Method for producing recombinant AAV particles

Abstract

A method for lysing mammalian cells producing recombinant AAV particles, comprising the step of bringing a mammalian cell culture broth into contact with an alkyl polyglucoside surfactant, preferably Triton CG 110, thereby lysing the mammalian cells producing recombinant AAV particles and releasing the produced recombinant AAV particles, wherein the mammalian cell culture broth comprises cultured mammalian cells producing recombinant AAV particles and a culture medium (used medium) used for culturing the mammalian cells producing recombinant AAV particles, has been reported.

Description

Technical Field

[0001] The present invention belongs to the field of gene therapy. More precisely, methods for releasing recombinant AAV particles from producer cells that lyse cells using Triton CG 110 are reported herein.

Background Art

[0002] Background Gene therapy, in a broad sense, refers to the therapeutic administration of genetic material to modify gene expression in living cells and thereby change their biological properties. After decades of research, gene therapy has advanced into the market and is expected to become increasingly important. Generally, gene therapy can be divided into either in vivo or ex vivo approaches.

[0003] Today, most in vivo therapies rely on DNA delivery by recombinant adeno-associated virus (rAAV) vectors. AAV is a small naturally occurring non-pathogenic parvovirus, which is composed of a non-enveloped icosahedral capsid. It contains a linear single-stranded DNA genome of approximately 4.7 kb. The genome of wild-type AAV vectors carries two genes, rep and cap, adjacent to the inverted terminal repeats (ITRs). The ITRs are cis-required for virus replication and packaging. The rep gene encodes four different proteins, and its expression is driven by two alternative promoters, P5 and P19. Furthermore, different forms are generated by alternative splicing. Rep proteins have multiple functions, such as DNA binding, endonuclease, and helicase activities. They play roles in gene regulation, site-specific integration, excision, replication, and packaging. The cap gene encodes three capsid proteins and one assembly activating protein. Differential expression of these proteins is achieved by alternative splicing and alternative start codon usage and is driven by a single promoter, P40, located in the coding region of the rep gene.

[0004] In the engineered therapeutic rAAV vector, the viral gene remains adjacent to the viral ITR but is replaced by a transgene expression cassette encoding the gene of interest under the control of a selected promoter. Unlike wild-type virus, the engineered rAAV vector does not undergo site-specific integration into the host genome and remains mainly episomal in the nucleus of transduced cells.

[0005] AAV is not itself replication-competent and requires the function of helper genes. These are naturally provided by co-infecting helper viruses such as, for example, adenovirus or herpes simplex virus. For example, five adenoviral genes, namely E1A, E1B, E2A, E4 and VA, are known to be essential for AAV replication. In contrast to other helper genes encoding proteins, VA is a small RNA gene.

[0006] For the production of rAAV vectors, DNA carrying the transgene adjacent to the ITR is introduced into a packaging host cell line that also contains the rep and cap genes, as well as the necessary helper genes. There are many ways to introduce these three groups of DNA elements into cells and ways to combine them on different DNA plasmids (e.g., Robert, M.A., et al. Biotechnol. J. 12 (2017) 1600193 (Non-Patent Document 1)).

[0007] Two common production methods are widely used. In the triple transfection method, an adenovirus helper plasmid carrying the necessary adenovirus helper genes is transiently co-transfected into a plasmid containing rep / cap and a plasmid containing the rAAV transgene. This process can be carried out using CHO or HEK cells. Alternatively, the rep / cap gene and the viral helper genes can be combined on one large plasmid (double transfection method). The second method involves the infection of insect cells (Sf9) with two baculoviruses, one carrying the rAAV genome and the other carrying rep and cap. In this system, the helper function is provided by the baculovirus plasmid itself. Similarly, herpes simplex virus is used in combination with HEK293 or BHK cells. More recently, Mietzsch et al. (Hum. Gene Ther. 25 (2014) 212 - 222 (Non-Patent Document 2); Hum. Gene Ther. Methods 28 (2017) 15 - 22 (Non-Patent Document 3)) engineered Sf9 cells in which rep and cap were stably integrated into the genome. In these cells, a single baculovirus with the rAAV transgene is sufficient to produce the rAAV vector. Clark et al. (Hum. Gene Ther. 6 (1995) 1329 - 1341 (Non-Patent Document 4)) generated a HeLa cell line in which the rep / cap gene and the rAAV transgene were integrated into its genome. By transfecting the cells with wild-type adenovirus, rAAV vector production is induced, and a mixed stock of rAAV vector and adenovirus is produced.

