Method for producing recombinant AAV particle preparation

Abstract

Herein is reported a method for producing a recombinant adeno-associated viral particle preparation (rAAVp), the method comprising the steps of culturing a mammalian cell comprising an expression cassette directed against: a non-adeno-associated viral gene interposed between two AAV inverted terminal repeats (ITRs), and thereby producing the rAAVp; 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; and an adeno-associated virus VA RNA gene, wherein the culturing is carried out at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value. The yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value is higher than the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.0 to pH 7.2 and including a terminal value, and the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value is lower than the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.0 to pH 7.2. And the rAAVp produced by the culture at a pH value in the range of pH 7.4 to pH 7.6 and including an endvalue has a higher percentage of intact particles than the rAAVp produced by the culture at a pH value in the range of pH 7.0 to pH 7.2 and including an endvalue.

Description

[0001] The present invention is in the field of gene therapy. More specifically, the present invention relates to a method for producing recombinant adeno-associated virus particles comprising a therapeutic transgene in mammalian cells, in particular CHO and HEK cells, at a higher pH value than the pH values commonly used in the art. BACKGROUND

[0002] Gene therapy brings unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing functional mutations by co-expressing the correct gene, the use of viral vectors is required to ensure the proper delivery of the therapeutic gene in order to restore the biological function. In this context, recombinant adeno-associated virus (rAAV) is the most widely used vector.

[0003] The biological handling of viruses is very complex and requires systematic and coordinated steps both in the upstream and downstream processing. However, the production of therapeutic viruses using traditional culture methods cannot support an efficient commercial manufacturing strategy. This is even more evident compared to therapeutic biomolecules such as e.g. antibodies, due to the larger size of viruses. In addition, viruses are much more complex.

[0004] The biological manufacturing process of therapeutic viruses requires the insertion of a therapeutic transgene into the recombinant AAV capsid shell (full rAAV particles, i.e. recombinant AAV particles comprising encapsidated nucleic acid). However, it is also possible that a certain percentage of rAAV is produced that does not contain the desired transgene (empty recombinant AAV particles, i.e. rAAV particles that do not comprise encapsidated nucleic acid), as well as partially filled rAAV (partially filled recombinant AAV particles). SUMMARY

[0005] The present invention is at least partially based on the finding that the productivity of mammalian cells producing recombinant adeno-associated virus particles can be increased when the cultivation is performed at an elevated pH value such as pH 7.4-7.6.

[0006] The present invention is at least partially based on the finding that the fraction of full recombinant AAV particles obtained from the cultivation of mammalian cells producing said recombinant adeno-associated virus particles can be increased when the cultivation is performed at an elevated pH value such as pH 7.4-7.6.

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

[0008] 1. A method for producing a preparation of recombinant adeno-associated viral particles (rAAVp), the method comprising the step of culturing a mammalian cell, and thereby producing the rAAVp, the mammalian cell comprising an expression cassette for a non- adeno-associated viral gene interposed between two AAV inverted terminal repeat sequences (ITRs), for an adeno-associated viral rep gene, for an adeno-associated viral cap gene, for an adeno-associated viral E1A gene, for an adeno-associated viral E1B gene, for an adeno-associated viral E2A gene, for an adeno-associated viral E4orf6, and optionally for an adeno-associated viral VA RNA gene,

[0009] wherein the culturing is performed at a pH value in the range of and including the end values of pH 7.4 to pH 7.6.

[0010] 2. A method for producing a preparation of recombinant adeno-associated viral particles (rAAVp), the method comprising the step of culturing a HEK cell, and thereby producing the rAAVp, the HEK cell comprising an expression cassette for a non- adeno-associated viral gene interposed between two AAV inverted terminal repeat sequences (ITRs), for an adeno-associated viral rep gene, for an adeno-associated viral cap gene, for an adeno-associated viral E2A gene, for an adeno-associated viral E4orf6, and optionally for an adeno-associated viral VA RNA gene,

[0011] wherein the culturing is performed at a pH value in the range of and including the end values of pH 7.4 to pH 7.6.

[0012] 3. The method according to any one of embodiments 1 or 2, wherein the mammalian cell is a HEK293 cell.

[0013] 4. The method according to any one of embodiments 1 to 3, wherein the genome titer of the rAAVp produced by the culturing performed at a pH value in the range of and including the end values of pH 7.4 to pH 7.6 is higher than the capsid titer of the rAAVp produced by a culturing performed at a pH value in the range of and including the end values of pH 7.0 to pH 7.2.

[0014] 5. The method according to embodiment 4, wherein the genome titer is at least 1.5 times higher.

[0015] 6. The method according to any one of embodiments 4 to 5, wherein the genome titer is at least 2 times higher.

[0016] 7. The method of any one of embodiments 4 to 6, wherein the genome titer is at least 6-fold higher.

[0017] 8. The method of any one of embodiments 4 to 7, wherein the genome titer is at least 10-fold higher.

[0018] 9. The method of any one of embodiments 1 to 8, wherein the capsid titer of the rAAVp produced by the culturing at a pH value in the range of and including the end values of pH 7.4 to pH 7.6 is higher than the capsid titer of the rAAVp produced by culturing at a pH value in the range of and including the end values of pH 7.0 to pH 7.2.

[0019] 10. The method of embodiment 9, wherein the capsid titer is at least 1.5-fold higher.

[0020] 11. The method of any one of embodiments 9 to 10, wherein the capsid titer is at least 2-fold higher.

[0021] 12. The method of any one of embodiments 9 to 11, wherein the capsid titer is at least 3-fold higher.

[0022] 13. The method of any one of embodiments 1 to 12, wherein the genome titer and the capsid titer of the rAAVp produced by the culturing at a pH value in the range of and including the end values of pH 7.4 to pH 7.6 is higher than the genome titer and the capsid titer of the rAAVp produced by culturing at a pH value in the range of and including the end values of pH 7.0 to pH 7.2.

[0023] 14. The method of embodiment 13, wherein the genome titer and the capsid titer is at least 1.5-fold higher.

[0024] 15. The method of any one of embodiments 13 to 14, wherein the genome titer and the capsid titer is at least 2-fold higher.

[0025] 16. The method of any one of embodiments 13 to 15, wherein the genome titer and the capsid titer is at least 3-fold higher.

[0026] 17. The method of any one of embodiments 13 to 16, wherein the genome titer is at least 4-fold higher and the capsid titer is at least 2-fold higher.

[0027] 18. The method of any one of embodiments 13-17, wherein the genome titer is at least 6-fold higher and the capsid titer is at least 2-fold higher.

[0028] 19. The method of any one of embodiments 1-20, wherein the rAAVp produced by the culturing at a pH value in the range of and including the end values of pH 7.4 to pH 7.6 has a higher percentage of full particles than rAAVp produced by culturing at a pH value in the range of and including the end values of pH 7.0 to pH 7.2.

[0029] 20. The method of embodiment 19, wherein the percentage of full particles is at least 1.5-fold higher.

[0030] 21. The method of any one of embodiments 19-20, wherein the percentage of full particles is at least 2-fold higher.

[0031] 22. The method of any one of embodiments 19-21, wherein the percentage of full particles is at least 4-fold higher.

[0032] 23. The method of any one of embodiments 1-22, wherein the rAAVp is a therapeutic rAAVp.

[0033] 24. The method of any one of embodiments 1-23, wherein the rAAVp is for transferring a nucleic acid that is transcribed into a polypeptide having a therapeutic effect into a target cell.

[0034] 25. The method of any one of embodiments 1-24, wherein the rAAVp is for transferring a nucleic acid having a therapeutic effect into a target cell.

[0035] 26. The method of any one of embodiments 1-25, wherein the rAAVp comprises a recombinant adeno-associated viral particle (rAAV) comprising at least one coding nucleic acid sequence interposed between two adeno-associated viral inverted terminal repeat sequences.

[0036] 27. The method of any one of embodiments 1-26, wherein the rAAV in the rAAVp is derived from a wild-type AAV particle 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, and variants thereof.

[0037] 28. The method of any one of embodiments 1-27, wherein the rAAV has a serotype of AAV2 or a variant thereof.

[0038] 29. The method of any one of embodiments 1-28, wherein the rAAV comprises one or both ITR sequences of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.

[0039] 30. The method of any one of embodiments 1-29, wherein culturing comprises inoculating and harvesting rAAVp from a bioreactor.

[0040] 31. The method of any one of embodiments 1-30, wherein culturing begins with inoculation of a bioreactor.

[0041] 32. The method of any one of embodiments 1-31, wherein one or more or all of the expression cassettes for the non-adenoviral gene inserted between two AAV ITRs, for the adeno-associated virus rep gene, for the adeno-associated virus cap gene, for the adeno-associated virus E2A gene, for the adeno-associated virus E4orf6, and for the adeno-associated virus VA RNA gene are introduced into the mammalian cell or HEK cell after inoculation of the bioreactor.

[0042] 33. The method of any one of embodiments 1 to 32, wherein one or more or all of the expression cassettes for the non-adenoviral gene inserted between two AAV ITRs, for the adeno-associated virus rep gene, for the adeno-associated virus cap gene, for the adeno-associated virus E2A gene, for the adeno-associated virus E4orf6 and optionally for the adeno-associated virus VA RNA gene are introduced into the mammalian cells or HEK cells after inoculation of the bioreactor, thereby co-transfecting at most three particles into the mammalian cells, wherein one of the plasmids comprises the expression cassette for the non-adenoviral gene inserted between two AAV ITRs, one of the plasmids comprises the expression cassette for the rep and cap genes, and one of the plasmids comprises the expression cassette for the adenoviral E2A, E4orf6 and VA RNA genes.

[0043] 34. The method of any one of embodiments 1 to 33, wherein expression of one or more or all of the non-adenoviral gene inserted between two AAV ITRs, the adeno-associated virus rep gene, the adeno-associated virus cap gene, the adeno-associated virus E2A gene, the adeno-associated virus E4orf6 and optionally the adeno-associated virus VA RNA gene is induced after inoculation of the bioreactor.

[0044] 35. The method of any one of embodiments 32 to 33, wherein the introducing is performed about 16 to 32 hours after inoculation of the bioreactor.

[0045] 36. The method of embodiment 34, wherein the inducing is performed about 16 to 32 hours after inoculation of the bioreactor.

