Method for producing recombinant AAV particle preparations

Abstract

A method for producing recombinant adeno-associated virus particle preparations (rAAVp) is reported herein, comprising the steps of culturing mammalian cells containing expression cassettes for non-adeno-associated virus genes, adeno-associated virus rep genes, adeno-associated virus cap genes, adeno-associated virus E1A genes, adeno-associated virus E1B genes, adeno-associated virus E2A genes, adeno-associated virus E4orf6, and adeno-associated virus VA RNA genes located between two AAV terminal inverted repeat sequences (ITRs), and producing rAAVp therefrom, wherein the culturing is performed at a pH value between pH 7.4 and pH 7.6. The yield of rAAVp produced by culturing at a pH value between pH 7.4 and pH 7.6 is higher than that of rAAVp produced by culturing at a pH value between pH 7.0 and pH 7.2, and the percentage of perfect particles is higher for rAAVp produced by culturing at a pH value between pH 7.4 and pH 7.6 than for rAAVp produced by culturing at a pH value between pH 7.0 and pH 7.2.

Description

Technical Field

[0001] The present invention belongs to the field of gene therapy. More specifically, the present invention is directed to a method for generating recombinant adeno-associated virus particles containing a therapeutic transgene at a higher pH value commonly used in the art in mammalian cells, particularly CHO and HEK cells.

Background Art

[0002] Background Gene therapy has opened up unprecedented opportunities for novel therapeutic approaches. Based on the concept of restoring a functional mutation by co-expressing the correct gene, the use of viral vectors is necessary to ensure proper delivery of the therapeutic gene in order to restore biological function. In this regard, recombinant adeno-associated virus (rAAV) is the most widely used vector.

[0003] The bioprocessing of viruses is complex and requires systematic and coordinated processes in both upstream and downstream processing. However, the use of traditional culture processes for the production of therapeutic viruses does not support an effective commercial manufacturing strategy. This is even more pronounced for large-sized viruses compared to therapeutic biomolecules such as antibodies. Furthermore, viruses are far more complex.

[0004] The biomanufacturing process of therapeutic viruses requires the insertion of a therapeutic transgene into a recombinant AAV capsid shell (fully rAAV particles, i.e., recombinant AAV particles containing a capsid-formed nucleic acid). However, the percentage of rAAV that does not contain the desired transgene (empty recombinant AAV particles, i.e., rAAV particles that do not contain a capsid-formed nucleic acid), as well as partially filled rAAV (partially filled recombinant AAV particles), can also be generated.

Summary of the Invention

[0005] Summary of Embodiments of the Present Invention This invention is at least in part based on the finding that the productivity of mammalian cells that produce recombinant adeno-associated virus particles can be increased when the culture is performed at a high pH value such as pH 7.4–7.6.

[0006] The present invention is at least in part based on the finding that the proportion of fully recombinant AAV particles obtained from a culture of mammalian cells that produce recombinant adeno-associated virus particles can increase when the culture is carried out at a high pH value such as pH 7.4–7.6.

[0007] The present invention includes at least the following embodiments:

[0008] 1. A method for producing recombinant adeno-associated virus particle preparations (rAAVp), comprising the steps of culturing mammalian cells containing an expression cassette for a non-adeno-associated virus gene, an adeno-associated virus rep gene, an adeno-associated virus cap gene, an adeno-associated virus E1A gene, an adeno-associated virus E1B gene, an adeno-associated virus E2A gene, an adeno-associated virus E4orf6 gene, and optionally an adeno-associated virus VA RNA gene, located between two AAV terminal inverted repeat sequences (ITRs), and thereby producing rAAVp, A method in which cultivation is performed at a pH value within the range of pH 7.4 to pH 7.6.

[0009] 2. A method for producing recombinant adeno-associated virus particle preparations (rAAVp), comprising the steps of culturing HEK cells containing an expression cassette for a non-adeno-associated virus gene, an adeno-associated virus rep gene, an adeno-associated virus cap gene, an adeno-associated virus E2A gene, an adeno-associated virus E4orf6 gene, and optionally an adeno-associated virus VA RNA gene, located between two AAV terminal inverted repeat sequences (ITRs), and thereby producing rAAVp, A method in which cultivation is performed at a pH value within the range of pH 7.4 to pH 7.6.

[0010] 3. The method according to Embodiment 1 or 2, wherein the mammalian cells are HEK293 cells.

[0011] 4. The method according to any one of Embodiments 1 to 3, wherein the genome titer of rAAVp produced by culturing at a pH value in the range of pH 7.4 to pH 7.6 is higher than the capsid titer of rAAVp produced by culturing at a pH value in the range of pH 7.0 to pH 7.2.

[0012] 5. The method according to Embodiment 4, wherein the genome titer is at least 1.5 times higher.

[0013] 6. The method according to any one of Embodiments 4 to 5, wherein the genome titer is at least twice as high.

[0014] 7. The method according to any one of Embodiments 4 to 6, wherein the genome titer is at least 6 times higher.

[0015] 8. The method according to any one of Embodiments 4 to 7, wherein the genome titer is at least 10 times higher.

[0016] 9. The method according to any one of Embodiments 1 to 8, wherein the capsid titer of rAAVp produced by culturing at a pH value in the range of pH 7.4 to pH 7.6 is higher than the capsid titer of rAAVp produced by culturing at a pH value in the range of pH 7.0 to pH 7.2.

[0017] 10. The method according to Embodiment 9, wherein the capsid titer is at least 1.5 times higher.

[0018] 11. The method according to any one of Embodiments 9 to 10, wherein the capsid titer is at least twice as high.

[0019] 12. The method according to any one of embodiments 9 to 11, wherein the capsid titer is at least three times higher.

[0020] 13. The method according to any one of Embodiments 1 to 12, wherein the genomic titer and capsid titer of rAAVp produced by culturing at a pH value within the range of pH 7.4 or higher and pH 7.6 or lower are higher than the genomic titer and capsid titer of rAAVp produced by culturing at a pH value within the range of pH 7.0 or higher and pH 7.2 or lower.

[0021] 14. The method according to Embodiment 13, wherein the genomic titer and capsid titer are at least 1.5 times higher.

[0022] 15. The method according to any one of Embodiments 13 to 14, wherein the genomic titer and capsid titer are at least 2 times higher.

[0023] 16. The method according to any one of Embodiments 13 to 15, wherein the genomic titer and capsid titer are at least 3 times higher.

[0024] 17. The method according to any one of Embodiments 13 to 16, wherein the genomic titer is at least 4 times higher and the capsid titer is at least 2 times higher.

[0025] 18. The method according to any one of Embodiments 13 to 17, wherein the genomic titer is at least 6 times higher and the capsid titer is at least 2 times higher.

[0026] 19. The method according to any one of Embodiments 1 to 20, wherein the percentage of complete particles of rAAVp produced by culturing at a pH value within the range of pH 7.4 or higher and pH 7.6 or lower is higher than that of rAAVp produced by culturing at a pH value within the range of pH 7.0 or higher and pH 7.2 or lower.

[0027] 20. The method according to Embodiment 19, wherein the percentage of complete particles is at least 1.5 times higher.

[0028] 21. The method according to any one of Embodiments 19 to 20, wherein the percentage of complete particles is at least 2 times higher.

[0029] 22. The method according to any one of embodiments 19 to 21, wherein the percentage of perfect particles is at least four times higher.

[0030] 23. The method according to any one of Embodiments 1 to 22, wherein rAAVp is a therapeutic rAAVp.

[0031] 24. The method according to any one of Embodiments 1 to 23, wherein rAAVp is used to transfer nucleic acids, which are transcribed into a polypeptide having a therapeutic effect, into target cells.

[0032] 25. The method according to any one of Embodiments 1 to 24, wherein rAAVp is used to transfer a therapeutic nucleic acid into target cells.

[0033] 26. The method according to any one of Embodiments 1 to 25, wherein the rAAVp comprises a recombinant adeno-associated virus particle (rAAV) having at least one coding nucleic acid sequence located between two adeno-associated virus terminal inverted repeat sequences.

[0034] 27. The method according to any one of Embodiments 1 to 26, wherein the rAAV in rAAVp is derived from wild-type AAV particles selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 2i8, AAV rh.74, AAV rh.10, and AAV 7m8.

[0035] 28. The method according to any one of Embodiments 1 to 27, wherein the rAAV is serotype AAV2 or a variant thereof.

[0036] 29. The method according to any one of Embodiments 1 to 28, wherein rAAV comprises one or two ITR sequences of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.

[0037] 30. The method according to any one of Embodiments 1 to 29, wherein culturing comprises inoculation into a bioreactor and recovery of rAAVp.

[0038] 31. The method according to any one of Embodiments 1 to 30, wherein culturing is initiated by inoculation into a bioreactor.

[0039] 32. The method according to any one of Embodiments 1 to 31, wherein one or more expression cassettes for the non-adeno-associated virus gene, adeno-associated virus rep gene, adeno-associated virus cap gene, adeno-associated virus E2A gene, adeno-associated virus E4orf6, and adeno-associated virus VA RNA gene present between two AAV ITRs are introduced into mammalian cells or HEK cells after inoculation into a bioreactor.

[0040] 33. The method according to any one of Embodiments 1 to 32, wherein one or more expression cassettes for a non-adeno-associated virus gene, an adeno-associated virus rep gene, an adeno-associated virus cap gene, an adeno-associated virus E2A gene, an adeno-associated virus E4orf6 gene, and optionally an adeno-associated virus VA RNA gene, present between two AAV ITRs, are introduced into mammalian cells or HEK cells after inoculation into a bioreactor, thereby co-transfecting up to three plasmids into the mammalian cells, thereby one of the plasmids containing an expression cassette for a non-adeno-associated virus gene present between two AAV ITRs, one of the plasmids containing an expression cassette for the rep gene and the cap gene, and one of the plasmids containing an expression cassette for the adenovirus E2A gene, the E4orf6 gene, and the VA RNA gene.

[0041] 34. The method according to any one of Embodiments 1 to 33, wherein the expression of one or more of the non-adeno-associated virus genes, adeno-associated virus rep gene, adeno-associated virus cap gene, adeno-associated virus E2A gene, adeno-associated virus E4orf6, and optionally adeno-associated virus VA RNA genes present between two AAV ITRs is induced after inoculation into a bioreactor.

[0042] 35. The method according to any one of embodiments 32 to 33, wherein the introduction occurs approximately 16 to 32 hours after inoculation into the bioreactor.

[0043] 36. The method according to Embodiment 34, wherein induction occurs approximately 16 to 32 hours after inoculation into the bioreactor.

[0044] 37. The method according to any one of embodiments 35 to 36, wherein introduction or induction occurs approximately 24 hours after inoculation into the bioreactor.

