclosed herein.

[0280] SEQ ID NO: 6 is an exemplary polynucleotide sequence of the minimum element suitable for use in the rep plasmid of this disclosure.

[0281] SEQ ID NO: 7 is an exemplary polynucleotide sequence of the rep plasmid disclosed herein.

[0282] SEQ ID NO: 8, 9, 10 and 11 are exemplary amino acid sequences of the rep78, rep68, rep52 and rep40 proteins, respectively.

[0283] SEQ ID NO: 12, 13 and 14 are exemplary amino acid sequences of VP1, VP2 and VP3 proteins, respectively.

[0284] SEQ ID NO: 15 and 16 are exemplary amino acid sequences of the AAP and X proteins, respectively.

[0285] SEQ ID NO: 17 is an exemplary amino acid sequence of the eGFP fluorescent reporter molecule used in the transgenic plasmid of this disclosure.

[0286] SEQ ID NO: 18 is an exemplary polynucleotide sequence containing a rep-binding element (RBE) derived from an ITR sequence or consisting of a rep-binding element (RBE) derived from an ITR sequence.

[0287] SEQ ID NO: 19 is an exemplary complete AAV2 genome.

[0288] SEQ ID NO: 20 is an exemplary polynucleotide sequence containing or consisting of rep-binding elements derived from the p5 promoter.

[0289] SEQ ID NO: 21 is an exemplary polynucleotide sequence of the rabbit β-globin poly(A) signal sequence.

[0290] SEQ ID NO: 22 is an exemplary amino acid sequence of the MAAP protein.

[0291] SEQ ID NO: 23, 24, and 25 are exemplary polynucleotide sequences of the p5, p19, and p40 promoters, respectively.

[0292] SEQ ID NO: 26 is an exemplary polynucleotide sequence of a natural poly(A) signal sequence from AAV.

[0293] SEQ ID NO: 27 is an exemplary polynucleotide sequence of the CMVie promoter.

[0294] SEQ ID NO: 28 is an exemplary polynucleotide sequence of the CellBioLabs© AAV2-derived pAAV-RC2 plasmid (CellBioLabs© Project No. VPK-422).

[0295] SEQ ID NO: 29 is an exemplary polynucleotide sequence of a control AAV2 Rep Cap plasmid with very weak rep expression.

[0296] SEQ ID NO: 30 is an exemplary polynucleotide sequence of an AAV2-derived rep plasmid disclosed herein.

[0297] SEQ ID NO: 31 is an exemplary polynucleotide sequence of an AAV8-derived rep plasmid of this disclosure.

[0298] SEQ ID NO: 32 is an exemplary polynucleotide sequence of the MMTV promoter.

[0299] SEQ ID NO: 33 is an exemplary polynucleotide sequence containing or composed of the following rep-binding elements derived from ITR sequences from AAV1, AAV2, AAV6, or AAV7.

[0300] SEQ ID NO: 34 is an exemplary polynucleotide sequence comprising or composed of the following rep-binding element, which is derived from the p5 promoter sequence from AAV1, AAV7, or AAV8.

[0301] SEQ ID NO: 35 is an exemplary polynucleotide sequence comprising or composed of the following rep-binding element, which is derived from an ITR sequence from AAV3.

[0302] SEQ ID NO: 36 is an exemplary polynucleotide sequence comprising or composed of the following rep-binding element, which is derived from an ITR sequence from AAV4.

[0303] SEQ ID NO: 37 is an exemplary polynucleotide sequence comprising or composed of the following rep-binding element, which is derived from the ITR sequence of AAV5.

[0304] SEQ ID NO: 38 is an exemplary polynucleotide sequence comprising or composed of the following rep-binding element, which is derived from the p5 promoter sequence from AAV5.

[0305] The following sequences consist of fewer than 10 nucleotides and are therefore not included in the sequence listing: SEQ ID NO: 39 (GAGTGAGC) is an exemplary polynucleotide sequence consisting of the following rep-binding elements derived from the p5 promoter sequence of AAV2, AAV3, AA4, or AAV6.

[0306] SEQ ID NO: 40 (AGYGAGC) is an exemplary common polynucleotide sequence of rep-binding element derived from an ITR sequence.

[0307] SEQ ID NO: 41 (AGYGAMC) is an exemplary common polynucleotide sequence of rep-binding element derived from the p5 promoter sequence.

[0308] Example

[0309] Example 1: AAVMax© cells (Thermo) were grown in Ambr15 at DO40, 630 rpm, pH 7.2–7.0 in HEK VIPNB medium (Sartorius Xell) for 4 days, maintaining cell densities between 0.5 and 4 million viable cells / mL. On day 5, at a density of 3.5–4 million viable cells / mL, cells were transfected with 1 μg plasmid per million viable cells and 1.8 μg PEIPro (PolyPlus) per million viable cells. The plasmid molar ratio was 1:1 (two-plasmid system) or 1:1:1 (three-plasmid system), and the transfection mixture volume was 15% v / v of the final volume. 24 hours post-transfection, cells were fed with 10% v / v FS (Sartorius Xell). 72 hours post-transfection, the cell suspension was lysed, digested with Benzonase, and subjected to ddPCR and capsid ELISA (Progen). The following plasmid systems were investigated: Plasmid system NO: 1 (PF HRC, PF ssAAV; Figure 1 ): PF HRC: A pDP2K plasmid derived from AAV2 (PlasmidFactory© Project No. PF2432) from PlasmidFactory©, containing rep, cap, and accessory genes. PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0310] Plasmid system NO: 2 (PF HRC, S ssAAV; Figure 1 ): PF HRC: A pDP2K plasmid derived from AAV2 (PlasmidFactory© Project No. PF2432) from PlasmidFactory©, containing rep, cap, and accessory genes. S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0311] Plasmid system NO: 3 (A H, T RC, PF ssAAV; Figure 2 ): T RC: CellBioLabs© pAAV-RC2 plasmid derived from AAV2, containing the rep and cap genes (CellBioLabs© Project No. VPK-422; SEQ ID NO: 28) AH: Aldevron© pHelper pALD-X80 plasmid PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0312] Plasmid system NO: 4 (A H, S RC, PF ssAAV; Figure 2 ): S RC: The rep plasmid of the present invention (SEQ ID NO: 7) comprises: The coding sequences of the rep proteins rep78, rep68, rep52 and rep40 (SEQ ID NO: 8, 9, 10 and 11), which are driven by their native p5 (SEQ ID NO: 23) and p19 (SEQ ID NO: 24) promoters; The coding sequences of the cap proteins V1, VP2, and VP3 (SEQ ID NO: 12, 13, and 14), which are driven by their native p40 promoter (SEQ ID NO: 25); o The coding sequences of accessory proteins AAP, X and MAAP (SEQ ID NO: 15, 16 and 22); o The polyadenylation signal sequence located downstream of the rep protein coding sequence (SEQ ID NO: 26); and o The smallest element (SEQ ID NO: 6) containing a rep-binding element (SEQ ID NO: 18) located downstream of the polyadenylation signal sequence.

