Method for producing a recombinant adeno-associated viral particle preparation, pharmaceutical composition and use of the method

Description

1 / 66 “METHOD FOR PRODUCING A RECOMBINANT ADENOASSOCIATED VIRAL PARTICLE PREPARATION, PHARMACEUTICAL COMPOSITION AND USE OF THE METHOD”

[0001] The present invention is in the field of gene therapy. More specifically, the present invention is directed to a method for producing recombinant adeno-associated viral particles comprising a therapeutic transgene in mammalian cells, especially CHO and HEK cells, at a pH value that is higher than that generally used in the art. Background

[0002] Gene therapy is opening up unprecedented opportunities for new therapeutic approaches. Based on the concept of rescuing functional mutations by co-expressing the correct gene, to allow biological functions to be restored, the use of viral vectors is required to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAVs) are the most widely used vectors.

[0003] Virus bioprocessing is complex and requires systematic and coordinated steps in both upstream and downstream processing. However, the use of traditional culture processes for the production of therapeutic viruses does not support effective commercial manufacturing strategies. This is further pronounced by the large size of viruses compared to therapeutic biomolecules, such as antibodies. In addition, viruses are much more complex.

[0004] The biomanufacturing process of therapeutic viruses requires the insertion of the therapeutic transgene into the capsid envelope of recombinant AAV (full rAAV particles, i.e., recombinant AAV particles comprising an encapsulated nucleic acid). However, a percentage of rAAV that do not contain the desired transgene (particles of Petition 870250084687, dated 09 / 19 / 2025, page 71 / 143 2 / 66 Empty recombinant AAVs (i.e., rAAV particles that do not comprise an encapsulated nucleic acid) can also be produced, as well as partially filled rAAVs (partially filled recombinant AAV particles). Brief Description of Embodiments of the Invention

[0005] The present invention is based, at least in part, on the discovery that the productivity of mammalian cells producing a recombinant adeno-associated viral particle can be increased when cultivation is carried out at elevated pH values, such as pH 7.4 to 7.6.

[0006] The present invention is based, at least in part, on the discovery that the fraction of filled recombinant AAV particles obtained from a culture of mammalian cells producing said recombinant adeno-associated viral particle can be increased when the culture is carried out at high pH values, such as pH 7.4 to 7.6.

[0007] The present invention comprises at least the following embodiments: 1. Method for producing a recombinant adeno-associated viral particle (rAAVp) preparation comprising the step of culturing a mammalian cell comprising expression cassettes for a non-adeno-associated viral gene, which is intercalated between two terminal inverted repeats (ITRs) of AAV, for an adeno-associated virus rep gene, for an adeno-associated virus cap gene, for an adeno-associated virus E1A gene, for an adeno-associated virus E1B gene, for an adeno-associated virus E2A gene, for an adeno-associated virus E4orf6 gene and, optionally, for an adeno-associated virus VA RNA gene, thereby producing the rAAVp, wherein the culture is at a pH value in the range of and including pH 7.4 to pH 7.6; 2. Method for producing a viral particle preparation Petition 870250084687, dated 09 / 19 / 2025, p. 72 / 143 3 / 66 recombinant adeno-associated (rAAVp) comprising the step of culturing a HEK cell comprising expression cassettes for a non-adeno-associated viral gene, which is intercalated between two terminal inverted repeats (ITRs) of AAV, for an adeno-associated virus rep gene, for an adeno-associated virus cap gene, for an adeno-associated virus E2A gene, for an adeno-associated virus E4orf6 gene and, optionally, for an adeno-associated virus VA RNA gene, thereby producing the rAAVp, wherein the culture is at a pH value in the range of and including pH 7.4 to pH 7.6; 3. Method, according to any of the realizations 1 to 2, where the mammalian cell is a HEK293 cell; 4. Method, according to any of the realizations 1 to 3, wherein the genomic titer of the rAAVp produced by cultivation at a pH value in the range of and including pH 7.4 to pH 7.6 is higher than the capsid titer of an rAAVp produced by cultivation at a pH value in the range of and including pH 7.0 to pH 7.2; 5. Method, according to embodiment 4, in which the genomic titer is at least 1.5 times higher; 6. Method, according to any of the realizations 4 to 5, where the genomic titer is at least 2 times higher; 7. Method, according to any of the realizations 4 to 6, where the genomic titer is at least 6 times higher; 8. Method, according to any of the realizations 4 to 7, in which the genomic titer is at least 10 times higher; 9. Method, according to any of the realizations 1 to 8, where the capsid titer of rAAVp produced by cultivation at a pH value in the range of and including pH 7.4 to pH 7.6 is higher than the capsid titer of an rAAVp produced by cultivation at a pH value in the range of and Petition 870250084687, dated 09 / 19 / 2025, page 73 / 143 4 / 66 including pH 7.0 to pH 7.2; 10. Method, according to embodiment 9, in which the capsid titer is at least 1.5 times higher; 11. Method, according to any of the realizations 9 to 10, in which the capsid titer is at least 2 times higher; 12. Method, according to any of the realizations 9 to 11, in which the capsid titer is at least 3 times higher; 13. Method, according to any of the realizations 1 to 12, wherein the genomic titer and capsid titer of rAAVp produced by cultivation at a pH value in the range of and including pH 7.4 to pH 7.6 is higher than the genomic titer and capsid titer of an rAAVp produced by cultivation at a pH value in the range of and including pH 7.0 to pH 7.2; 14. Method, according to embodiment 13, in which the genomic titer and capsid titer are at least 1.5 times higher; 15. Method, according to any of the realizations 13 to 14, in which the genomic titer and the capsid titer are at least 2 times higher; 16. Method, according to any of the realizations 13 to 15, in which the genomic titer and the capsid titer are at least 3 times higher; 17. Method, according to any of the realizations 13 to 16, in which the genomic titer is at least 4 times higher and the capsid titer is at least 2 times higher; 18. Method, according to any of the realizations 13 to 17, in which the genomic titer is at least 6 times higher and the capsid titer is at least 2 times higher; 19. Method, according to any of the realizations 1 to 20, where the rAAVp produced by cultivation at a pH value in the range of e Petition 870250084687, dated 09 / 19 / 2025, page 74 / 143 5 / 66 including pH 7.4 to pH 7.6 has a higher percentage of filled particles than a rAAVp produced by a culture at a pH value in the range of and including pH 7.0 to pH 7.2; 20. Method, according to embodiment 19, in which the percentage of filled particles is at least 1.5 times higher; 21. Method, according to any of the embodiments 19 to 20, in which the percentage of filled particles is at least 2 times higher; 22. Method, according to any of the embodiments 19 to 21, in which the percentage of filled particles is at least 4 times higher; 23. Method, according to any of the realizations 1 to 22, where rAAVp is a therapeutic rAAVp; 24. Method, according to any of the realizations 1 to 23, where rAAVp is for the transfer of a nucleic acid that is transcribed into a polypeptide with a therapeutic effect on target cells; 25. Method, according to any of the realizations 1 to 24, where rAAVp is for the transfer of a nucleic acid that has a therapeutic effect on target cells; 26. Method, according to any of the realizations 1 to 25, wherein rAAVp comprises recombinant adeno-associated viral particles (rAAVs) comprising at least one coding nucleic acid sequence interspersed between two adeno-associated viral inverted repeats; 27. Method, according to any of the realizations 1 to 26, wherein rAAV in 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, AAV2i8, AAV Petition 870250084687, dated 09 / 19 / 2025, p. 75 / 143 6 / 66 rh.74, AAV rh.10 and AAV 7m8, as well as variants thereof; 28. Method, according to any of the realizations 1 to 27, where rAAV is of the AAV2 serotype or a variant thereof; 29. Method, according to any of the realizations 1 to 28, wherein rAAV comprises one or two ITR sequences from a wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12; 30. Method, according to any of the realizations 1 to 29, in which cultivation includes inoculation of the bioreactor and harvesting of rAAVp; 31. Method, according to any of the realizations 1 to 30, in which cultivation begins with the inoculation of the bioreactor; 32. Method, according to any of the realizations 1 to 31, in which one or more or all of the expression cassettes for the non-adeno-associated viral gene, which is intercalated 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 gene, and for the adeno-associated virus VA RNA gene are introduced into the mammalian cell or HEK cell after bioreactor inoculation; 33. Method, according to any of the realizations 1 to 32, wherein one or more or all of the expression cassettes for the non-adeno-associated viral gene, which is intercalated 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 gene, and optionally for the adeno-associated virus VA RNA gene are introduced into the mammalian cell or HEK cell after bioreactor inoculation, whereby up to three plasmids are co-transfected into the mammalian cell, whereby one of the plasmids comprises the expression cassette for the non-adeno-associated viral gene, which is intercalated between two Petition 870250084687, dated 09 / 19 / 2025, page 76 / 143 7 / 66 In AAV ITRs, one of the plasmids comprises the expression cassettes for the rep and cap genes, and one of the plasmids comprises the expression cassettes for the RNA VA, adenoviral E2A, and E4orf6 genes. 34. Method, according to any of the realizations 1 to 33, in which the expression of one or more or all of the non-adeno-associated viral gene, which is intercalated 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 gene and, optionally, the adeno-associated virus VA RNA gene, is induced after inoculation of the bioreactor; 35. Method, according to any of the embodiments 32 to 33, in which the introduction is approximately 16 to 32 hours after inoculation of the bioreactor; 36. Method, according to embodiment 34, in which induction occurs approximately 16 to 32 hours after inoculation of the bioreactor; 37. Method, according to any of the embodiments 35 to 36, in which the introduction or induction is approximately 24 hours after inoculation of the bioreactor; 38. Method, according to any of the realizations 1 to 37, wherein the method further comprises, after the cultivation step, the step of isolating the rAAV from the cells and / or the culture medium and, optionally, purifying the rAAV; 39. Method, according to embodiment 38, in which purification is carried out by one or more column chromatography steps and / or a centrifugation step in a CsCl or iodixanol gradient; 40. Method, according to any of the embodiments 38 to 39, in which the first chromatography step is an affinity chromatography step; 41. Method, according to any of the realizations 38 Petition 870250084687, dated 09 / 19 / 2025, page 77 / 143 8 / 66 to 40, where purification is by a sequence of chromatography steps, the first being affinity chromatography, followed by anion exchange chromatography or cation exchange chromatography and optional size exclusion chromatography; 42. Pharmaceutical composition comprising the rAAVp obtained by a method, as defined in any of embodiments 1 to 41; 43. Pharmaceutical composition comprising rAAVp obtained by a method, as defined in any of embodiments 1 to 41, and a pharmaceutically acceptable excipient; 44. Use of the method, as defined in any of embodiments 1 to 41, to increase the yield of a recombinantly produced rAAVp; 45. Use of the method, as defined in any of embodiments 1 to 41, to increase the percentage of filled particles in a rAAVp.

[0008] In addition to the various embodiments represented and claimed, the disclosed subject matter also extends to other embodiments that have other combinations of the features disclosed and claimed herein. Thus, the particular features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, so that the disclosed subject matter includes any suitable combination of the features disclosed herein. A prior description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed. Brief Description of the Figures

[0009] Figure 1: visualization of the genomic titer in samples collected from Set 1 (collected 120 hours post-transfection, without lysis). Petition 870250084687, dated 09 / 19 / 2025, page 78 / 143 9 / 66

[0010] Figure 2: visualization of capsid titer in samples collected from Set 1 (collected 120 hours post-transfection, without lysis).

[0011] Figure 3: visualization of the ratio of full to empty cells in samples collected from Set 1 (collected 120 hours post-transfection, without lysis). Detailed Description

[0012] The present invention is based, at least in part, on the discovery that the productivity of mammalian cells producing a recombinant adeno-associated viral particle can be increased when cultivation is carried out at elevated pH values, such as pH 7.4 to 7.6. Definitions

[0013] 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 skilled in the art. In addition, unless otherwise required by the context, singular terms shall include plurals and plural terms shall include the singular.

[0014] Useful methods and techniques for carrying out the present invention are described in, for example, Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture - A Practical Approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987). The content of which is incorporated into this document by reference.

[0015] The use of recombinant DNA technology allows for Petition 870250084687, dated 09 / 19 / 2025, p. 79 / 143 10 / 66 generation of nucleic acid derivatives. Such derivatives may, for example, be modified at individual or multiple nucleotide positions by substitution, alteration, exchange, deletion, or insertion. Modification or derivatization may, for example, be performed by means of site-directed mutagenesis. Such modifications may be easily performed by a 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, BD, and Higgins, SG, Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0016] It should be noted that, as used in this document and the accompanying claims, the singular forms a, an, and the include plural reference unless the context clearly indicates 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. Furthermore, the terms a, one or more, and at least one may be used interchangeably in this document. It is also to be noted that the terms comprising, including, and having may be used interchangeably.