[0045] 38. The method according to any one of Embodiments 1 to 37, further comprising the steps of isolating rAAV from cells and / or culture medium after the culturing step, and optionally purifying rAAV.

[0046] 39. The method according to Embodiment 38, wherein purification is performed by one or more column chromatography steps and / or CsCl or iodixanol gradient centrifugation steps.

[0047] 40. The method according to Embodiment 38 or 39, wherein the first chromatography step is an affinity chromatography step.

[0048] 41. The method according to any one of Embodiments 38 to 40, wherein purification is performed by a series of chromatographic steps, the first being affinity chromatography, followed by anion exchange chromatography or cation exchange chromatography, and then any size exclusion chromatography.

[0049] 42. A pharmaceutical composition comprising rAAVp obtained by the method of any one of Embodiments 1 to 41.

[0050] 43. A pharmaceutical composition comprising rAAVp obtained by the method of any one of Embodiments 1 to 41, and a pharmaceutically acceptable excipient.

[0051] 44. Use of the method according to any one of Embodiments 1 to 41 to increase the yield of recombinant rAAVp.

[0052] 45. Use of the method according to any one of Embodiments 1 to 41 to increase the percentage of perfect particles in rAAVp.

[0053] In addition to the various embodiments described and claimed, the subject matter of this disclosure also covers other embodiments having other combinations of the features disclosed and claimed herein. Accordingly, specific features presented herein may be combined with each other in other ways within the scope of the subject matter of this disclosure so that the subject matter of this disclosure includes any preferred combination of the features disclosed herein. The foregoing description of specific embodiments of the subject matter of this disclosure is provided for illustrative and explanatory purposes only. It is not intended to be exhaustive or to limit the disclosed subject matter to the disclosed embodiments. [Brief explanation of the drawing]

[0054] [Figure 1] Visualization of genome titers in the recovered sample under setting 1 (recovered 120 hours after transfection, without lysis). [Figure 2] Visualization of capsid titer in the recovered sample under setting 1 (recovered 120 hours after transfection, without dissolution). [Figure 3] Visualization of the ratio of complete airtightness in the recovered sample under setting 1 (recovered 120 hours after transfection, without dissolution). [Modes for carrying out the invention]

[0055] Detailed explanation This invention is at least in part based on the finding that the productivity of mammalian cells that produce recombinant adeno-associated virus particles can be increased when the culture is performed at a high pH value such as pH 7.4–7.6.

[0056] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specifically required by context, singular terms shall include plural forms and plural terms shall include singular forms.

[0057] Useful methods and techniques for carrying out the present invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); and Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987). These contents are incorporated herein by reference.

[0058] Recombinant DNA technology enables the creation of nucleic acid derivatives. Such derivatives can be modified at individual or several nucleotide positions, for example, by substitution, alteration, exchange, deletion, or insertion. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications are readily available to those skilled in the art (see, for example, Sambrook, J., et al., Molecular Cloning: A Laboratory Manual (1999), Cold Spring Harbor Laboratory Press, New York, USA; and Hames, BD, and Higgins, SG, Nucleic Acid Hybridization - A Practical Approach (1985), IRL Press, Oxford, England).

[0059] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless otherwise explicitly stated in the context. Thus, for example, a reference to “a cell” includes multiple such cells and their equivalents known to those skilled in the art, and so on. Similarly, the terms “a” (or “an”), “one or more,” and “at least one” may be used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” may be used interchangeably.

[0060] The term "approximately" means a range of ±20% of the following number. In certain embodiments, the term "approximately" means a range of ±10% of the following number. In certain embodiments, the term "approximately" means a range of ±5% of the following number.

[0061] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” and “contain(s),” and their variations, when used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. The term “comprising” also encompasses the term “consisting of.” This disclosure also contemplates other embodiments of “comprising,” “consisting of,” and “consisting essentially of” the embodiments or elements presented herein, whether expressly described or not.

[0062] The terms “empty recombinant AAV particles” and “empty rAAV” refer to protein shells composed of adeno-associated capsid polypeptides that do not contain capsid-formed / packaged functional nucleic acids internally (rAAV = recombinant adeno-associated virus particles). That is, empty rAAVs may contain no capsid-formed nucleic acids, or nucleic acids or portions thereof that are not transcribed at all or not transcribed into a functional transcript. Therefore, empty rAAVs do not function to transfer nucleic acids that encode a functional protein or are transcribed into a functional transcript of interest into target cells. In all aspects and certain embodiments of the set, the functional protein or functional transcript of interest has a therapeutic effect.

[0063] The term "endogenous" indicates that either is naturally present, for example, within cells, or is naturally produced by cells.

[0064] The term "exogenous" means that either, for example, a nucleotide sequence, does not originate from the same entity in which it exists. For example, a nucleic acid is exogenous to a particular cell if it has been introduced into the cell by a DNA delivery method such as transfection, electroporation, or transduction. Similarly, a nucleic acid is exogenous to an AAV particle if it does not originate from the same AAV particle or serotype. Therefore, an exogenous nucleotide sequence is an artificial sequence, either in an isolated form or within a cell or rAAV, and artificiality can arise, for example, from a combination of partial sequences of different origins, e.g., a combination of a recombinase recognition sequence and the coding sequence for the SV40 promoter and green fluorescent protein, or a combination of an AAV ITR from a first serotype and a capsid polypeptide from a second serotype, or a combination of an AAV ITR and a non-AAV nucleic acid, or from a deletion of a part of a sequence, e.g., a sequence encoding only the extracellular domain of a membrane-bound receptor or cDNA, or from a mutation of a nucleic acid base in an endogenous nucleic acid sequence. This does not preclude the possibility that an "exogenous" nucleotide sequence may have an "endogenous" counterpart with the same base composition, but it also does not preclude the possibility that a sequence becomes an "exogenous" sequence through combination with exogenous regulatory elements such as exogenous secretory signals or promoters.

[0065] The terms “complete recombinant AAV particle” or “complete rAAV,” which can be used interchangeably, refer to a non-covalent complex formed from an adeno-associated capsid polypeptide and a protein shell composed of a capsidized / packaged functional nucleic acid sequence within it. That is, a complete rAAV contains a nucleic acid to be transcribed into a functional transcript. Thus, a complete rAAV functions to transfer a nucleic acid to a target cell that encodes a protein or is transcribed into a transcript of interest. In certain embodiments, the functional nucleic acid comprises at least one coding nucleic acid sequence located between two adeno-associated virus terminal inverted repeat sequences (ITRs).

[0066] The term "perfect-to-empty ratio" refers to the mathematical ratio of the number of perfect recombinant AAV particles (perfect rAAVs) to the total number of recombinant AAV particles (the sum of perfect rAAVs and empty rAAVs) in a sample or recombinant AAV particle preparation (rAAVp). Since the number of perfect rAAVs can never exceed the total number of rAAVs, the ratio is at most 1. Generally, the ratio is less than 1 and is expressed as a percentage. The number of perfect rAAVs is determined by determining the number of nucleic acid sequences present between two AAV ITRs in the sample or preparation. This can be done by PCR, particularly digital droplet PCR (ddPCR) or quantitative PCR (qPCR). The total number of rAAVs is determined by determining the number of protein shells formed by adeno-associated capsid polypeptides in the sample or preparation. This can be done by ELISA, particularly capsid protein-specific ELISA.

[0067] The term "in vitro" refers to either such an artificial environment, or the process or reaction taking place within such an artificial environment.

[0068] The term "in vivo" refers to a compound taking place in its natural environment (e.g., an animal or cell), or a process or reaction taking place within that natural environment.

[0069] The terms “recombinant AAV vector” or “transgene,” as interchangeable in this specification, mean nucleic acids derived from the wild-type genome of adeno-associated virus, in which all endogenous AAV nucleic acids, except for the ITR (adeno-associated virus terminal inversion sequence) sequence, are replaced by one or more exogenous nucleic acids. For example, such exogenous nucleic acids may be nucleic acids transcribed into a transcript of interest, or nucleic acids encoding a therapeutic protein or therapeutic nucleic acid. Typically, for recombinant AAV vectors, one or both ITR sequences of the wild-type AAV genome are retained. Thus, recombinant AAV vectors can be distinguished from wild-type AAV vectors because all or at least part of the viral genome is replaced with non-virally derived (i.e., exogenous) nucleic acids. Therefore, the incorporation of non-derived nucleic acids defines an AAV vector as a “recombinant” vector. It should be noted that the serotype of the ITR in a recombinant AAV vector does not need to be the same as the serotype of the adeno-associated capsid polypeptide that forms the shell of the recombinant AAV particle containing the recombinant AAV vector.

[0070] In principle, any non-AAV nucleic acid can be packaged into a shell consisting of an adeno-associated capsid polypeptide that results in “recombinant AAV particles” for subsequent infection (transduction) of cells, for example, ex vivo, in vitro, or in vivo.

[0071] As used herein, the term “serotype” is used to classify different wild-type and recombinant AAV particles based on the amino acid sequence of the polypeptide that forms the protein shell (capsid) of each AAV particle. Originally, serological specificity was determined based on the lack of cross-reactivity between antibodies against a given AAV particle compared to another AAV particle. Such differences in cross-reactivity are usually due to differences in the capsid polypeptide sequence and each antigenic determinant (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). AAV variants containing capsid variants differ from the reference, wild-type, or other AAV serotypes in at least one amino acid residue, even though they may not be serologically distinguishable from the reference AAV, wild-type AAV, or other AAV serotypes.

[0072] Under the conventional definition, a serotype means that the virus of interest has been tested against serums specific to all existing and characterized serotypes for neutralizing activity, and no antibodies neutralizing the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid variants are generated, they may or may not be serologically different from any of the currently existing serotypes. Therefore, if a new AAV particle has no serological differences, this new AAV particle is a subgroup or variant of the corresponding wild-type serotype. Often, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are other serotypes according to the conventional definition of serotypes.

[0073] The term "vector" refers to a larger nucleic acid, such as a recombinant plasmid, that is ultimately packaged, encapsulated, or capsid-formed into a protein shell composed of adeno-associated virus capsid polypeptide, either directly, in single-stranded form, or in RNA form, in order to form recombinant AAV particles. When recombinant AAV particles are constructed or manufactured using a recombinant plasmid, the viral particles do not contain any "plasmid" portion that does not correspond to the vector portion of the recombinant plasmid. For example, in the case of rAAV, the recombinant vector contains that portion of the recombinant plasmid that lies between two AAV ITRs. The non-vector portion of the recombinant plasmid is called the "plasmid backbone." The plasmid backbone is essential for the processes required for plasmid cloning and amplification, proliferation, and recombinant virus production, but it is not packaged, encapsulated, or capsid-formed into recombinant AAV particles by itself. Therefore, "vector" refers to the nucleic acid that is packaged, encapsulated, or capsid-formed into rAAV by a protein shell composed of adeno-associated virus capsid polypeptide.