[0046] 37. The method of any one of embodiments 35 to 36, wherein the introducing or inducing is performed about 24 hours after inoculation of the bioreactor.

[0047] 38. The method of any one of embodiments 1 to 37, wherein the method further comprises a step of isolating the rAAV from the cells and / or culture medium and optionally purifying the rAAV after the culturing step.

[0048] 39. The method of embodiment 38, wherein the purifying is performed by one or more column chromatography steps and / or CsCl or iodixanol gradient centrifugation steps.

[0049] 40. The method according to any one of embodiments 38 or 39, wherein the first chromatography step is an affinity chromatography step.

[0050] 41. The method according to any one of embodiments 38 to 40, wherein the purification is performed by a series of chromatography steps, wherein first is an affinity chromatography, followed by an anion exchange chromatography or a cation exchange chromatography, and optionally a size exclusion chromatography.

[0051] 42. A pharmaceutical composition comprising a rAAVp obtained by the method according to any one of embodiments 1 to 41.

[0052] 43. A pharmaceutical composition comprising a rAAVp obtained by the method according to any one of embodiments 1 to 41, and a pharmaceutically acceptable excipient.

[0053] 44. Use of the method according to any one of embodiments 1 to 41 for increasing the yield of recombinantly produced rAAVp.

[0054] 45. Use of the method according to any one of embodiments 1 to 41 for increasing the percentage of full particles in the rAAVp.

[0055] The disclosed subject matter is directed to other embodiments having other combinations of the features disclosed and claimed herein in addition to the various embodiments depicted and claimed. Accordingly, particular features presented herein can be combined in other ways to achieve other embodiments of the disclosed subject matter, and the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to the precise forms disclosed. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 Visualization of genomic titers in harvest samples of Group 1 (harvested at 120 hours post transfection, no lysis).

[0057] Figure 2 Visualization of capsid titers in harvest samples of Group 1 (harvested at 120 hours post transfection, no lysis).

[0058] Figure 3 Visualization of full to empty particle ratio in harvest samples of Group 1 (harvested at 120 hours post transfection, no lysis). DETAILED DESCRIPTION

[0059] The present invention is based, at least in part, on the discovery that the productivity of mammalian cells producing recombinant adeno-associated viral particles can be increased when cultured at elevated pH values, such as pH 7.4-7.6.

[0060] Definitions

[0061] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0062] Methods and techniques applicable to the practice of the present invention are described in, for example, Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I-III (1997); Glover, N.D. and Hames, B.D. eds., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J.D. et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J. ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, Second Edition, Alan R. Liss, Inc., N.Y. (1987). The contents of these texts are incorporated herein by reference.

[0063] Using recombinant DNA technology it is possible to generate derivatives of nucleic acids. Such derivatives can be modified, for example, at one or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can be performed, for example, by means of site-directed mutagenesis. Such modifications can be readily performed by the person 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; Hames, B.D. and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0064] It must be noted that, as used herein and in the appended claims, singular articles such as "a", "an" and "the" can refer to plural constituents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "one" or "a" or "one or more" and "at least one" are used interchangeably herein. It is further noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0065] The term "about" denotes a range of + / - 20% of the number value it is connected to. In certain embodiments, the term about denotes a range of + / - 10% of the number value it is connected to. In certain embodiments, the term about denotes a range of + / - 5% of the number value it is connected to.

[0066] The terms "comprise", "include", "have", "possess", "may", "contain", and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The term "comprising" also encompasses the terms "including", "containing", "having" and "possessing". The disclosure also contemplates other embodiments "comprising", "consisting of", and "consisting essentially of", the embodiments or elements presented herein, whether explicitly set forth or not.

[0067] The terms "empty recombinant AAV particle" and "empty rAAV" are used interchangeably to denote a protein shell composed of adeno-associated capsid polypeptides, wherein no functional nucleic acid is encapsidated / packaged. That is, an empty rAAV can contain no encapsidated nucleic acid or contain a little nucleic acid or parts thereof that is not transcribed or does not transcribe into a functional transcript. Thus, an empty rAAV does not transfer a nucleic acid encoding a functional protein or a nucleic acid transcribed into a functional transcript of interest into a target cell. In certain embodiments of all aspects and embodiments, the functional protein or the functional transcript of interest has a therapeutic effect.

[0068] The term "endogenous" denotes that something is naturally produced, e.g., within a cell or naturally produced by a cell, e.g.

[0069] The term "exogenous" denotes that something, e.g., a nucleotide sequence, does not originate from the same entity in which it is present. For example, if a nucleic acid has been introduced into a particular cell by a DNA delivery method, such as, e.g., by transfection, electroporation or transduction, the nucleic acid is exogenous to the cell. Likewise, if a nucleic acid is not derived from the same AAV particle or serotype, the nucleic acid is exogenous to the AAV particle. Thus, an exogenous nucleotide sequence is an artificial sequence, which can be in isolated form or within a cell or rAAV, wherein the artificial sequence can originate from a combination of sub-sequences from different sources, e.g., a combination of a recombinase recognition sequence with a SV40 promoter and a green fluorescent protein coding sequence, or a combination of an AAV ITR from a first serotype with a capsid polypeptide of a second serotype, or a combination of an AAV ITR with a non- AAV nucleic acid; or from a deletion of parts 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 nucleobase in an endogenous nucleic acid sequence. This does not exclude that an "exogenous" nucleotide sequence can have an "endogenous" counterpart of identical base composition, but the sequence becomes "exogenous" by its combination with exogenous regulatory elements, such as an exogenous secretion signal or a promoter.

[0070] The terms "intact recombinant AAV particle" or "intact rAAV" are used interchangeably to denote a non-covalent complex of a protein shell composed of adeno-associated capsid polypeptides and a functional nucleic acid sequence encapsidated / packaged therein. That is, an intact rAAV contains a nucleic acid transcribed into a functional transcript. Thus, the function of an intact rAAV is to transfer a nucleic acid encoding a protein or a nucleic acid transcribed into a transcript of interest into a target cell. In certain embodiments, the functional nucleic acid comprises at least one coding nucleic acid sequence interposed between two adeno-associated virus inverted terminal repeat sequences (ITRs).

[0071] The term "intact to empty particle ratio" denotes the mathematical ratio of the number of intact recombinant AAV particles (intact rAAV) to the total number of recombinant AAV particles (sum of intact rAAV and empty rAAV) in a sample or in a preparation of recombinant AAV particles (rAAVp). Since the number of intact rAAV can at most be identical to the total number of rAAV, the ratio can at most be 1. Usually, the ratio is less than 1 and is expressed as a percentage. The number of intact rAAV is determined by determining the number of nucleic acid sequences intercalated between two AAV ITRs in the sample or preparation. This can be done by PCR, in particular by digital droplet PCR (ddPCR) or quantitative PCR (qPCR). The total number of rAAV 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, in particular by capsid polypeptide specific ELISA.

[0072] The term "in vitro" denotes an artificial environment per se or a process or reaction performed in such an artificial environment.

[0073] The term "in vivo" denotes the natural environment of a compound (e.g. an animal or a cell) or a process or reaction performed within its natural environment.

[0074] The terms "recombinant AAV vector" or "transgene" are used interchangeably herein to denote a nucleic acid derived from the wild-type genome of an adeno-associated virus, wherein all endogenous AAV nucleic acids except for the ITR (adeno-associated virus inverted terminal repeat) sequences are replaced by one or more exogenous nucleic acids. For example, such exogenous nucleic acids can be nucleic acids transcribed into a transcript of interest or nucleic acids encoding a therapeutic protein or a therapeutic nucleic acid. Typically, for a recombinant AAV vector, one or both ITR sequences of the wild-type AAV genome are retained. Thus, a recombinant AAV vector can be distinguished from a wild-type AAV vector in that all or at least a portion of the viral genome has been replaced by a non-native (i.e. exogenous) nucleic acid with respect to the virus. Thus, the incorporation of a non-native nucleic acid defines the AAV vector as a "recombinant" vector. It has to be pointed out that the serotype of the ITR in the recombinant AAV vector does not need to be identical to the serotype of the adeno-associated capsid polypeptide forming the shell of the recombinant AAV particle comprising said recombinant AAV vector.

[0075] In principle, any non-AAV nucleic acid can be packaged into a shell composed of adeno-associated capsid polypeptides, resulting in a "recombinant AAV particle", e.g. for subsequent infection (transduction) ex vivo, in vitro or in vivo.

[0076] 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 polypeptides forming the protein shell (capsid) of the respective AAV particle. Initially, serological uniqueness was determined based on the lack of cross-reactivity of antibodies between one AAV particle compared to another AAV particle. Such cross-reactivity differences are typically due to differences in the capsid polypeptide sequences and corresponding antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). While an AAV variant including a capsid variant can not be serologically distinct from a reference or wild-type AAV or other AAV serotype, it differs by at least one amino acid residue compared to the reference or wild-type or other AAV serotype.

[0077] Under the traditional definition, a serotype refers to a target virus that has been tested against sera specific for all existing and characterized serotypes for neutralization activity and no antibody was found that neutralizes the target virus. As more naturally occurring viral isolates are discovered and / or capsid mutants are generated, there can or can not be serological differences from any of the currently existing serotypes. Thus, in the case where a new AAV particle does not have a serological difference, the new AAV particle will be a subset or variant of the corresponding wild-type serotype. In many cases, serological testing for neutralization activity has not been performed for mutant viruses having capsid sequence modifications to determine whether they have another serotype according to the traditional definition of serotype.

[0078] The term "vector" denotes the portion of a larger nucleic acid (e.g., a recombinant plasmid) that is ultimately packaged or encapsidated or encapsulated directly or in single-stranded form or in RNA form into a protein shell composed of adeno-associated virus capsid polypeptides to form a recombinant AAV particle. In the case where a recombinant plasmid is used to construct or make a recombinant AAV particle, the viral particle does not include a "plasmid" portion that does not correspond to the vector portion of the recombinant plasmid. For example, in the case of rAAV, the recombinant vector includes that portion of the recombinant plasmid that is inserted between two AAV ITRs. The non-vector portion of the recombinant plasmid is referred to as the "plasmid backbone." The plasmid backbone is important for cloning and amplification of the plasmid, a process required for propagation and recombinant virus production, but is not itself packaged or encapsidated or encapsulated into a recombinant AAV particle. Thus, "vector" refers to the nucleic acid that is packaged or encapsidated or encapsulated by a protein shell composed of adeno-associated virus capsid polypeptides, i.e., in rAAV.