[0017] The term “approximately” denotes a range of + / - 20% of the following numerical value. In certain realizations, the term approximately denotes a range of + / - 10% of the subsequent numerical value. In certain realizations, the term approximately denotes a range of + / - 5% of the subsequent numerical value.

[0018] The terms “comprises”, “includes”, “having”, “has”, “may”, “contains” and variants thereof, as used in this document, 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 term “consisting of”. This disclosure also contemplates other realizations “comprising”, Petition 870250084687, dated 09 / 19 / 2025, pp. 80 / 143 11 / 66 “consisting of” and “consisting essentially of” the accomplishments or elements presented in this document, whether explicitly stated or not.

[0019] The terms “empty recombinant AAV particle” and “empty rAAV,” which may be used interchangeably, denote a protein shell composed of adeno-associated capsid polypeptides without a functional nucleic acid encapsulated / packaged within it (rAAV = recombinant Adeno-associated Virus particle). That is, an empty rAAV may be free of encapsulated nucleic acid or comprise a nucleic acid or part thereof that is not transcribed in whole or is not transcribed into a functional transcript. Consequently, an empty rAAV does not function to transfer a nucleic acid that encodes a functional protein or is transcribed into a functional transcript of interest to a target cell. In certain all-aspect embodiments and realizations, the functional protein or functional transcript of interest has a therapeutic effect.

[0020] The term “endogenous” denotes that something occurs naturally, for example, within a cell, or is produced naturally, for example, by a cell.

[0021] The term “exogenous” denotes that something, for example, a nucleotide sequence, does not originate from the same entity in which it is present. For example, a nucleic acid is exogenous to a specific cell if it has been introduced into that cell by a method of DNA delivery, such as transfection, electroporation, or transduction. Similarly, a nucleic acid is exogenous to an AAV particle if it does not originate from the same AAV particle or serotype. Thus, an exogenous nucleotide sequence is an artificial sequence, either in isolated form or within a cell or rAAV, where the artificiality may originate, for example, from the combination of subsequences of different origin, for example, a Petition 870250084687, dated 09 / 19 / 2025, p. 81 / 143 12 / 66 combination of a recombinase recognition sequence with an SV40 promoter and a green fluorescent protein coding sequence or a combination of AAV ITRs from a first serotype with the capsid polypeptides of a second serotype or a combination of AAV ITRs with a non-AAV nucleic acid, or the deletion of parts of a sequence, for example, a sequence encoding only the extracellular domain of a membrane-bound receptor or a cDNA, or the mutation of nucleobases in an endogenous nucleic acid sequence. This does not exclude that an “exogenous” nucleotide sequence may have an “endogenous” counterpart that is identical in base composition, but where the sequence is becoming an “exogenous” sequence by its combination with exogenous regulatory elements, such as a secretion signal or exogenous promoter.

[0022] The terms “recombinant full AAV particle” or “rAAV full,” which may be used interchangeably, denote a non-covalent complex formed by a protein shell composed of adeno-associated capsid polypeptides and a functional nucleic acid sequence encapsulated / packaged therein. That is, a full rAAV comprises a nucleic acid that is transcribed into a functional transcript. Thus, the full rAAV functions to transfer a nucleic acid that encodes a protein or is transcribed into a transcript of interest to a target cell. In certain embodiments, a functional nucleic acid comprises at least one coding nucleic acid sequence interspersed between two adeno-associated viral inverted terminal repeats (ITRs).

[0023] The term “full-to-empty ratio” denotes the mathematical ratio between the number of filled recombinant AAV particles (rAAV filled) and the total number of recombinant AAV particles (sum of rAAV filled and rAAV empty) in a sample or in a recombinant AAV particle preparation (rAAVp). As the number of rAAV filled can be at most Petition 870250084687, dated 09 / 19 / 2025, p. 82 / 143 13 / 66, the same as the total number of rAAVs, the ratio can be at most 1. Generally, the ratio is less than 1 and is expressed as a percentage. The full rAAV number is determined by determining the number of nucleic acid sequences interspersed between two AAV ITRs in the sample or preparation. This can be done by PCR, especially digital droplet PCR (ddPCR) or quantitative PCR (qPCR). The total number of rAAVs is determined by determining the number of protein coats formed by adeno-associated capsid polypeptides in the sample or preparation. This can be done by ELISA, especially by a capsid polypeptide-specific ELISA.

[0024] The term in vitro denotes an artificial environment as such, or that a process or reaction is carried out within such an artificial environment.

[0025] The term in vivo denotes the natural environment (e.g., an animal or a cell) of a compound, or that a process or reaction takes place within its natural environment.

[0026] The terms “recombinant AAV vector” or “transgene,” which may be used interchangeably in this document, denote a nucleic acid derived from a wild-type genome of an adeno-associated virus, in which, except for the ITR (Inverted Terminal Repeat of adeno-associated virus) sequences, all endogenous AAV nucleic acids are replaced by one or more exogenous nucleic acid(s). For example, such an exogenous nucleic acid may be a nucleic acid transcribed in a transcript of interest or 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 since all or at least part of the viral genome has been replaced by a non-exogenous nucleic acid. Petition 870250084687, dated 09 / 19 / 2025, p. 83 / 143 14 / 66 native (i.e., exogenous) relative to the virus. The incorporation of a non-native nucleic acid therefore defines the AAV vector as a recombinant vector. It should be noted that the serotype of the ITRs in the recombinant AAV vector need not be the same as the serotype of the adeno-associated capsid polypeptides that form the shell of the recombinant AAV particle comprising said recombinant AAV vector.

[0027] 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, for example, for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo.

[0028] As used in this document, the term serotype is used to classify different wild-type and recombinant AAV particles based on the amino acid sequence of the polypeptides that form the protein coat (capsid) of the respective AAV particle. Originally, serological distinction was determined based on the lack of cross-reactivity between antibodies to one AAV particle compared to another AAV particle. These cross-reactivity differences are usually due to differences in capsid polypeptide sequences and their respective antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes).Although AAV variants, including capsid variants, may not be serologically distinct from a reference or wild-type AAV or another AAV serotype, they differ in at least one amino acid residue compared to the reference or wild-type AAV serotype or another.

[0029] Under the traditional definition, a serotype means that the virus of interest has been tested against specific serum for all serotypes. Petition 870250084687, dated 09 / 19 / 2025, page 84 / 143 15 / 66 existing and characterized for neutralizing activity, and no antibody was found that neutralizes the virus of interest. As more isolates of naturally occurring viruses are discovered and / or capsid mutants are generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new AAV particle has no serological difference, this new AAV particle would be a subgroup or variant of the corresponding wild-type serotype. In many cases, serological tests for neutralizing activity still have to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype, according to the traditional definition of serotype.

[0030] The term “vector” denotes the portion of a larger nucleic acid, for example, from a recombinant plasmid, that is ultimately packaged or encapsulated or encapsulated directly or in single-strand or RNA form within a protein shell composed of adeno-associated virus capsid polypeptides to form a recombinant AAV particle. In cases where recombinant plasmids are used to construct or manufacture recombinant AAV particles, the viral particle does not include the portion of the plasmid that does not correspond to the vector portion of the recombinant plasmid. For example, in the case of a rAAV, the recombinant vector comprises that portion of the recombinant plasmid that is interspersed 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 plasmid cloning and amplification, a process necessary for the propagation and production of recombinant viruses, but it is not packaged, encapsulated, or encapsulated within the recombinant AAV particle. Thus, a “vector” refers to the nucleic acid that is packaged or encapsulated or encapsulated by a protein coat composed of adeno-associated virus capsid polypeptides, i.e., within a recombinant AAV. Petition 870250084687, dated 09 / 19 / 2025, page 85 / 143 16 / 66 General methods for producing rAAV particles

[0031] WO 1999 / 11764 reported methods for generating high-titer auxiliary-free preparations of recombinant AAV vectors. Additionalally grown, undefined AAV-producing cells in suspension in bioreactors were infected with Adenovirus Type 5 (Ad5) at a multiplicity of infection (MOI) of 10⁻⁶ in 1.5 L low-serum media at different pH values. 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 can be calculated (75% of the nominal value) to be approximately 1.125 L. Therefore, the total number of particles corresponds 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.

[0032] Document WO 2000 / 14205 reported the production of AAV particles in an undefined cell type denoted as JL14 cells, by co-infection with adenoviral helper virus, in which, at a pH value of 7.4, the highest number of AAV particles (sum of intracellular and secreted AAV particles), at a pH value of 8, AAV particles with the highest infectivity and at a pH of 7.6, the highest ratio of number of AAV particles to infectivity were obtained. The culture was performed in a medium volume of 1.5 L, and thus the total number of particles corresponds 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 infectivity data provided, it can be assumed that the full / empty ratio of the rAAV particles thus produced is less than 1%.

[0033] This is based on the following calculation of the data in the Figures. Petition 870250084687, dated 09 / 19 / 2025, p. 86 / 143 17 / 66 2B and 3B of document WO 2000 / 14205 (3 days post-infection): Table 1 Day 3 pH Total DRPs (=vp) => vp / ml 7.2 4.50 E+12 3.00 E+09 7.4 1.95 E+13 1.30 E+10 7.6 1.85 E+13 1.23 E+10 7.8 5.00 E+12 3.33 E+09 8 1.65 E+13 1.10 E+10 Table 2 Day 3 pH Total RUs => RU / ml = RU / ml / vp / ml 7.2 2.00 E+09 1.33 E+06 0.044% 7.4 7.40 E+09 4.93 E+06 0.038% 7.6 5.00 E+09 3.33 E+06 0.027% 7.8 2.75 E+09 1.83 E+06 0.055% 8 7.50 E+09 5.00 E+06 0.045%

[0034] Piras, BA, et al. (Mol. Ther. Meth. Clin. Dev. 3 (2016) (16015) compared the distribution of AAV8 in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found an increase in viral production up to day 6, with the proportion of viral particles in the media increasing from 76% on day 3 to 94% on day 7. Large-scale productions showed that the ratio of filled to empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. AAV-FVIII showed an increase in production when the culture was extended from day 3 (1.1 χ¹ E+13 ± 9.2 χ¹ E+11 and 3.6 χ¹ E+13 ± 2.5 χ¹ E+12 total capsids in lysate and media, respectively) to day 5. (6.7 χ 1 E+12 ± 6.7 χ 1 E+11 and 4.5 χ 1 E+13 ± 2.6 χ 1 E+12 total capsids in lysate and media, respectively), day 6 (5.0 χ 1 Petition 870250084687, dated 09 / 19 / 2025, page 87 / 143 18 / 66 E+12 ± 1.9 x 1 E+11 and 5.3 x 1 E+13 ± 3.3 x 1 E+12 total capsids in lysate and media, respectively) and day 7 (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 total capsids in lysate and media, respectively). Piras et al. employed adherent HEK293T / 17 cells which were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum supplemented with 2 mmol / l GlutaMAX (Life Technologies, Grand Island, NY). AAV was produced by transfecting two plasmids using PEIpro™ (Polyplus-transfection SA, Illkirch, France) 1 day after seeding the cells at a density of 7.26 x 1 E+04 cells / cm².

[0035] Powers, AD, et al. (Hum. Gene Ther. Meth. 27 (2016) 112121) reported the development and optimization of hFIX AAV particle production by transient transfection in an iCELLis(R) fixed-bed bioreactor. A yield as high as 9 E+14 viral particles per square meter of fixed bed was obtained. On day 3 after inoculation with HEK293T / 17 cells, the vessel was transfected with scAAV-LP1-hFIXco-helpv3 plasmid and CR21+LTAAV help 2-8 plasmid at a plasmid mass ratio of 3:1, respectively, using polyethyleneimine (PEIpro™ Transfection Reagent Cat #115-375; Polyplus) in IMDM (Lonza) or DMEM supplemented with 10% FBS and 6 mM GlutaMAX™. The PEI and DNA solutions were combined at a 2:1 ratio.

[0036] Poulain, A., et al. (j. Biotechnol. 255 (2017) 16-27 reported rapid protein production from stable CHO cell pools using a plasmid vector and cumate gene exchange. Cells were transfected using linear polyethyleneimine (PEIpro™) from Polyplus-Transfection (Illkirch, France). On the day of transfection, cells were suspended at a density of 2 x 1 E+06 cells / ml in CD DG44 medium (Life Technologies Inc., Burlington, ON, Canada), supplemented with 4 mM glutamine and 0.1% Kolliphor® P 188. The cell suspension was distributed into 6-well plates (1.8 ml / well). Petition 870250084687, dated 09 / 19 / 2025, page 88 / 143 19 / 66 DNA:PEIpro™ complexes were prepared at a 1:5 (w:w) ratio, with a total of 2 μg of DNA per well to transfect in 100 μg of complete culture medium.