[0074] General method for generating rAAV particles International Publication No. 1999 / 11764 reported a method for preparing high-titer, helper-free preparations of recombinant AAV vectors. Undefined AAV producer cells, grown in a suspension in a bioreactor, were infected with adenovirus type 5 (Ad5) at a multiplicity of infection (MOI) of 10 in 1.5 L volumes of low-serum medium at different pH values. Total particles of 4.7 E+12 were obtained at culture pH 7.2, 1.95 E+13 at culture pH 7.4, 1.84 E+13 at culture pH 7.6, and 1.63 E+13 at culture pH 8.0. The culture was performed in a 1.5 L bioreactor, and the culture volume (75% of the nominal value) can be calculated to be approximately 1.125 L. Therefore, the total particle counts correspond to 4.2 E+09vp / mL (pH 7.2), 1.7 E+10vp / mL (pH 7.4), 1.6 E+10vp / mL (pH 7.6), and 1.5 E+10vp / mL (pH 8), respectively.

[0075] International Publication No. 2000 / 14205 reports the generation of AAV particles in an undefined cell type, referred to as JL-14 cells, upon co-infection with adenovirus helper virus, thereby obtaining the highest number of AAV particles (total of intracellular and secreted AAV particles) at a pH of 7.4, the highest infectivity of AAV particles at a pH of 8, and the highest ratio of the number of AAV particles to infectivity at a pH of 7.6. Since the culture was performed in 1.5 L of medium, the total particle counts correspond to 3.0 E+09 vp / mL (pH 7.2), 1.3 E+10 vp / mL (pH 7.4), 1.2 E+10 vp / mL (pH 7.6), 3.3 E+9 vp / mL (pH 7.8), and 1.1 E+10 vp / mL (pH 8), respectively. Based on the provided infectivity data, it can be assumed that the complete / empty ratio of the rAAV particles generated is less than 1%.

[0076] This is based on the following calculations from the data in Figures 2B and 3B of International Publication No. 2000 / 14205 (3 days after infection): TIFF2026511015000001.tif57128TIFF2026511015000002.tif61137

[0077] Piras, BA, et al. (Mol.Ther.Meth.Clin.Dev.3(2016)16015) compared the distribution of AAV8 in cell culture media and lysates on days 3, 5, 6, and 7 after transfection, finding an increase in viral production up to day 6, with the percentage of viral particles in the medium increasing from 76% on day 3 to 94% on day 7. Large-scale production showed that the ratio of fully airtight AAV particles was similar in the medium and lysates, and that AAV recovered on day 6 after transfection provided similar functions in mice compared to AAV recovered on day 3. AAV-FVIII showed increased production when the culture was extended from day 3 (total capsids of 1.1 × 1 E+13 ± 9.2 × 1 E+11 and 3.6 × 1 E+13 ± 2.5 × 1 E+12 in lysate and medium, respectively) to day 5 (total capsids of 6.7 × 1 E+12 ± 6.7 × 1 E+11 and 4.5 × 1 E+13 ± 2.6 × 1 E+12 in lysate and medium, respectively), day 6 (total capsids of 5.0 × 1 E+12 ± 1.9 × 1 E+11 and 5.3 × 1 E+13 ± 3.3 × 1 E+12 in lysate and medium, respectively), and day 7 (total capsids of 3.0 × 1 E+12 ± 1.3 × 1 E+10 and 5.0 × 1 E+13 ± 1.9 × 1 E+12 in lysate and medium, respectively). Piras et al. used adherent HEK293T / 17 cells cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum supplemented with 2 mmol / l GlutaMAX (Life Technologies, Grand Island, NY). The cell density was 7.26 × 1 E+04 cells / cm². 2 AAV was generated one day after seeding cells at a density of [density] by two plasmid transfections using PEIpro® (Polyplus-transfection SA, Illkirch, France).

[0078] Powers, AD, et al. (Hum. Gene Ther. Meth. 27 (2016) 112-121) reported the development and optimization of AAV hFIX particle generation by transient transfection in an iCELLis(R) fixed-bed bioreactor. Yields of up to 9 E+14 viral particles per square meter of fixed bed were obtained. Three days after inoculation of HEK293T / 17 cells, containers were transfected with plasmid scAAV-LP1-hFIXco-helpv3 and plasmid CR21+LTAAVhelp 2-8, respectively, using polyethyleneimine (PEIpro(trademark) Transfection Reagent catalog no. 115-375; Polyplus) in IMDM (Lonza) or DMEM supplemented with 10% FBS and 6 mM GlutaMAX(trademark). PEI and DNA solutions were combined in a 2:1 ratio.

[0079] Poulain, A., et al. (j. Biotechnol. 255 (2017) 16-27) reported rapid protein production from a stable CHO cell pool using plasmid vectors and chemate gene switches. Cells were transfected using linear polyethyleneimine (PEIpro®) from Polyplus-Transfection (Illkirch, France). On the day of transfection, cells were suspended at a density of 2 × 1 E+06 cells / mL in CD DG44 medium (Life Technologies Inc., Burlington, ON, Canada) supplemented with 4 mM glutamine and 0.1% Kolliphor® P 188. The cell suspension was distributed into 6-well plates (1.8 mL / well). DNA:PEIpro® complexes were prepared in a 1:5 (w:w) ratio using a total of 2 μg of DNA per well for transfection in 100 μL of complete culture medium.

[0080] International Publication No. 2017 / 096039 reported a scalable method for generating recombinant AAV vectors in serum-free suspension cell culture systems suitable for clinical use. rAAV vector generation was performed in a bioreactor containing HEK293F cells at a temperature of 37°C and a pH of 7.2, with a cell density of 1 E+06 cells / mL (1,000,000 cells / mL), using a plasmid ratio of 1:1:1 and a PEI-based transfection reagent (PEI / DNA weight ratio of 2:1, with half of the PEI being free PEI).

[0081] Nyamay'antu, A., et al. (Cell Gen.Ther.Ins. 4(2018)71-79) reported that PEI is widely used in both adherent and suspension cells grown in serum-free medium due to its availability and high DNA delivery efficiency. PEIpro® is suitable for small to large-scale generation of various viruses, particularly AAV particles. In a stirred-tank bioreactor using HEK293 or HEK293T cells, titers in the range of 0.8–1.5 E+09–E+10 vg / mL can be obtained.

[0082] Koo, T., et al. (Nat.Commun. 9(2018)1855) reported that CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration. To generate AAV vectors, they were pseudotyped in the AAV9 capsid. HEK293T cells (ATCC, CRL-3216) were transfected with pAAV-ITR-LbCpf1-crRNA, pAAV2 / 9 encoding AAV2rep and AAV9cap, and a helper plasmid. HEK293T cells were cultured in DMEM containing 2% FBS. Recombinant pseudotyped AAV vector stocks were prepared using PEI co-precipitation with PEIpro® (Polyplus transfection) and triple transfection with plasmids in a 1:1:1 molar ratio in HEK293T cells. After 72 hours of incubation, the cells were lysed and the particles were purified by iodixanol-step gradient ultracentrifugation.

[0083] Rep proteins derived from AAV2 are commonly and almost exclusively used in the production of rAAVs derived from serotypes AAV1-AAV13 (Daya, S., and Berns, KI, Clin. Microbiol. Rev. 21 (2008) 583-593; Zincarelli, C., et al., Mol. Ther. 16 (2008) 1073-1080).

[0084] International Publication No. 2019 / 094253 reported means and methods for preparing viral vectors and their use. Adherent HEK293 cells were cultured in a bioreactor at a pH of 7.23 and triple-transfected with PEI / DNA at a PEI-plasmid ratio of approximately 1:1 by weight (plasmid ratio 1:1:1).

[0085] Collaud, F. et al. (Mol.Ther.Meth.Clin.Dev.12(2019)157-174) reported the titers of rAAV8 particles at 6.0±1.89 E+04vg / cell and 1.77±1.37 E+04vg / cell for (single-stranded) and (self-complementary) AAV, respectively, in adherent HEK293 cells. A fully scalable method based on triple transfection of HEK293 cells cultured in suspension was also reported. Triple transfection of HEK293 cells was performed directly in a 10 L bioreactor using polyethyleneimine (PEIpro®, Polyplus). The AAV vector was recovered from both the supernatant and cells by mild surfactant lysis followed by AVB Sepharose affinity column purification. The purified vector was then concentrated and tested for quality and potency. Information regarding pH values ​​and obtained titers is not provided.

[0086] Nyamay'antu, A., et al. (Cell Gen.Ther.Ins. 6(2020) 655-661) reported that the efficiency of the delivery process is essential for obtaining a large number of producible cells. Among existing transfection methods, the use of PEI-based transfection reagents is dominant in gene therapy because it combines the availability and suitability for transfection of adherent and suspension cells. Compared to PEIpro®, the gold standard used in viral vector production, FectoVIR®-AAV was found to significantly improve both the rAAV2 production yield of rAAV2-GFP in suspension cells, up to 10-fold compared to PEIMax® and up to 2-fold compared to PEIpro®, respectively, when each transfection reagent was used under recommended conditions. More specifically, suspension HEK293T cells were transfected using each transfection reagent under recommended conditions. rAAV2-GFP was harvested 72 hours after transfection. The titers obtained with VectoVIR™ range from 1 E+04 to 4.5 E+04 vg / cell, depending on the volume of complexation used (1% to 10%) equivalent to 1 E+12 vg / mL. Each functional titer is approximately 2 to 8 E+08 TU / mL. The results are largely independent of the culture medium used.

[0087] In a blog post titled "Optimization of AAV production for high-yield and scalable GMP processes with Catalent" (www.polyplus-transfection.com), different transfection reagent ratios to DNA were tested using two serotypes of AAV9 (1:1 and 2:1) and AAV2 (3:1.5 and 5:2.5). The yield of the AAV2 vector was not significantly affected by the 4-5-fold increase in vector genome titer and the 3-6-fold increase in viral particle titer with FectoVIR®-AAV compared to PEIpro®. These results indicate that yield improvements may vary depending on the AAV serotype. Further studies comparing additional AAV2 and AAV5 vectors (different from the previously mentioned AAV2 and AAV5 vectors) and optimizing them using a DoE approach involved experiments with various transfection reagent-to-DNA ratios (3:2, 3:1.5) and plasmid DNA molar ratios (1:1:1, 2:1:2, 1:2:1). Compared to PEIpro®, FectoVIR®-AAV-AAV resulted in a 3- to 5-fold increase in vector genome titer for AAV2 and a 1.1- to 1.6-fold increase for AAV5. Viral particle titers increased 3.5- to 4.5-fold for AAV2 and 2.5- to 3.75-fold for AAV5. Reagent-to-DNA ratios of 2:1 and 1.5:1 and plasmid ratios of 1:1:1, 2:1:2, and 1:2:1 were used. Titers obtained with VectoVIR® ranged from 4 E+11 to 1 E+12 vg / mL.