[0079] General methods for producing rAAV particles

[0080] WO 1999 / 11764 reports a method for generating high-titer, helper-free preparations of recombinant AAV vectors. A non-further defined AAV producer cell was infected with adenovirus type 5 (Ad5) at a multiplicity of infection (MOI) of 10 in low serum medium at a scale of 1.5 L at different pH values in a bioreactor with suspension growth. At a culture pH of 7.2, 4.7 E+12 total particles were obtained; at a culture pH of 7.4, 1.95 E+13 total particles were obtained; at a culture pH of 7.6, 1.84 E+13 total particles were obtained; and at a culture pH of 8.0, 1.63 E+13 total particles were obtained. The cultivation was carried out in a 1.5 L bioreactor and, thus, the cultivation volume (75% of the nominal value) can be calculated to be about 1.125 L. Thus, the total particle numbers correspond to 4.2 E+09 vp / mL (pH 7.2), 1.7 E+10 vp / mL (pH 7.4), 1.6 E+10 vp / mL (pH 7.6), and 1.5 E+10 vp / mL (pH 8), respectively.

[0081] WO 2000 / 14205 reports the production of AAV particles in a non-defined cell type called JL-14 cells by co-infection with an adenovirus helper virus, wherein the number of AAV particles (sum of intracellular and secreted AAV particles) is highest at a pH value of 7.4, the infectivity of AAV particles is highest at a pH value of 8, and the ratio of AAV particle number to infectivity is highest at a pH of 7.6. The cultivation was carried out in a medium with a volume of 1.5 L and, thus, the total particle numbers 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 full / empty particle ratio of the thus produced rAAV particles is below 1%.

[0082] This is based on the following calculations from the data of WO 2000 / 14205, Fig. 2B and Fig. 3B (3 days post infection):

[0083]

[0084]

[0085] Piras, B.A., et al. (Mol. Ther. Meth. Clin. Dev. 3 (2016) 16015) compared the distribution of AAV8 in cell culture medium and lysate at days 3, 5, 6, and 7 post transfection and found that virus production increased continuously up to day 6, with the proportion of viral particles in the medium increasing from 76% at day 3 to 94% at day 7. Larger scale production indicated that the ratio of intact to empty AAV particles was similar in the medium and lysate, and that AAV harvested at day 6 post transfection provided the same functionality in mice as AAV harvested at day 3. When the culture was extended from day 3 (total capsids in lysate and medium were 1.1 x 1 E+13 ± 9.2 x 1 E+11 and 3.6 x 1 E+13 ± 2.5 x 1 E+12, respectively) to day 5 (total capsids in lysate and medium were 6.7 x 1 E+12 ± 6.7 x 1 E+11 and 4.5 x 1 E+13 ± 2.6 x 1 E+12, respectively), day 6 (total capsids in lysate and medium were 5.0 x 1 E+12 ± 1.9 x 1 E+11 and 5.3 x 1 E+13 ± 3.3 x 1 E+12, respectively), and day 7 (total capsids in lysate and medium were 3.0 x 1 E+12 ± 1.3 x 1 E+10 and 5.0 x 1 E+13 ± 1.9 x 1 E+12, respectively), AAV-FVIII exhibited an increase in production. 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). One day after seeding the cells at a density of 7.26 x 1 E+04 cells / cm2, AAV was produced by double plasmid transfection using PEIpro(TM) (Polyplus-transfection SA, Illkirch, France). 2 AAV was produced by double plasmid transfection using PEIpro(TM) (Polyplus-transfection SA, Illkirch, France) one day after seeding the cells at a density of 7.26 x 1 E+04 cells / cm2.

[0086] Powers, A.D., et al. (Hum. Gene Ther. Meth. 27 (2016) 112-121) report the development and optimization of AAV hFIX particle production by transient transfection in iCELLis(R) fixed-bed bioreactors. Virus particle yields of up to 9 E+14 per square meter of fixed bed were obtained. Three days after seeding of HEK293T / 17 cells, the vessels were transfected with polyethylenimine (PEIpro(TM) transfection reagent cat. no. 115-375; Polyplus) in IMDM (Lonza) or DMEM supplemented with 10% FBS and 6 mM GlutaMAX(TM) at a plasmid mass ratio of 3:1 of plasmid scAAV-LPl-hFIXco-helpv3 and plasmid CR21+LTA A V help 2-8, respectively. PEI and DNA solutions were combined at a ratio of 2:1.

[0087] Poulain, A., et al. (j. Biotechnol. 255 (2017) 16-27 report the rapid production of proteins from stable CHO cell pools using plasmid vectors and a cumate gene switch. Cells were transfected using linear polyethylenimine (PEIpro(TM)) from Polyplus-Transfection (Illkirch, France). On the day of transfection, cells were suspended in CD DG44 medium (Life Technologies Inc., Burlington, ON, Canada) supplemented with 4 mM glutamine and 0.1% Kolliphor® P 188 at a density of 2 x 1 E+06 cells / mL. Cell suspensions were distributed in 6-well plates (1.8 mL / well). DNA:PEIpro(TM) complexes were prepared at a ratio of 1:5 (w:w) with a total of 2 pg DNA per well to transfect in 100 pL of complete medium.

[0088] WO 2017 / 096039 reports a scalable method for producing recombinant AAV vectors in a serum-free suspension cell culture system suitable for clinical use. rAAV vector production was performed using triple transfection in a bioreactor containing HEK293F cells at a cell density of 1 E+06 cells / mL (1,000,000 cells / mL), a 1:1:1 plasmid ratio, and a PEI-based transfection reagent (PEI / DNA weight ratio of 2:1, with ½ of the PEI being free PEI) at 37°C and a pH of 7.2.

[0089] Nyamay'antu, A., et al. (Cell Gen. Ther. Ins. 4 (2018) 71-79) report that PEI is widely used for both adherent and suspension cells grown in serum-free media due to its affordability and high DNA delivery efficiency. PEIpro(TM) is suitable for small- to large-scale production of various viruses, particularly AAV particles. Cell titers ranging from 0.8 to 1.5 E+09-E+10 vg / mL can be achieved in stirred-tank bioreactors using HEK293 or HEK293T cells.

[0090] Koo, T., et al. (Nat. Commun. 9 (2018) 1855) reported that CRISPR-LbCpf1 prevented choroidal neovascularization in a mouse model of age-related macular degeneration. To generate AAV vectors, they were pseudotyped in AAV9 capsids. HEK293T cells (ATCC, CRL-3216) were transfected with pAAV-ITR-LbCpf1-crRNA, pAAV2 / 9 encoding the AAV2 rep and AAV9 cap, and helper plasmids. HEK293T cells were cultured in DMEM supplemented with 2% FBS. Recombinant pseudotyped AAV vector stocks were generated by coprecipitation with PEI and PEIpro™ (Polyplus transfection) and triple transfection with plasmids at a 1:1:1 molar ratio in HEK293T cells. After 72 h of incubation, cells were lysed and particles were purified by iodixanol step-gradient ultracentrifugation.

[0091] Rep proteins from AAV2 are commonly and almost exclusively used for the production of rAAV derived from serotypes AAV1 to AAV13 (Daya, S., and Berns, K.I., Clin. Microbiol. Rev. 21 (2008) 583-593; Zincarelli, C., et al., Mol. Ther. 16 (2008) 1073-1080).

[0092] WO 2019 / 094253 reports means and methods for the preparation of viral vectors and uses thereof. Adherent HEK293 cells were cultivated in a bioreactor at a pH value of 7.23 and triple transfected with PEI / DNA at a PEI-plasmid ratio of about 1 :1 by weight (plasmid ratio 1 :1 :1).

[0093] Collaud, F. et al. (Mol. Ther. Meth. Clin. Dev. 12 (2019) 157-174) report for adherent HEK293 cells a titer of 6.0 ± 1.89 E+04 vg / cell and 1.77 ± 1.37 E+04 vg / cell for (single stranded) and (self-complementary) AAV, respectively, for rAAV8 particles. A fully scalable method based on triple transfection of HEK293 cells cultivated in suspension is also reported. Triple transfection of HEK293 cells was performed directly in a 10 L bioreactor with polyethyleneimine (PEIpro(TM), Polyplus). AAV vectors were recovered from both supernatant and cells by mild detergent lysis followed by purification over an AVB Sepharose affinity column. Purified vectors were then concentrated and tested for quality and potency. No information is provided about the pH value and the titer obtained.

[0094] Nyamay'antu, A., et al. (Cell Gen. Ther. Ins. 6 (2020) 655-661) report that the efficiency of the delivery process is crucial for obtaining large numbers of producer cells. Among the existing transfection methods, the use of PEI-based transfection reagents dominates in gene therapy because it combines the economy and compatibility of transfection of adherent and suspension cells. When each transfection reagent was used under the recommended conditions, FectoVIR(TM)-AAV was found to significantly improve both viral genome production and packaging efficiency in rAAV2 production from rAAV2-GFP in suspension cells compared to the gold standard PEIpro(TM) used for viral vector manufacturing, up to 10-fold compared to PEIMax(TM) and up to 2-fold compared to PEIpro(TM), respectively. In more detail, suspension HEK293T cells were transfected using the respective transfection reagents under the recommended conditions. rAAV2-GFP was harvested 72 hours post transfection. The titers obtained using VectoVIR(TM) ranged from 1 E+04 to 4.5 E+04 vg / cell depending on the complex volume used (1%-10%) corresponding to 1 E+12 vg / mL. The respective functional titers were about 2-8 E+08 TU / mL. The results were almost independent of the culture medium used.