[0037] Document WO 2017 / 096039 reported scalable methods for producing recombinant AAV vectors in serum-free suspension cell culture systems suitable for clinical use. Production of rAAV vectors was performed in bioreactors with HEK293F cells using triple transfection at a cell density of 1 E+06 cells / ml (1,000,000 cells / ml) with a plasmid ratio of 1:1:1 and a PEI-based transfection reagent (PEI / DNA weight ratio of 2:1 with ½ PEI as free PEI) at a temperature of 37 °C and a pH value of 7.2.

[0038] Nyamay'antu, A., et al. (Cell Gen. Ther. Ins. 4 (2018) 7179) reported that PEI is widely used due to its accessibility and high efficiency in delivering DNA, both in adherent cells and in suspensions grown in serum-free media. PEIpro(TM) is suitable for small to large-scale production of various viruses, particularly AAV particles. In stirred tank bioreactors using HEK293 or HEK293T cells, titers in the range of 0.8-1.5 E+09-E+10 vg / ml can be obtained.

[0039] Koo, T., et al. (Nat. Commun. 9 (2018) 1855) reported that CRISPR-LbCpf1 prevents choroidal neovascularization in a mouse model of age-related macular degeneration. To produce AAV vectors, they were pseudotyped into AAV9 capsids. HEK293T cells (ATCC, CRL-3216) were transfected with pAAV-ITR-LbCpf1crRNA, pAAV2 / 9 encoding for AAV2rep and AAV9cap, and helper plasmid. HEK293T cells were cultured in DMEM with 2% FBS. Recombinant pseudotyped AAV vector stocks were generated using coprecipitation of PEI with PEIpro™ (Polyplus transfection) and triple transfection with plasmids at a molar ratio of 1:1:1 in HEK293T cells. After Petition 870250084687, dated 09 / 19 / 2025, page 89 / 143 After 20 / 66 h of incubation, the cells were lysed and the particles were purified by ultracentrifugation in an iodixanol gradient.

[0040] Rep proteins from AAV2 are commonly and almost exclusively used in the production of rAAVs derived from serotypes AAV1 to AAV13 (Daya, S. and Berns, KI, Clin. Microbiol. Rev. 21 (2008) 583-593; Zincarelli, C., et al., Mol. Ther. 16 (2008) 1073-1080).

[0041] Document WO 2019 / 094253 reported means and methods for preparing viral vectors and their uses. Adherent HEK293 cells were cultured in bioreactors at a pH of 7.23 and triply transfected (plasmid ratio 1:1:1) with PEI / DNA at a PEI-plasmid ratio of approximately 1:1 by weight.

[0042] Collaud, F. et al. (Mol. Ther. Meth. Clin. Dev. 12 (2019) 157174) reported titers for rAAV8 particles of 6.0 ± 1.89 E+04 vg / cell and 1.77 ± 1.37 E+04 vg / cell for AAV (single-stranded) and (autocomplementary), respectively, for adherent HEK293 cells. A fully scalable method based on triple transfection of HEK293 cells in which suspension culture was performed was also reported. Triple transfection of HEK293 cells was performed with polyethyleneimine (PEIpro(TM), Polyplus) directly in 10 L bioreactors. AAV vectors were recovered from both supernatant and cells by mild detergent lysis followed by purification on an AVB Sepharose affinity column. The purified vectors were then concentrated and tested for quality and potency. No information on the pH value and titers obtained is provided.

[0043] Nyamay'antu, A., et al. (Cell Gen. Ther. Ins. 6 (2020) 655661) reported that the efficiency of the delivery process is essential to obtain a high number of producing cells. Of the existing transfection methods, the use of PEI-based transfection reagent is predominant in gene therapy, as it combines accessibility and compatibility for transfection of Petition 870250084687, dated 09 / 19 / 2025, pp. 90 / 143 21 / 66 adherent cells in suspension. Compared to the gold standard PEIpro™ used for viral vector fabrication, FectoVIR™-AAV was found to significantly improve the yield of rAAV2 production, both in viral genome production and in the efficiency of packaging rAAV2-GFP into suspension cells, by up to 10 times compared to PEIMax™ and up to 2 times compared to PEIpro™, respectively, when each transfection reagent is used under recommended conditions. In more detail, HEK293T cells in suspension were transfected using the respective transfection reagent under recommended conditions. rAAV2-GFP were harvested 72 hours post-transfection. The titer obtained with VectoVIR™ is in the range of 1 E+0⁴ to 4.5 E+0⁴ vg / cell depending on the complexation volume used (1% to 10%), corresponding to 1 E+12 vg / ml. The respective functional titers are approximately 2-8 E+0⁸ TU / ml. The results are almost independent of the culture medium used.

[0044] In a blog post titled “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 with the two AAV9 (1:1 and 2:1) and AAV2 (3:1.5 and 5:2.5) serotypes. The AAV2 vector yield was not significantly affected, with a 4- to 5-fold increase in vector genome titer and a 3- to 6-fold increase in viral particle titer with FectoVIR™ AAV compared to PEIpro™. These results show that the improvement in yield can vary with 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 conducted varying the transfection reagent-to-DNA ratios (3:2, 3:1.5) and plasmid DNA molar ratios (1:1:1, Petition 870250084687, dated 09 / 19 / 2025, pp. 91 / 143 22 / 66 2:1:2, 1:2:1). A 3- to 5-fold increase for AAV2 and a 1.1- to 1.6-fold increase for AAV5 in vector genome titer with FectoVIR™-AAV compared to PEIpro™ were observed. Viral particle titer increased 3.5- to 4.5-fold for AAV2 and 2.5- to 3.75-fold for AAV5. Reagent ratios for DNA of 2:1 and 1.5:1 and plasmid ratios of 1:1:1 to 2:1:2 to 1:2:1 were used. The titer obtained with VectoVIR™ was in the range of 4 E+11 to 1 E+12 vg / ml.

[0045] Rossi, A. and Peigné, CM. (Cell Culture Dish Article 17 May 2021) described that, normally, AAV production titers are around 1 E+11 to 1 E+12 in vg / ml and 1 E+08 to 1 E+09 TU / ml.

[0046] To put the numbers into perspective, it is important to keep in mind that two AAV serotypes are unlikely to give the same yield, even when using the same production process.

[0047] In more detail, AAV production yields vary depending on the serotype and gene of interest. Generally, to increase the production of a given AAV, parameters that directly impact the yield are optimized: plasmid DNA, transfection reagent, cells, and medium. PEI-based transfection processes, for example, can reduce the amount of DNA by 10 times and can be used to transfect cells grown in the presence or absence of serum.

[0048] Wosnitzka, K., et al. (Cell Gen. Ther. Ins. 7 (2021) 1-7) reported that physical titer analysis revealed a 3-fold increase in viral particles (VP) and viral genome (VG) per ml of cell culture when using FectoVIR(TM)-AAV transfection reagent compared with PEIpro(TM).

[0049] Porte, M., et al. (poster entitled “Next-Generation Transfection Reagent for Large Scale AAV Manufacturing”, Polyplus, Illkirch, France) reported the transfection of HEK293T cells in suspension with the Petition 870250084687, dated 09 / 19 / 2025, pp. 92-143 23 / 66 Ideal conditions for the other PEI-based reagent (1.5 μg / million cells, DNA:PEI ratio of 1 μg:4 μl) and FectoVIR™-AAV (1 μg / million cells, DNA:reagent ratio of 1 μg:1 μl) were used following the recommended protocol for each reagent. A titer of approximately 5 E+11 vg / ml versus 1.5 E+11 vg / ml using FectoVIR™ and the PEI-based transfection reagent, respectively, with packing efficiencies of 20% vs. approximately 13.5%, respectively, was obtained. RECOMBINANT CELL

[0050] Generally, for efficient and large-scale production of a rAAV, a cell that expresses and, if possible, also secretes said rAAV is used. This cell is called a “recombinant producer cell” or, in short, a “producer cell”.

[0051] For the generation of a recombinant producer cell, a suitable mammalian cell is transfected with the nucleic acids required to produce said rAAV, including the required AAV ancillary functions.

[0052] Generally, for the expression of a coding sequence, i.e., an open reading frame, additional regulatory elements, such as a promoter and a polyadenylation signal (sequence), are required. Thus, for functional transcription, an open reading frame must be and is operatively linked to said additional regulatory elements. This can be achieved by combining these parts into a so-called expression cassette. The minimum regulatory elements required for an expression cassette to be functional in a mammalian cell are a functional promoter in said mammalian cell, which is located upstream, i.e., 5', to the open reading frame, and a functional polyadenylation signal (sequence) in said mammalian cell, which is located downstream, i.e., 3', to the open reading frame. In addition, a terminator sequence may Petition 870250084687, dated 09 / 19 / 2025, pp. 93 / 143 24 / 66 must be present 3' to the polyadenylation signal (sequence). For expression, the promoter, the open reading frame / coding region, and the polyadenylation signal sequence must be arranged in an operatively linked form.

[0053] Similarly, a nucleic acid that is transcribed into a non-protein coding RNA is called an “RNA gene”. Also for the expression of an RNA gene, additional regulatory elements, such as a promoter and a transcription termination signal or polyadenylation signal (sequence), are required. The nature and location of such elements depend on the RNA polymerase that is intended to control the expression of the RNA gene. Thus, an RNA gene is also normally integrated into an expression cassette.

[0054] In the case of an rAAV, which is composed of different capsid polypeptides (monomeric) and a single-stranded DNA molecule encapsulated within it, and which, moreover, requires other viral auxiliary functions for production and encapsulation, a multitude of expression cassettes differing in the coding sequences / open reading frames contained within are required. In this case, at least one expression cassette is required for each of the transgenes, for the polypeptides that form the rAAV capsid, and for the required viral auxiliary functions. Thus, individual expression cassettes are required for at least each of the auxiliary functions E1A, E1B, E2A, E4orf6, the rep and cap genes. HEK293 cells constitutively express the auxiliary functions E1A and E1B. Adeno-associated virus (AAV)

[0055] For a general review of AAVs and the auxiliary functions of adenovirus or herpes, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The AAV genome is described in Srivastava et al., J. Virol., 45 (1983) 555-564. In US document 4,797,368, design considerations for constructing recombinant AAV vectors are Petition 870250084687, dated 09 / 19 / 2025, pp. 94 / 143 25 / 66 described (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. The construction of recombinant AAV vectors is described in US document 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. Academic. Sci. USA 81 (1984) 64666470; Samulski et al. J. Virol. 63 (1989) 3822-3828.

[0056] An AAV is a replication-deficient 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, poxviruses like vaccinia. However, an AAV can replicate in virtually any cell line of human, simian, or rodent origin, provided the appropriate helper viral functions are present.

[0057] Without helper viral genes present, an AAV establishes latency in its host cell. Its genome integrates at a specific site on chromosome 19 [(Chr) 19 (q13.4)], which is called the adeno-associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV2, other integration sites have been found, for example, on chromosome 5 [(Chr) 5 (p13.3)], called AAVS2, and on chromosome 3 [(Chr) 3 (p24.3)], called AAVS3.

[0058] AAVs are categorized into different serotypes. These were allocated based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 12 different serotypes and more than one hundred sequences corresponding to different AAV clades have been identified.

[0059] The type and symmetry of the capsid protein determine the tissue tropism of the respective AAV. For example, AAV2, AAV4, and AAV5 are Petition 870250084687, dated 09 / 19 / 2025, pp. 95 / 143 26 / 66 specific for retina, AAV2, AAV5, AAV8, AAV9 and AAV-rh.10 are specific for brain, AAV1, AAV2, AAV6, AAV8 and AAV9 are specific for heart tissue, AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 are specific for liver, AAV1, AAV2, AAV5 and AAV9 are specific for lung.

[0060] Pseudotyping denotes a process that involves the cross-packaging of the AAV genome between several serotypes, i.e., the genome is packaged with capsid proteins of different origin.

[0061] The wild-type AAV genome is approximately 4.7 kb in size. The AAV genome additionally comprises two overlapping genes named rep and cap, which comprise multiple open reading frames (see, for example, 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 frame encoding the Rep protein provides four proteins of different sizes, which are named Rep78, Rep68, Rep52, and Rep40. These are involved in AAV replication, rescue, and integration. The open reading frame encoding the Cap protein provides four proteins, which are named VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the proteinaceous capsid of AAV particles. The open reading frames of the combined rep and cap are flanked at their 5' and 3' ends by so-called inverted terminal repeats (ITRs).For replication, an AAV requires, in addition to the Rep and Cap proteins, the products of the E1A, E1B, E4orf6, E2A, and VA genes of an adenovirus or corresponding factors from another helper virus.

[0062] In the case of an AAV of serotype 2 (AAV2), for example, the ITRs each have a length of 145 nucleotides and flank a coding sequence region of approximately 4470 nucleotides. Of the 145 ITR nucleotides, 125 nucleotides have a palindromic sequence and Petition 870250084687, dated 09 / 19 / 2025, pp. 96 / 143 27 / 66 can form a T-shaped hairpin structure. This structure functions as a primer during viral replication. The remaining 20 unpaired nucleotides are denoted as sequence D.