[0088] Rossi, A. and Peigne, C. (Cell Culture Dish Article May 17, 2021) outlined that typically, AAV production titers are approximately 1 E+11–1 E+12 (vg / mL) and 1 E+08–1 E+09 TU / mL.

[0089] When looking at the numbers as a whole, it is important to note that the two AAV serotypes are most likely not to yield the same yield even when using the same production process.

[0090] More specifically, AAV production yield varies depending on the target serotype and gene. Generally, to increase the production of a given AAV, parameters that directly affect the yield, namely plasmid DNA, transfection reagents, cells, and culture medium, are optimized. PEI-based transfection processes can, for example, reduce the amount of DNA by a factor of ten and can be used to transfect cells grown in or without serum.

[0091] Wosnitzka, K., et al. (Cell Gen.Ther.Ins.7(2021)1-7) reported that physical titer analysis revealed a threefold increase in both viral particles (VP) and viral genome (VG) per ml of cell culture when using the FectoVIR®-AAV transfection reagent compared to PEIpro®.

[0092] Porte, M., et al. (poster entitled "Next-Generation Transfection Reagent for Large Scale AAV Manufacturing," Polyplus, Illkirch, France) reported the transfection of suspension-HEK293T cells under optimal conditions for other PEI-based reagents (1.5 μg / 1 million cells, DNA:PEI ratio of 1 μg:4 μL) and FectoVIR®-AAV (1 μg / 1 million cells, DNA:reagent ratio of 1 μg:1 μL), following the recommended protocols for each reagent. Using FectoVIR® and PEI-based transfection reagents, titers of approximately 5 E+11 vg / mL and 1.5 E+11 vg / mL were obtained, respectively, with packaging efficiencies of approximately 20% vs. 13.5%, respectively.

[0093] Recombinant cells Generally, for the efficient and large-scale production of rAAV, cells that express and, if possible, secrete the rAAV particles are used. Such cells are called "recombinant producer cells" or short "producer cells".

[0094] To generate recombinant producer cells, appropriate mammalian cells are transfected with nucleic acids necessary for generating rAAV, including the required AAV helper function.

[0095] The expression of a coding sequence, i.e., an open reading frame, requires additional regulatory elements such as a promoter and a polyadenylation signal (sequence). Therefore, the open reading frame must be operably linked to these additional regulatory elements for functional transcription. This can be achieved by incorporating these parts into a so-called expression cassette. The minimum regulatory elements required for an expression cassette to be functional in mammalian cells are a promoter functional in the mammalian cell, located upstream (i.e., at the 5' end) of the open reading frame, and a polyadenylation signal (sequence) functional in the mammalian cell, located downstream (i.e., at the 3' end) of the open reading frame. Furthermore, a terminator sequence may be present at the 3' end of the polyadenylation signal (sequence). For expression, the promoter, open reading frame / coding region, and polyadenylation signal sequence must be arranged in an operably linked form.

[0096] Similarly, nucleic acids transcribed into non-protein-coding RNA are called "RNA genes." RNA gene expression also requires additional regulatory elements, such as promoters and transcription termination signals or polyadenylation signals (sequences). The nature and localization of these elements depend on the RNA polymerase intended to drive RNA gene expression. Therefore, RNA genes are typically incorporated into expression cassettes as well.

[0097] In the case of rAAV, which is composed of different (monomer) capsid polypeptides and the single-stranded DNA molecules that form the capsid within them, and which also requires other viral helper functions for generation and capsid formation, a number of expression cassettes with different open reading frame / coding sequences are required. In this case, at least an expression cassette is required for each transgene relating to the polypeptide that forms the rAAV capsid for the required viral helper function. Therefore, at least individual expression cassettes are required for each of the helper function E1A, E1B, E2A, E4orf6, rep, and cap genes. HEK293 cells constitutively express E1A and E1B helper functions.

[0098] Adeno-associated virus (AAV) For a general review of AAV and the helper functions of adenoviruses or herpes, see Berns and Bohensky, Advances in Virus Research, Academic Press, 32(1987)243-306. The AAV genome is described in Srivastava et al., J. Virol., 45(1983)555-564. U.S. Patent No. 4,797,368 describes design considerations for constructing recombinant AAV vectors (see also International Publication No. 93 / 24641). Further references describing AAV vectors are West et al., Virol. 160(1987)38-47; Kotin, Hum. Gene Ther. 5(1994)793-801; and Muzyczka J. Clin. Invest. 94(1994)1351. The construction of recombinant AAV vectors is described in U.S. Patent No. 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8 (1988) 3988-3996; Tratschin et al., Mol. Cell. Biol. 5 (1985) 3251-3260; Tratschin et al., Mol. Cell. Biol. 4 (1994) 2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81 (1984) 6466-6470; Samulski et al. J. Virol. 63 (1989) 3822-3828.

[0099] AAV is a replication-deficient parvovirus. It can only replicate in cells where specific viral function is provided by co-infecting helper viruses such as adenoviruses, herpesviruses, and, in some cases, poxviruses such as vaccinia. Nevertheless, AAV can replicate in substantially any cell line of human, monkey, or rodent origin, provided that appropriate helper viral function is present.

[0100] If the helper virus gene is absent, AAV establishes a latent period in its host cell. Its genome is integrated into a specific site on chromosome 19 [(Chr)19(q13.4)], called adeno-associated virus integration site 1 (AAVS1). For certain serotypes, such as AAV2, other integration sites have been found, such as on chromosome 5 [(Chr)5(p13.3)] called AAVS2 and on chromosome 3 [(Chr)3(p24.3)] called AAVS3.

[0101] AAV is classified into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 12 different serotypes and more than 100 sequences corresponding to different clades of AAV have been identified.

[0102] The type and symmetry of the capsid protein determine the tissue tropism of each AAV. For example, AAV2, AAV4, and AAV5 are retinal specific; AAV2, AAV5, AAV8, AAV9, and AAV-rh.10 are brain specific; AAV1, AAV2, AAV6, AAV8, and AAV9 are cardiac tissue specific; AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10 are liver specific; and AAV1, AAV2, AAV5, and AAV9 are lung specific.

[0103] Pseudotyping involves the cross-packaging of AAV genomes across different serotypes, meaning the genome is packaged with capsid proteins of different origins.

[0104] The wild-type AAV genome is approximately 4.7 kb in size. The AAV genome further contains two duplicate genes called rep and cap, which contain multiple open reading frames (see, e.g., Srivastava et al., J. Viral., 45(1983) 555-564; Hermonat et al., J. Viral. 51(1984) 329-339; Tratschin et al., J. Viral., 51(1984) 611-619). The Rep proteins, which encode the open reading frames, provide four proteins of different sizes called Rep78, Rep68, Rep52, and Rep40. These are involved in AAV replication, rescue, and integration. The open reading frames, which encode the Cap proteins, provide four proteins called VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the proteinaceous capsid of the AAV particle. The combined open reading frames of rep and cap are flanked at their 5' and 3' ends by so-called terminal inversion repeat sequences (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of adenovirus genes E1A, E1B, E4orf6, E2A, and VA, or corresponding factors from another helper virus.

[0105] For example, in the case of AAV serotype 2 (AAV2), each ITR is 145 nucleotides long and flanks a coding region of approximately 4470 nucleotides. Of the 145 nucleotides in the ITR, 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure functions as a primer during viral replication. The remaining 20 unpaired nucleotides are shown as the D sequence.

[0106] The wild-type AAV genome possesses three transcription promoters, P5, P19, and P40, for the expression of rep and cap genes (Laughlin et al., Proc. Natl. Acad. Sci. USA 76(1979) 5567-5571).

[0107] ITR sequences must be cis relative to the coding region. ITRs provide functional origins of replication (ori), signals necessary for integration into the target cell genome, and efficient excision and rescue from host cell chromosomes or recombinant plasmids. ITRs further contain origin-of-replication-like elements such as Rep protein binding sites (RBS) and terminal dissociation sites (TRS). It has been found that ITRs themselves can function as transcription promoters (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0108] The replication of the viral single-stranded DNA genome and the formation of the capsid both require trans-organization of the rep gene product and the cap gene product, respectively.

[0109] The rep locus contains two internal promoters called P5 and P19. It contains open reading frames for four proteins. Promoter P5 is operably ligated to nucleic acid sequences that provide a 4.2kb unsplicing RNA encoding the Rep protein Rep78 (a chromatin niccasse for cell cycle arrest) and a 3.9kb splicing mRNA encoding the Rep protein Rep68 (a site-specific endonuclease). Promoter P19 is operably ligated to nucleic acid sequences that provide a 3.3kb unsplicing mRNA encoding the Rep protein Rep52 and a 3.3kb splicing mRNA encoding the Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0110] The two larger Rep proteins, Rep78 and Rep68, appear to be essential for AAV double-stranded DNA replication, while the smaller Rep proteins, Rep52 and Rep40, seem to be essential for offspring and single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173(1989)120-128).

[0111] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit predetermined enzymatic activity required to degrade replication at the AAV terminus. Expression of Rep78 or Rep68 may be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68(1994)7169-7177 and 69(1995)6880-6885).

[0112] All Rep proteins, mainly Rep78 and Rep68, are thought to exhibit regulatory activity such as induction and repression of AAV genes and inhibitory effects on cell growth (Tratschin et al., Mol.Cell.Biol.6(1986)2884-2894; Labow et al., Mol.Cell.Biol.,7(1987)1320-1325; Khleif et al., Virology,181(1991)738-741).

[0113] Recombinant overexpression of Rep78 results in a phenotype characterized by reduced cell proliferation due to the induction of DNA damage. This causes host cells to arrest in the S phase, thereby promoting latent viral infection (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102(2005)13634-13639).

[0114] Tratschin et al. reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, very low expression has been reported in certain cell lines after stable incorporation of AAV (see, e.g., Mendelson et al., Virol. 166 (1988) 154-165).

[0115] The cap locus contains a single promoter called P40. Promoter P40 is operably linked to a nucleic acid sequence providing 2.6 kb of mRNA encoding the Cap proteins VP1 (87 kDa, unspliced ​​mRNA transcript), VP2 (72 kDa from spliced ​​mRNA transcript), and VP3 (61 kDa from alternative start codon) through alternative splicing and the use of an alternative start codon. VP1-VP3 constitute the building blocks of the viral capsid. The capsid has the function of binding to cell surface receptors and enabling intracellular transport of the virus. VP3 accounts for approximately 90% of the total viral particle proteins. Nevertheless, all three proteins are essential for effective capsid formation.