[0313] AH: Aldevron© pHelper pALD-X80 plasmid PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451) like Figure 1As shown, using a dual plasmid system, the PlasmidFactory©AAV2-derived pDP2K plasmid (PF HRC) was combined with the PlasmidFactory© pAAV-ssGFP transgenic plasmid (PF ssAAV) to produce an ssAAV capsid (pAAV-ssGFP). Similarly, using a dual plasmid system, the PlasmidFactory©AAV2-derived pDP2K plasmid (PF HRC) was combined with the transgenic plasmid of the present invention (S ssAAV; SEQ ID NO: 3) to produce an ssAAV capsid (optimized pAAV-ssGFP). In the dual plasmid system (plasmid system NO: 2) containing the improved transgenic plasmid of the present invention, the measured genomic titer was unexpectedly increased. Simultaneously, the percentage of intact capsids also increased.

[0314] like Figure 2 As shown, using a three-plasmid system, the CellBioLabs© AAV2-derived pAAV-RC2 plasmid (T RC) was combined with the Aldevron© pHelper plasmid (AH) and the PlasmidFactory© pAAV-ssGFP transgenic plasmid (PF ssAAV) to produce an ssAAV capsid (pAAV RC2). Similarly, using a three-plasmid system, the present invention's rep plasmid (S RC; SEQ ID NO: 7) was combined with the PlasmidFactory© pAAV-ssGFP transgenic plasmid (PF ssAAV) and the Aldevron© pHelper plasmid (AH) to produce an ssAAV capsid (optimized pAAV RC2). In the three-plasmid system (plasmid system NO: 4) containing the present invention's rep plasmid, the measured genomic titer and intact capsid were substantially improved.

[0315] This demonstrates that the transgenic plasmids and rep plasmids of the present invention can be advantageously used in rAAV production processes because replication and packaging in the capsid are more efficient compared to conventional plasmids.

[0316] Example 2: AAVMax© cells (Thermo) or HEK293 clones C1, C2, C3, or genetically modified HEK293 (H-GMO) cells were grown in shake flasks in TF medium (Sartorius Xell) under the following conditions: 80% relative humidity, 37°C, 5% CO2, and 185 rpm, with cell density maintained between 0.5 and 4 million viable cells / mL. At a density of 3.5–4 million viable cells / mL, transfection was performed using 1 μg plasmid per million viable cells and 1.8 μg PEIPro (PolyPlus) per million viable cells. The plasmid molar ratio was 1:1:1, and the transfection mixture volume was 15% v / v of the final volume. Twenty-four hours post-transfection, cells were fed with 10% v / v FS (Sartorius Xell). 72 hours after transfection, the cell suspension was lysed, digested with Benzonase, and subjected to ddPCR and capsid ELISA (Progen). In some cases, GFP-based potency assays were performed (transduced suspension of HEK cells).

[0317] like Figure 3 As shown, a three-plasmid system was used in shake flasks with Thermo AAVMax© cells. The following three-plasmid system was investigated: Plasmid system NO: 5 (A H, T RC, PF ssAAV; Figure 3 ): T RC: CellBioLabs© pAAV-RC2 plasmid derived from AAV2, containing the rep and cap genes (CellBioLabs© Project No. VPK-422; SEQ ID NO: 28) AH: Aldevron© pHelper pALD-X80 plasmid PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0318] Plasmid system NO: 6 (A H, T RC, S ssAAV; Figure 3 ): T RC: CellBioLabs© pAAV-RC2 plasmid derived from AAV2, containing the rep and cap genes (CellBioLabs© Project No. VPK-422; SEQ ID NO: 28) AH: Aldevron© pHelper pALD-X80 plasmid S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0319] Plasmid system NO: 7 (A H, T RC, S scAAV; Figure 3 ): T RC: CellBioLabs© pAAV-RC2 plasmid derived from AAV2, containing the rep and cap genes (CellBioLabs© Project No. VPK-422; SEQ ID NO: 28) AH: Aldevron© pHelper pALD-X80 plasmid S scAAV: The transgenic plasmid of the present invention (SEQ ID NO: 5), comprising: o 5' ITR (SEQ ID NO: 4); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0320] Plasmid system NO: 8 (A H, S RC, PF ssAAV; Figure 3 ): S RC: The rep plasmid of the present invention (SEQ ID NO: 7) comprises: The coding sequences of the rep proteins rep78, rep68, rep52 and rep40 (SEQ ID NO: 8, 9, 10 and 11), which are driven by their native p5 (SEQ ID NO: 23) and p19 (SEQ ID NO: 24) promoters; The coding sequences of the cap proteins V1, VP2, and VP3 (SEQ ID NO: 12, 13, and 14), which are driven by their native p40 promoter (SEQ ID NO: 25); o The coding sequences of accessory proteins AAP, X and MAAP (SEQ ID NO: 15, 16 and 22); o The polyadenylation signal sequence located downstream of the rep protein coding sequence (SEQ ID NO: 26); and o The smallest element (SEQ ID NO: 6) containing a rep-binding element (SEQ ID NO: 18) located downstream of the polyadenylation signal sequence.