[0063] The wild-type AAV genome harbors 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.

[0064] ITR sequences must be present cis to the coding region. ITRs provide a functional origin of replication (ori), signals required for integration into the target cell genome, and efficient excision and rescue of host cell chromosomes or recombinant plasmids. ITRs additionally comprise the origin of replication-like elements such as a Rep protein binding site (RBS) and a terminal resolution site (TRS). ITRs themselves have been found to function as a transcription promoter (Flotte et al., J. Biol. Chem. 268 (1993) 3781-3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163-10167).

[0065] For replication and encapsulation, respectively, of the viral single-stranded DNA genome, a trans arrangement of the rep and cap gene products is required.

[0066] The rep gene locus comprises two internal promoters, designated P5 and P19. It comprises open reading frames for four proteins. The P5 promoter is operatively linked to a nucleic acid sequence that provides a 4.2 kb splice-free mRNA encoding the Rep protein Rep78 (chromatin nickase for cell cycle arrest) and a 3.9 kb splice-protected mRNA encoding the Rep protein Rep68 (site-specific endonuclease). The P19 promoter is operatively linked to a nucleic acid sequence that provides a splice-free mRNA encoding the Rep protein Rep52 and a 3.3 kb splice-protected mRNA encoding the Rep protein Rep40 (DNA helicases for accumulation and packaging). Petition 870250084687, dated 09 / 19 / 2025, pp. 97 / 143 28 / 66

[0067] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex DNA replication, while 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).

[0068] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit defined enzyme activities, which are required to resolve replication at the AAV terminals. Expression of Rep78 or Rep68 may be sufficient for the formation of infectious particles (Holscher, C., et al. J. Virol. 68 (1994) 71697177 and 69 (1995) 6880-6885).

[0069] It is considered that all Rep proteins, especially Rep78 and Rep68, exhibit regulatory activities, such as induction and suppression of AAV genes, as well as inhibitory effects on cell development (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virologia, 181 (1991) 738-741).

[0070] Recombinant overexpression of Rep78 results in a phenotype with reduced cell development due to the induction of DNA damage. In this way, the host cell is arrested in the S phase, in which latent infection by the virus is facilitated (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0071] Tratschin et al. reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, only very low expression was reported for certain cell lines after stable AAV integration (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0072] The cap gene locus comprises a promoter, called P40. The P40 promoter is operatively linked to a sequence Petition 870250084687, dated 09 / 19 / 2025, pp. 98 / 143 29 / 66 of nucleic acid provides mRNA 2,6, which, through alternative splicing and the use of alternative start codons, encodes the Cap proteins VP1 (87 kDa, unjoined mRNA transcript), VP2 (72 kDa, joined mRNA transcript), and VP3 (61 kDa, alternative start codon). VP1 to VP3 constitute the building blocks of the viral capsid. The capsid functions to bind to a cell surface receptor and allow intracellular trafficking of the virus. VP3 accounts for approximately 90% of the total viral particle proteins. However, all three proteins are essential for effective capsid production.

[0073] It has been reported that inactivation of all three capsid proteins VP1 to VP3 prevents the accumulation of single-stranded progeny AAV DNA. Mutations at the amino-terminal VP1 (Lip-negative or Inf-negative) still allow the assembly of single-stranded DNA into viral particles, where the infectious titer is significantly reduced.

[0074] The open reading frame of AAP encodes the activation assembly protein (AAP). It is approximately 22 kDa in size and transports native VP proteins to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0075] In individual AAV particles, only one single-stranded DNA molecule is contained. This can be either the positive or negative strand. AAV particles containing one DNA molecule are infectious. Inside the infected cell, the infectious parental single-stranded DNA is converted into double-stranded DNA, which is subsequently amplified. The amplification results in a large pool of double-stranded DNA molecules, from which single strands are displaced and packaged into capsids.

[0076] Adeno-associated viral vectors (AAVs) can transduce dividing cells as well as resting cells. It can be assumed that a transgene introduced using an AAV vector into a target cell will be Petition 870250084687, dated 09 / 19 / 2025, pp. 99 / 143 30 / 66 expressed for a long period. A disadvantage of using an AAV vector is the limitation on the size of the transgene that can be introduced into cells.

[0077] Parvovirus particles, including AAV serotypes and variants thereof, provide a means for ex vivo, in vitro, and in vivo delivery of nucleic acid, which encodes proteins, into cells so that infected cells express the encoded protein. AAVs are useful viruses as gene therapy vectors because they can penetrate cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material can be stably maintained in infected cells. Because AAVs are not associated with pathogenic diseases in humans, AAVs are able to deliver heterologous polynucleotide sequences (e.g., proteins and therapeutic agents) to human patients without causing pathogenesis or substantial AAV-related disease.

[0078] AAV particles used as vehicles for effective gene delivery possess a number of desirable characteristics for such applications, including tropism for dividing and non-dividing cells. Initial clinical experience with these vectors also demonstrated no sustained toxicity, and immune responses were minimal or undetectable. AAVs are known to infect a wide variety of cell types in vivo and in vitro by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans, targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0079] Recombinant AAV particles typically do not include viral genes associated with pathogenesis. These particles typically comprise a genome in which one or more wild-type AAV genes have been deleted in whole or in part, for example, rep and / or cap genes, but retain at least one functional flanking ITR sequence, as Petition 870250084687, dated 09 / 19 / 2025, pp. 100 / 143 31 / 66 is required for the rescue, replication, and packaging of the recombinant vector into a rAAV. Thus, an AAV vector includes sequences required in cis for replication and packaging (i.e., functional ITR sequences).

[0080] Recombinant AAV particles, as well as methods and uses thereof, may be based on any wild-type AAV genome or serotype, or a combination thereof. As a non-limiting example, a rAAV may be based on any wild-type AAV genome, i.e., comprise the respective ITR sequences, such as AAV1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, rh.74, rh.10, or 7m8, for example. Such particles may be based on the same strain or serotype (or subgroup or variant) or be different from one another. As a non-limiting example, a wild-type-based rAAV may be identical to or different from one or more of the capsid proteins that package the vector. In addition, a recombinant AAV vector can be based on a wild-type AAV serotype genome (e.g., AAV2) distinct from one or more AAV capsid proteins that package the vector.For example, the AAV vector may be based on AAV2, while at least one of the three capsid proteins may be AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10, or AAV7m8, or a variant thereof. AAV variants include variants and chimeras of the capsids AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.74, AAV-rh.10, and AAV-7m8.

[0081] In certain embodiments of all aspects and embodiments of the invention, 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, as well as variants (e.g., variants Petition 870250084687, dated 09 / 19 / 2025, pp. 101 / 143 32 / 66 of the capsid, such as insertions, additions, substitutions and deletions of amino acids) thereof, for example, as set forth in WO 2013 / 158879, WO 2015 / 013313 and US 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0082] In certain embodiments of all aspects and embodiments of the invention, rAAV comprises capsid polypeptides with an amino acid sequence having 70% or more sequence identity with a capsid sequence of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV-rh.10, AAV-rh.74 or AAV7m8 wild type.

[0083] In certain embodiments of all aspects and embodiments of the invention, the rAAV particle comprises one or two ITR sequences with 70% or more sequence identity with an ITR sequence of wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12.

[0084] Recombinant AAV particles can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for, among other things, administration and delivery to an individual in vivo or ex vivo. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicle or excipient. Such excipients include any pharmaceutical agent that does not induce a harmful immune response in the individual receiving the composition and that can be delivered without undue toxicity.

[0085] Protocols for the generation of adenoviral vectors were described in documents US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810 and WO 94 / 23744, which are incorporated herein by reference in their entirety. Recombinant adeno-associated viral particles (rAAV particles)

[0086] Different methods are known in the art for generating Petition 870250084687, dated 09 / 19 / 2025, pp. 102 / 143 33 / 66 recombinant AAV particles. For example, transfection with an AAV vector comprising a plasmid and a plasmid comprising AAV helper sequences (rep and cap) in conjunction with co-infection with an AAV helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) or transfection with a recombinant 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 documents 6,001,650, 6,004,797, WO 2017 / 096039, and WO 2018 / 226887. Following the production of rAAV (i.e., particle generation in cell culture systems), rAAV can be obtained from host cells and / or cell culture supernatant and purified.

[0087] For the generation of recombinant AAV particles, expression of the Rep and Cap proteins, the auxiliary proteins E1A, E1B, E2A and E4orf6, as well as optionally adenoviral VA RNA in a single mammalian cell is required. The auxiliary proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), especially the CMV IE promoter. Thus, any promoter can be operatively linked to the aforementioned genes for functional expression.

[0088] Generally, to produce rAAV, different and complementary plasmids are cotransfected into a host cell. One of the plasmids comprising the transgene is sandwiched between the two cis-acting AAV ITRs. The missing AAV elements required for replication and subsequent packaging of recombinant progeny genomes, namely the open reading frames for the Rep and Cap proteins, are contained in trans in a second plasmid. Overexpression of Rep proteins results in inhibitory effects on cell development (Li, J., et al., J. Virol. 71 (1997) 5236-5243). In addition, a third plasmid comprising the Petition 870250084687, dated 09 / 19 / 2025, pp. 103 / 143 34 / 66 genes from a helper virus, namely E1, E4orf6, E2A and VA from adenovirus, are required for the production of rAAV.

[0089] To reduce the number of plasmids required, rep, cap and adenovirus helper genes can be combined into a single plasmid.

[0090] Alternatively, the host cell may already stably express the E1 gene products. Such a cell is a HEK293 cell. The human embryonic kidney clone denoted as 293 was generated in 1977 by the integration of adenoviral DNA into human embryonic kidney cells (HEK cells) (Graham, FL, et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line comprises base pairs 1 to 4344 of the adenovirus serotype 5 genome. This includes the E1A and E1B genes, as well as the adenoviral packaging signals (Louis, N., et al., Virology 233 (1997) 423-429).

[0091] Using HEK293 cells, the absent E2A, E4orf6 and VA genes can be introduced by coinfection with an adenovirus or by cotransfection with a plasmid expressing E2A, E4orf6 and VA (see, e.g., Sam, RJ, Virolski, R.J., Virolski et al., 63 (1989) 3822-3828; J. Virol 71 (1997) 6816-6822; Conway, JE, et al., J. Virol 71 (1997) 8780-8789; al., Hum Ther 9 (1998) 695-706; J. Virol 72 (1998) 2224-2232; 10 (1999) 2527-2537). Alternatively, hybrid adenovirus / AAV or herpes simplex virus / AAV vectors can be used (see, for example, Conway, JE, et al., J. Virol.71 (1997) 8780-8789; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, A.J., et al., Gene Ther. 2 (1995) 481-485; Fisher, JK, et al., Hum. Gene Ther. 7 (1996). Petition 870250084687, dated 09 / 19 / 2025, pp. 104 / 143 35 / 66 2079-2087; Johnston, KM, et al., Hum. Gene Ther. 8 (1997) 359-370).

[0092] To limit transgene activity to specific tissues, i.e., to limit the site of action, the transgene can be operatively linked to an inducible or tissue-specific promoter (see, for example, Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0093] The E1A and E1B coding sequences (open reading frames) can be derived from a human adenovirus, such as, for example, human adenovirus serotype 2 or serotype 5. An exemplary sequence of human Ad5 (adenovirus serotype 5) is found in GenBank entries X02996, AC_000008 and that of an exemplary human Ad2 in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequences encoding E1A and E1B of human adenovirus serotype 5. The pSTK146 plasmid, as reported in EP 1 230 354, as well as the pGS119 and pGS122 plasmids, as reported in WO 2007 / 056994, can also be used as a source for the open reading frames of E1A and E1B.

[0094] E1A is the first viral helper gene that is expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes the 12S and 13S proteins, which are based on the same E1A mRNA by alternative splicing. Expression of the 12S and 13S proteins results in the activation of the other viral functions E1B, E2, E3, and E4. Furthermore, expression of the 12S and 13S proteins forces the cell to enter the S phase of the cell cycle. If only the proteins derived from E1A are expressed, the cell will die (apoptosis).

[0095] E1B is the second viral helper gene that is expressed. It is activated by proteins derived from E1A 12S and 13S. The mRNA derived from the E1B gene can undergo splicing in two different ways, resulting in a first transcript of 55 kDa and a second transcript of 19 kDa. The 55 kDa E1B protein is involved in cell cycle modulation, in the prevention of Petition 870250084687, dated 09 / 19 / 2025, pp. 105 / 143 36 / 66 cellular mRNA transport in the late phase of infection and in the prevention of E1A-induced apoptosis. The 19 kDa E1B protein is involved in the prevention of E1A-induced apoptosis of cells.

[0096] The E2 gene encodes different proteins. The E2A transcript encodes the single-strand binding protein (SSBP), which is essential for AAV replication.