[0116] Inactivation of all three capsid proteins VP1-VP3 has been reported to prevent the accumulation of single-stranded progeny AAV DNA. Mutations at the amino terminus of VP1 ("Lip-negative" or "Inf-negative") still allow the assembly of single-stranded DNA into the viral particle, thereby significantly reducing the infectivity titer.

[0117] The AAP open reading frame encodes the assembly activation protein (AAP). It is approximately 22 kDa in size and transports the native VP protein to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein-coding sequence.

[0118] Each AAV particle contains only single-stranded DNA molecules. These can be either the "positive" or "negative" strand. AAV particles containing DNA molecules are infectious. Inside an infected cell, the parental infectious single-stranded DNA is converted to double-stranded DNA and then amplified. Amplification results in a large pool of double-stranded DNA molecules from which single strands are substituted and packaged into a capsid.

[0119] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. Transgenes introduced into target cells using AAV vectors are thought to be expressed for extended periods. One drawback of using AAV vectors is the limited size of the transgenes that can be introduced into cells.

[0120] Parvovirus particles, including AAV serotypes and their variants, provide a means for delivering protein-encoding nucleic acids to cells ex vivo, in vitro, and in vivo, so that infected cells express the encoded proteins. AAV is a useful virus as a gene therapy vector because it can penetrate cells and introduce nucleic acids / genetic material so that the nucleic acids / genetic material can be stably maintained within the infected cells. Since AAV is not associated with pathogenic disease in humans, AAV can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and drugs) to human patients without causing substantial AAV-related pathogenesis or disease.

[0121] AAV particles, used as vehicles for effective gene delivery, possess several desirable characteristics for such applications, including directivity to dividing and non-dividing cells. Early clinical experience with these vectors has shown no persistent toxicity and minimal or undetectable immune responses. AAVs are known to infect a wide variety of cell types in vivo and in vitro via receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0122] Recombinant AAV particles typically do not contain the viral genes associated with the pathogenesis. Such particles typically have one or more wild-type AAV genes, e.g., the rep and / or cap genes, deleted entirely or partially, but retain at least one functional flanking ITR sequence as needed for the rescue, replication, and packaging of the recombinant vector into rAAV. Therefore, the AAV vector contains the sequences necessary for cis (i.e., functional ITR sequences) for replication and packaging.

[0123] Recombinant AAV particles, as well as methods and uses thereof, may be based on any wild-type AAV genome or serotype or combination thereof. As a non-limiting example, rAAVs may be based on any wild-type AAV genome and contain various ITR sequences, such as AAV1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, rh.74, rh.10, or 7m8. Such particles may be based on the same strain or serotype (or subgroup or variant) or may be different from one another. As a non-limiting example, rAAVs based on one wild-type genome may be identical or different from one or more of the capsid proteins packaging the vector. Furthermore, recombinant AAV vectors may be based on AAV (e.g., AAV2) wild-type serotype genomes that are different from one or more of the AAV capsid proteins packaging the vector. For example, an AAV vector may be based on AAV2, but at least one of the three capsid proteins may be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10, or AAV-7m8 or a variant thereof. Examples of AAV variants include variants and chimeras of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10, and AAV-7m8 capsids.

[0124] In all aspects and certain embodiments of the present invention, the rAAV particles are derived from wild-type AAV particles selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10, and AAV 7m8, as well as their variants (e.g., capsid variants, e.g., insertions, additions, substitutions, and deletions of amino acids), as described, for example, in International Publication No. 2013 / 158879, International Publication No. 2015 / 013313, and U.S. Patent Application Publication No. 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0125] In all aspects and certain embodiments of the present invention, rAAV includes a capsid polypeptide having an amino acid sequence having 70% or more sequence identity with the wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.10, AAV-rh.74, or AAV-7m8 capsid sequence.

[0126] In all aspects and certain embodiments of the present invention, the rAAV particles contain one or two ITR sequences having 70% or more sequence identity with wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 ITR sequences.

[0127] Recombinant AAV particles may be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for administration and delivery to subjects in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical that does not induce an adverse immune response in the individual receiving the composition and can be administered without excessive toxicity.

[0128] Protocols for the production of adenovirus vectors are described in U.S. Patent Nos. 5,998,205; 6,228,646; 6,093,699; 6,100,242; International Publication Nos. 94 / 17810 and International Publication Nos. 94 / 23744, which are incorporated herein by reference in their entirety.

[0129] Recombinant adeno-associated virus particles (rAAV particles) Numerous methods for generating recombinant AAV particles are known in the art. For example, transfection using AAV plasmids and AAV helper sequences (rep and cap) in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpesvirus, or vacciniavirus), or transfection with recombinant AAV plasmids, AAV helper plasmids, and helper functional plasmids. Non-limiting methods for generating rAAV particles are described, for example, in U.S. Patent No. 6,001,650, U.S. Patent No. 6,004,797, International Publication No. 2017 / 096039, and International Publication No. 2018 / 226887. After rAAV generation (i.e., particle generation in a cell culture system), rAAV can be obtained and purified from host cells and / or cell culture supernatant.

[0130] The generation of recombinant AAV particles requires the expression of Rep and Cap proteins, helper proteins E1A, E1B, E2A, and E4orf6, and optionally adenovirus VA RNA, in a single mammalian cell. Helper proteins E1A, E1B, E2A, and E4orf6 can be expressed using any promoter, particularly the CMV IE promoter, as shown by Matsushita et al. (Gene Ther. 5(1998) 938-945). Thus, any promoter can be operably ligated to the genes for functional expression.

[0131] Generally, different complementary plasmids are co-transfected into host cells to generate rAAV. One of the plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The missing AAV elements necessary for replication and subsequent packaging of the progeny recombinant genome, namely the open reading frames of the Rep and Cap proteins, are included trans on the second plasmid. Overexpression of the Rep protein results in an inhibitory effect on cell proliferation (Li, J., et al., J. Virol. 71 (1997) 5236-5243). Furthermore, a third plasmid containing helper virus genes, namely adenovirus-derived E1, E4orf6, E2A, and VA, is required for AAV replication.

[0132] To reduce the number of plasmids required, the rep, cap, and adenovirus helper genes may be combined on a single plasmid.

[0133] Alternatively, the host cell may already be stably expressing the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone, designated as 293, was created in 1977 by incorporating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, FL, et al., J. Gen. Virol. 36(1977) 59-74). The HEK293 cell line contains base pairs 1-4344 of the adenovirus serotype 5 genome. This includes the E1A and E1B genes as well as the adenovirus packaging signal (Louis, N., et al., Virology 233(1997) 423-429).

[0134] When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with E2A, E4orf6, and VA expression plasmids (e.g., Samulski, RJ, et al., J.Virol. 63 (1989) 3822-3828; Allen, JM, et al., J.Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum.Gene Ther. 7 (1996) 507-513; Flotte, TR, et al., Gene Ther. 2 (1995) 29-37; Conway, JE, et al., J.Virol. 71 (1997) 8780-8789; Chiorini, JA Ther.6(1995)1531-1541;Ferrari,FK,et al.,J.Virol.70(1996)3227-3234;Salvetti,A.,et al.,Hum.Gene Ther.9(1998)695-706;Xiao,X.,et al., J. Virol. 72 (1998) 2224-2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors may be used (see, for example, Conway, JE, et al., J. Virol. 71 (1997) 8780-8789; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, AJ, et al., Gene Ther. 2 (1995) 481-485; Fisher, JK, et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370).

[0135] To restrict the activity of a transgene to a specific tissue, i.e., to restrict the site of action, the transgene can be operably linked to an inducible promoter or a tissue-specific promoter (see, for example, Yang, Y., et al. Hum. Gene. Ther. 6(1995) 1203-1213).

[0136] The coding sequences for E1A and E1B (open reading frames) may be derived from human adenoviruses, such as human adenovirus serotype 2 or serotype 5. An exemplary sequence for human Ad5 (adenovirus serotype 5) is found in GenBank entry X02996, AC_000008, and an exemplary sequence for human Ad2 is found in GenBank entry AC_000007. Nucleotides 505-3522 contain the nucleic acid sequences encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146, reported in European Patent No. 1230354, and plasmids pGS119 and pGS122, reported in International Publication No. 2007 / 056994, can also be used as sources for E1A and E1B open reading frames.

[0137] E1A is the first viral helper gene expressed after adenovirus DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins based on the same E1A mRNA through alternative splicing. Expression of 12S and 13S proteins leads to the activation of other viral functions E1B, E2, E3, and E4. Furthermore, expression of 12S and 13S proteins pushes the cell into the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cell dies (apoptosis).

[0138] E1B is the second viral helper gene that is expressed. It is activated by E1A-derived proteins 12S and 13S. E1B gene-derived mRNA can be spliced ​​in two different ways, resulting in a first 55kDa transcript and a second 19kDa transcript. The E1B 55kDa protein is involved in regulating the cell cycle, preventing the transport of cellular mRNA in the later stages of infection, and preventing E1A-induced apoptosis. The E1B 19kDa protein is involved in preventing E1A-induced apoptosis in cells.

[0139] The E2 gene codes for various proteins. The E2A transcript codes for single-strand binding proteins (SSBPs), which are essential for AAV replication.

[0140] The E4 gene also codes for several proteins. The 34kDa protein derived from the E4 gene (E4orf6), along with the E1B 55kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the cell nucleus to the cytoplasm.

[0141] Virus-associated RNA (VA RNA) is the non-coding RNA of adenoviruses (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed from the type II polymerase III promoter by RNA polymerase III (e.g., Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289). For the generation of recombinant AAV particles, the adenovirus VA RNA gene can be driven by any promoter.

[0142] The structure, function, and evolution of adenovirus-associated RNAs using a phylogenetic approach were investigated by Ma, Y. and Mathews, MB (J. Virol. 70(1996) 5083-5099). They provided alignment and consensus VA RNA sequences based on 47 known human adenovirus serotypes. This disclosure is incorporated in its entirety by reference.

[0143] VA RNA, VAI, and VAII consist of 157 to 160 nucleotides (nt).

[0144] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is thought to play the role of the dominant provirus, while VA RNAII may partially compensate for the absence of VA RNAI (Vachon, VKand Conn, GL, Virus Res. 212 (2016) 39-52).

[0145] VA RNA is not essential, but it plays a crucial role in efficient viral replication by overcoming the cell's antiviral mechanisms. In other words, although VA RNA is not essential for viral replication, VA RNA-deficient adenoviruses may not be able to replicate during the initial stages of vector generation, when only a few copies of the viral genome exist per cell, because other viral genes that block the cell's antiviral mechanisms may not be sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0146] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient generation of an adenovirus vector lacking the gene for virus-associated RNA that disrupts the cellular RNAi mechanism. In this study, HEK293 cells constitutively and highly expressing flipper jelly combinase were infected, and VA RNA-deficient adenoviruses were obtained by FLP recombinase-mediated excision of the VA RNA locus.