[0321] AH: Aldevron© pHelper pALD-X80 plasmid PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0322] Plasmid system NO: 9 (A H, S RC, S ssAAV; Figure 3 ): S RC: The rep plasmid of the present invention according to plasmid system NO: 8 (SEQ ID NO: 7) AH: Aldevron© pHelper pALD-X80 plasmid S ssAAV: The transgenic plasmid of the present invention according to plasmid system NO: 6 (SEQ ID NO: 3)

[0323] Plasmid system NO: 10 (A H, S RC, S scAAV; Figure 3 ): S RC: The rep plasmid of the present invention according to plasmid system NO: 8 (SEQ ID NO: 7) AH: Aldevron© pHelper pALD-X80 plasmid S scAAV: The transgenic plasmid of the present invention according to plasmid system NO: 7 (SEQ ID NO: 5)

[0324] Plasmid system NO: 11 (A H, weak RC, PF ssAAV; Figure 3 ): Weak RC: Control AAV2 Rep Cap plasmid with very weak rep expression (SEQ ID NO: 29) AH: Aldevron© pHelper pALD-X80 plasmid PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0325] Plasmid system NO: 12 (A H, weak RC, S ssAAV; Figure 3 ): Weak RC: Control AAV2 Rep Cap plasmid with very weak rep expression (SEQ ID NO: 29) AH: Aldevron© pHelper pALD-X80 plasmid S ssAAV: The transgenic plasmid of the present invention according to plasmid system NO: 6 (SEQ ID NO: 3)

[0326] Plasmid system NO: 13 (A H, weak RC, S scAAV; Figure 3 ): Weak RC: Control AAV2 Rep Cap plasmid with very weak rep expression (SEQ ID NO: 29) AH: Aldevron© pHelper pALD-X80 plasmid S scAAV: The transgenic plasmid of the present invention according to plasmid system NO: 7 (SEQ ID NO: 5) These examples demonstrate that incorporating the transgenic plasmids of the present invention advantageously increases genomic titer compared to prior art PF ssAAV transgenic plasmids (plasmid systems NO: 6, 7, 9, and 10). Even when rep expression is weak (weak RC), resulting in undetectable genomic titer with conventional plasmids, increased titers can be obtained by using the transgenic plasmids provided by the present invention (plasmid systems NO: 12 and 13). Furthermore, increased titers are obtained when the rep plasmid (S RC) of the present invention is combined with the conventional transgenic plasmid PFssAAV (plasmid system NO: 8). Further increased titers are provided when the rep plasmid (S RC) of the present invention is combined with either the S ssAAV or S scAAV transgenic plasmids of the present invention (plasmid systems NO: 9 and 10). Most notably, plasmid systems using the rep plasmids and scAAV transgenic (S RC and S scAAV) plasmids of the present invention achieve a synergistic effect in terms of genomic titer and intact capsid.

[0327] exist Figure 4 The paper also presents a summary of various HEK cell (banks or clones) tested in shake flasks using two-plasmid and three-plasmid systems.

[0328] The following two plasmid systems were studied: Plasmid system NO: 14 (PF HRC, PF ssAAV; Figure 4 ): PF HRC: A pDP2K plasmid derived from PlasmidFactory©AAV2 (PlasmidFactory© Project No. PF2432), containing rep, cap, and accessory genes. PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0329] Plasmid system NO: 15 (PF HRC, S ssAAV; Figure 4 ): PF HRC: A pDP2K plasmid derived from PlasmidFactory©AAV2 (PlasmidFactory© Project No. PF2432), containing rep, cap, and accessory genes. S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0330] Plasmid system NO: 16 (PF HRC, S scAAV; Figure 4 ): PF HRC: A pDP2K plasmid derived from PlasmidFactory©AAV2 (PlasmidFactory© Project No. PF2432), containing rep, cap, and accessory genes. S scAAV: The transgenic plasmid of the present invention (SEQ ID NO: 5), comprising: o 5' ITR (SEQ ID NO: 4); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0331] The following three-plasmid systems were studied: Plasmid system NO: 17 (A H, S RC, S ssAAV; Figure 4 ): S RC: The rep plasmid of the present invention (SEQ ID NO: 7) comprises: The coding sequences of the rep proteins rep78, rep68, rep52 and rep40 (SEQ ID NO: 8, 9, 10 and 11), which are driven by their native p5 (SEQ ID NO: 23) and p19 (SEQ ID NO: 24) promoters; The coding sequences of the cap proteins V1, VP2, and VP3 (SEQ ID NO: 12, 13, and 14), which are driven by their native p40 promoter (SEQ ID NO: 25); o The coding sequences of accessory proteins AAP, X and MAAP (SEQ ID NO: 15, 16 and 22); o The polyadenylation signal sequence located downstream of the rep protein coding sequence (SEQ ID NO: 26); and o The smallest element (SEQ ID NO: 6) containing a rep-binding element (SEQ ID NO: 18) located downstream of the polyadenylation signal sequence.

[0332] AH: Aldevron© pHelper pALD-X80 plasmid S ssAAV: The transgenic plasmid of the present invention according to plasmid system NO: 15 (SEQ ID NO: 3)

[0333] Plasmid system NO: 18 (A H, S RC, S scAAV; Figure 4 ): S RC: The rep plasmid of the present invention according to plasmid system NO: 17 (SEQ ID NO: 7) AH: Aldevron© pHelper pALD-X80 plasmid S scAAV: The transgenic plasmid of the present invention according to plasmid system NO: 16 (SEQ ID NO: 5) It has been demonstrated that the scAAV and ssAAV transgenic plasmids of the present invention advantageously increase genomic titers even when used in plasmid systems containing conventional rep / cap plasmids (plasmid systems NO: 15 and 16). Combinations of the rep plasmid (SRC) of the present invention with either the ssAAV or scAAV transgenic plasmid of the present invention further yield even higher titers, and a synergistic effect on genomic titers is again observed in plasmid systems containing the rep plasmid and the scAAV transgenic plasmid of the present invention (plasmid systems NO: 17 and 18).