[0097] In addition, the E4 gene encodes several proteins. The 34 kDa protein derived from the E4 gene (E4orf6) prevents the accumulation of cellular mRNAs in the cytoplasm along with the 55 kDa E1B protein, but also promotes the transport of viral RNAs from the cell nucleus to the cytoplasm.

[0098] Viral-associated RNA (VARNA) is a non-coding RNA of adenovirus (Ad) that regulates translation. The adenoviral genome comprises two independent copies: VAI (RNAI VA) and VAII (RNAII VA). Both are transcribed by RNA polymerase III (see, for example, Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289) from a type 2 polymerase III promoter. For recombinant AAV particle production, the adenoviral VARNA gene can be controlled by either promoter.

[0099] The structure, function, and evolution of adenovirus-associated RNA using a phylogenetic approach were investigated by Ma, Y. and Mathews, MB (J. Virol. 70 (1996) 5083-5099). They provided alignments as well as consensus VA RNA sequences based on 47 known human adenovirus serotypes. The aforementioned disclosure is incorporated herein by reference in its entirety in this application.

[00100] VA, VAI and VAII RNAs consist of 157-160 nucleotides (nt).

[00101] Depending on the serotype, adenoviruses contain one or two RNA VA genes. RNAI VA is believed to play the dominant proviral role, while RNAII VA may partially compensate for the absence of Petition 870250084687, dated 09 / 19 / 2025, pp. 106 / 143 37 / 66 VAI RNA (Vachon, VK and Conn, GL, Virus Res. 212 (2016) 39-52).

[00102] VA RNAs are not essential, but they play an important role in efficient viral development by overcoming the cellular antiviral mechanism. That is, although VA RNAs are not essential for viral development, adenovirus deleted with VA RNA cannot develop during the initial stage of vector generation, where only a few copies of the viral genome are present per cell, possibly because viral genes other than VA RNAs that block the cellular antiviral mechanism may not be sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[00103] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenovirus vector lacking virus-associated RNA genes that disrupt the cellular RNAi mechanism, in which HEK293 cells constitutively and highly expressing flippase recombinase were infected to obtain RNA VA deleted adenovirus by FLP-mediated excision of the RNA VA locus.

[00104] The RNAI VA of human adenovirus 2 corresponds to nucleotides 10586 to 10810 of the GenBank entry sequence AC_000007. The RNAI VA of human adenovirus 5 corresponds to nucleotides 10579 to 10820 of the GenBank entry sequence AC_000008. General Description of Recombinant AAV Particle Production

[00105] After entering the nucleus of the host cell, AAV can follow one of two distinct and interchangeable pathways in its life cycle: the lytic or the lysogenic pathway. The former develops in cells infected with a helper virus, such as Ad or the herpes simplex virus (HSV), while the latter establishes itself in host cells in the absence of a helper virus.

[00106] When a latently infected cell is superinfected with a helper virus, the AAV gene expression program Petition 870250084687, dated 09 / 19 / 2025, pp. 107 / 143 38 / 66 is activated, leading to AAV Rep-mediated rescue (i.e., excision) of proviral DNA from the host cell chromosome, followed by replication and packaging of the viral genome. Finally, upon helper virus-induced cell lysis, the newly assembled virions (particles) are released. Thus, the lytic phase of the AAV life cycle is induced.

[00107] Therefore, in the presence of Ad auxiliary functions, the rAAV vector is subjected to wild-type AAV lytic processes when rescued from the plasmid main strand, replicated and packaged into preformed AAV capsids as single-stranded molecules (Gonçalves, MAFV, Virol. J., 2 (2005) 43).

[00108] The generation of a recombinant AAV particle involves replacing most of the wild-type AAV genome with a desired transgene and providing the viral genes that are essential for trans-packaging of viruses into a separate plasmid. After all components are transfected together into a packaging cell line, the recombinant AAV particles are assembled using the cell's cellular mechanisms. The viral assembly and encapsulation process takes approximately two days, after which the cells are lysed to release the rAAV for further purification and concentration (https: / / old.abmgood.com / marketing / knowledge_base / Adeno_Associated_Virus_Production_and_Modification_of_AAV.php).

[00109] AAV is not released very efficiently by cells, although large differences have been observed between serotypes (see, for example, Strobel, B., et al., Lamla T. Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant AdenoAssociated Viral Vectors for Preclinical Applications. Hum. Gene Ther. Methods 26 (2015) 147-157). When harvesting the culture, a cell disruption method is usually applied to recover vectors trapped in the cells. Petition 870250084687, dated 09 / 19 / 2025, pp. 108 / 143 39 / 66

[00110] Historically, the fabrication of rAAVs was performed by double transfection of a plasmid containing the rep and cap ORFs and a plasmid with the gene of interest flanked by ITRs. Then, a helper virus, typically Adenovirus, was co-infected (see, for example, Aponte-Ubillus, JJ, et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Muzyczka, N., Curr. Top. Microbiol. Immunol. 158 (1992) 97-129). In this scenario, separation of the helper virus from the final product was difficult, but a critical element to avoid inducing inflammatory responses after injection in patients (see, for example, Schnell, MA, et al., Mol. Ther. 3 (2001) 708-722.). Therefore, the production of rAAVs has currently evolved to an adenovirus-free approach, using triple transfection (see, for example, Large, EE, et al., Viruses 13 (2021) 1336).To this end, three components are required: a plasmid encoding the genes for Rep and Cap without the ITRs, a second plasmid with the transgene of interest flanked by ITRs, and a helper plasmid to provide the helper virus genes (see, for example, Aponte-Ubillus, JJ, et al., Appl. Microbiol. Biotechnol. 102 (2018) 1045-1054; Farris, KD and Pintel, DJ, Hum. Gene Ther. 19 (2008) 1421-1427; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Ferrari, FK, et al., Nat. Med. 3 (1997) 1295-1297). For example, adenovirus helper carries the minimum required adenoviral genes E2A, E4, and VA. It is important to note that human embryonic kidney cells 293 (HEK293) constitutively express the adenoviral genes E1A / B, which are also required for rAAV production. Therefore, HEK293 cells are classic rAAV production cells. Other cell types require E1A / B supplementation.

[00111] Carter et al. showed that all open reading frames of rep and cap in the wild-type AAV genome can be deleted and replaced by a transgene (Carter, BJ, in Handbook of Parvoviruses, ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Additionally, it was reported Petition 870250084687, dated 09 / 19 / 2025, pp. 109 / 143 40 / 66 that ITRs need to be maintained to retain the function of replication, rescue, packaging and integration of the transgene into the target cell genome.

[00112] When cells containing the respective viral helper genes are transduced by an AAV vector, or vice versa, when cells containing an integrated AAV provirus are transduced by a suitable helper virus, then the AAV provirus is activated and enters a lytic infection cycle again (Clark, KR, et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[00113] The producing cells contain the rep and cap gene sequences, as well as the transgene cassette flanked by ITR sequences on one or more plasmids that are retained, for example, through drug selection. rAAV production in these cell lines generally occurs after their infection with the required helper functions. Therefore, the cells are infected with a replication-competent adenovirus (usually wild-type Ad5) or a plasmid comprising the respective helper genes to supply helper virus proteins and initiate rAAV production. A packaging cell line differs from a producing cell line in that it contains only the rep and cap genes.

[00114] More generally, cells transfected or transduced with DNA for the recombinant production of AAV particles may be referred to as a recombinant cell. Such a cell may be any mammalian cell that has been used as a recipient of a nucleic acid (plasmid) encoding packaging proteins, such as AAV packaging proteins, a nucleic acid (plasmid) encoding auxiliary proteins, 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 the progeny of the original cell that was transduced or transfected. It is understood that the progeny of a single cell Petition 870250084687, dated 09 / 19 / 2025, pp. 110 / 143 41 / 66 parental cells may not necessarily be completely identical in morphology or in genomic or total nucleic acid complement as the original parental cell, due to natural, accidental, or deliberate mutation.

[00115] Numerous cell development media suitable for sustaining cell viability or providing cell development and / or proliferation are commercially available. Examples of such media include serum-free eukaryotic development media, such as media for sustaining viability or providing development of mammalian (e.g., human) cells. Non-limiting examples include Ham F12 or F12K medium (Sigma-Aldrich), FreeStyle (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. These media may be supplemented with trace vitamins and / or minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[00116] To produce rAAV, three plasmids are cotransfected into a mammalian cell. The transgene plasmid encodes the expression cassette, which is cloned between the AAV ITRs, while the rep and cap genes are provided trans by cotransfection of a second packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also referred to as the helper plasmid, contains the minimal helper viral factors, commonly the E2A, E4orf6, and adenoviral VA genes, but lacks AAV ITRs.

[00117] Various methods for transferring DNA into mammalian cells have been reported in the art. These are all useful in the methods according to the present invention. In certain embodiments of all aspects and embodiments, electroporation, nucleofection, or microinjection for nucleic acid transfer / transfection are used. In certain embodiments of all aspects and embodiments, an inorganic substance (such as, for example, an inorganic substance) is used. Petition 870250084687, dated 09 / 19 / 2025, pp. 111 / 143 42 / 66 example, calcium phosphate / DNA coprecipitation), a cationic polymer (such as, for example, polyethyleneimine, DEAE-dextran) or a cationic lipid (lipofection) is used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection for nucleic acid transfer on larger scales (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), of which polyethyleneimine is preferred.

[00118] Development in serum-free suspension culture and improved efficiency and reproducibility of transfection conditions using PEI as a transfection reagent enable rapid scalding of AAV production using shake flask, wave, or stirred tank bioreactors.

[00119] The composition may additionally comprise plasmids and / or cells. Such plasmids and cells may be in contact with free PEI.

[00120] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA, a short-chain branched fatty acid, inhibits histone deacetylase activity. For this reason, it is commonly added to mammalian cell culture as an enhancer of recombinant protein production.

[00121] Encoded AAV packaging proteins include, in certain embodiments of all aspects and embodiments, AAV rep and / or AAV cap proteins. Such AAV packaging proteins include, in certain embodiments of all aspects and embodiments, AAV rep and / or AAV cap proteins of any AAV serotype.

[00122] Encoded helper proteins include, in certain embodiments of all aspects and embodiments, adenovirus E1A and E1B, adenovirus E2 and / or E4, VA RNA and / or non-AAV helper proteins.

[00123] Cultivation can be carried out using the conditions Petition 870250084687, dated 09 / 19 / 2025, pp. 112 / 143 43 / 66 wells are generally used for the cultivation of eukaryotic cells at approximately 37 °C, 95% humidity, and 8% CO2 by volume. Cultivation can be carried out in serum-containing or serum-free media, in adherent culture or in suspension culture. Suspension culture can be carried out in any fermentation vessel, such as stirred tank reactors, wave reactors, swing bioreactors, vibrating vessels, or rotating vessels, or so-called rolling flasks. Transfection can be carried out in high-throughput and screening formats, respectively, for example, in a 96-well or 384-well format.

[00124] The methods according to the present invention may include AAV particles of any serotype or a variant thereof. In certain embodiments of all aspects and embodiments, a recombinant AAV particle comprises any of the AAV serotypes 1 to 12, an AAV capsid protein VP1, VP2 and / or VP3, or a modified or variant AAV capsid protein VP1, VP2 and / or VP3, or a wild-type AAV capsid protein VP1, VP2 and / or VP3. In certain embodiments of all aspects and embodiments, an AAV particle comprises an AAV serotype or an AAV pseudotype, wherein the AAV pseudotype comprises an AAV capsid serotype other than an ITR serotype.

[00125] Expression control elements include constitutive or adjustable control elements, such as a tissue-specific expression control element or promoter.

[00126] ITRs may be any of the AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. AAV particles may include any VP1, VP2 and / or VP3 capsid protein with 75% or more sequence identity with any of the capsid proteins of 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, or comprise a Petition 870250084687, dated 09 / 19 / 2025, pp. 113 / 143 44 / 66 VP1, VP2 and / or VP3 capsid protein modified or selected variant from any of the AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9, AAV-2i8, AAV-rh.10, AAV-rh.74 and AAV-7m8.

[00127] Following the production of recombinant viral particles (e.g., AAV), if desired, the viral particles (e.g., rAAV) can be purified and / or isolated from host cells using a variety of conventional methods. Such methods include column chromatography, CsCl gradients, iodixanol gradients, and the like.

[00128] For example, a plurality of column purification steps, such as purification on an anion exchange column, an affinity column, and / or a cation exchange column, can be used (see, for example, WO 02 / 12455 and US 2003 / 0207439). Alternatively, or in addition, iodixanol or CsCl gradient steps can be used (see, for example, US 2012 / 0135515; and US 2013 / 0072548). Additionally, if infectious virus is used to express the packaging and / or auxiliary proteins, the residual virus can be inactivated using various methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, for example, 20 minutes or more. This treatment effectively inactivates the helper virus, since AAV is heat-stable, while the helper adenovirus is heat-labile.