[0095] In a blog post entitled “Optimization of AAV production for high-yielding and scalable GMP processes with Catalent” (www.polyplus-transfection.com), different transfection reagent to DNA ratios were tested using two serotypes of AAV9 (1 : 1 and 2: 1) and AAV2 (3: 1.5 and 5:2.5). AAV2 vector yields were not significantly affected, with vector genome titers with FectoVIR(TM)-AAV increased by 4-5 fold and viral particle titers increased by 3-6 fold compared to PEIpro(TM). These results suggest that the increase in yield can vary depending on the AAV serotype. In a further study comparing additional AAV2 and AAV5 vectors (different from the previous AAV2 and AAV5 vectors) and using a DoE approach for optimization, experiments were performed with different transfection reagent to DNA ratios (3:2, 3: 1.5) and plasmid DNA molar ratios (1 : 1 : 1, 2: 1 :2, 1 :2: 1). Vector genome titers with FectoVIR(TM)-AAV were observed to increase by 3-5 fold for AAV2 and 1.1-1.6 fold for AAV5 compared to PEIpro(TM). Viral particle titers increased by 3.5-4.5 fold for AAV2 and 2.5-3.75 fold for AAV5. Reagent to DNA ratios of 2: 1 and 1.5: 1 and plasmid ratios of 1 : 1 : 1 to 2: 1 :2 to 1 :2: 1 were used. Titers obtained with VectoVIR(TM) were in the range of 4 E+11 to 1 E+12 vg / mL.

[0096] Rossi, A. and Peigné, C-M. (Cell Culture Dish Article, May 17, 2021) state that AAV production titers are typically around 1 E+11 to 1 E+12 (in vg / mL) and 1 E+08 to 1 E+09 TU / mL.

[0097] To put these numbers into perspective, it is important to remember that even with the same production process, two AAV serotypes are likely to not produce the same yield.

[0098] In more detail, AAV production yields vary according to the serotype and the gene of interest. Generally, to increase the yield of a given AAV, it is necessary to optimize parameters that directly affect the yield: plasmid DNA, transfection reagent, cells and culture medium. For example, PEI-based transfection processes can reduce the amount of DNA by a factor of 10 and can be used to transfect cells grown in the presence or absence of serum.

[0099] Wosnitzka, K., et al. (Cell Gen. Ther. Ins. 7 (2021) 1-7) report that physical titer analysis showed a 3-fold increase in both viral particles (VP) and viral genomes (VG) per milliliter of cell culture when using FectoVIR(TM)-AAV transfection reagent compared to PEIpro(TM).

[0100] Porte, M., et al. (Poster entitled “Next-Generation Transfection Reagent for Large Scale AAV Manufacturing”, Polyplus, Illkirch, France) report transfecting suspension-HEK293T cells following the recommended protocol for each reagent using optimal conditions for other PEI-based reagents (1.5 pg per million cells, ratio DNA: PEI 1 pg: 4 pL) and FectoVIR(TM)-AAV (1 pg per million cells, ratio DNA: reagent 1 pg: 1 pL). Titers of about 5 E+11 vg / mL with FectoVIR(TM) and 1.5 E+11 vg / mL with PEI-based transfection reagent were obtained, with packaging efficiencies of 20% and about 13.5%, respectively.

[0101] Recombinant cells

[0102] Generally, to produce rAAV efficiently and at a large scale, cells that express and, if possible, also secrete said rAAV are used. Such cells are referred to as “recombinant production cells” or simply “production cells”.

[0103] To generate recombinant production cells, a suitable mammalian cell is transfected with the nucleic acids required for the production of said rAAV, including the required AAV helper functions.

[0104] In general, for the expression of a coding sequence (i.e. an open reading frame), additional regulatory elements are required, such as a promoter and a polyadenylation signal (sequence). Thus, for functional transcription, the open reading frame has to be and is operably linked to said additional regulatory elements. This can be achieved by combining these parts into so-called expression cassettes. The minimum regulatory elements required for a functional expression cassette in a mammalian cell are a promoter functional in said mammalian cell, which is located upstream (i.e. 5') of the open reading frame, and a polyadenylation signal (sequence) functional in this mammalian cell, which is located downstream (i.e. 3') of the open reading frame. In addition, a terminator sequence can be present 3' of the polyadenylation signal (sequence). For expression, the promoter, the open reading frame / coding region and the polyadenylation signal sequence have to be arranged in operable linkage.

[0105] Likewise, a nucleic acid which is transcribed into a non-protein-coding RNA is called "RNA gene". In addition, for the expression of an RNA gene, additional regulatory elements are required, such as a promoter and a transcription termination signal or a polyadenylation signal (sequence). The nature and positioning of such elements depends on the RNA polymerase which is intended to drive the expression of the RNA gene. Thus, RNA genes are usually also integrated into expression cassettes.

[0106] If an rAAV is intended (which is composed of different (monomeric) capsid polypeptides and a capsidized single-stranded DNA molecule therein, and which additionally requires other viral helper functions for production and capsidization), a number of expression cassettes is required, which comprise open reading frames / coding sequences which are different. In this case, for each transgene, for the polypeptides forming the capsid of the rAAV, at least an expression cassette for the required viral helper functions is required. Thus, at least a separate expression cassette is required for each of the helper functions E1A, E1B, E2A, E4orf6, rep and cap genes. HEK293 cells express the E1A and E1B helper functions constitutively.

[0107] Adeno-associated virus (AAV)

[0108] For a general review of AAV and adenovirus or herpesvirus helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. Design considerations for constructing recombinant AAV vectors are described in US 4,797,368 (see also WO 93 / 24641). Additional 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. Construction of recombinant AAV vectors is described in US 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.

[0109] AAV is a replication-defective parvovirus. It can only replicate in cells where certain viral functions are provided by a co-infecting helper virus, such as adenovirus, herpesvirus, and in some cases poxvirus, such as vaccinia. Nonetheless, AAV can replicate in virtually any cell line of human, simian, or rodent origin, provided that the appropriate helper virus functions are present.

[0110] If no helper virus genes are present, AAV establishes a latent phase in its host cell. Its genome integrates into a specific site in chromosome 19 [(Chr) 19 (q13.4)], called the adeno-associated virus integration site 1 (AAVS1). For certain serotypes, such as AAV2, other integration sites have been found, such as for example on chromosome 5 [(Chr) 5 (p13.3)], called AAVS2, and on chromosome 3 [(Chr) 3 (p24.3)], called AAVS3.

[0111] AAV are classified into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity and DNA sequence homology. So far, 12 different serotypes and more than 100 sequences corresponding to different branches of AAV have been identified.

[0112] The type and symmetry of the capsid proteins determine the tissue tropism of the respective AAV. For example, AAV2, AAV4 and AAV5 are specific for the retina, AAV2, AAV5, AAV8, AAV9 and AAV-rh.10 are specific for the brain, AAV1, AAV2, AAV6, AAV8 and AAV9 are specific for cardiac tissue, AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 are specific for the liver, AAV1, AAV2, AAV5 and AAV9 are specific for the lung.

[0113] Pseudotyping denotes a method which includes the cross-packaging of AAV genomes between various serotypes, i.e. the packaging of genomes with capsid proteins of different origin.

[0114] The wild-type AAV genome has a size of about 4.7 kb. The AAV genome further comprises two overlapping genes, called rep and cap, which comprise 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. Virol., 51 (1984) 611-619). The open reading frames encoding the Rep proteins provide four proteins of different size, which are called Rep78, Rep68, Rep52 and Rep40. These are involved in replication, rescue and integration of AAV. The open reading frames encoding the Cap proteins provide four proteins, which are called VP1, VP2, VP3 and AAP. VP1, VP2 and VP3 are part of the protein coat of the AAV particle. The combined rep and cap open reading frames are flanked at their 5' and 3' ends by so-called inverted terminal repeat sequences (ITRs). For replication, AAV needs, in addition to the Rep and Cap proteins, the products of the genes E1A, E1B, E4orf6, E2A and VA of adenovirus or corresponding factors of another helper virus.

[0115] For example, in the case of AAV of serotype 2 (AAV2), the ITRs each have a length of 145 nucleotides and flank a coding sequence region of about 4470 nucleotides. Of the 145 nucleotides of the ITR, 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure has the function of a primer during viral replication. The remaining 20 unpaired nucleotides are denoted as D-sequence.

[0116] The wild-type AAV genome contains three transcription promoters P5, P19 and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of the rep and cap genes.

[0117] ITR sequences must be present in cis to the coding region. ITRs provide a functional replication origin (ori), signals required for integration into the target cell genome, and efficient excision and rescue from host cell chromosomes or recombination plasmids. ITRs further contain replication origin-like elements such as Rep protein binding sites (RBS) and terminal resolution sites (TRS). It has been found that ITRs can themselves have the function of a transcriptional promoter (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0118] For the separate replication and encapsidation of the viral single-stranded DNA genome, the rep and cap gene products are required in trans.

[0119] The rep locus contains two internal promoters, designated P5 and P19. It comprises the open reading frames for four proteins. The promoter P5 is operably linked to nucleic acid sequences that provide a non-spliced 4.2 kb mRNA encoding the Rep protein Rep78, a chromatin- nicking enzyme that blocks the cell cycle, and a spliced 3.9 kb mRNA encoding the Rep protein Rep68, a site-specific endonuclease. The promoter P19 is operably linked to nucleic acid sequences that provide a non-spliced mRNA encoding the Rep protein Rep52 and a spliced 3.3 kb mRNA encoding the Rep protein Rep40, a DNA helicase for accumulation and packaging.

[0120] The two larger Rep proteins Rep78 and Rep68 are essential for AAV duplex DNA replication, whereas the smaller Rep proteins Rep52 and Rep40 appear to be essential for progeny and single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0121] The larger Rep proteins Rep68 and Rep78 can bind specifically to the hairpin conformation of the AAV ITRs. They exhibit defined enzymatic activities that are required to resolve the AAV termini for replication. Expression of Rep78 or Rep68 can be sufficient to form infectious particles (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0122] It is assumed that all Rep proteins, primarily Rep78 and Rep68, exhibit regulatory activities such as induction and repression of AAV genes and inhibition of 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).

[0123] Recombinant overexpression of Rep78 leads to a phenotype of reduced cell growth caused by induction of DNA damage. Thereby, the host cell is arrested in the S phase, thus promoting the latent infection of the virus (Berthet, C. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0124] Tratschin et al. report that the P5 promoter is negatively auto-regulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effect of Rep protein expression, it is reported that expression is only very low for certain cell lines after stable integration of AAV (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0125] The cap locus comprises a promoter, called P40. The promoter P40 is operably linked to a nucleic acid sequence providing 2.6 kb mRNA, which encodes the Cap proteins VP1 (87 kDa, non-spliced mRNA transcript), VP2 (72 kDa, from spliced mRNA transcript) and VP3 (61 kDa, from alternative start codon) by alternative splicing and use of alternative start codons. VP1 to VP3 constitute the building blocks of the viral capsid. The capsid has the function of binding to cell surface receptors and allowing intracellular transport of the virus. VP3 accounts for about 90% of the total protein of the viral particle. However, all three proteins are necessary for efficient capsid production.