[0334] Example 3: Sartorius HEK293 clone 3 was grown in shake flasks in TF medium (Sartorius Xell) under conditions of 80% relative humidity, 37°C, 5% CO2, and 185 rpm, with cell densities maintained between 0.5 and 4 million viable cells / mL. At densities of 3.5–4 million viable cells / mL, transfection was performed using 1 μg plasmid per million viable cells and 1 μg PEIMax (Polysciences) per million viable cells. The plasmid molar ratio was 1:1:1, and the transfection mixture volume was 10% v / v of the final volume. 72 hours post-transfection, the cell suspension was lysed, digested with Benzonase, and ddPCR was performed. The following plasmid systems were investigated: Plasmid system NO: 20 (PF HRC, PF ssAAV; Figure 5 ): PF HRC: A pDP2K plasmid derived from PlasmidFactory©AAV2 (PlasmidFactory© Project No. PF2432), containing rep, cap, and accessory genes. PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0335] Plasmid system NO: 21 (A H, S RC, S ssAAV; Figure 5 ): S RC: The rep plasmid of the present invention (SEQ ID NO: 7) comprises: The coding sequences of the rep proteins rep78, rep68, rep52 and rep40 (SEQ ID NO: 8, 9, 10 and 11), which are driven by their native p5 (SEQ ID NO: 23) and p19 (SEQ ID NO: 24) promoters; The coding sequences of the cap proteins V1, VP2, and VP3 (SEQ ID NO: 12, 13, and 14), which are driven by their native p40 promoter (SEQ ID NO: 25); o The coding sequences of accessory proteins AAP, X and MAAP (SEQ ID NO: 15, 16 and 22); o The polyadenylation signal sequence located downstream of the rep protein coding sequence (SEQ ID NO: 26); and o The smallest element (SEQ ID NO: 6) containing a rep-binding element (SEQ ID NO: 18) located downstream of the polyadenylation signal sequence.

[0336] AH: Aldevron© pHelper pALD-X80 plasmid S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0337] like Figure 5 As shown, the combination of the rep plasmid (S RC) of the present invention and the ssAAV transgenic plasmid of the present invention yielded a higher titer (plasmid system NO: 21). These examples also demonstrate that the addition of the rep plasmid and transgenic plasmid of the present invention advantageously increases the genome titer compared to prior art plasmid systems.

[0338] Example 4: Thermo EXPI293F ICs were grown in shake flasks in Freestyle 293 expression medium (Thermo) under conditions of 80% relative humidity, 37°C, 8% CO2, and 130 rpm, with cell densities maintained between 0.2 and 3 million viable cells / mL. Two days after inoculation, at a density of 1.3–1.5 million viable cells / mL, transfection was performed using 0.55 μg plasmid per million viable cells and 0.55 μl FectoVir (PolyPlus) per million viable cells. The plasmid molar ratio in Freestyle 293 expression medium was 4.5:1 (ssAAV:HRC), and the transfection mixture volume was 5% v / v of the final culture volume. 72 hours after transfection, the cell suspension was lysed, digested with Denarase, and subjected to ddPCR and capsid ELISA (Progen). The following plasmid systems were tested in duplicate (replica 1 and replica 2): Plasmid system NO: 22 (PF HRC, PF ssAAV; Figure 6 ): PF HRC: PlasmidFactory© AAV8-derived plasmid pDP8K (PlasmidFactory© Project No. PF2438), containing rep, cap, and helper genes. PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0339] Plasmid system NO: 23 (PF HRC, S ssAAV; Figure 6 ): PF HRC: PlasmidFactory© AAV8-derived plasmid pDP8K (PlasmidFactory© Project No. PF2438), containing rep, cap, and helper genes. S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0340] like Figure 6As shown, compared with the prior art ssAAV transgenic plasmid (PF ssAAV), the transgenic plasmid (S ssAAV) of the present invention advantageously increases the genome titer.

[0341] Example 5: Thermo EXPI293F ICs were grown in shake flasks in Freestyle 293 expression medium (Thermo) under conditions of 80% relative humidity, 37°C, 8% CO2, and 130 rpm, with cell densities maintained between 0.2 and 3 million viable cells / mL. Two days after inoculation, at densities of 1.3–1.5 million viable cells / mL, transfection was performed using 0.6 μg plasmid per million viable cells and 0.6 μl FectoVir (PolyPlus) per million viable cells. The plasmid molar ratio in Freestyle 293 expression medium was 4.5:1 (ssAAV:HRC), and the transfection mixture volume was 5% v / v of the final culture volume. 72 hours post-transfection, the cell suspension was lysed, digested with Denarase, and subjected to ddPCR and capsid ELISA (Progen). The following plasmid systems were investigated: Plasmid system NO: 24 (PF HRC, PF ssAAV; Figure 7 ): PF HRC: PlasmidFactory© AAV8-derived plasmid pDP8K (PlasmidFactory© Project No. PF2438), containing rep, cap, and helper genes. PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0342] Plasmid system NO: 25 (PF HRC, S ssAAV; Figure 7 ): PF HRC: PlasmidFactory© AAV8-derived plasmid pDP8K (PlasmidFactory© Project No. PF2438), containing rep, cap, and helper genes. S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0343] like Figure 7 As shown, compared with the prior art ssAAV transgenic plasmid (PF ssAAV; plasmid system NO: 24), the transgenic plasmid of the present invention (S ssAAV, plasmid system NO: 25) advantageously increases the genome titer.