[00129] One objective in rAAV production and purification systems is to implement strategies to minimize / control the generation of production-related impurities, such as proteins, nucleic acids, and vector-related impurities, including wild-type / pseudo-wild-type (wtAAV) AAV species and residual DNA impurities encapsulated in AAVs.

[00130] Considering that rAAV represents only a small fraction of the biomass, rAAV needs to be purified to a level of purity that Petition 870250084687, dated 09 / 19 / 2025, pp. 114 / 143 45 / 66 can be used as a clinical human gene therapy product (see, for example, Smith PH, et al., Mo. Therapy 7 (2003) 8348; Chadeuf G., et al, Mo. Therapy 12 (2005) 744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[00131] In certain embodiments of all aspects and embodiments of the method according to the present invention, as an initial step, typically the cultured cells that produce rAAV particles are harvested, optionally in combination with the harvesting of the cell culture supernatant (medium) in which the cells (suspension or adherent) that produce recombinant AAV particles were cultured. The harvested cells and, optionally, the cell culture supernatant can be used as is, as appropriate, lysed or concentrated. Additionally, if the infection is employed to express auxiliary functions, the residual auxiliary virus can be inactivated. For example, adenovirus can be inactivated by heating to temperatures of approximately 60 °C for, for example, 20 minutes or more, which inactivates only the auxiliary virus, since AAV is heat-stable, while the auxiliary adenovirus is heat-labile.

[00132] Cells in the harvested culture broth can be lysed using methods now in the art, such as, for example, detergent lysis or freeze-thaw cycles, to release rAAV particles. Simultaneously during cell lysis or subsequently after cell lysis, a nuclease, such as, for example, benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell residues, for example, by filtration or centrifugation, to provide a clarified cell lysate. In one particular example, the lysate is filtered with a micron-diameter pore size filter (such as a 0.1 to 10.0 µm pore size filter, for example, a 0.45 µm filter and / or a 0.2 µm pore size filter) to produce a clarified lysate. Petition 870250084687, dated 09 / 19 / 2025, pp. 115 / 143 46 / 66

[00133] The lysate (optionally clarified) contains recombinant AAV particles (comprising both filled and empty rAAVs) and production / process-related impurities, such as soluble cellular components from host cells which may include, inter alia, cellular proteins, lipids and / or nucleic acids and cell culture medium components. The optionally clarified lysate is then subjected to purification steps to purify the rAAV (comprising rAAV vectors) from impurities using chromatography. The clarified lysate may be diluted or concentrated with an appropriate buffer prior to the first chromatography step.

[00134] After cell lysis, optional clarification, and optional dilution or concentration, a plurality of subsequent and sequential chromatography steps can be used to purify rAAV.

[00135] The first chromatography step is preferably an affinity chromatography step that uses an AAV affinity chromatography ligand.

[00136] If the first chromatography step is affinity chromatography, the second chromatography step may be anion exchange chromatography. Thus, in certain all-aspect embodiments and embodiments, the purification of rAAV is done by affinity chromatography, followed by purification by anion exchange chromatography and / or cation exchange chromatography and / or size exclusion chromatography, in any order, sequence or combination.

[00137] The removal of empty capsids from filled capsids, for example, during downstream processing, is based on their different isoelectric points (pI) in anion-exchange chromatography. The average pI calculated across all serotypes is 5.9 for filled capsids and 6.3 for empty capsids (Venkatakrishnan, B., et al., J. Virol. 87 (2013) 4974-4984). Petition 870250084687, dated 09 / 19 / 2025, pp. 116 / 143 47 / 66

[00138] Cation exchange chromatography is used to separate AAV from cellular and other components present in the clarified lysate and / or column eluate from affinity 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, as indicated by the presence of the sulfonate functional group, 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 DOWEX®, AMBERLITE®, and AMBERLYST® families of commercial resins available from Aldrich Chemical Company (Milliwaukee, WI, USA). Weak cation exchange resins include, without limitation, any carboxylic acid-based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on the phosphate functional group), methyl sulfonate (S), and sulfopropyl (SP) resins.

[00139] Anion exchange chromatography is used to separate rAAV from proteins, cellular components, and other elements present in the clarified lysate and / or column eluate from affinity, cation exchange, or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thus control the amount of empty rAAV in the eluate. For example, an anion exchange column with both filled and empty rAAV attached can be washed with a solution comprising NaCl at a modest concentration (e.g., about 100 to 125 mM, such as 110 to 115 mM), and a portion of the empty rAAV can be eluted in the stream without substantial elution of the filled rAAV. Subsequently, the filled rAAV attached to the column... Petition 870250084687, dated 09 / 19 / 2025, pp. 117 / 143 48 / 66 anion exchange can be eluted using a solution comprising NaCl at a higher concentration (e.g., about 130 to 300 mM NaCl), thus producing a column eluate with reduced or depleted amounts of empty rAAVs and proportionally increased amounts of filled rAAV comprising an rAAV vector.

[00140] Exemplary anion exchange resins include, without limitation, those based on polyamine resins and other resins. Examples of strong anion exchange resins include those generally based on the quaternized nitrogen atom, including, without limitation, quaternary ammonium salt resins, such as trialkylbenzyl ammonium resins.Suitable exchange chromatography materials include, without limitation, MACRO PREP Q (strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (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 50PI (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).

[00141] A commercial manufacturing process for purifying recombinant AAV particles intended as a product for treating Petition 870250084687, dated 09 / 19 / 2025, pp. 118 / 143 49 / 66 human diseases must meet the following objectives: 1) consistent purity, potency, and safety of particles; 2) scalability of the manufacturing process; and 3) acceptable manufacturing cost.

[00142] Exemplary processes for purification of recombinant AAV particles are reported in document WO 2019 / 006390.

[00143] Methods for determining infectious titer of rAAV particles containing a transgene are known in the art (see, for example, Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for examining empty rAAV and rAAV filled with packaged transgenes are known (see, for example, Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[00144] To determine the presence or quantity of degraded / denatured capsid, purified rAAV can be subjected to SDS-polyacrylamide gel electrophoresis, which consists of any gel capable of separating the three capsid proteins, for example, a gradient gel, then run the gel until the sample is separated and blot the gel onto nylon or nitrocellulose membranes. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (see, for example, Wobus et al., J. Viral. 74 (2000) 9281-9293). A secondary antibody that binds to the primary antibody contains a means to detect the primary antibody. The binding between the primary and secondary antibodies is detected semi-quantitatively to determine the amount of capsids. Another method would be analytical HPLC with an analytical SEC or ultracentrifuge column. Description of Specific Embodiments of the Invention

[00145] The present invention is based, at least in part, on the discovery that the productivity of mammalian cells that produce a recombinant adeno-associated viral particle can be increased when the Petition 870250084687, dated 09 / 19 / 2025, pp. 119 / 143 50 / 66 cultivation is carried out at high pH values, such as pH 7.4 to 7.6.

[00146] Although optimizing transfection parameters, DNA / reagent ratio, VCD in transfection, and complexation time can improve rAAV productivity, such an increase is far outweighed by the effect that the pH value according to the present invention has.

[00147] The reference value, or base value, in each table is indicated by the identifier “(100%)”.

[00148] It has been found that for transient rAAV particle production in HEK293 cells, increasing the culture pH value from the commonly used pH of 7.2 to a pH value of 7.4 or even 7.6 increases particle yield as well as genomic yield. Since the increase in genomic yield is higher than the increase in titer yield, the full-to-empty ratio is also improved.

[00149] For example, for the transient production of rAAV2 particles in serum-free medium using a suspension-developing HEK293 cell, increasing the culture pH value from the commonly used pH of 7.2 to a pH value of 7.4 or even 7.6 increases the particle yield by more than 3 times and the genomic yield by more than 10 times.

[00150] The following tables provide illustrative data for an HEK293 cell adapted for development in suspension in serum-free culture medium, showing the effect of the method according to the present invention (see also Figures 1, 2 and 3). It can be observed that the genomic titer (vg / ml) is increased more than 10 times if the culture pH value is increased from pH 7.2 to pH 7.4 or pH 7.6. Concomitantly, the capsid titer (vp / ml) is increased 2 to 3 times. Thus, as the increase in capsid titer is smaller compared to the increase in genomic titer, the ratio of full to empty cells increases more than 4 times. Petition 870250084687, dated 09 / 19 / 2025, pp. 120 / 143 51 / 66 Table 3 pH value (sample collected 72 hours after transfection) vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution 7.0 4.23 E+08 88% 8.10 E+09 32% 4.96% 263% #1 #1 7.2 (100%) 4.80 E+08 100% 2.50 E+10 100% 1.89% 100% #1 #2 7.4 8.95 E+09 1865% 9.60 E+10 384% 8.53% 452% #1 #3 7.6 5.33 E+09 1110% 5.90 E+10 236% 8.28% 439% #1 #4

[00151] Yield is independent of growing time for pH 7.2, pH 7.4 and pH 7.6, while at a pH value of 7.0 the yield decreases with increasing cultivation time. This is shown in Table 4 below. Thus, at pH values ​​of 7.4 and 7.6, the process is more robust and results in increased yields compared to pH 7 or pH 7.2, respectively. Table 4 pH 7.0 vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution collection 72 hours after transfection (100%) 4.23 E+08 100% 8.10 E+09 100% 5.22% 100% #1 #1 collection 96 hours after transfection 2.04 E+08 48% 5.50 E+09 68% 3.71% 71% #1 #1 collection 120 hours after transfection 1.30 E+08 31% 4.60 E+09 57% 2.83% 54% #1 #1 pH 7.2 vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution collection 72 hours after transfection (100%) 4.80 E+08 100% 2.50 E+10 100% 1.92% 100% #1 #2 collection 96 hours after transfection 5.01 E+08 104% 2.30 E+10 92% 2.18% 113% #1 #2 collection 120 hours after transfection 3.75 E+08 78% 2.40 E+10 96% 1.56% 81% #1 #2 Petition 870250084687, dated 09 / 19 / 2025, pp. 121 / 143 52 / 66 pH 7.4 vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution harvest 72 hours after transfection (100%) 8.95 E+09 100% 9.60 E+10 100% 9.33% 100% #1 #3 harvest 96 hours after transfection 8.86 E+09 99% 9.40 E+10 98% 9.43% 101% #1 #3 harvest 120 hours after transfection 9.02 E+09 101% 9.60 E+10 100% 9.39% 101% #1 #3 pH 7.6 vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution harvest 72 hours after transfection (100%) 5.33 E+09 100% 5.90 E+10 100% 9.03% 100% #1 #4 harvest 96 hours after transfection 5.10 E+09 96% 5.90 E+10 100% 8.64% 96% #1 #4 harvest 120 hours after transfection 5.15 E+09 97% 5.80 E+10 98% 8.87% 98% #1 #4

[00152] The effect of changing the pH value from pH 7.2 to pH 7.4 or pH 7.6 far exceeds the increase in titer obtained by optimizing process conditions, such as changing the transfection reagent or adding feed, as shown in Table 5 below. The increase in titer due to a concomitant change in the culture pH value, the transfection reagent, and the addition of feed is shown in the third row of data. Petition 870250084687, dated 09 / 19 / 2025, pages 122 / 143 53 / 66 Table 5 Harvest 72h post-transfection vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution pH 7.2, transfection with PEI, no feed (100%) 4.80 E+08 100% 2.50 E+10 100% 1.92% 100% #1 #2 pH 7.2, transfection with FectoVIR(TM), feed 9.39 E+08 195% 1.20 E+10 48% 7.83% 407% #3 #5 pH 7.4, transfection with FectoVIR(TM), feed (average of #3#1, #3#2, #4#1, #4#2) 1.22 E+10 2539% 1.15 E+11 460% 10.62% 553%

[00153] For a direct comparison, the titer increase resulting from a change in the culture pH value under optimized transfection reagent conditions and with the addition of a feed is shown in Table 6 below. Table 6 vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution pH 7.2, transfection with FectoVIR™, feeding (100%) 9.39 E+08 100% 1.20 E+10 100% 7.83% 100% #3 #5 pH 7.4, transfection with FectoVIR™, feeding (average of #3#1, #3#2, #4#1, #4#2) 1.22 E+10 1299% 1.15 E+11 958% 10.62% 136%

[00154] Due to the optimization of growing conditions, the crop becomes less robust at a pH value of 7.2. However, at a pH value of 7.4, the process maintains its robustness. That is, increasing the pH value from pH 7.2 Petition 870250084687, dated 09 / 19 / 2025, pp. 123 / 143 54 / 66 for pH 7.4, the loss in process robustness due to optimization of reaction conditions can be countered. This is shown in Table 7 below. Table 7 pH 7.2, transfection with FectoVIR™, food vg / ml (absolute) vg / ml (relative) vp / ml (absolute) 1 vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution collection 72 hours post-transfection (100%) 9.39 E+08 100% 1.20 E+10 100% 7.83% 100% #3 #5 collection 96 hours post-transfection 5.35 E+08 57% 6.40 E+09 53% 8.36% 107% #3 #5 pH 7.4, transfection with FectoVIR™, food vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution harvest 72 hours post-transfection (100%) (average of #3#1, #3#2, #4#1, #4#2) 1.22 E+10 100% 1.15 E+11 100% 10.62% 100% harvest 96 hours post-transfection 1.10 E+10 90% 9.90 E+10 86% 11.08% 104% #4 #2