[0008] Arvind Srivastava et al. reported on the manufacturing challenges of AAV viral vectors and rational formulation development (J. Pharm. Sci. 110 (2021) 2609 - 2624 (Non-Patent Document 5)).

[0009] Mafalda Moleirinho et al. reported on the clinical-grade purification of oncolytic adenoviruses using polysorbate 20 as an alternative to cell lysis (Curr. Gene Ther. 18 (2018) 1-9 (Non-Patent Document 6)). WO 2022 / 003565 (Patent Document 1) reported on surfactants and methods for purifying biological therapeutics.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0012] The present invention is at least partially based on the finding that the recovery rate of recombinant AAV particles in AAV affinity chromatography, i.e., the yield (both capsid-based yield and genome-based yield), is affected by / dependent on the surfactant used to lyse the cells that produced the recombinant AAV particles prior to AAV affinity chromatography.

[0013] The present invention further relates to the finding that the ratio of full recombinant AAV particles to empty recombinant AAV particles obtained by AAV affinity chromatography is affected by / dependent on the surfactant used to lyse the cells that produced the recombinant AAV particles prior to AAV affinity chromatography, at least in part.

[0014] It has been found that by using an alkyl polyglucoside surfactant to lyse the cells that produced the recombinant AAV particles prior to the AAV affinity chromatography step, both the yield (capsid-based as well as genome-based) and the ratio of full recombinant AAV particles to empty recombinant AAV particles in AAV affinity chromatography can be increased.

[0015] The prior art does not suggest using alkyl polyglucoside (APG) for lysis of AAV-producing cells, let alone using it to solve the problem of providing a method for increasing the yield and the ratio of full recombinant AAV particles to empty recombinant AAV particles.

[0016] Accordingly, the present invention encompasses the following embodiments.

[0017] 1. A method for lysing mammalian cells producing recombinant AAV particles, comprising: - contacting a mammalian cell culture broth with an alkyl polyglucoside surfactant thereby lysing the mammalian cells producing recombinant AAV particles, A method in which a mammalian cell culture broth contains cultured mammalian cells producing recombinant AAV particles and a culture medium (used medium) used for culturing the mammalian cells producing recombinant AAV particles.

[0018] 2. A method for releasing recombinant AAV particles from mammalian cells producing recombinant AAV particles, comprising: - a step of bringing the mammalian cell culture broth into contact with an alkyl polyglucoside surfactant thereby lysing the mammalian cells producing recombinant AAV particles, and a mammalian cell culture broth contains cultured mammalian cells producing recombinant AAV particles and a culture medium (used medium) used for culturing the mammalian cells producing recombinant AAV particles. A method in which a mammalian cell culture broth contains cultured mammalian cells producing recombinant AAV particles and a culture medium (used medium) used for culturing the mammalian cells producing recombinant AAV particles.

[0019] 3. A method for purifying recombinant AAV particles, comprising: - a step of bringing the mammalian cell culture broth into contact with an alkyl polyglucoside surfactant; - a step of removing cell debris from the mixture; - a step of purifying the recombinant AAV particles by AAV affinity chromatography thereby purifying the recombinant AAV particles, and a mammalian cell culture broth contains cultured mammalian cells producing recombinant AAV particles and a culture medium (used medium) used for culturing the mammalian cells producing recombinant AAV particles. A method in which a mammalian cell culture broth contains cultured mammalian cells producing recombinant AAV particles and a culture medium (used medium) used for culturing the mammalian cells producing recombinant AAV particles.

[0020] 4. A method for purifying recombinant AAV particles, comprising: - a step of releasing recombinant AAV particles from the producing mammalian cells by contacting each mammalian cell culture broth with an alkyl polyglucoside surfactant; - a step of purifying the recombinant AAV particles by AAV affinity chromatography thereby purifying the recombinant AAV particles, and a mammalian cell culture broth contains cultured mammalian cells producing recombinant AAV particles and a culture medium (used medium) used for culturing the mammalian cells producing recombinant AAV particles. A method in which a mammalian cell culture broth comprises cultured recombinant AAV particle-producing mammalian cells and a culture medium (spent medium) used for culturing the recombinant AAV particle-producing mammalian cells.