[0126] It has been reported that inactivation of all three capsid proteins, VP1 to VP3, prevents accumulation of single-stranded progeny AAV DNA. Mutations at the amino-terminal end of VP1 ("Lip negative" or "Inf negative") still allow single-stranded DNA to assemble into virus particles, thereby greatly reducing the infectious titer.

[0127] The AAP open reading frame encodes an assembly-activating protein (AAP). It has a size of about 22 kDa and transports the native VP proteins to the nucleolar region for capsid assembly. The open reading frame is located upstream of the VP3 protein coding sequence.

[0128] In a single AAV particle, there is only one single-stranded DNA molecule. This can be either the "plus" or "minus" strand. An AAV particle containing a DNA molecule is infectious. Within the infected cell, the parental infecting single-stranded DNA is converted to double-stranded DNA, which is then amplified. Amplification results in a large number of double-stranded DNA molecules, from which single-stranded and packaged into capsids.

[0129] Adeno-associated virus (AAV) vectors can transduce dividing and quiescent cells. It can be assumed that a transgene introduced to a target cell using an AAV vector will be expressed long-term. One disadvantage of using AAV vectors is that the size of a transgene that can be introduced to a cell is limited.

[0130] Parvovirus particles, including AAV serotypes and variants thereof, provide a tool for delivering a nucleic acid encoding a protein ex vivo, in vitro, and in vivo into a cell such that the infected cell expresses the encoded protein. AAVs are viruses that can be used as gene therapy vectors because they can penetrate cells and introduce nucleic acid / genetic material such that the nucleic acid / genetic material can be stably maintained in the infected cell. Because AAVs are not associated with pathogenic diseases in humans, AAVs are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and pharmaceutical agents) to human patients without causing substantial AAV-related pathogenesis or disease.

[0131] AAV particles used as vehicles for efficient gene delivery have many of the features required for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors has also shown no persistent toxicity and a small or undetectable immune response. AAVs are known to infect a variety of cell types in vivo and in vitro by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans to target the retinal epithelium, liver, skeletal muscle, airway, brain, joints, and hematopoietic stem cells.

[0132] Recombinant AAV particles typically do not comprise viral genes associated with pathogenesis. Such particles typically comprise a genome in which one or more of the wild-type AAV genes (e.g., rep and / or cap genes) have been deleted in whole or in part, but retain at least one functional flanking ITR sequence, which is necessary for rescue, replication, and packaging of the recombinant vector into rAAV. Thus, the AAV vector comprises the cis sequences (i.e., functional ITR sequences) required for replication and packaging.

[0133] Recombinant AAV particles, methods and uses thereof can be based on any wild-type AAV genome or serotype or combinations thereof. As non-limiting examples, rAAV can be based on any wild-type AAV genome, i.e., comprising the corresponding ITR sequences, such as, for example, AAV1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-2i8, AAV-rh.74, AAV-rh.10, or AAV-7m8. Such particles can be based on the same strain or serotype (or subgroup or variant), or different from each other. As non-limiting examples, rAAV based on one wild-type genome can be the same or different from the one or more capsid proteins packaging the vector. Further, the recombinant AAV vector can be based on a different AAV (e.g., AAV2) wild-type serotype genome from the one or more AAV capsid proteins of the packaging vector. For example, the AAV vector genome can be based on AAV2, while at least one of the three capsid proteins can be AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10, or AAV-7m8 or variants thereof. AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10, and AAV 7m8 capsids.

[0134] In certain embodiments of all aspects and embodiments of the application, the rAAV particle is derived from a wild-type AAV particle 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, and variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions, and deletions), e.g., as described in WO 2013 / 158879, WO 2015 / 013313, and US2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0135] In certain embodiments of all aspects and embodiments of the application, the rAAV comprises a capsid polypeptide having an amino acid sequence that has 70% or more sequence identity to a 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.

[0136] In certain embodiments of all aspects and embodiments of the application, the rAAV particle comprises one or two ITR sequences having 70% or more sequence identity to a wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 ITR sequence.

[0137] The recombinant AAV particles can be incorporated into a pharmaceutical composition. Such pharmaceutical compositions are particularly useful for administration and delivery to a subject, either in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of an immune response in the individual to which it is administered, and which is not toxic to the individual at the dosages administered.

[0138] Protocols for generating adenoviral vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810; and WO 94 / 23744, the entire contents of which are incorporated herein by reference.

[0139] Recombinant adeno-associated virus particles (rAAV particles)

[0140] Different methods for producing recombinant AAV particles are known in the art. For example, transfection is performed with an AAV vector comprising a plasmid and a plasmid comprising AAV helper sequences (rep and cap) in combination with co-infection with one AAV helper virus (e.g. adenovirus, herpesvirus or vaccinia virus), or transfection is performed with a recombination AAV vector comprising a plasmid, an AAV helper plasmid (comprising rep and cap) and a helper function plasmid. Non-limiting methods for generating rAAV are described, for example, in US 6,001,650, US 6,004,797, WO 2017 / 096039 and WO 2018 / 226887. After production of rAAV (i.e. generation of particles in a cell culture system), rAAV can be obtained from the host cells and / or cell culture supernatant and purified.

[0141] For the generation of recombinant AAV particles, Rep and Cap proteins, the helper proteins E1A, E1B, E2A and E4orf6 and optionally the adenoviral VA RNA have to be expressed in a single mammalian cell. The helper proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), in particular the CMV IE promoter. Thus, any promoter can be operably linked to the genes for functional expression.

[0142] Generally, for the production of rAAV, different complementary plasmids are co-transfected into a host cell. One of the plasmids comprises a transgene sandwiched between two cis-acting AAV ITRs. The missing AAV components (i.e. the open reading frames for Rep and Cap proteins) required for replication and subsequent packaging of the progeny recombinant genome are contained in trans in a second plasmid. Overexpression of Rep proteins causes an inhibitory effect on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). In addition, rAAV production requires a third plasmid comprising helper virus genes, i.e. E1, E4orf6, E2A and VA from adenovirus.

[0143] To reduce the number of required plasmids, rep, cap and adenoviral helper genes can be combined on a single plasmid.

[0144] Alternatively, the host cell can have stably expressed the El gene products. Such cells are HEK293 cells. The human embryonic kidney clone denoted 293 was generated as early as 1977 by integrating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, F.L., et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line contains base pairs 1 to 4344 of the adenovirus serotype 5 genome. This includes the El A and El B genes as well as the adenovirus packaging signal (Louis, N., et al., Virology 233 (1997) 423-429).

[0145] When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with plasmids expressing E2A, E4orf6, and VA (see, e.g., Samulski, R.J., et al., J. Virol. 63 (1989) 3822-3828; Allen, J.M., et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, T.R., et al., Gene Ther. 2 (1995) 29-37; Conway, J.E., et al., J. Virol. 71 (1997) 8780-8789; Chiorini, J.A., et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, F.K., 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 can be used (see, e.g., Conway, J.E., et al., J. Virol. 71 (1997) 8780-8789; Johnston, K.M., et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, A.J., et al., Gene Ther. 2 (1995) 481-485; Fisher, J.K., et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, K.M., et al., Hum. Gene Ther. 8 (1997) 359-370).

[0146] To restrict the transgene activity to a specific tissue, i.e. to limit the site of action, the transgene can be operably linked to an inducible or tissue-specific promoter (see, e.g., Yang, Y. et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0147] The coding sequences (open reading frames) of E1A and E1B can be derived from a human adenovirus, such as, for example, in particular human adenovirus serotype 2 or serotype 5. Exemplary sequences of human Ad5 (adenovirus serotype 5) are found in GenBank entries X02996, AC_000008, and exemplary sequences of human Ad2 are found in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. The plasmid pSTK146 as reported in EP 1 230 354 and the plasmids pGS119 and pGS122 as reported in WO 2007 / 056994 can also be used as a source for the E1A and E1B open reading frames.

[0148] E1A is the first viral helper gene expressed after entry of the adenoviral DNA into the nucleus. The E1A gene encodes 12S and 13S proteins, which are based on the same E1A mRNA by alternative splicing. The expression of the 12S and 13S proteins leads to the activation of the other viral functions E1B, E2, E3 and E4. In addition, the expression of the 12S and 13S proteins forces the cell into the S phase of the cell cycle. If only E1A derived proteins are expressed, the cell will die (apoptosis).

[0149] E1B is the second viral helper gene expressed. It is activated by the E1A derived proteins 12S and 13S. The E1B gene derived mRNA can be spliced in two different ways, resulting in a first 55 kDa transcript and a second 19 kDa transcript. The E1B 55 kDa protein is involved in the modulation of the cell cycle, in the prevention of the transport of post-infection cellular mRNA and in the prevention of E1A induced apoptosis. The E1B 19 kDa protein is involved in the prevention of E1A induced apoptosis.

[0150] The E2 gene encodes different proteins. The E2A transcript encodes the single-stranded binding protein (SSBP), which is essential for AAV replication

[0151] Furthermore, the E4 gene encodes multiple proteins. The 34 kDa protein derived from the E4 gene (E4orf6) prevents cellular mRNA accumulation in the cytoplasm together with the ElB 55 kDa protein, but also facilitates the transport of viral RNA from the nucleus to the cytoplasm.

[0152] Virus-associated RNA (VA RNA) is a non-coding RNA of the adenovirus (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed by RNA polymerase III (see, e.g., Machitani, M. et al., J. Contr. Rel. 154 (2011) 285-289) from a type 2 polymerase III promoter. For recombinant AAV particle production, the adenovirus VA RNA genes can be driven by any promoter.

[0153] Ma, Y. and Mathews, M.B. (J. Virol. 70 (1996) 5083-5099) investigated the structure, function, and evolution of adenovirus-associated RNA using phylogenetic methods. They provided an alignment based on 47 known human adenovirus serotypes and a consensus VA RNA sequence. The disclosure is hereby incorporated by reference in its entirety into the present application.

[0154] VA RNA, VAI, and VAII consist of 157 to 160 nucleotides (nt).

[0155] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is thought to exert the major pro-viral effect, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, V.K. and Conn, G.L., Virus Res. 212 (2016) 39-52).