[0147] Human adenovirus 2 VA RNAI corresponds to nucleotides 10586-10810 of the GenBank entry AC_000007 sequence. Human adenovirus 5 VA RNAI corresponds to nucleotides 10579-10820 of the GenBank entry AC_000008 sequence.

[0148] General explanation of recombinant AAV particle generation After entering the host cell nucleus, AAV can follow one of two different, interchangeable life cycles: lytic or lysogenic. The former occurs in cells infected with helper viruses such as Ad or herpes simplex virus (HSV), while the latter is established in host cells in the absence of helper viruses.

[0149] When latent infected cells become over-infected with the helper virus, the AAV gene expression program is activated, leading to AAV Rep-mediated rescue (i.e., excision) of proviral DNA from the host cell chromosome, followed by replication and packaging of the viral genome. Finally, newly assembled virions (particles) are released during helper virus-induced cell lysis. Thus, the lysis phase of the AAV life cycle is induced.

[0150] Therefore, in the presence of Ad helper function, the rAAV vector is rescued from the plasmid backbone, replicated, and packaged as a single-stranded molecule into a pre-formed AAV capsid, thereby being subjected to the wild-type AAV lysis process (Goncalves, MAFV, Virol.J.,2(2005)43).

[0151] The production of recombinant AAV particles involves replacing a large portion of the wild-type AAV genome with a desired transgene, providing the viral genes essential for transpackaging the virus onto a separate plasmid. Once all components are transfected together into a packaging cell line, the recombinant AAV particles are assembled using the cell's cellular mechanisms. The viral assembly and encapsulation process takes approximately two days, after which the cells are lysed to release rAAV for further purification and enrichment (https: / / old.abmgood.com / marketing / knowledge_base / Adeno_Associated_Virus_Production_and_Modification_of_AAV.php).

[0152] AAVs are not released very efficiently from cells, but significant differences have been observed between serotypes (see, for example, Strobel, B., et al., Lamla T. Comparative Analysis of Cesium Chloride-and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications. Hum. Gene Ther. Methods 26(2015) 147-157). When recovering cultures, cell disruption is usually applied to recover the vectors captured by the cells.

[0153] Historically, rAAV production was carried out by double transfection of plasmids containing rep and cap ORFs with plasmids containing the target gene flanked by the ITR. This was followed by co-infection with a helper virus, typically adenovirus (see, e.g., Aponte-Ubillus, JJ, et al., Appl. Microbiol. Biotechnol. 102(2018) 1045-1054; Muzyczka, N., Curr. Top. Microbiol. Immunol. 158(1992) 97-129). In this context, isolation of the helper virus from the final product was difficult, but it was an important factor in avoiding induction of an inflammatory response after injection into the patient (see, e.g., Schnell, MA, et al., Mol. Ther. 3(2001) 708-722). Therefore, the generation of rAAV has now shifted towards adenovirus-free approaches by utilizing triple transfection (see, for example, Large, EE, et al., Viruses 13(2021)1336). For this purpose, three components are required: one plasmid encoding the Rep and Cap genes without ITR, a second plasmid containing the desired transgene adjacent to the ITR, and a helper plasmid to provide the helper gene for the helper virus (see, for example, Aponte-Ubillus, JJ, et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Farris, K. D. and Pintel, DJ, Hum. Gene Ther. 19 (2008) 1421-1427; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Ferrari, FK, et al., Nat. Med. 3 (1997) 1295-1297). For example, the adenovirus helper has the minimum necessary adenovirus genes E2A, E4, and VA. It is important to note that human embryonic kidney cell 293 (HEK293) constitutively expresses the adenovirus gene E1A / B, which is also required for rAAV generation.Therefore, HEK293 cells are the classic producer cells for rAAV and its production. Other cell types require E1A / B supplementation.

[0154] Carter et al. demonstrated that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, BJ, in "Handbook of Parvoviruses", ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Furthermore, it has been reported that the ITR must be maintained to preserve the functions of replicating, rescuing, packaging, and incorporating the transgene into the target cell genome.

[0155] When cells containing each viral helper gene are transduced by an AAV vector, or vice versa, when cells containing the incorporated AAV provirus are transduced by the appropriate helper virus, the AAV provirus is activated and enters the lytic infection cycle again (Clark, KR, et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0156] Producer cells contain rep and cap gene sequences, as well as a transgene cassette adjacent to one or more ITR sequences on plasmids, which are retained, for example, via drug selection. rAAV production in these cell lines generally occurs after their infection with the necessary helper function. Therefore, cells are infected with either a replicating-competent adenovirus (usually wild-type Ad5) or a plasmid containing the respective helper gene to supply the helper viral protein and initiate rAAV production. Packaging cell lines differ from producer cell lines because they contain only the rep and cap genes.

[0157] More generally, cells transfected or transfected with DNA for the recombination generation of AAV particles may be called “recombinant cells.” Such cells can be any mammalian cell used as a recipient of nucleic acids (plasmids) encoding packaging proteins such as AAV packaging proteins, nucleic acids (plasmids) encoding helper proteins, and nucleic acids (plasmids) that are transcribed into a protein-coding or desired transcript, i.e., an transgene placed between two AAV ITRs. The term includes offspring of the transfected or transfected original cell. It is understood that offspring of a single parental cell may not necessarily be morphologically or genomically or in terms of whole nucleic acid complements, due to natural, accidental, or intentional mutations.

[0158] Numerous cell growth media are commercially available that are suitable for maintaining cell viability or providing cell growth and / or proliferation. Examples of such media include serum-free eukaryotic growth media, such as media for maintaining viability or media for providing growth of mammalian (e.g., human) cells. Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. Such media may be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0159] To generate rAAV, three plasmids are co-transfected into mammalian cells. The transgene plasmid encodes an expression cassette cloned between the AAV ITRs, while the rep and cap genes are provided trans-transfected by co-transfecting a second packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also called a helper plasmid, contains minimal helper viral factors, generally adenovirus E2A, E4 or F6, and VA genes, but lacks the AAV ITR.

[0160] A variety of methods for DNA transfer into mammalian cells have been reported in the art. All of these are useful in the methods according to the present invention. In all aspects and in certain embodiments of the embodiments, electroporation, nucleofection, or microinjection is used for nucleic acid transfer / transfection. In all aspects and in certain embodiments of the embodiments, inorganic substances (e.g., calcium phosphate / DNA coprecipitation), cationic polymers (e.g., polyethyleneimine, DEAE-dextran), or cationic lipids (lipofection) are used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection for nucleic acid transfer on a larger scale (e.g., Baldi et al., Biotechnol. Lett. 29(2007) 677-684), with polyethyleneimine being preferred.

[0161] Improving the efficiency and reproducibility of growth conditions in serum-free suspension cultures and transfection conditions using PEI as a transfection reagent enables easy scale-up of AAV production using shaking flasks, wave or agitated tank bioreactors.

[0162] The composition may include further plasmids and / or cells. Such plasmids and / or cells may be in contact with free PEI.

[0163] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA is a branched short-chain fatty acid that inhibits histone deacetylase activity. For this reason, it is commonly added to mammalian cell cultures as an enhancer of recombinant protein production.

[0164] In all aspects and in certain embodiments of the embodiments, the encoded AAV packaging protein includes AAV rep and / or AAV cap proteins. In all aspects and in certain embodiments of the embodiments, such an AAV packaging protein includes AAV rep and / or AAV cap proteins of any AAV serotype.

[0165] The encoded helper proteins include, in all aspects and in certain embodiments of the set, adenovirus E1A and E1B, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0166] Culture can be carried out using conditions commonly used for eukaryotic cell culture, such as approximately 37°C, 95% humidity, and 8% CO2 by volume. Culture can be carried out in serum-containing or serum-free medium, in adherent culture or suspension culture. Suspension culture can be carried out in any fermentation vessel, such as a stirred tank reactor, wave reactor, agitated bioreactor, shaker or spinner vessel, or so-called roller bottle. Transfection can be carried out in high-throughput and screening formats, for example, in 96 or 384-well formats.

[0167] The method according to the present invention may include AAV particles of any serotype or variant thereof. In all embodiments and in certain embodiments of the embodiment, the recombinant AAV particles include any of the following: AAV serotypes 1-12, AAV VP1, VP2 and / or VP3 capsid proteins, or modified or variant AAV VP1, VP2 and / or VP3 capsid proteins, or wild-type AAV VP1, VP2 and / or VP3 capsid proteins. In all embodiments and in certain embodiments of the embodiment, the AAV particles include an AAV serotype or an AAV pseudotype, and the AAV pseudotype includes an AAV capsid serotype different from the ITR serotype.

[0168] Expression regulatory elements include tissue-specific expression regulatory elements or constitutive or modulotable regulatory elements such as promoters.

[0169] ITR may be any of the AAV2, AAV6, AAV8, or AAV9 serotypes, or a combination thereof. AAV particles may contain any VP1, VP2, and / or VP3 capsid proteins having 75% or more sequence identity to any of the VP1, VP2, and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV12, AAV 2i8, AAV rh.10, AAV rh.74, or AAV 7m8, or may contain modified or variant VP1, VP2, and / or VP3 capsid proteins selected from any of the AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-2i8, AAV-rh.10, AAV-rh.74, and AAV-7m8.

[0170] After the generation of recombinant virus (e.g., AAV) particles, the virus (e.g., rAAV) particles can, if desired, be purified and / or isolated from host cells using a variety of conventional methods. Such methods include column chromatography, CsCl gradients, and iodixanol gradients.

[0171] For example, multiple column purification steps may be used, such as purification by anion exchange column, affinity column and / or cation exchange column (see, e.g., International Publication 02 / 12455 and U.S. Patent Application Publication 2003 / 0207439). Alternatively or additionally, iodixanol or CsCl gradient steps may be used (see, e.g., U.S. Patent Application Publication 2012 / 0135515 and U.S. Patent Application Publication 2013 / 0072548). Furthermore, if infectious viruses are used for packaging and / or expression of helper proteins, residual viruses may be inactivated by various methods. For example, adenoviruses can be inactivated by heating to a temperature of approximately 60°C for, for example, 20 minutes or more. This treatment effectively inactivates helper viruses because AAVs are heat-stable, while helper adenoviruses are heat-unstable.

[0172] The objective of the rAAV generation and purification system is to implement strategies to minimize / control the generation of generation-related impurities, such as wild-type / pseudowild-type AAV species (wtAAV) and AAV-encapsulated residual DNA impurities, as well as protein, nucleic acid, and vector-related impurities.

[0173] Given that rAAV represents a very small biomass, it needs to be purified to a level of purity suitable for use as a clinical human gene therapy product (see, for example, Smith PH, et al., Mo. Therapy 7(2003)8348; Chadeuf G., et al, Mo. Therapy 12(2005)744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005,183989 / 2004).