[0021] 5. A method for producing recombinant AAV particles comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest, comprising: (i) providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) providing a plasmid comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest intervening between AAV ITRs; (iii) contacting one or more mammalian cells with the provided plasmid, further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture; or providing physical means such as an electric current to introduce the nucleic acid into the cells; (iv) culturing the transfected cells; (v) recovering the cultured cells and the culture medium to produce a mammalian cell culture broth; (vi) lysing the cells by contacting the mammalian cell culture broth with an alkyl polyglucoside surfactant to produce a mammalian cell culture broth lysate; (vii) optionally, isolating recombinant AAV particles from the culture broth lysate using AAV affinity chromatography, thereby producing recombinant AAV particles comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest.

[0022] 6. A method for producing recombinant AAV particles comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest, comprising: (i) Providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) Providing a plasmid comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcript of interest; (iii) (a) Generating stably transfected cells by contacting one or more mammalian cells with the plasmid provided in (i), further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture, or by providing physical means such as an electric current to introduce the nucleic acid into the cells; selecting a first stably transfected cell; contacting the selected first stably transfected cell with the plasmid provided in (ii), further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cells; or (b) Generating transiently transfected cells by contacting one or more mammalian cells with the plasmids provided in (i) and (ii), further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cells wherein in either of the above steps, transfected cells are thereby generated; (iv) Culturing the transfected cells of (iii); (v) Recovering the cultured cells and the culture medium to produce a mammalian cell culture broth; (vi) Lysing the cells by contacting the mammalian cell culture broth with an alkyl polyglucoside surfactant to produce a mammalian cell culture broth lysate; (vii) Optionally, isolating recombinant AAV particles from the mammalian cell culture broth lysate using AAV affinity chromatography. A method for producing recombinant AAV particles comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest.

[0023] 7. A method for purifying recombinant AAV particles, comprising: a) recovering a cell culture supernatant containing cultured recombinant AAV particle-producing mammalian cells and rAAV particles to produce a mammalian cell culture broth; b) optionally, concentrating the recovery product produced in step (a) to produce a concentrated mammalian cell culture broth; c) lysing the mammalian cells contained in the mammalian cell culture broth produced in step (a) or the concentrated mammalian cell culture broth produced in step (b) by bringing the broth into contact with an alkyl polyglucoside surfactant to produce a lysate of the mammalian cell culture broth; d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; e) optionally, filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting the clarified lysate to produce a diluted clarified lysate; f) subjecting the nucleic acid-reduced lysate obtained in step (d), or the clarified lysate or diluted clarified lysate produced in step (e), to AAV affinity chromatography to produce a column eluate containing recombinant AAV particles, thereby separating the rAAV particles from protein impurities or other production / process-related impurities, and optionally concentrating the column eluate to produce a concentrated column eluate. A method for purifying recombinant AAV particles thereby.

[0024] 8. The method according to embodiment 7, further comprising: g) To produce a second column eluate containing rAAV particles, subjecting the column eluate or concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC), thereby separating the rAAV particles from protein impurities or other production / process-related impurities, and optionally diluting the second column eluate to produce a diluted second column eluate; h) Optionally, to produce a third column eluate containing rAAV particles, subjecting the second column eluate or diluted second column eluate produced in step (g) to anion exchange chromatography, thereby separating the rAAV particles from protein impurities or other production / process-related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; i) filtering the second column eluate or diluted second column eluate produced in step (g), or filtering the third column eluate or concentrated third column eluate produced in step (h); thereby purifying recombinant AAV particles.

[0025] 9. The method according to embodiment 7, further comprising: g) To produce a second column eluate containing rAAV particles, subjecting the column eluate or diluted column eluate produced in step (f) to cation exchange column chromatography, thereby separating the rAAV particles from protein impurities or other production / process-related impurities, and optionally diluting the column eluate to produce a diluted second column eluate; h) To produce a third column eluate containing rAAV particles, subjecting the column eluate or diluted column eluate produced in step (g) to anion exchange chromatography, thereby separating the rAAV particles from protein impurities or production / process-related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate. A method for purifying recombinant AAV particles.