[0156] VA RNA is not essential, but plays an important role in efficient virus growth by overcoming cellular anti-viral mechanisms. That is, while VA RNA is not essential for virus growth, VA RNA-deleted adenoviruses fail to grow during the initial steps of vector production, where only a few copies of the viral genome exist per cell, possibly because viral genes other than VA RNA that block cellular anti-viral mechanisms can not be expressed sufficiently (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0157] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenoviral vectors lacking the virus-associated RNA gene that disrupts the cellular RNAi machinery, where HEK293 cells constitutively and highly expressing the Flippase recombinase were infected to obtain VA RNA-deleted adenoviruses by FLP recombinase-mediated excision of the VA RNA locus.

[0158] Human adenovirus 2 VA RNAI corresponds to nucleotides 10586-10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5 VA RNAI corresponds to nucleotides 10579-10820 of the sequence of GenBank entry AC_000008.

[0159] Overview of recombinant AAV particle production

[0160] Upon entry into the host cell nucleus, AAV can follow one of two different and interchangeable pathways of its life cycle: the lytic pathway or the lysogenic pathway. The former develops in cells infected with a helper virus such as Ad or herpes simplex virus (HSV), while the latter is established in host cells in the absence of a helper virus.

[0161] When a latently infected cell is superinfected with a helper virus, the AAV gene expression program is activated, leading to AAV Rep-mediated rescue (i.e. excision) of the proviral DNA from the host cell chromosome, followed by replication and packaging of the viral genome. Finally, after helper virus-induced cell lysis, newly assembled virions (particles) are released. Thus, the lytic phase of the AAV life cycle is induced.

[0162] Therefore, in the presence of Ad helper functions, the rAAV vector undergoes the wild-type AAV lytic process by being rescued from the plasmid backbone, replicated and packaged into preformed AAV capsids as a single-stranded molecule (Gonçalves, M.A.F.V., Virol. J., 2 (2005) 43).

[0163] Generation of recombinant AAV particles involves replacing most of the AAV wild-type genome with the desired transgene and providing the viral genes necessary for packaging the virus in trans on separate plasmids. Once all components are transfected together into a packaging cell line, the recombinant AAV particles are assembled using the cell’s cellular machinery. The process of viral assembly and encapsidation takes approximately two days, after which the cells are lysed to release the rAAV for further purification and concentration (https: / / old.abmgood.com / marketing / knowledge_base / Adeno_Associated_Virus_Production_and_Modification_of_AAV.php).

[0164] Although major differences are observed between serotypes, AAV is not very efficiently released from the cells (see, e.g., 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). At the time of harvest of the culture, a cell disruption method is usually applied to recover the vectors entrapped in the cells.

[0165] Historically, rAAV production was performed by double transfection of a plasmid containing the rep and cap ORFs and a plasmid of the gene of interest flanked by ITRs. Then, a helper virus, usually an adenovirus, was co-infected (see, e.g., Aponte-Ubillus, J.J., et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Muzyczka, N., Curr. Top. Microbiol. Immunol. 158 (1992) 97-129). In this case, the separation of the helper virus from the final product is difficult but a key factor to avoid the induction of inflammatory reactions after injection into the patient (see, e.g., Schnell, M.A., et al., Mol. Ther. 3 (2001) 708-722.). Therefore, the production of rAAV now shifted to an adenovirus-free approach by making use of triple transfection (see, e.g., Large, E.E., et al., Viruses 13 (2021) 1336). For this, three components are needed: one plasmid encoding the Rep and Cap genes, but without ITRs, a second plasmid with the gene of interest flanked by ITRs, and a helper plasmid providing the helper virus with the helper genes (see, e.g., Aponte-Ubillus, J.J., et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Farris, K.D. and Pintel, D.J., Hum. Gene Ther. 19 (2008) 1421-1427; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Ferrari, F.K., et al., Nat. Med. 3 (1997) 1295-1297). For example, the adenovirus helper carries the minimal adenovirus genes E2A, E4, and VA needed. Notably, human embryonic kidney cells 293 (HEK293) constitutively express the adenovirus gene E1A / B, which is also required for the production of rAAV. Therefore, HEK293 cells are the classical producer cells for rAAV and manufacturing. Other cell types need to be supplemented with E1A / B.

[0166] Carter et al. have shown that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with a transgene (Carter, B. J., in "Handbook of Parvoviruses", P. Tijssen, ed., CRC Press, pp. 155-168 (1990)). Furthermore, it was reported that the ITRs must be maintained to preserve the functions of replication, rescue, packaging, and integration of the transgene into the target cell genome.

[0167] When cells containing the respective viral helper genes are transduced by the AAV vector, or vice versa, when cells containing the integrated AAV provirus are transduced by the appropriate helper virus, then the AAV provirus is activated and re-enters the lytic infectious cycle (Clark, K. R., et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, R. J., Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0168] Production cells contain rep and cap gene sequences, as well as a transgene cassette flanked by ITR sequences on one or more plasmids that are retained, e.g., via drug selection. Production of rAAV in these cell lines generally occurs after the provision of helper functions required for infection of the cells. Thus, the cells are infected with a replication-competent adenovirus (usually wild-type Ad5) or a plasmid containing the respective helper genes to provide the helper virus proteins and initiate rAAV production. A packaging cell line differs from a production cell line in that it contains only rep and cap genes.

[0169] More generally, a cell that is transfected or transduced with DNA for recombinant production of AAV particles can be referred to as a "recombinant cell." Such a cell can be any mammalian cell that has been rendered a recipient of a nucleic acid (plasmid) encoding a packaging protein such as an AAV packaging protein, a nucleic acid (plasmid) encoding a helper protein, and a nucleic acid (plasmid) encoding a protein or transcribed into a transcript of interest (i.e., a transgene placed between two AAV ITRs). The term includes descendants of the original cell that has been transduced or transfected. It will be appreciated that descendants of a single parent cell can not necessarily be exactly identical in morphology or genome or total nucleic acid complement due to natural, accidental, or intentional mutations.

[0170] A number of cell growth media suitable for maintaining cell viability or providing cell growth and / or proliferation are commercially available. Examples of such media include serum-free eukaryotic growth media, such as media for maintaining mammalian (e.g., human) cell viability or providing mammalian (e.g., human) cell growth. Non-limiting examples include Ham's F12 or F12K media (Sigma-Aldrich), FreeStyle (FS) F17 media (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as the essential amino acids for mammalian (e.g., human) cells.

[0171] To produce rAAV, three plasmids are co-transfected into mammalian cells. The transgene plasmid encodes an expression cassette cloned between AAV ITRs, while the rep and cap genes are provided in trans by co-transfecting a second packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. A third plasmid, also called helper plasmid, contains minimal helper viral factors (typically adenovirus E2A, E4orf6 and VA genes), but lacks AAV ITRs.

[0172] A variety of methods for transferring DNA into mammalian cells have been reported in the art. These can all be used in the methods according to the present application. In certain embodiments of all aspects and embodiments, nucleic acid transfer / transfection is performed using electroporation, nucleofection or microinjection. In certain embodiments of all aspects and embodiments, nucleic acid transfer / transfection is performed using inorganic substances such as, for example, calcium phosphate / DNA co-precipitation, cationic polymers such as, for example, polyethylenimine, DEAE-dextran or cationic lipids (lipofection). Calcium phosphate and polyethylenimine are the most commonly used reagents in transfection for larger scale nucleic acid transfer (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), with polyethylenimine being preferred.

[0173] Growth in serum-free suspension culture and use of PEI as transfection reagent to improve efficiency and reproducibility of transfection conditions allows to scale up AAV production in real-time using shake flasks, wave or stirred tank bioreactors.

[0174] The composition can further comprise plasmids or / and cells. Such plasmids and cells can be contacted with free PEI.

[0175] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA is a branched short-chain fatty acid and inhibits histone deacetylase activity. For this reason, it is often added to mammalian cell cultures as an enhancer of recombinant protein production.

[0176] In certain embodiments of all aspects and embodiments, the encoded AAV packaging proteins include AAV rep and / or AAV cap. In certain embodiments of all aspects and embodiments, such AAV packaging proteins include AAV rep and / or AAV cap proteins of any AAV serotype.

[0177] In certain embodiments of all aspects and embodiments, the encoded helper proteins include adenovirus ElA and ElB, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0178] Culturing can be performed using common conditions for culturing eukaryotic cells of about 37°C, 95% humidity, and 8% CO2 by volume. Culturing can be performed in serum-containing or serum-free media, adherent cultures, or suspension cultures. Suspension culturing can be performed in any fermentation vessel, such as, for example, in a stirred tank reactor, a wave reactor, a rocking bioreactor, a shaker vessel, or a rotating vessel, or so-called roller bottles. Transfection can be performed in high-throughput format and screening, respectively, for example, in 96- or 384-well format.

[0179] The methods according to the present application can include AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles comprise any of AAV serotypes 1 to 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 certain embodiments of all aspects and embodiments, the AAV particles comprise an AAV serotype or an AAV pseudotype, wherein the AAV pseudotype includes an AAV capsid serotype that is different from the ITR serotype.

[0180] Expression control elements include constitutive or regulatable control elements, such as tissue-specific expression control elements or promoters.

[0181] The ITRs can be any of AAV2 or AAV6 or AAV8 or AAV9 serotypes, or combinations thereof. The AAV particle can comprise any VP1, VP2 and / or VP3 capsid proteins having 75% or more sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV12, AAV 2i8, AAV rh.10, AAV rh.74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or comprise a modified or variant VP1, VP2 and / or VP3 capsid protein selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-2i8, AAV-rh.10, AAV-rh.74 and AAV-7m8 AAV serotypes.

[0182] Following production of recombinant viral (e.g., AAV) particles, the viral (e.g., rAAV) particles can be purified and / or isolated from the host cells, if desired, using a variety of conventional methods. Such methods include column chromatography, CsCl gradients, iodixanol gradients, and the like.

[0183] For example, multiple column purification steps can be used, such as purification by an anion exchange column, an affinity column, and / or a cation exchange column. (See, e.g., WO 02 / 12455 and US 2003 / 0207439). Alternatively or additionally, an iodixanol or CsCl gradient step can be used. (See, e.g., US 2012 / 0135515; and US 2013 / 0072548). Further, if an infectious virus is used to express the packaging and / or helper proteins, various methods can be used to inactivate residual virus. For example, adenovirus can be inactivated via heating to a temperature of about 60°C for, e.g., 20 minutes or more. This treatment effectively inactivates the helper virus, as AAV is heat stable, while the helper adenovirus is heat labile.