[00155] For example, for the transient production of rAAV2 particles in serum-free medium using the commercially available HEK 293 Expi cell under the optimized conditions as described above, the particle yield can be further increased by about 1.7 times and the genomic yield by about 1.8 times, i.e., by 80%. The data are shown in Table 8 below. Table 8 pH 7.4, transfection with FectoVIR™, food vg / ml (absolute) vg / ml (relative) vp / ml (absolute) 1 vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set execution HEK293 serum-free suspension collected 72 hours post-transfection (100%) (average of #3#1, #3#2, #4#1, #4#2) 1.22 E+10 100% 1.15 E+11 100% 10.60% 100% Petition 870250084687, dated 09 / 19 / 2025, pp. 124 / 143 55 / 66 pH 7.4, transfection with FectoVIR™, feed vg / ml (absolute) vg / ml (relative) vp / ml (absolute) vp / ml (relative) full / empty ratio (absolute) full / empty ratio (relative) set run HEK commercial 293 Expi harvest 72 hours post-transfection (average of #3#3, #3#4, #4#3, #4#4) 2.21 E+10 181% 1.98 E+11 172% 11.20% 106%

[00156] The examples and figures are provided to aid understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications may be made to the procedures set forth without departing from the spirit of the invention. Examples Materials Cell lines

[00157] Commercially available HEK293 cells were used to produce AAV particles using transient transfection with three plasmids. Growing materials

[00158] Culture media and supplements were used according to the supplier's operating instructions. Media and feed were stored at 4 °C in the dark and consumed according to the manufacturer's instructions. Corrective agents were stored at room temperature (glucose solution; sodium carbonate solution; antifoaming solution). Example 1 Culture of HEK293 cells and production of recombinant AAV preparations.

[00159] Generally, cultivation methods were adapted from Petition 870250084687, dated 09 / 19 / 2025, pages 125 / 143 56 / 66 standard protocols (see e.g. Lindl, T., “Zell- und Gewebekultur: Einführung in die Grundlagen sowie ausgewãhlte Methoden und Anwendungen”, Spektrum Akademischer Verlag GmbH, Heidelberg / Berlin, 2002) and operating instructions from the respective supplier. Pre-cultivation

[00160] HEK cells were thawed and propagated in vibration flasks at 37 °C, 85% humidity, 5% pCO2, and a vibration frequency of 120 rpm for two to three weeks in culture medium. Cells were divided every three to four days and expanded in medium to the volume required for inoculation of the production culture. PRODUCTION CULTIVATION

[00161] To produce recombinant AAV particles, culture was carried out in the respective pre-cultured HEK293 cells in the respective reactor in a batch or fed-batch process under the indicated conditions.

[00162] Set 1 - rAAV particle preparation with particles comprising a capsid variant derived from AAV2 serotype and a therapeutic transgene: reactor: Ambr250; Cell line: HEK293 suspension adapted for serum-free medium; Culture medium: HEK ViP NB + 8 mM glutamine + insulin; Feeding: absent (batch); temperature 37 °C; Speed: ~ 450 rpm; Cultivation time after inoculation: 144 hours; Transfection: transient; three plasmids; ratio ~ 1:2.5:2 (transgene:rep / cap:auxiliary); Transfection: ~ 24 hours post-inoculation; Petition 870250084687, dated 09 / 19 / 2025, pages 126 / 143 57 / 66 transfection reagent: PEIpro(TM); Transfection reagent ratio: DNA: ~ 2:1; DNA concentration: ~ 3 pg / ml; VCD transfection: ~ 30 E+05 cells / ml; Transfection: transfection mixture in ½ of fresh culture medium; Lise: no. Table 9 Set of parameters: execution time, post-transfection, pH, Genomic titer [vg / ml], Capsid titer [vp / ml], Full / Empty [%] #1 #1 72 7 4.23 E+08 8.10 E+09 5.22 #1 #1 96 7 2.04 E+08 5.50 E+09 3.71 #1 #1 120 7 1.30 E+08 4.60 E+09 2.83 #1 #2 72 7.2 4.80 E+08 2.50 E+10 1.92 #1 #2 96 7.2 5.01 E+08 2.30 E+10 2.18 #1 #2 120 7.2 3.75 E+08 2.40 E+10 1.56 #1 #3 72 7.4 8.95 E+09 9.60 E+10 9.33 #1 #3 96 7.4 8.86 E+09 9.40 E+10 9.43 #1 #3 120 7.4 9.02 E+09 9.60 E+10 9.39 #1 #4 72 7.6 5.33 E+09 5.90 E+10 9.03 #1 #4 96 7.6 5.10 E+09 5.90 E+10 8.64 #1 #4 120 7.6 5.15 E+09 5.80 E+10 8.87

[00163] Set 2 - Preparation of rAAV particle with particles comprising capsid variant derived from AAV2 serotype and therapeutic transgene: reactor: Ambr250; Cell line: HEK293 suspension adapted for serum-free medium; Culture medium: HEK ViP NB + 8 mM glutamine + insulin; Feeding: absent (batch); Petition 870250084687, dated 09 / 19 / 2025, pp. 127 / 143 58 / 66 temperature 37 °C; Speed: ~ 450 rpm; Cultivation time after inoculation: 120 hours; Transfection: transient; three plasmids; ratio ~ 1:2.5:2 (transgene:rep / cap:auxiliary); Transfection: ~ 24 hours post-inoculation; transfection reagent: PEIpro(TM); Transfection reagent ratio: DNA: ~ 2:1; DNA concentration: ~ 3 pg / ml; VCD transfection: ~ 30 E+05 cells / ml; Transfection: transfection mixture in % of fresh culture medium; Lise: no. Table 10 Set of parameters: execution time, post-transfection, pH, genomic titer [vg / ml], capsid titer [vp / ml], full / empty [%] #2 #1 96 7 4.00 E+08 5.50 E+09 7.27 #2 #2 96 7.4 7.70 E+09 9.90 E+10 7.78 Sets 3 and 4 - DoE:

[00164] Preparation of rAAV particles: 1) with particles comprising a capsid variant derived from an AAV2 serotype and a therapeutic transgene; 2) with particles comprising a wild-type AAV2 capsid and a green fluorescent protein (GFP) transgene: reactor: Ambr15; Cell lineage: 1) HEK293 suspension adapted for serum-free medium; 2) HEK293 Expi; Culture medium: HEK ViP NB + 8 mM glutamine + insulin; Feeding: 1) absent (batch); 2) glucose medium and feeding post-transfection; Petition 870250084687, dated 09 / 19 / 2025, pages 128 / 143 59 / 66 temperature 37 °C; Speed: ~ 450 rpm; Cultivation time after inoculation: 120 hours; Transfection: transient; three plasmids; ratio 1) ~ 1:2.5:2 or 2) ~ 1:1:1 (transgene:rep / cap:auxiliary); Transfection: 24 hours post-inoculation; Transfection reagent: 1) PEI + free PEI + valproic acid; 2) FectoVIR™-AAV; Transfection reagent ratio:DNA: 1) ~ 2.5:1; 2) ~ 1.5:1; DNA concentration: 1) ~ 3 pg / ml; 2) ~ 2 pg / ml; VCD transfection: ~ 30 E+05 cells / ml; Transfection: transfection mixture in % of fresh culture medium; Lise: 1) no; 2) yes. Table 11 Set of execution time post-transfection pH AAV Lineage AaI Feed ratio (lysis transfection 1 Plasmid concentration Γ. .« / «,11 reagent / DN ratio Genomic titer [vg / ml] Capsid titer [vp / ml] Full / Empty [%] #3 #1 72 7.4 1) 1) 1) 2) 1) 2) 1) 2) 1.18 E+10 1.1 E+11 10.75 #3 #1 96 7.4 1) 1) 1) 2) 1) 2) 1) 2) 1.05 E+10 9.4 E+10 11.20 #3 #2 72 7.4 1) 1) 1) 2) 1) 2) 1) 2) 1.24 E+10 1.1 E+11 11.28 #3 #2 96 7.4 1) 1) 1) 2) 1) 2) 1) 2) 1.19 E+10 9.9 E+10 12.04 #3 #3 72 7.4 1) 2) 1) 2) 1) 2) 1) 2) 2.07 E+10 1.7 E+11 12.19 #3 #3 96 7.4 1) 2) 1) 2) 1) 2) 1) 2) 2.06 E+10 1.6 E+11 12.87 #3 #4 72 7.4 1) 2) 1) 2) 1) 2) 1) 2) 2.29 E+10 2.0 E+11 11.47 Petition 870250084687, dated 09 / 19 / 2025, pp. 129 / 143 60 / 66 transfection time execution set pH AAV Strain AaI Feed ratio lysis transfection Γ plasmid conc. .« / «,11 reagent / DN ratio Genomic titer [vg / ml] Capsid titer [vp / ml] Full / Empty [%] #3 #4 96 7.4 1) 2) 1) 2) 1) 2) 1) 2) 2.52 E+10 1.9 E+11 13.27 #3 #5 72 7.2 1) 1) 1) 2) 1) 2) 1) 2) 9.39 E+08 1.2 E+10 7.83 #3 #5 96 7.2 1) 1) 1) 2) 1) 2) 1) 2) 5.35 E+08 6.4 E+09 8.36 #3 #6 72 7.0 1) 1) 1) 1) 1) 2) 1) 2) 6.29 E+08 4.4 E+09 14.30 #3 #6 96 7.0 1) 1) 1) 1) 1) 2) 1) 2) 3.46 E+08 2.5 E+09 13.85 #3 #7 72 7.2 1) 2) 1) 2) 1) 2) 1) 2) 1.39 E+10 1.1 E+11 12.61 #3 #7 96 7.2 1) 2) 1) 2) 1) 2) 1) 2) 1.60 E+10 1.0 E+11 16.01 #3 #8 72 ​​7.0 1) 2) 1) 1) 1) 2) 1) 2) 1.94 E+10 1.0 E+11 19.43 #3 #8 96 7.0 1) 2) 1) 1) 1) 2) 1) 2) 2.01 E+10 9.0 E+10 22.30 #3 #9 72 7.4 1) 2) 2) 2) 1) 1) 2) 1) 5.66 E+09 6.6 E+10 8.57 #3 #9 72 7.4 1) 2) 2) 2) 2) 1) 2) 1) 1.43 E+10 9.7 E+10 14.76 #3 #10 72 7.0 2) 1) 1) 1) 1) 2) 1) 2) 1.88 E+09 1.6 E+10 11.73 #3 #10 96 7.0 2) 1) 1) 1) 1) 2) 1) 2) 1.67 E+09 1,7 E+10 9.82 #3 #11 72 7.4 2) 1) 1) 2) 1) 2) 1) 2) 6.81 E+09 4.7 E+10 14.48 #3 #11 96 7.4 2) 1) 1) 2) 1) 2) 1) 2) 4.43 E+09 6.7 E+10 6.61 #3 #12 72 7.4 2) 1) 2) 2) 1) 1) 2) 1) 2.29 E+09 3.3 E+10 6.95 #3 #12 72 7.4 2) 1) 2) 2) 2) 1) 2) 1) 8.27 E+10 5.9 E+11 14.01, Petition 870250084687, dated 09 / 19 / 2025, pages 130 / 143 61 / 66 Table 12 set of execution time post-transfection pH AAV Cell Line Feed ratio lysis transfection Conc. Plasmid [µg / ml] reagent / DNA ratio Genomic titer [vg / ml] Capsid titer [vp / ml] Full / Empty [%] #4 #1 72 7.4 1) 1) 1) 2) 1) 2) 1) 2) 1.11 E+10 1.1 E+11 10.13 #4 #1 96 7.4 1) 1) 1) 2) 1) 2) 1) 2) 1.01 E+10 9.3 E+10 10.82 #4 #2 72 7.4 1) 1) 1) 2) 1) 2) 1) 2) 1.34 E+10 1.3 E+11 10.32 #4 #2 96 7.4 1) 1) 1) 2) 1) 2) 1) 2) 1.13 E+10 1.1 E+11 10.26 #4 #3 72 7.4 1) 2) 1) 2) 1) 2) 1) 2) 2.26 E+10 2.2 E+11 10.28 #4 #3 96 7.4 1) 2) 1) 2) 1) 2) 1) 2) 2.45 E+10 2.1 E+11 11.65 #4 #4 72 7.4 1) 2) 1) 2) 1) 2) 1) 2) 2.20 E+10 2.0 E+11 10.98 #4 #4 96 7.4 1) 2) 1) 2) 1) 2) 1) 2) 2.27 E+10 2.4 E+11 9.44 #4 #5 72 7.4 1) 1) 2) 2) 1) 1) 2) 1) 2.52 E+09 6.2 E+10 4.07 #4 #5 72 7.4 1) 1) 2) 2) 2) 1) 2) 1) 4.61 E+09 7.9 E+10 5.84 #4 #6 72 7.0 2) 2) 2) 1) 1) 1) 2) 1) 1.14 E+08 6.2 E+08 18.45 #4 #6 72 7.0 2) 2) 2) 1) 2) 1) 2) 1) 1.52 E+09 1.9 E+10 7.98 #4 #7 72 7.0 2) 2) 1) 1) 1) 2) 1) 2) 1.25 E+09 1,2 E+10 10.45 #4 #7 96 7.0 2) 2) 1) 1) 1) 2) 1) 2) 5.48 E+08 7.6 E+09 7.21 #4 #8 72 ​​7.0 1) 1) 2) 1) 1) 1) 2) 1) 7.44 E+08 7.6 E+09 9.79 #4 #8 72 ​​7.0 1) 1) 2) 1) 2) 1) 2) 1) 2.84 E+09 5.0 E+10 5.68 #4 #9 72 7.4 2) 2) 1) 2) 1) 2) 1) 2) 4.35 E+09 5.0 E+10 8.70 #4 #9 96 7.4 2) 2) 1) 2) 1) 2) 1) 2) 6.60 E+09 7.1 E+10 9.30 #4 #10 72 7.0 2) 1) 2) 1) 1) 1) 2) 1) 1.10 E+09 1.4 E+10 7.86, Petition 870250084687, dated 09 / 19 / 2025, pages 131 / 143 62 / 66 set of execution time post-transfection pH AAV Cell Line Feed ratio lysis transfection Conc. Plasmid [µg / ml] reagent / DNA ratio Genomic titer [vg / ml] Capsid titer [vp / ml] Full / Empty [%] #4 #10 72 7.0 2) 1) 2) 1) 2) 1) 2) 1) 4.31 E+09 7.7 E+10 5.60 #4 #11 72 7.0 1) 2) 2) 1) 1) 1) 2) 1) not determined 4.3 E+09 not determined #4 #11 72 7.0 1) 2) 2) 1) 2) 1) 2) 1) not determined 4.6 E+10 not determined #4 #12 72 7.4 2) 2) 2) 2) 1) 1) 2) 1) 4.26 E+09 2.0 E+10 21.31 #4 #12 72 7.4 2) 2) 2) 2) 2) 1) 2) 1) 6.86 E+09 8.2 E+10 8.36 Example 2 Lise