[0344] Example 6: Thermo EXPI293F IC was grown in Freestyle 293 expression medium (Thermo) in shake flasks at 80% relative humidity, 37°C, 8% CO2, and 130 rpm, or in a 10L stirred tank bioreactor at 201 rpm (15 W / m²). 3 Cells were grown in Freestyle 293 expression medium (Thermo) at 40% DO, with cell densities maintained between 0.2 and 3 million viable cells / mL. For shake-flask cultures, transfection was performed 2 days after inoculation at a density of 1.3–1.5 million viable cells / mL using 0.5–1.0 μg plasmid per million viable cells and 0.5–1.0 μl FectoVir (PolyPlus) per million viable cells. In Freestyle 293 expression medium, the plasmid molar ratio was 2.8–4.2:1 (ssAAV:HRC), and the transfection mixture volume was 5% v / v of the final culture volume. Specifically, the following transfection compositions (TX Mix 1–3) were used for the transfection reaction: TX Mix 1: 0.5 μg plasmid and 0.5 μl FectoVir (PolyPlus) per million live cells, with a plasmid molar ratio of 2.8:1 (ssAAV:HRC). TX Mix 2: 0.8 μg plasmid per million live cells and 0.8 μl FectoVir (PolyPlus) per million live cells, with a plasmid molar ratio of 4.2:1 (ssAAV:HRC). TX Mix 3: 1 μg plasmid per million live cells and 1 μl FectoVir (PolyPlus) per million live cells, with a plasmid molar ratio of 4.2:1 (ssAAV:HRC). For stirred-tank bioreactor culture, transfection was performed 2 days after inoculation at a density of 1.3–1.5 million viable cells / mL using 1.0 μg plasmid per million viable cells and 1.0 μl FectoVir (PolyPlus) per million viable cells. In Freestyle 293 expression medium, the plasmid molar ratio was 4.2:1 (ssAAV:HRC), and the transfection mixture volume was 5% v / v of the final culture volume. In shake-flask culture, cell suspensions were lysed and digested with Denarase at 48 and 72 hours post-transfection, followed by ddPCR and capsid ELISA (Progen) analysis. In stirred-tank bioreactor culture, cell suspensions were lysed and digested with Denarase at 48, 72, and 96 hours post-transfection, followed by ddPCR and capsid ELISA (Progen) analysis. The following plasmid systems were investigated: Plasmid system NO: 26 (PF HRC, PF ssAAV; Figure 8 ): PF HRC: A pDP9 plasmid derived from AAV9 by PlasmidFactory© (PlasmidFactory© Project No. PF0439), containing rep, cap, and accessory genes. PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0345] Plasmid system NO: 27 (PF HRC, S ssAAV; Figure 8 , 9 ): PF HRC: A pDP9 plasmid derived from AAV9 by PlasmidFactory© (PlasmidFactory© Project No. PF0439), containing rep, cap, and accessory genes. S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0346] The results of shake flask culture are as follows Figure 8As shown, the present invention's transgenic plasmid (S ssAAV, plasmid system NO: 27) advantageously provides improved genome titer (vg / L) and capsid titer (cp / L) compared to the prior art ssAAV transgenic plasmid (PF ssAAV; plasmid system NO: 26). Furthermore, Figure 8 The increased concentration of transgenic plasmids, particularly the transgenic plasmids of the present invention, is shown to increase genome and capsid titers (TX Mix3).

[0347] The results of stirred tank culture are shown in Figure 9 The invention demonstrates that rAAV can be produced in a larger volume in a stirred tank bioreactor using the transgenic plasmid (platinum system NO:27) of the present invention.

[0348] Example 7: HEK293 cells in Ambr ® Cells were grown in TF medium (Sartorius Xell) in an Advanced Microbioreactor System at densities maintained between 0.5 and 4 million viable cells / mL. At densities of 3.5–4 million viable cells / mL, transfection was performed using 1 μg plasmid per million viable cells and 1.8 μg PEIPro (PolyPlus) or 1 μl FectoVir per million viable cells. The plasmid molar ratio was 1:1:1, and the transfection mixture volume was 15% v / v of the final culture volume. Twenty-four hours post-transfection, cells were fed with 10% v / v FS (Sartorius Xell). Seventy-two hours post-transfection, the cell suspension was lysed, digested with Benzonase, and subjected to ddPCR and capsid ELISA (Progen). The following plasmid systems were investigated: Plasmid system NO: 28 (PF HRC, PF ssAAV; Figure 10 ): PF HRC: A pDP2K plasmid derived from AAV2 (PlasmidFactory© Project No. PF2432) from PlasmidFactory©, containing rep, cap, and accessory genes. PF ssAAV: PlasmidFactory© pAAV-ssGFP transgenic plasmid (PlasmidFactory© Project No. PF1451)

[0349] Plasmid system NO: 29 (PPH, SRC, SssAAV, FectoVir; Figure 10 ,11): S RC: The AAV2-derived rep plasmid of the present invention (SEQ ID NO: 30), comprising: o The coding sequences of rep78 and rep68 (SEQ ID NO: 8 and 9) operatively linked to the heterologous MMTV promoter (SEQ ID NO: 32). The coding sequences of the rep proteins rep52 and rep40 (SEQ ID NO: 10 and 11), which are driven by their native p19 promoter (SEQ ID NO: 24); The coding sequences of the cap proteins V1, VP2, and VP3 (SEQ ID NO: 12, 13, and 14), which are driven by their native p40 promoter (SEQ ID NO: 25); o The coding sequences of accessory proteins AAP, X and MAAP (SEQ ID NO: 15, 16 and 22); o The first natural polyadenylation signal sequence located downstream of the rep protein coding sequence (SEQ ID NO: 26); o The smallest element (SEQ ID NO: 23) containing the rep-binding element (SEQ ID NO: 20) located downstream of the first natural polyadenylation signal sequence; and o The second strong polyadenylation signal sequence located downstream of the smallest element (SEQ ID NO: 21).

[0350] PP H: pPLUS® AAV-helper plasmid S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0351] Plasmid system NO: 30 (PPH, SRC, SssAAV, PEIPro; Figure 10 , 11 ): S RC: The rep plasmid of the present invention according to plasmid system NO: 29 PP H: pPLUS® AAV-helper plasmid S ssAAV: The transgenic plasmid of the present invention according to plasmid system NO: 29 (SEQ ID NO: 3) like Figure 10 As shown, compared with the existing plasmid system (platinum system NO: 28), the combination of the rep plasmid (SRC) and the transgenic plasmid (S ssAAV) of the present invention achieved higher genomic titers (platinum systems NO: 29 and 30). Furthermore, improved genomic titers were also obtained when using different transfection reagents (FectoVir or PEIPro).

[0352] Figure 11 The combination of the present invention's rep plasmid (SRC) and the present invention's transgenic plasmid (S ssAAV) shows an increase in the percentage of intact capsids compared to prior art plasmid systems (plasmid systems NO: 29 and 30) (plasmid systems NO: 28). This effect was also observed when using different transfection reagents (FectoVir or PEIPro).

[0353] Example 8: Thermo EXPI293F ICs were grown in shake flasks in Freestyle 293 expression medium (Thermo) under conditions of 80% relative humidity, 37°C, 8% CO2, and 130 rpm, with cell densities maintained between 0.2 and 3 million viable cells / mL. For the dual-plasmid system, transfection was performed 2 days post-inoculation at a density of 1.3–1.5 million viable cells / mL using 0.6 μg plasmid per million viable cells and 0.6 μl FectoVir (PolyPlus) per million viable cells. The plasmid molar ratio in Freestyle 293 expression medium was 4.5:1 (ssAAV:HRC), and the transfection mixture volume was 5% v / v of the final culture volume.