[0174] In all aspects of the method according to the present invention and in certain embodiments of the embodiments, as a first step, typically, cultured cells that produce rAAV particles are recovered, optionally in combination with the recovery of the cell culture supernatant (medium) in which the recombinant rAAV particle-producing cells (suspension or adherent) were cultured. The recovered cells and optionally the cell culture supernatant can be used as is, lysed, or concentrated as needed. Furthermore, if infection is used to express helper function, residual helper viruses can be inactivated. For example, adenoviruses can be inactivated by heating to a temperature of approximately 60°C for, for example, 20 minutes or more, which inactivates only the helper viruses because AAV is heat-stable while helper adenoviruses are heat-unstable.

[0175] Cells in the recovered culture broth may be lysed using current methods in the art, such as detergent dissolution or freeze-thaw cycles, to release rAAV particles. During or after cell lysis, a nuclease, such as benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtering or centrifugation, to give a clarified cell lysate. In specific cases, the lysate is filtered through a micron-diameter pore filter (e.g., a filter with a pore size of 0.1–10.0 μm, e.g., a filter with a pore size of 0.45 μm and / or 0.2 μm) to produce a clarified lysate.

[0176] The lysate (optionally clarified) contains recombinant AAV particles (including complete rAAV vectors and empty rAAVs) and production / process-related impurities, such as soluble cellular components from host cells, which may include, among other things, cellular proteins, lipids and / or nucleic acids, as well as components of the cell culture medium. The clarified lysate is then optionally subjected to a purification step to purify the rAAV (including the rAAV vector) from impurities using chromatography. The clarified lysate may be diluted or concentrated with appropriate buffer before the first chromatography step.

[0177] After cell lysis, any clarification, and any dilution or concentration, rAAV particles can be purified using a series of subsequent chromatography steps.

[0178] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0179] If the first chromatography step is affinity chromatography, the second chromatography step may be anion exchange chromatography. Thus, in all aspects and certain embodiments of the model, the purification of rAAV is performed by affinity chromatography, followed by purification by anion exchange chromatography and / or cation exchange chromatography and / or size exclusion chromatography, in any order, sequence, or combination.

[0180] For example, the removal of empty capsids from complete capsids during downstream processing is based on their different isoelectric points (pI) in anion exchange chromatography. The average pI calculated across all serotypes is 5.9 for complete capsids and 6.3 for empty capsids (Venkatakrishnan, B., et al., J. Virol. 87(2013) 4974-4984).

[0181] Cation exchange chromatography functions to separate AAV from cellular and other components present in the clarified lysate and / or column eluate from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins that can bind to rAAV over a wide pH range include, but are not limited to, any sulfonic acid resins, including aryl and alkyl-substituted sulfonates such as sulfopropyl or sulfoethyl resins, indicated by the presence of sulfonate functional groups. Typical matrices include, but are not limited to, POROS HS, POROS HS 50, POROS XS, POROS SP, and POROS S (strong cation exchangers available from Thermo Fisher Scientific, Inc., Waltham, MA, USA). Further examples include Capto S, Capto S ImpAct, and Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), as well as the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® families of resins from Aldrich Chemical Company (Milliwaukee, WI, USA). Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resins. Exemplary cation exchange resins include carboxymethyl (CM), phospho (phosphate-based), methyl sulfonate (S), and sulfopropyl (SP) resins.

[0182] Anion exchange chromatography functions to separate rAAV from proteins, cellular components, and other components present in the clarified lysate and / or column eluate from affinity chromatography, cation exchange chromatography, or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and control the amount of empty rAAV in the eluate. For example, an anion exchange column bound with complete and empty rAAV can be washed with a solution containing a moderate concentration of NaCl (e.g., about 100–125 mM, e.g., 110–115 mM), allowing some of the empty rAAV to flow through without substantially eluting the complete rAAV. Subsequently, the complete rAAV bound to the anion exchange column can be eluted using a solution containing a higher concentration of NaCl (e.g., about 130–300 mM NaCl) to produce a column eluate containing a reduced or depleted amount of empty rAAV and a proportionally increased amount of complete rAAV containing the rAAV vector.

[0183] Examples of anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include, but are not limited to, those based on quaternary nitrogen atoms, including quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion exchangers available from GE Healthcare, Marlborough, MA, USA); DEAE Examples include SEPHAROSE (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA) and Q SEPHAROSE (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Further exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0184] The manufacturing process for purifying recombinant AAV particles intended as a product for treating human diseases should achieve the following objectives: 1) consistent particle purity, efficacy, and safety; 2) scalability of the manufacturing process; and 3) acceptable manufacturing costs.

[0185] An exemplary process for purifying recombinant AAV particles is described in International Publication No. 2019 / 006390.

[0186] Methods for determining the infectivity titer of rAAV particles containing transgenes are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15(2004) 709). Methods for assaying empty rAAV and complete rAAV with transgenes packaged are also known (see, e.g., Grimm et al., Gene Therapy 6(1999) 1322-1330; Sommer et al., Malec. Ther. 7(2003) 122-128).

[0187] To determine the presence or amount of degraded / denatured capsids, purified rAAV can be subjected to SDS-polyacrylamide gel electrophoresis using any gel capable of separating the three capsid proteins, such as a gradient gel, and the gel can then be run until the sample is separated, and the gel can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody to bind to the denatured capsid proteins (see, e.g., Wobus et al., J.Viral. 74(2000) 9281-9293). The secondary antibody, which binds to the primary antibody, includes means for detecting the primary antibody. The amount of capsids is determined by semi-quantitatively detecting the binding between the primary and secondary antibodies. Another method is analytical HPLC using an SEC column or analytical ultracentrifuge.

[0188] Description of specific embodiments of the present invention This invention is at least in part based on the finding that the productivity of mammalian cells that produce recombinant adeno-associated virus particles can be increased when the culture is performed at a high pH value such as pH 7.4–7.6.

[0189] While optimizing transfection parameters, DNA / reagent ratio, VCD during transfection, and complex formation time can improve rAAV productivity, such increases are far outweighed by the effect of the pH value according to the present invention.

[0190] In the following, the reference value, or baseline value, for each table is indicated by the identifier "(100%)".

[0191] For transient generation of rAAV particles in HEK293 cells, increasing the culture pH from the commonly used pH 7.2 to pH 7.4 or even 7.6 was found to increase particle yield and genome yield. The ratio of complete to empty cells also improved, as the increase in genome yield was greater than the increase in titer yield.

[0192] For example, in transient generation of rAAV2 particles in serum-free medium using HEK293 cells grown in suspension, increasing the culture pH from the commonly used pH 7.2 to pH 7.4 or even 7.6 resulted in a more than 3-fold increase in particle yield and a more than 10-fold increase in genome yield.

[0193] The following table provides exemplary data of HEK293 cells adapted to grow in suspension in serum-free culture medium, demonstrating the effect of the method according to the present invention (see also Figures 1, 2, and 3). It can be seen that increasing the culture pH from pH 7.2 to pH 7.4 or pH 7.6 results in a more than 10-fold increase in genomic titer (vg / mL). Simultaneously, the capsid titer (vp / mL) increases 2-3 times. Therefore, the lower the increase in capsid titer compared to the increase in genomic titer, the more than 4-fold the ratio of complete capsids increases. TIFF2026511015000003.tif85141

[0194] The yield is independent of incubation time for pH 7.2, pH 7.4, and pH 7.6, but at pH 7.0, the yield decreases with increasing incubation time. This is shown in the table below. Therefore, the process is more robust at pH values ​​of 7.4 and 7.6, resulting in increased yields compared to pH 7 and pH 7.2, respectively. TIFF2026511015000004.tif174141TIFF2026511015000005.tif176141

[0195] The effect of changing the pH value from pH 7.2 to pH 7.4 or pH 7.6 far exceeds the increase in titer obtained by optimizing process conditions, such as changing the transfection reagent or adding feed, as shown in the table below. The increase in titer due to simultaneous changes in culture pH, transfection reagent, and feed addition is shown in the third data row. TIFF2026511015000006.tif128140

[0196] For direct comparison, the following table shows the increase in titer due to changes in culture pH and the addition of feed under optimized transfection reagent conditions. TIFF2026511015000007.tif103146

[0197] Optimizing the culture conditions reduces the robustness of the culture at a pH of 7.2. However, at a pH of 7.4, the process maintains its robustness. In other words, increasing the pH from 7.2 to 7.4 can counteract the loss of process robustness caused by the optimization of reaction conditions. This is shown in the table below. TIFF2026511015000008.tif154143

[0198] For example, transient generation of rAAV2 particles in serum-free medium using commercially available HEK 293 Expi cells under the optimized conditions described above can further increase particle yield by approximately 1.7 times and genome yield by approximately 1.8 times, or 80%. The respective data are shown in the table below. TIFF2026511015000009.tif124143

[0199] Examples and drawings are provided to aid in understanding the invention, and its true scope is set forth in the appended claims. It is understood that modifications to the described procedures can be made without departing from the spirit of the invention. [Examples]

[0200] material cell line Commercially available HEK293 cells were used to generate AAV particles using transient transfection with three plasmids.

[0201] Culture material The growing medium and supplements were used according to the supplier's operating instructions. The medium and feed were stored in the dark at 4°C and consumed according to the manufacturer's instructions. The corrective agents were stored at room temperature (glucose solution; sodium carbonate solution; antifoaming agent solution).

[0202] Example 1 Culture of HEK293 cells and generation of recombinant AAV preparations Generally, the culture method is adapted from standard protocols (e.g., Lindl, T.,''Zell-und Gewebekultur:Einfuhrung in die Grundlagen sowie ausgewahlte Methoden und Anwendungen'', Spektrum Akademischer Verlag GmbH, Heidelberg / Berlin, 2002) and the operating instructions of each supplier.

[0203] Preculture HEK cells were thawed and grown in culture medium at 37°C, 85% humidity, 5% pCO2, and a shaking frequency of 120 rpm in a shaking flask for 2-3 weeks. The cells were divided every 3-4 days and expanded in the culture medium to the volume required for inoculation into the progenitor culture.

[0204] production culture To generate recombinant AAV particles, each pre-cultured HEK293 cell was cultured in its respective reactor under the indicated conditions using a batch or fed-batch process.