[00165] If lysis was included in the process, it was performed as follows: To release AAV particles into the cell culture broth, 5% (v / v) lysis buffer (10% Triton CG 110, 40 mM MgCl2) was added to the culture broth. Additionally, 100 U / ml of Benzonase™ nuclease (Merck) was added. The cell culture broth was then incubated for approximately one hour at 37 °C with agitation, without aeration and pH control. After the respective incubation, 5 M NaCl solution was added and the lysate was sterile filtered. Example 3 AAV PARTICLE PURIFICATION

[00166] For the affinity chromatography step, a column comprising 10.5 ml of Thermo Fisher AAVX resin was used in an Akta Avant 25 chromatography system. The system was run at a flow rate of approximately 300 cm³ / h. After equilibration with buffer A (1x PBS, pH 7.4, 0.001% Pluronic F-68), 200 ml of lysed culture broth were applied to Petition 870250084687, dated 09 / 19 / 2025, pages 132 / 143 63 / 66 column, followed by 2 washing steps with equilibrium buffer and 0.5 M NaCl, pH 6.0, respectively. The AAV particles were eluted with 0.1 M sodium citrate solution, pH 2.4. The pH of the eluate was adjusted to pH 7.5 by adding 2 M Tris, pH 10. Table 13 Buffer stage column volumes [CV] equilibrium 1xPBS, pH 7.4 3 loading lysate wash I 1xPBS, pH 7.4 4 wash II 0.5 M sodium chloride, pH 6.0 4 wash III 1xPBS, pH 7.4 4 elution 0.1 M sodium citrate, pH 2.4 3 Example 4: Analytical Methods Enzyme-linked immunosorbent assay (ELISA) for total titer determination

[00167] For determination of AAV capsid titer, the PROGEN kit (Cat. No. PRAAV8) was used in accordance with the manufacturer's instructions.

[00168] In summary, this assay is a sandwich ELISA that uses a recombinant AAV capsid-specific antibody as the capture antibody and a biotin-labeled detection antibody.

[00169] The wells of the pre-coated multititer plate (MTP) were incubated overnight with 100 µl of standard, sample, or control, respectively, at 4 °C. The following day, the wells were washed three times with ASSB buffer (1x), as supplied in the kit. Then, 100 µl per well of a solution comprising the biotinylated detection antibody was added. Petition 870250084687, dated 09 / 19 / 2025, pages 133 / 143 64 / 66 (diluted according to the manufacturer's instructions) were added and incubated for two hours at room temperature with vibration. Subsequently, the wells were washed three times with ASSB buffer (1x) as supplied in the kit. In the next step, 100 μI of a solution comprising streptavidin-conjugated horseradish peroxidase were added to each well and incubated for 30 minutes at room temperature with vibration. Subsequently, the wells were washed three times with ASSB buffer (1x) as supplied in the kit. For the color reaction, 100 μI of a solution comprising ABTS prepared according to the manufacturer's instructions were added to each well and incubated with vibration. The color intensity was determined by triggering an MTP-ELISA Versa Max reader (Molecular Devices) at 405 nm with a reference wavelength of 490 nm until the difference in extinction between vacuum and the standard with the highest concentration reached approximately 1.5.

[00170] Each sample, standard and control were measured in duplicate.

[00171] The number of capsids (capsids / ml) was calculated based on a standard curve determined by a 4-parameter adjustment, for example, according to the Wiemer-Rodbard algorithm, using the average values ​​of the standards. Digital droplet polymerase chain reaction (ddPCR) for GENOMIC TITER DETERMINATION

[00172] Reagents for enzymatic treatment of samples: 1) DNase I Buffer (NEB): 100 mM Tris-HCl, pH 7.6, 25 mM MgSO4, 5 mM CaCl2; 2) DNase I (NEB): 0.2 U / μl; 3) Proteinase K (NEB; approx. 20 mg / ml = 800 U / ml): 16 U / ml; 4) Proteinase K buffer (BioRad): 400 mM Tris-HCl, 20 mM Petition 870250084687, dated 09 / 19 / 2025, pages 134 / 143 65 / 66 EDTA, 2000 mM NaCl, 1% SDS, pH 8; 5) Sodium dodecyl sulfate (SDS) solution: 10% (w / v).

[00173] Enzymatic treatment of samples: - Mix 30 μL of H2O, 5 μL of DNase I buffer, 5 μL of DNase I, and 10 μL of sample; - incubate at 37 °C for 30 min; - Heat to 75 °C for 15 min. to obtain an incubated DNase I mixture; - short cooling and centrifugation; - Mix 42 μL of H2O + 2 μL of proteinase K + 5 μL of proteinase K buffer + 1 μL of 10% SDS solution and add to the incubated DNase I mixture; - incubate for 60 min. at 50°C; - Heat to 95 °C for 15 minutes; - cooling to 4 °C. ddPCR:

[00174] For viral genome titration, a ddPCR duplexing assay was performed. The primer and probes were designed against the CMV promoter used and against the poIyA / 3'UTR sequence. The PCR mastermix was prepared according to Table 14 below (Bio-Rad digital drop PCR guide). Table 14 Components Volume per well [μL] Final concentration Supermix (2x) 11 1x 20 μM CMV primer dir. 0.99 900 nM 20 μM CMV primer rev 0.99 900 nM 20 μM CMV probe 0.275 250 nM model 5.5 - Petition 870250084687, dated 09 / 19 / 2025, pp. 135 / 143 66 / 66 Components: Volume per well [μI], Final water concentration 0.99 - Total 22

[00175] The prepared mastermix was pipetted into a 96-well plate with 16.5 μl per well. Then, dilution series of the pretreated samples were conducted: 10 μl of samples were transferred with Tips LoRetention was diluted to 90 μL of water in LoBind tubes and thoroughly mixed. Then, 5.5 μL of the samples were added to the mastermix solution in the 96-well plate in 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. Using an automatic dropper generator, which removes 20 μL of the PCR mixture from each well, up to 20,000 drops per well were produced and transferred to another 96-well plate. After sealing the dropper plate at 180 °C, a PCR run was performed. The respective conditions are shown in Table 15 below. Table 15 Number of cycles: denaturation, annealing, final elongation, end. 1 94 °C, 10 min. 39 94 °C, 30 sec. 58 °C, 1 min. 1 98 °C, 10 min. constant at 12 °C

[00176] In a dropper reader, the fluorescence signal was measured for each drop. QuantaSoft software processed the reader data and calculated the number of copies per 20 μl well for the target sequences. Initial sample titers can be determined with the following equation (1): Icó piasi number of copies I----—I ,, | copies |sa'daIpoço de 20 μΐjsI a Amostra de μΐ I [ poço de 20 μΐ ] • fator de diluição · 1000 |μj (1) Petition 870250084687, dated 09 / 19 / 2025, pages 136 / 143

Claims

1 / 3 Claims 1. METHOD FOR PRODUCING A RECOMBINANT ADENOASSOCIATED VIRAL PARTICLE PREPARATION (rAAVp), characterized by comprising the step of culturing a HEK293 cell comprising expression cassettes for a non-adenoassociated viral gene, which is intercalated between two terminal inverted repeats (ITRs) of AAV, an adenoassociated virus rep gene, an adenoassociated virus cap gene, an adenoassociated virus E1A gene, an adenoassociated virus E1B gene, an adenoassociated virus E2A gene, an adenoassociated virus E4orf6 gene and, optionally, an adenoassociated virus VA RNA gene, thereby producing the rAAVp, wherein the culture is at a pH value in the range of and including pH 7.4 to pH 7.

6.

2. METHOD, according to claim 1, characterized in that the yield of rAAVp produced by cultivation 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 cultivation at a pH value in the range of and including pH 7.0 to pH 7.

2.

3. METHOD, according to any one of claims 1 to 2, characterized in that the rAAVp produced by cultivation at a pH value in the range of and including pH 7.4 to pH 7.6 has a higher percentage of filled particles than an rAAVp produced by cultivation at a pH value in the range of and including pH 7.0 to pH 7.

2.

4. METHOD, according to any one of claims 1 to 3, characterized in that rAAVp is a therapeutic rAAVp.

5. METHOD, according to any one of claims 1 to 4, characterized by rAAVp comprising recombinant adeno-associated viral particles (rAAVs) comprising at least one coding nucleic acid sequence interspersed between two adeno-associated viral inverted terminal repeats.

6. METHOD, according to any one of claims 1 to 5, characterized in that rAAV is of serotype AAV2 or a variant thereof.

7. METHOD, according to any one of claims 1 to 6, characterized by cultivation encompassing bioreactor inoculation and harvesting of rAAVp.

8. METHOD, according to any one of claims 1 to 7, characterized by one or more or all of the expression cassettes for the non-adeno-associated viral gene, which is intercalated between two AAV ITRs, for the rep gene of the adeno-associated virus, for the cap gene of the adeno-associated virus, for the E2A gene of the adeno-associated virus, for the E4orf6 gene of the adeno-associated virus and, optionally, for the VA RNA gene of the adeno-associated virus being introduced into the mammalian cell after inoculation of the bioreactor.

9. METHOD, according to any one of claims 1 to 8, characterized in that the method further comprises, after the cultivation step, the step of isolating the rAAV from the cells and / or the culture medium and, optionally, purifying the rAAV.

10. METHOD, according to claim 9, characterized in that the purification is by a sequence of chromatographic steps, wherein the first is an affinity chromatography, followed by an anion exchange chromatography or a cation exchange chromatography and an optional size exclusion chromatography.

11. PHARMACEUTICAL COMPOSITION, characterized by comprising the rAAVp obtained by a method as defined in any one of claims 1 to 10.

12. PHARMACEUTICAL COMPOSITION, characterized by Petition 870250084687, dated 09 / 19 / 2025, page 138 / 143 3 / 3 comprising the rAAVp obtained by a method, as defined in any of claims 1 to 10, and a pharmaceutically acceptable excipient.

13. USE OF THE METHOD, as defined in any one of claims 1 to 10, characterized in that it is for increasing the yield of a recombinantly produced rAAVp.

14. USE OF THE METHOD, as defined in any one of claims 1 to 10, characterized by being for increasing the percentage of filled particles in a rAAVp. Petition 870250084687, dated 09 / 19 / 2025, pp. 139 / 143

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