[0354] For the three-plasmid system, transfection was performed 2 days post-inoculation at a density of 1.3–1.5 million viable cells / mL using 0.9 μg or 1.4 μg plasmid per million viable cells and 0.9 μl or 1.4 μl FectoVir (PolyPlus) per million viable cells. The plasmid molar ratio in Freestyle 293 expression medium was 1:1:1 (PPH:SRC:ssAAV), and the transfection mixture volume was 5% v / v of the final culture volume. Specifically, the following transfection compositions were used for the transfection reactions (TX Mix 1 and 2): TX Mix 1: 0.9 μg plasmid per million live cells and 0.9 μl FectoVir (PolyPlus) per million live cells, with a plasmid molar ratio of 1:1:1 (PP H:S RC:ssAAV). TX Mix 2: 1.4 μg plasmid per million live cells and 1.4 μl FectoVir (PolyPlus) per million live cells, with a plasmid molar ratio of 1:1:1 (PP H:S RC:ssAAV). For both two-plasmid and three-plasmid systems, 72 hours post-transfection, the lysed cell suspension was digested with Denarase and subjected to ddPCR and capsid ELISA (Progen). The following plasmid systems were investigated: Plasmid system NO: 31 (PF HRC, S ssAAV; Figure 12 ): PF HRC: PlasmidFactory© AAV8-derived plasmid pDP8K (PlasmidFactory© Project No. PF2438), containing rep, cap, and helper genes. S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0355] Plasmid system NO: 32 (PPH, SRC, SssAAV, Figure 12 ): S RC: The AAV8-derived rep plasmid of the present invention (SEQ ID NO: 31), comprising: o The coding sequences of rep78 and rep68 (SEQ ID NO: 8 and 9) that are operatively linked to the heterologous MMTV promoter (SEQ ID NO: 32); The coding sequences of the rep proteins rep52 and rep40 (SEQ ID NO: 10 and 11), which are driven by their native p19 promoter (SEQ ID NO: 24); The coding sequences of the cap proteins V1, VP2, and VP3 (SEQ ID NO: 12, 13, and 14), which are driven by their native p40 promoter (SEQ ID NO: 25); o The coding sequences of accessory proteins AAP, X and MAAP (SEQ ID NO: 15, 16 and 22); o The first natural polyadenylation signal sequence located downstream of the rep protein coding sequence (SEQ ID NO: 26); o The smallest element (SEQ ID NO: 23) containing the rep-binding element (SEQ ID NO: 20) located downstream of the first natural polyadenylation signal sequence; and o The second strong polyadenylation signal sequence located downstream of the smallest element (SEQ ID NO: 21).

[0356] pPLUS® AAV helper plasmid (PPH) S ssAAV: The transgenic plasmid of the present invention (SEQ ID NO: 3), comprising: o 5' ITR (SEQ ID NO: 1); o 3' ITR (SEQ ID NO: 2); o CMVie promoter (SEQ ID NO: 26); o eGFP transgene (SEQ ID NO: 17); and o SV40 polyA signal.

[0357] The study of plasmid system NO: 31 was repeated twice, and the study of plasmid system NO: 32 was repeated three times for TX Mix 1.

[0358] Figure 12 The combination of the present invention's rep plasmid (SRC) and the present invention's transgenic plasmid (S ssAAV) demonstrates improved genome and capsid titers compared to prior art plasmid systems (platinum system NO: 31) (platinum system NO: 32). Furthermore, Figure 12 The results show that increased transfection plasmid concentration leads to increased genome and capsid titers (see TX Mix 2).

[0359] These experiments confirmed the production of suitable rAAV using the rep plasmid and the transgenic plasmid of the present invention. As described above, the rep plasmid and transgenic plasmid of the present invention unexpectedly yielded higher genome titers, capsid titers, and the percentage of intact capsids, thereby increasing rAAV yield. The increased genome titers, capsid titers, and percentage of intact capsids shown with different transfection reagents (PEIpro, PEIMax, FectoVir) demonstrate the robustness of the plasmids.

[0360] It should be understood from the foregoing that while specific embodiments have been illustrated and shown, various modifications are possible and contemplated herein. The invention is not intended to be limited to the specific embodiments provided in the specification. Although the invention has been shown with reference to the foregoing specification, the description and illustration of preferred embodiments herein are not intended to be limiting. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions presented herein, and depend on various conditions and variables. Various modifications in form and detail of embodiments of the invention will be apparent to those skilled in the art. Therefore, it is contemplated that the invention will also cover any such modifications, variations, and equivalents. The following claims are intended to define the scope of the invention, and the methods and structures within the scope of these claims, and their equivalents, are thereby covered. Attached Figure Description

[0361] Figure 1 The results show the viral genome concentration [vg / L (column)] and percentage of intact capsids [integrity % (point)] measured when using a dual plasmid system (PF HRC, PF ssAAV) containing the transgenic plasmid of the present invention, compared to a dual plasmid system (PF HRC, S ssAAV) using conventional plasmids.

[0362] Figure 2 The results show the measured viral genome concentration [vg / L (column)] and percentage of intact capsid [integrity % (point)] when using a three-plasmid system (AH, T RC, PF ssAAV) containing the rep plasmid of the present invention, compared to a three-plasmid system (AH, S RC, PF ssAAV) using conventional plasmids.

[0363] Figure 3 The results show the viral genome concentration [vg / L (column)] and percentage of intact capsid [integrity % (point)] measured when using a three-plasmid system with various combinations of conventional rep-cap plasmid (T RC or weak RC), conventional transgenic plasmid (PF ssAAV), the rep plasmid of the present invention (S RC), and the transgenic plasmid of the present invention (S ssAAV, S scAAV).