[0184] One goal of rAAV production and purification systems is to implement strategies to minimize / control production-related impurities, such as protein, nucleic acid, and vector-related impurities, including wild type / pseudo wild type AAV species (wtAAV) and AAV encapsidated residual DNA impurities.

[0185] Given that rAAV represents only a small fraction of the biomass, rAAV needs to be purified to purity levels that can be used as a clinical human gene therapy product (see e.g. Smith P.H. et al., Mo. Therapy 7 (2003) 8348; Chadeuf G. et al., Mo. Therapy 12 (2005) 744; Report from CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0186] In certain embodiments of all aspects and embodiments of the method according to the application, as an initial step, the culture cells producing rAAV particles are typically harvested, optionally combined with the cell culture supernatant (culture medium) in which the cells producing the recombinant AAV particles (suspended or adherent cells) have been cultivated. The harvested cells and optional cell culture supernatant can be used as such, lysed or concentrated as appropriate. Furthermore, if infection is utilized for expression of helper functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to a temperature of about 60°C for e.g. 20 minutes or more, which inactivates only the helper virus, as AAV is heat stable, while the helper adenovirus is heat labile.

[0187] The cells in the harvested culture broth can be lysed to release the rAAV particles using methods available in the art, such as e.g. detergent lysis or freeze-thaw cycles. Simultaneously with the cell lysis or after the cell lysis, a nuclease such as e.g. benzonase is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, e.g. by filtration or centrifugation, to obtain a clarified cell lysate. In particular examples, the lysate is filtered with a micrometer diameter pore size filter such as a 0.1-10.0 pm pore size filter, e.g. a 0.45 pm and / or a 0.2 pm pore size filter, to produce a clarified lysate.

[0188] The lysate, optionally clarified, contains the recombinant AAV particles (including intact and empty rAAV) and production / process related impurities such as soluble cellular components from the host cells, which can include inter alia cellular proteins, lipids and / or nucleic acids, and cell culture medium components. The optional clarified lysate is then subjected to purification steps, using chromatography to purify the rAAV (comprising the rAAV vector) from the impurities. Prior to the first chromatography step, the clarified lysate can be diluted or concentrated with appropriate buffers.

[0189] Following cell lysis, optional clarification, and optional dilution or concentration, multiple subsequent and sequential chromatography steps can be used to purify rAAV.

[0190] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0191] If the first chromatography step is affinity chromatography, the second chromatography step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV purification is performed via affinity chromatography followed by purification via anion exchange chromatography or / and cation exchange chromatography or / and size exclusion chromatography in any order or sequence or combination.

[0192] For example, during downstream processing, empty capsids are removed from full capsids based on their different isoelectric points (pi) in anion exchange chromatography. The average pi calculated value for full capsids is 5.9 and for empty capsids is 6.3 for all serotypes (Venkatakrishnan, B. et al., J. Virol. 87 (2013) 4974-4984).

[0193] The role of cation exchange chromatography is to separate AAV particles from cells 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 capable of binding rAAV over a wide pH range include, but are not limited to, any sulfonic acid-based resin indicated by the presence of sulfonate functional groups, including aryl and alkyl substituted sulfonates, such as sulfopropyl or sulfoethyl resins. Representative 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). Additional examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers, available from GE Healthcare, Marlborough, MA, USA), and the commercial DOWEX®, AMBERLITE®, and AMBERLYST® series of resins available 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) resins, phosphate resins (based on phosphate functional groups), methylsulfonate (S) resins, and sulfopropyl (SP) resins.

[0194] Anion exchange chromatography serves to separate rAAV from proteins, cells, and other components present in clarified lysate and / or column eluate from affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thereby control the amount of empty rAAV in the eluate. For example, an anion exchange column to which both intact and empty rAAV are bound can be washed with a solution comprising an appropriate concentration of NaCl (e.g., about 100-125 mM, such as 110-115 mM), and a portion of the empty rAAV can be eluted in a flow that does not significantly elute intact rAAV. Subsequently, intact rAAV bound to the anion exchange column can be eluted using a solution comprising a higher concentration of NaCl (e.g., about 130-300 mM NaCl), thereby producing a column eluate having a reduced or depleted amount of empty rAAV and a proportionally increased amount of intact rAAV comprising rAAV vector.

[0195] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those typically based on quaternized nitrogen atoms, including but not limited to quaternary ammonium salt resins, such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (strong anion exchanger, available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion exchanger, available from BioRad, Hercules, CA, USA); POROS 50 HQ (strong anion exchanger, available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion exchanger, available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion exchanger, available from Applied Biosystems, Foster City, CA, USA); POROS 50 PI (weak anion exchanger, available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion exchanger, available from GE healthcare, Marlborough, MA, USA); DEAE SEPHAROSE (weak anion exchanger, available from Amersham Biosciences, Piscataway, NJ, USA); Q SEPHAROSE (strong anion exchanger, available from Amersham Biosciences, Piscataway, NJ, USA). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0196] A commercial manufacturing process for purified recombinant AAV particles intended as a product for treating human disease should achieve the following goals: 1) consistent particle purity, potency, and safety; 2) manufacturing process scalability; and 3) acceptable manufacturing cost.

[0197] WO 2019 / 006390 reports an exemplary process for purification of recombinant AAV particles.

[0198] Methods of determining the infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for assaying empty rAAV and full rAAV with packaged transgenes are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0199] To determine the presence or amount of degraded / denatured capsids, purified rAAV can be subjected to SDS-polyacrylamide gel electrophoresis consisting of any gel that is capable of separating the three capsid proteins, such as a gradient gel, then run the gel until the samples are separated and blot the gel onto a nylon or nitrocellulose membrane. Anti-AAV capsid antibodies are then used as the primary antibody that binds to the denatured capsid proteins (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody that binds the primary antibody contains the means to detect the primary antibody. The binding between the primary and secondary antibodies is semi-quantitatively detected to determine the amount of capsid. Another method is analytical HPLC using SEC columns or analytical ultracentrifuge.

[0200] Description of specific embodiments of the invention

[0201] The present application is based, at least in part, on the discovery that the productivity of mammalian cells producing recombinant adeno-associated virus particles can be increased when the incubation is performed at an elevated pH value, such as pH 7.4-7.6.

[0202] While optimization of transfection parameters, DNA / reagent ratios, VCD at the time of transfection, and complexation times can increase rAAV productivity, such increases are far surpassed by the effect that pH values according to the present application have.

[0203] In the following, the reference value, i.e. the base value, in each table is indicated by the identifier "(100%)".

[0204] It has been found that for the transient production of rAAV particles in HEK293 cells, increasing the incubation pH value from the commonly used pH 7.2 to a pH value of 7.4 or even 7.6 increases the particle yield as well as the genome yield. Since the increase in genome yield is higher than the increase in titer yield, the full to empty particle ratio is also improved.

[0205] For example, for the transient production 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 a pH value of 7.4 or even 7.6 increases the particle yield by more than a factor of 3 and the genome yield by more than a factor of 10.

[0206] The following table provides exemplary data for HEK293 cells grown in suspension in serum-free medium showing the effect of the method according to the application (see also Figures 1, 2 and 3). It can be seen that if the culture pH value is increased from pH 7.2 to pH 7.4 or pH 7.6, the genome titer (vg / mL) is increased by more than a factor of 10. At the same time, the capsid titer (vp / mL) is increased by a factor of 2 to 3. Thus, when the increase in capsid titer is lower than the increase in genome titer, the ratio of full to empty particles is increased by more than a factor of 4.

[0207]

[0208] For pH 7.2, pH 7.4 and pH 7.6, the yield is independent of the culture time, while at a pH value of 7.0, the yield decreases with increasing culture time. This is shown in the following table. Thus, at a pH value of 7.4 and 7.6, the process is more robust and the yield is higher compared to pH 7 or pH 7.2.

[0209]

[0210]

[0211] The effect of changing the pH value from pH 7.2 to pH 7.4 or pH 7.6 far exceeds the titer increase obtained by optimizing the process conditions such as, for example, changing the transfection reagent or adding a feed, as shown in the following table. The third data row shows the titer increase caused by the concomitant change of the culture pH value, the transfection reagent and the feed addition.

[0212]

[0213] For a direct comparison, the titer increase caused by the change of the culture pH value and the addition of a feed under optimized transfection reagent conditions is shown in the following table.

[0214]

[0215] By optimizing the culture conditions, the culture at pH 7.2 became less robust. However, at pH 7.4, the process still maintained its robustness. That is, by increasing the pH value from pH 7.2 to pH 7.4, the loss of process robustness due to the optimization of the reaction conditions can be offset. This is shown in the table below.

[0216]

[0217] For example, for the transient production of rAAV2 particles in serum-free medium using commercially available HEK 293 Expi cells under the optimized conditions as described above, the particle yield can be further increased by about 1.7-fold and the genome yield can be further increased by about 1.8-fold, i.e. by 80%. This data is shown in the table below.

[0218]

[0219] ***

[0220] Examples and figures are provided to assist in understanding the present invention, and the true scope of the invention is set forth in the appended claims. It should be understood that modifications can be made to the procedures set forth without departing from the spirit of the present invention.

[0221] ***

[0222] Examples

[0223] Materials

[0224] Cell lines

[0225] Commercially available HEK293 cells were used for the production of AAV particles using transient transfection with three plasmids.

[0226] Culture materials

[0227] The culture media and supplements were used according to the supplier’s instructions. The culture media and feed were stored at 4°C in the dark and consumed according to the manufacturer’s instructions. The correction agents were stored at room temperature (glucose solution; sodium carbonate solution; antifoam solution).

[0228] Example 1

[0229] Culture of HEK293 cells and production of recombinant AAV preparations

[0230] In general, the cultivation methods were adapted from standard protocols (see, e.g., Lindl, T., "Zell- und Gewebekultur: Einführung in die Grundlagen sowie ausgewählte Methoden und Anwendungen", Spektrum Akademischer Verlag GmbH, Heidelberg / Berlin, 2002) and corresponding supplier's instructions.

[0231] Pre-culture

[0232] The HEK cells were thawed and propagated in shake flasks for two to three weeks at 37°C, 85% humidity, 5% pCO2 and a shaking frequency of 120 rpm. The cells divided every three to four days and were expanded in culture to the volume required for seeding of the production culture.