[0026] 10. The method according to embodiment 7, further comprising g) subjecting the column eluate or diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or production / process-related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; h) subjecting the column eluate or diluted column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process-related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate, A method for purifying recombinant AAV particles.

[0027] 11. Use of an alkyl polyglucoside surfactant for lysing recombinant AAV particle-producing mammalian cells to increase the yield of recombinant AAV particles obtained in subsequent AAV affinity chromatography.

[0028] 12. Use of an alkyl polyglucoside surfactant for lysing recombinant AAV particle-producing mammalian cells to increase the ratio of complete recombinant AAV particles (in the eluate fraction) obtained in subsequent AAV affinity chromatography to empty recombinant AAV particles.

[0029] 13. Use of an alkyl polyglucoside surfactant for increasing the yield of recombinant AAV particles, wherein the AAV particle-producing mammalian cells are lysed with the alkyl glucoside surfactant and the recombinant AAV particles are obtained in a subsequent AAV affinity chromatography step.

[0030] 14. Use of an alkyl polyglucoside surfactant for increasing the yield of recombinant AAV particles, wherein mammalian cells producing the recombinant AAV particles are lysed with the alkyl polyglucoside surfactant and the yield is determined after a subsequent AAV affinity chromatography step.

[0031] 15. Use of an alkyl polyglucoside surfactant for increasing the ratio of intact recombinant AAV particles (in the eluate fraction) obtained by subsequent AAV affinity chromatography to empty recombinant AAV particles, wherein mammalian cells producing the recombinant AAV particles are lysed with the alkyl polyglucoside surfactant prior to AAV affinity chromatography.

[0032] 16. The method and use according to any one of the preceding embodiments, wherein the alkyl polyglucoside surfactant is a mixture of 58.0 - 62.0 (w / v)% D-glucopyranose, oligomers, decyloctyl glucoside and 38.0 - 42.0 (w / v)% water.

[0033] 17. The method and use according to any one of the preceding embodiments, wherein the alkyl polyglucoside surfactant has the CAS number 68515-73-1.

[0034] 18. The method and use according to any one of the preceding embodiments, wherein the mammalian cell culture broth is a crude mammalian cell culture broth.

[0035] 19. The method and use according to any one of the preceding embodiments, wherein bringing into contact with, or contacting, the alkyl polyglucoside surfactant is incubating with the alkyl polyglucoside surfactant for a defined time at a defined concentration of the alkyl polyglucoside surfactant.

[0036] 20. The method and use according to any one of the preceding embodiments, wherein the alkyl polyglucoside surfactant is in solution.

[0037] 21. The method and use according to any one of the preceding embodiments, wherein the mammalian cell culture broth is combined with a solution containing an alkyl polyglucoside surfactant at 2.5% to 20% (2.5% (v / v) to 20% (v / v)) of its volume.

[0038] 22. The method and use according to any one of the preceding embodiments, wherein the mammalian cell culture broth is combined with a solution containing an alkyl polyglucoside surfactant at 5% to 10% (5% (v / v) to 10% (v / v)) of its volume.

[0039] 23. The method and use according to any one of embodiments 20 to 22, wherein the solution containing an alkyl polyglucoside surfactant contains the alkyl polyglucoside surfactant at a concentration of 5% to 20%.

[0040] 24. The method and use according to any one of embodiments 20 to 22, wherein the solution containing an alkyl polyglucoside surfactant contains the alkyl polyglucoside surfactant at a concentration of 7.5% to 15%.

[0041] 25. The method and use according to any one of embodiments 20 to 22, wherein the solution containing an alkyl polyglucoside surfactant preferably contains the alkyl polyglucoside surfactant at a concentration of about 10%.

[0042] 26. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting the alkyl polyglucoside surfactant is for 30 minutes to 90 minutes.

[0043] 27. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting the alkyl polyglucoside surfactant is for 45 minutes to 75 minutes.

[0044] 28. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting the alkyl polyglucoside surfactant is preferably for about 60 minutes.

[0045] 29. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting with an alkyl polyglucoside surfactant is carried out at a temperature of 25°C to 45°C.