[0364] Figure 4 The results show the viral genome concentration [vg / L (column)] and percentage of intact capsid [integrity % (point)] measured when using various combinations of dual-plasmid systems or triple-plasmid systems with conventional rep-cap plasmid (PF HRC), conventional transgenic plasmid (PF ssAAV), the rep plasmid of the present invention (S RC), and the transgenic plasmid of the present invention (S ssAAV, S scAAV).

[0365] Figure 5 The viral genome concentration [vg / L] is shown when using a two-plasmid system with a conventional rep-cap plasmid (PF HRC) and a conventional transgenic plasmid (PF ssAAV), and a three-plasmid system containing the rep plasmid (S RC) and the transgenic plasmid (S ssAAV) of the present invention.

[0366] Figure 6 The results show the viral genome concentration [vg / L] and the percentage of intact capsids [integrity % (points)] measured when using a dual-plasmid system with the conventional rep-cap plasmid (PF HRC) and the conventional transgenic plasmid (PF ssAAV) or the transgenic plasmid of the present invention (S ssAAV).

[0367] Figure 7 The results show the viral genome concentration [vg / L] and capsid concentration [cp / L] measured when using a dual plasmid system containing a conventional rep-cap plasmid (PF HRC) and a conventional transgenic plasmid (PF ssAAV) or the transgenic plasmid of the present invention (S ssAAV).

[0368] Figure 8 The viral genome concentration [vg / L] and capsid concentration [cp / L] are shown when using a dual-plasmid system containing a conventional rep-cap plasmid (PF HRC) and a conventional transgenic plasmid (PF ssAAV) or the transgenic plasmid of the present invention (S ssAAV). For different transfection compositions (TX Mix 1-3), viral genome and capsid concentrations were measured at different time points post-transfection (days post-transfection, dPTX).

[0369] Figure 9 The viral genome concentration [vg / L] and capsid concentration [cp / L] are shown when transfected cells are cultured in shake flasks or bioreactors using a dual-plasmid system comprising a conventional rep-cap plasmid (PF HRC) and the transgenic plasmid of this invention (SssAAV). Viral genome and capsid concentrations were determined at different time points post-transfection (days post-transfection, dPTX).

[0370] Figure 10The viral genome concentrations [vg / L] measured using either a two-plasmid system or a three-plasmid system are shown. The two-plasmid system comprises a standard rep-cap plasmid (PF HRC) and a standard transgenic plasmid (PF ssAAV). The three-plasmid system comprises a standard helper plasmid (PP H), the rep plasmid of this invention (S RC), and the transgenic plasmid of this invention (SssAAV). Transfection with two different transfection reagents (FectoVir and PEIPro) was investigated using the three plasmid systems. The viral genome concentrations of two HEK293 clones (HEK293#1 and HEK293#2) were determined.

[0371] Figure 11 The percentage of intact capsids [intact %] is shown when using a two-plasmid system or a three-plasmid system. The two-plasmid system comprises a conventional rep-cap plasmid (PF HRC) and a conventional transgenic plasmid (PF ssAAV). The three-plasmid system comprises a conventional helper plasmid (PP H), the rep plasmid of this invention (S RC), and the transgenic plasmid of this invention (SssAAV). Transfection with two different transfection reagents (FectoVir and PEIPro) was investigated using the three plasmid systems. Viral genome concentrations of two HEK293 clones (HEK293#1, HEK293#2) were determined.

[0372] Figure 12 The viral genome concentration [vg / L] and capsid concentration [cp / L] measured using a dual-plasmid system or a triple-plasmid system are shown. The dual-plasmid system comprises a conventional rep-cap plasmid (PF HRC) and the transgenic plasmid of the present invention (SssAAV). The triple-plasmid system comprises a conventional helper plasmid (PP H), the rep plasmid of the present invention (S RC), and the transgenic plasmid of the present invention (S ssAAV). Viral genome and capsid concentrations were measured for different transfection compositions (TX Mix 1 and 2).

[0373] Figure 13 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 13 The rep plasmid shown contains p5, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), a polyA signal sequence, and a minimal element containing a rep-binding element (RBE). The minimal element containing the RBE is located downstream of the polyA signal sequence.

[0374] Figure 14 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 14The rep plasmid shown contains p5, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), a polyA signal sequence, and a minimal element containing a rep-binding element (RBE). The minimal element containing the RBE is located downstream of the polyA signal sequence and is in reverse orientation; that is, the polynucleotide sequence of the minimal element is... Figure 13 The reverse complementary sequence of the polynucleotide sequence of the smallest element shown.

[0375] Figure 15 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 15 The rep plasmid shown contains p5, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), a polyA signal sequence, and a minimal element containing a rep-binding element (RBE). The minimal element containing the RBE is located upstream of the polyA signal sequence.

[0376] Figure 16 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 16 The rep plasmid shown contains p5, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), a polyA signal sequence, and a minimal element containing a rep-binding element (RBE). The minimal element containing the RBE is located upstream of the polyA signal sequence and is in reverse orientation; that is, the polynucleotide sequence of the minimal element is... Figure 15 The reverse complementary sequence of the polynucleotide sequence of the smallest element shown.

[0377] Figure 17 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 17 The rep plasmid shown contains p5, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), coding sequences for cap proteins (VP1 to VP3), coding sequences for accessory proteins (MAAP, AAP, and X), a polyA signal sequence, and a minimal element containing a rep-binding element (RBE). The minimal element containing the RBE is located downstream of the polyA signal sequence.

[0378] Figure 18 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 18The rep plasmid shown contains p5, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), coding sequences for cap proteins (VP1 to VP3), coding sequences for accessory proteins (MAAP, AAP, and X), a polyA signal sequence, and a minimal element containing a rep-binding element (RBE). The minimal element containing the RBE is located downstream of the polyA signal sequence and in the opposite orientation; that is, the polynucleotide sequence of the minimal element is... Figure 17 The reverse complementary sequence of the polynucleotide sequence of the smallest element shown.

[0379] Figure 19 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 19 The rep plasmid shown contains p5, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), coding sequences for cap proteins (VP1 to VP3), coding sequences for accessory proteins (MAAP, AAP, and X), a polyA signal sequence, and a minimal element containing a rep-binding element (RBE). The minimal element containing the RBE is located upstream of the polyA signal sequence.