[0233] Production culture

[0234] For the production of recombinant AAV particles, the respective pre-cultured HEK293 cells were cultivated in batch or fed-batch processes in the respective reactor under the indicated conditions.

[0235] Group 1 - rAAV particle preparation whose particles comprise a capsid variant derived from the AAV2 serotype and a therapeutic transgene:

[0236] Reactor: Ambr250

[0237] Cell line: Suspension HEK293 adapted to serum-free culture medium

[0238] Culture medium: HEK ViP NB + 8 mM glutamine + insulin

[0239] Feed: None (batch)

[0240] Temperature 37°C

[0241] Speed: about 450 rpm

[0242] Culture time after seeding: 144 hours

[0243] Transfection: transient; three plasmids; ratio about 1 :2.5:2 (transgene: rep / cap: helper)

[0244] Transfection: about 24 hours after seeding

[0245] Transfection reagent: PEIpro (TM)

[0246] Transfection reagent:DNA ratio: about 2:1

[0247] DNA concentration: about 3 pg / mL

[0248] Transfection VCD: about 30 E+05 cells / mL

[0249] Transfection: Transfection mix was diluted in 1 / 3 fresh medium

[0250] Lysis: No

[0251]

[0252] Group 2 - rAAV particle preparation whose particles comprise a capsid variant derived from the AAV2 serotype and a therapeutic transgene:

[0253] Reactor: Ambr250

[0254] Cell line: Suspension HEK293 adapted to serum-free medium

[0255] Culture medium: HEK ViP NB + 8 mM glutamine + insulin

[0256] Feeding: No (batch)

[0257] Temperature 37°C

[0258] Speed: about 450 rpm

[0259] Culture time after seeding: 120 hours

[0260] Transfection: Transient; three plasmids; ratio about 1:2.5:2 (transgene: rep / cap: helper)

[0261] Transfection: About 24 hours after seeding

[0262] Transfection reagent: PEIpro (TM)

[0263] Transfection reagent:DNA ratio: about 2:1

[0264] DNA concentration: about 3 pg / mL

[0265] Transfection VCD: about 30 E+05 cells / mL

[0266] Transfection: Transfection mix was diluted in 1 / 3 fresh medium

[0267] Lysis: No

[0268]

[0269] Group 3 and 4 - DoE:

[0270] rAAV particle preparation: 1) whose particles comprise a capsid variant derived from AAV2 serotype and a therapeutic transgene; 2) whose particles comprise AAV2 wild-type capsid and a green fluorescent protein (GFP) transgene:

[0271] Reactor: Ambr15

[0272] Cell line: 1) suspension HEK293 adapted to serum-free medium; 2) HEK293 Expi

[0273] Culture medium: HEK ViP NB + 8 mM glutamine + insulin

[0274] Feed: 1) none (batch); 2) medium and glucose feed after transfection

[0275] Temperature 37°C

[0276] Speed: about 450 rpm

[0277] Culture time after inoculation: 120 hours

[0278] Transfection: transient; three plasmids; ratio 1) about 1:2.5:2 or 2) about 1:1:1 (transgene:rep / cap:helper)

[0279] Transfection: 24 hours after inoculation

[0280] Transfection reagent: 1) PEI + free PEI + valproic acid; 2) FectoVIR(TM)-AAV

[0281] Transfection reagent: DNA ratio: 1) about 2.5:1; 2) about 1.5:1

[0282] DNA concentration: 1) about 3 µg / mL; 2) about 2 µg / mL

[0283] Transfection VCD: about 30 E+05 cells / mL

[0284] Transfection: transfection mix in 1 / 3 fresh medium

[0285] Lysis: 1) no; 2) yes

[0286]

[0287]

[0288] Example 2

[0289] lysis

[0290] If the process reports false lysis, the following is done: To release the AAV particles into the cell culture broth, 5% (v / v) lysis buffer (10% Triton CG 110, 40 mM MgCl2) is added to the culture broth. In addition, 100 U / ml Benzonase™ Nuclease (Merck) is added. The cell culture broth is then incubated at 37°C for about one hour under stirring without aeration and pH control. After the respective incubation, a 5 M NaCl solution is added and the lysate is sterile filtered.

[0291] Example 3

[0292] AAV particle purification

[0293] For the affinity chromatography step, a column containing 10.5 mL AAVX resin obtained from ThermoFisher was used on an Akta Avant 25 chromatography system. The system was run at a flow rate of about 300 cm / h. After equilibration with buffer A (lx PBS, pH 7.4, 0.001% Pluronic F-68), 200 mL of the lysed culture broth was applied to the column, followed by 2 washing steps with equilibration buffer and 0.5 M NaCl (pH 6.0), respectively. The AAV particles were eluted with a 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).

[0294]

[0295] Example 4

[0296] Analytical methods

[0297] Enzyme-linked immunosorbent assay (ELISA) for total titer determination

[0298] For AAV capsid titer determination, a kit obtained from PROGEN (cat. no. PRAAV8) was used according to the manufacturer’s instructions.

[0299] In brief, the assay is a sandwich ELISA using a recombinant AAV capsid specific antibody and a biotin-labeled detection antibody as capture antibody.

[0300] Precoated multi-dilution plates (MTP) wells were incubated with 100 mΐ of standard, sample or control, respectively, overnight at 4°C. The next day, the wells were washed three times with ASSB buffer (lx) as provided in the kit. Thereafter, 100 mΐ of a solution containing biotinylated detection antibody (diluted according to manufacturer’s instructions) was added per well and incubated for two hours at room temperature under shaking. Then, the wells were washed three times with ASSB buffer (lx) as provided in the kit. In the next step, 100 mΐ of a solution containing horseradish peroxidase conjugated to streptavidin was added to each well and incubated for 30 minutes at room temperature under shaking. Then, the wells were washed three times with ASSB buffer (lx) as provided in the kit. For the color development reaction, 100 mΐ of a solution containing ABTS prepared according to the manufacturer’s instructions was added to each well and incubated under shaking. The color intensity was determined using a MTP-ELISA-Reader Versa Max (Molecular Devices) at 405 nm (reference wavelength 490 nm) until the extinction difference between the blank and the standard with the highest concentration reached about 1.5.

[0301] Each sample, standard and control was measured in duplicate.

[0302] Based on the standard curve determined by a 4-parameter fit (e.g. according to Wiemer Rodbard algorithm), the amount of capsid (capsid / mL) was calculated using the average of the standards.

[0303] Digital droplet polymerase chain reaction (ddPCR) for genome titer determination

[0304] Reagents for enzymatic sample treatment:

[0305] 1) DNAse I buffer (NEB): 100 mM Tris-HCl, pH 7.6, 25 mM MgS04, 5 mM CaCl2

[0306] 2) DNAse I (NEB): 0.2 U / pL

[0307] 3) Proteinase K (NEB; approx. 20 mg / mL = 800 U / mL): 16 U / mL

[0308] 4) Proteinase K buffer (BioRad): 400 mM Tris-HCl, 20 mM EDTA, 2000 mM NaCl, 1% SDS, pH 8

[0309] 5) Sodium dodecyl sulfate (SDS) solution: 10% (w / v)

[0310] Enzymatic sample treatment:

[0311] - Mix 30 pL H20, 5 pL DNase I buffer, 5 pL DNase I, 10 pL sample

[0312] - Incubate at 37°C for 30 minutes

[0313] - Heat to 75°C for 15 minutes to obtain incubated DNase I-mixture

[0314] - Cool down shortly and centrifuge

[0315] - Mix 42 pL H20 + 2 pL Proteinase K + 5 pL Proteinase K buffer + 1 pL 10% SDS solution and add to incubated DNase I-mixture

[0316] - Incubate at 50°C for 60 minutes

[0317] - Heat to 95°C for 15 minutes

[0318] - Cool down to 4°C

[0319] ddPCR:

[0320] For viral genome titration, a duplex ddPCR assay was performed. Primers and probes were designed against the used CMV promoter and against the polyA / 3'UTR sequence. PCR master mix was prepared according to the table below (Droplet Digital PCR Guide - Bio-Rad).

[0321]

[0322] The prepared master mix was moved into a 96-well plate at 16.5 μΐ^ per well. Then, serial dilutions were performed on the pre-treated samples: 10 μΐ^ of sample was transferred into 90 μΐ^ of water in a LoBind tube using LoRentention Tips and mixed well. Thereafter, 5.5 μΐ^ of sample was added to the master mix solution in a 96-well plate through several dilution steps. The plate was sealed at 180°C, vortexed at 2,200 rpm for 1 minute and centrifuged at 1,000 rpm for another 1 minute. 20 μΐ^ of PCR mix was taken from each well using an automated droplet generator device, producing up to 20,000 droplets per well, and transferred to another 96-well plate. After sealing the droplet plate at 180°C, a PCR run was performed. The corresponding conditions are shown in the table below.

[0323]

[0324] In a microdroplet analyzer, the fluorescence signal of each microdroplet was measured. QuantaSoft software processed the reader data and calculated the copy number of the target sequence per 20 μΐ^ well. The initial sample titer can be determined with the following formula:

[0325]

Claims

1. A method for producing a preparation of recombinant adeno-associated viral particles (rAAVp), the method comprising the steps of: HEK293 cells are cultured and from them the rAAVp is produced, the HEK293 cells comprising expression cassettes for: a non-adenoviral gene inserted between two AAV inverted terminal repeats (ITRs); 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; and optionally an adeno-associated virus VA RNA gene, wherein the culturing is performed at a pH value in the range of and including pH 7.4 to pH 7.

6.

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

2.

3. The method of any one of claims 1 to 2, wherein the rAAVp produced by the culturing performed at a pH value in the range of and including pH 7.4 to pH 7.6 has a higher percentage of full particles than rAAVp produced by culturing performed at a pH value in the range of and including pH 7.0 to pH 7.

2.

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

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

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

7. The method of any one of claims 1 to 6, wherein the culturing comprises inoculating a bioreactor and harvesting the rAAVp.

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

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

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

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

12. A pharmaceutical composition comprising: a rAAVp obtained by the method according to 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 for increasing the yield of recombinantly produced rAAVp.

14. Use of the method according to any one of claims 1 to 10 for increasing the percentage of full particles in the rAAVp.

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