[0205] rAAV particle preparation containing particles with setting 1-AAV2 serotype and capsid variants derived from therapeutic transgenes: Reactor: Ambr250 Cell line: HEK293 suspension suitable for serum-free medium Culture medium: HEK ViP NB + 8mM glutamine + insulin Supply: None (batch) Temperature: 37℃ Speed: about 450rpm Culture time after inoculation: 144 hours Transfection: transient; 3 plasmids; ratio approximately 1:2.5:2 (transgene:rep / cap:helper) Transfection: (Approximately 24 hours after vaccination) Transfection reagent: PEIpro (trademark) Transfection reagent:DNA ratio:approximately 2:1 DNA concentration: approx. 3μg / mL Transfection VCD: Approximately 30 E+0 5 cells / mL Transfection: Transfection mix in 1 / 3 fresh culture medium Dissolution: None TIFF2026511015000010.tif122153

[0206] rAAV particle preparation containing particles with setting 2-AAV2 serotype and capsid variants derived from therapeutic transgenes: Reactor: Ambr250 Cell line: HEK293 suspension suitable for serum-free medium Culture medium: HEK ViP NB + 8mM glutamine + insulin Supply: None (batch) Temperature: 37℃ Speed: about 450rpm Incubation time after inoculation: 120 hours Transfection: transient; 3 plasmids; ratio approximately 1:2.5:2 (transgene:rep / cap:helper) Transfection: (Approximately 24 hours after vaccination) Transfection reagent: PEIpro (trademark) Transfection reagent:DNA ratio:approximately 2:1 DNA concentration: approx. 3μg / mL Transfection VCD: Approximately 30 E+0 5 cells / mL Transfection: Transfection mix in 1 / 3 fresh culture medium Dissolution: None TIFF2026511015000011.tif36152

[0207] Settings 3 and 4 - DoE: rAAV particle preparations: 1) Particles containing capsid variants derived from AAV2 serotypes and therapeutic transgenes; 2) Particles containing AAV2 wild-type capsids and green fluorescent protein (GFP) transgenes: Reactor: Ambr15 Cell lines: 1) HEK293 suspension suitable for serum-free medium; 2) HEK293 Expi Culture medium: HEK ViP NB + 8mM glutamine + insulin Supplies: 1) None (batch); 2) Post-transfection culture medium and glucose supply Temperature 37℃ Speed: about 450rpm Incubation time after inoculation: 120 hours Transfection: transient; 3 plasmids; ratio 1) approximately 1:2.5:2 or 2) approximately 1:1:1 (transgene: rep / cap: helper) Transfection: 24 hours after vaccination Transfection reagents: 1) PEI + free PEI + valproic acid; 2) FectoVIR(TM)-AAV Transfection reagent:DNA ratio: 1) approx. 2.5:1; 2) approx. 1.5:1 DNA concentration: 1) approx. 3 μg / mL; 2) approx. 2 μg / mL Transfection VCD: Approximately 30 E+0 5 cells / mL Transfection: Transfection mix in 1 / 3 fresh culture medium Dissolution: 1) None; 2) Yes TIFF2026511015000012.tif214166TIFF2026511015000013.tif232163

[0208] Example 2 Dissolution When lysis was included in the process, it was performed as follows: To release AAV particles into the cell culture broth, 5% (v / v) lysis buffer (10% Triton CG 110, 40 mM MgCl2) was added to the culture broth. In addition, 100 U / ml of Benzonase® nuclease (Merck) was added. The cell culture broth was then incubated at 37°C for approximately 1 hour with agitation, without aeration or pH control. After each incubation, a 5 M NaCl solution was added, and the lysate was filtered to sterile.

[0209] Example 3 AAV particle purification For the affinity chromatography step, a column containing 10.5 mL of Thermo Fisher AAVX resin was used in an AKTA Avant 25 chromatography system. The system was operated at a flow rate of approximately 300 cm / h. After equilibration with buffer A (1 × PBS, pH 7.4, 0.001% Pluronic F-68), 200 mL of dissolved culture broth was applied to the column, followed by two washes each with equilibration buffer and 0.5 M NaCl, pH 6.0. AAV particles were eluted with 0.1 M sodium citrate solution (pH 2.4). The pH of the eluate was adjusted to pH 7.5 by adding 2 M Tris (pH 10). TIFF2026511015000014.tif61151

[0210] Example 4 Analysis method Enzyme-linked immunosorbent assay (ELISA) for total titer measurement For AAV capsid titer measurement, the PROGEN kit (catalog number PRAAV8) was used according to the manufacturer's instructions.

[0211] In short, this assay is a sandwich ELISA that uses recombinant AAV capsid-specific antibodies and biotin-labeled detection antibodies as capture antibodies.

[0212] Each well of a pre-coated multi-titer plate (MTP) was incubated overnight at 4°C with 100 μL of standard, sample, or control. The following day, the wells were washed three times with ASSB buffer (1×) as provided in the kit. Then, 100 μL / well of a solution containing biotinylation detection antibody (diluted according to the manufacturer's instructions) was added, and the wells were incubated at room temperature for 2 hours with shaking. The wells were then washed three times with ASSB buffer (1×) as provided in the kit. In the next step, 100 μL of a solution containing horseradish peroxidase conjugated to streptavidin was added to each well, and the wells were incubated for 30 minutes. The wells were then washed three times with ASSB buffer (1×) as provided in the kit. For the color reaction, 100 μL of a solution containing ABTS, prepared according to the manufacturer's instructions, was added to each well, and the wells were incubated with shaking. Color intensity was determined using a 405 nm MTP-ELISA-Reader Versa Max (Molecular Devices) at a reference wavelength of 490 nm until the extinction difference between the blank and the highest concentration standard reached approximately 1.5.

[0213] Each sample, standard, and control were measured in a double-row system.

[0214] The amount of capsid (capsid / mL) was calculated using the standard mean value, based on a standard curve determined by four-parameter fitting, for example, according to the Wiemer-Rodbard algorithm.

[0215] Digital droplet polymerase chain reaction (ddPCR) for genome titer determination Reagents for enzyme sample processing: 1) DNase I buffer (Promega): 400 mM Tris-HCl, pH 8, 100 mM MgSO4, 10 mM CaCl2 2) DNase I (NEB): 0.2 U / μL 3) Proteinase K (NEB; approximately 20 mg / mL = 800 U / mL): 16 U / mL 4) Proteinase K buffer (BioRad): 400 mM Tris-HCl, 20 mM EDTA, 2000 mM NaCl, 1% SDS, pH 8 5) Sodium dodecyl sulfate (SDS) solution: 10% (w / v) Enzyme sample processing: Mix 30 μL H2O, 5 μL DNase I buffer, 5 μL DNase I, and 10 μL sample. Incubate at -37°C for 30 minutes. Heat at -75°C for 15 minutes to obtain incubated DNase I-Mix. -Short-term cooling and centrifugal separation - Mix 42 μL H2O + 2 μL proteinase K + 5 μL proteinase K buffer + 1 μL 10% SDS solution, and add incubated DNase I-Mix. Incubate at -50°C for 60 minutes. Heat to -95°C for 15 minutes. Cool to -4°C

[0216] ddPCR: A dual ddPCR assay was performed for viral genome titration. Primers and probes were designed for the CMV promoter and poly(A / 3'UTR) sequence used. PCR master mixes were prepared according to the following table (Droplet Digital PCR Guide - Bio-Rad). TIFF2026511015000015.tif69157

[0217] The prepared master mix was pipetteed into a 96-well plate at a rate of 16.5 μL / well. Next, a dilution series of the pre-treated samples was performed: 10 μL of sample was transferred to 90 μL of water in a LoBind tube with LoRentention Tips and thoroughly mixed. Then, 5.5 μL of sample was added to the master mix solution in the 96-well plate at several dilution stages. The plate was sealed at 180°C, vortexed at 2,200 rpm for 1 minute, and then centrifuged at 1,000 rpm for another 1 minute. An automated droplet generator was used to produce up to 20,000 droplets per well, each containing 20 μL of PCR mix, which were then transferred to another 96-well plate. After sealing the droplet plates at 180°C, PCR was performed. The conditions for each procedure are shown in the table below. TIFF2026511015000016.tif45138

[0218] A droplet reader was used to measure the fluorescence signal for each droplet. QuantaSoft software processed the reader data and calculated copy number per 20 μL well for the target sequence. The initial sample titer can be determined using the following formula. TIFF2026511015000017.tif16128

Claims

1. A method for producing recombinant adeno-associated virus particle preparations (rAAVp), comprising the steps of culturing HEK293 cells containing an expression cassette for a non-adeno-associated virus gene, an adeno-associated virus rep gene, an adeno-associated virus cap gene, an adeno-associated virus E1A gene, an adeno-associated virus E1B gene, an adeno-associated virus E2A gene, an adeno-associated virus E4orf6 gene, and optionally an adeno-associated virus VA RNA gene, between two AAV terminal inverted repeat sequences (ITRs), and thereby producing the rAAVp, A method in which cultivation is performed at a pH value within the range of pH 7.4 to pH 7.

6.

2. The method according to claim 1, wherein the yield of rAAVp produced by culturing at a pH value within the range of pH 7.4 to pH 7.6 is higher than the yield of rAAVp produced by culturing at a pH value within the range of pH 7.0 to pH 7.

2.

3. The method according to claim 1 or 2, wherein the rAAVp produced by culturing at a pH value within the range of pH 7.4 to pH 7.6 has a higher percentage of perfect particles than the rAAVp produced by culturing at a pH value within the range of pH 7.0 to pH 7.

2.

4. The method according to any one of claims 1 to 3, wherein the rAAVp is therapeutic rAAVp.

5. The method according to any one of claims 1 to 4, wherein the rAAVp comprises a recombinant adeno-associated virus particle (rAAV) having at least one coding nucleic acid sequence located between two adeno-associated virus terminal inverted repeat sequences.

6. The method according to any one of claims 1 to 5, wherein the rAAV is of serotype AAV2 or a variant thereof.

7. The method according to any one of claims 1 to 6, wherein the culturing includes inoculation into a bioreactor and recovery of the rAAVp.

8. The method according to any one of claims 1 to 7, wherein one or more or all of the expression cassettes for the non-adeno-associated virus gene, the adeno-associated virus rep gene, the adeno-associated virus cap gene, the adeno-associated virus E2A gene, the adeno-associated virus E4orf6 gene, and optionally the adeno-associated virus VA RNA gene, which are located between two AAV ITRs, are introduced into mammalian cells after inoculation into a bioreactor.

9. The method according to any one of claims 1 to 8, further comprising the steps of isolating the rAAV from the cells and / or culture medium after the culturing step, and optionally purifying the rAAV.

10. The method according to claim 9, wherein the purification is performed by a series of chromatographic steps, the first being affinity chromatography, followed by anion exchange chromatography or cation exchange chromatography, and then any size exclusion chromatography.

11. A pharmaceutical composition comprising the rAAVp obtained by the method according to any one of claims 1 to 10.

12. A pharmaceutical composition comprising the rAAVp obtained by the method of any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

13. Use of the method according to any one of claims 1 to 10 to increase the yield of rAAVp produced by recombination.

14. Use of the method according to any one of claims 1 to 10 to increase the percentage of perfect particles in rAAVp.

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