[0380] Figure 20 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 20 The rep plasmid shown contains p5, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), coding sequences for cap proteins (VP1 to VP3), coding sequences for accessory proteins (MAAP, AAP, and X), a polyA signal sequence, and a minimal element containing a rep-binding element (RBE). The minimal element containing the RBE is located upstream of the polyA signal sequence and is in reverse orientation; that is, the polynucleotide sequence of the minimal element is... Figure 19 The reverse complementary sequence of the polynucleotide sequence of the smallest element shown.

[0381] Figure 21 A schematic diagram of an exemplary embodiment of the rep plasmid disclosed herein is shown. Figure 21The rep plasmid shown contains MMTV, p19, and p40 promoters, coding sequences for long rep proteins (rep68 and rep78) and short rep proteins (rep40 and rep52), coding sequences for cap proteins (VP1 to VP3), coding sequences for accessory proteins (MAAP, AAP, and X), a native polyA signal sequence, a minimal element containing a rep-binding element (RBE) (p5 promoter), and a strong heterologous polyA signal sequence. The minimal element containing the RBE is located downstream of the native polyA signal sequence and upstream of the strong heterologous polyA signal sequence.

[0382] Figure 22 A schematic diagram of an exemplary embodiment of the transgenic plasmid disclosed herein is shown. Figure 22 The transgenic plasmid shown contains 5' ITR and 3' ITR sequences, a promoter, a transgenic coding sequence, and a polyA signaling sequence.

[0383] Figure 23 A schematic diagram of an exemplary embodiment of the transgenic plasmid disclosed herein is shown. Figure 23 The transgenic plasmid shown contains 5' ITR and 3' ITR sequences, a promoter, a transgenic coding sequence, and a polyA signaling sequence. The 5' ITR is deleted compared to the natural terminal inverted repeat sequence.

[0384] Figure 24 A schematic diagram of an exemplary embodiment of the transgenic plasmid disclosed herein is shown. Figure 24 The transgenic plasmid shown contains 5' ITR (flipped orientation) and 3' ITR (forward and reverse orientation) sequences, a CMVie promoter, a transgenic coding sequence encoding the eGFP reporter gene, and an SV40 polyA signal sequence.

Claims

1. A rep plasmid comprising: (i) at least one adeno-associated virus replication protein coding sequence encoding at least one functional rep protein; (ii) at least one polyadenylation signal sequence located downstream of the adeno-associated virus replication protein coding sequence; and (iii) The smallest element located downstream of the polyadenylation signal sequence, comprising one or more recombinant rep binding elements.

2. The rep plasmid according to claim 1, wherein one or more rep binding elements act as binding sites for rep68 and / or rep78.

3. The rep plasmid according to claim 1 or 2, wherein the rep binding element comprises or consists of a sequence selected from or composed of the following sequences: SEQ ID NO: 18, 20 and / or 33-41, or a sequence having at least 80% identity with one of the SEQ ID NOs, preferably a sequence having at least 85% identity with one of the SEQ ID NOs, more preferably a sequence having at least 90% identity with one of the SEQ ID NOs, even more preferably a sequence having at least 95% identity with one of the SEQ ID NOs, and most preferably a sequence having at least 99% identity with one of the SEQ ID NOs.

4. The rep plasmid according to any one of claims 1 to 3, wherein the rep binding element is derived from a terminal inverted repeat sequence, preferably from a 3' terminal inverted repeat sequence derived from J01901.1 AAV2.

5. The rep plasmid according to any one of claims 1 to 4, wherein the smallest element comprises SEQ ID NO: 6 or a sequence having at least 60%, at least 70%, or at least 80% identity with the SEQ ID NO, preferably a sequence having at least 85% identity with the SEQ ID NO, more preferably a sequence having at least 90% identity with the SEQ ID NO, even more preferably a sequence having at least 95% identity with the SEQ ID NO, and most preferably a sequence having at least 99% identity with the SEQ ID NO.

6. A plasmid system for producing adeno-associated virus particles, comprising: (i) transgenic plasmids; and (ii) The rep plasmid according to any one of claims 1 to 5.

7. The plasmid system of claim 6, wherein the system further comprises an auxiliary plasmid, wherein the auxiliary plasmid preferably comprises one or more coding sequences encoding E2A, E4orf6, or E4orf7, or any combination thereof.

8. The plasmid system according to claim 6 or 7, wherein the transgenic plasmid comprises a promoter, a transgene, a polyadenylation signal sequence, and 5' and 3' terminal inverted repeat sequences. The transgenic plasmids mentioned above are selected from one of the following: (i) Conventional single-stranded recombinant adeno-associated virus, or (ii) Self-complementary genomic recombinant adeno-associated virus.

9. The plasmid system of claim 8, wherein the 3' and 5' terminal inverted repeat sequences are derived from J01901.1AAV2, and / or at least one terminal inverted repeat sequence, preferably the 5' terminal inverted repeat sequence, has a deletion compared to the natural terminal inverted repeat sequence.

10. A transgenic plasmid comprising: CMVie promoter, transgene, SV40 PolyA, 5' and 3' terminal inverted repeat sequences derived from J01901.1 AAV2; The transgenic plasmids mentioned above are selected from one of the following: (i) Conventional single-stranded recombinant adeno-associated virus, or (ii) Self-complementary genomic recombinant adeno-associated virus.

11. The transgenic plasmid according to claim 10, wherein the transgenic plasmid comprises the following structure: 5' ITR (inverted)-CMVie promoter-transgenic-SV40 PolyA-ITR (forward and reverse) 3'.

12. A stable or transient cell expression system comprising a plasmid system and cell line according to any one of claims 6 to 9.

13. A cell comprising the rep plasmid according to any one of claims 1 to 5, the plasmid system according to any one of claims 6 to 9, or the transgenic plasmid according to claim 10 or 11.

14. A kit comprising a stable or transient cell expression system according to claim 12 or cells according to claim 13, and a cell culture medium.

15. A method for producing a recombinant adeno-associated virus vector, the method comprising: (i) Transfecting cells with the rep plasmid according to any one of claims 1 to 5, the plasmid system according to any one of claims 6 to 9, or the transgenic plasmid according to claim 10 or 11; (ii) Culturing transfected cells to produce the adeno-associated virus vector; and (iii) Isolate the recombinant adeno-associated virus vector.

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