[0046] 30. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting with an alkyl polyglucoside surfactant is carried out at a temperature of 28°C to 42°C.

[0047] 31. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting with an alkyl polyglucoside surfactant is carried out at a temperature of 32°C to 40°C.

[0048] 32. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting with an alkyl polyglucoside surfactant is preferably carried out at a temperature of about 37°C.

[0049] 33. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting with an alkyl polyglucoside surfactant is carried out while stirring or while shaking.

[0050] 34. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting with an alkyl polyglucoside surfactant is carried out without aeration.

[0051] 35. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting with an alkyl polyglucoside surfactant is carried out without pH adjustment during the contacting or bringing into contact.

[0052] 36. After bringing into contact with or contacting with an alkyl polyglucoside surfactant respectively, adding diatomaceous earth and incubating the mixture for 5 to 20 minutes, the method and use according to any one of the preceding embodiments.

[0053] 37. After bringing into contact with or contacting with an alkyl polyglucoside surfactant or diatomaceous earth respectively, centrifuging the mixture and / or passing it through a sterile filter, the method and use according to any one of the preceding embodiments.

[0054] 38. The method and use according to any one of embodiments 20 to 37, wherein the solution containing an alkyl polyglucoside surfactant contains a buffer salt.

[0055] 39. The method and use according to any one of embodiments 20 to 38, wherein the solution containing an alkyl polyglucoside surfactant contains TRIS.

[0056] 40. The method and use according to any one of embodiments 20 to 39, wherein the solution containing an alkyl polyglucoside surfactant contains a buffer salt having a concentration of 100 mM to 1000 mM.

[0057] 41. The method and use according to any one of embodiments 20 to 40, wherein the solution containing an alkyl polyglucoside surfactant contains a buffer salt having a concentration of 250 mM to 750 mM.

[0058] 42. The method and use according to any one of embodiments 20 to 41, wherein the solution containing an alkyl polyglucoside surfactant contains a buffer salt having a concentration of 400 mM to 600 mM.

[0059] 43. The method and use according to any one of embodiments 20 to 42, wherein the solution containing an alkyl polyglucoside surfactant contains a buffer salt having a concentration of about 500 mM.

[0060] 44. The method and use according to any one of embodiments 20 to 43, wherein the solution containing the alkyl polyglucoside surfactant contains an ionic regulator salt.

[0061] 45. The method and use according to any one of embodiments 20 to 44, wherein the solution containing the alkyl polyglucoside surfactant preferably contains magnesium (II) chloride as the ionic regulator salt.

[0062] 46. The method and use according to any one of embodiments 20 to 45, wherein the solution containing the alkyl polyglucoside surfactant contains an ionic regulator salt having a concentration of 5 mM to 200 mM.

[0063] 47. The method and use according to any one of embodiments 20 to 46, wherein the solution containing the alkyl polyglucoside surfactant contains an ionic regulator salt having a concentration of 7.5 mM to 150 mM.

[0064] 48. The method and use according to any one of embodiments 20 to 47, wherein the solution containing the alkyl polyglucoside surfactant contains an ionic regulator salt having a concentration of 10 mM to 50 mM.

[0065] 49. The method and use according to any one of embodiments 20 to 48, wherein the solution containing the alkyl polyglucoside surfactant preferably contains an ionic regulator salt having a concentration of 10 mM to 40 mM.

[0066] 50. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting the alkyl polyglucoside surfactant is at a pH value of 6.5 to 9.0.

[0067] 51. The method and use according to any one of the preceding embodiments, wherein bringing into contact with or contacting the alkyl polyglucoside surfactant is at a pH value of 6.5 to 8.0.

[0068] 52. The method and use according to any one of the preceding embodiments, wherein being in a contact state with or contacting an alkyl polyglucoside surfactant is at a pH value of 7.0 to 7.5.

[0069] 53. The method and use according to any one of the preceding embodiments, wherein being in a contact state with or contacting an alkyl polyglucoside surfactant is at a pH value of 6.5 to 9.0, and the pH value is adjusted to 7.0 to 7.5 after being in a contact state with or contacting respectively.

[0070] 54. The method and use according to any one of the preceding embodiments, wherein being in a contact state with or contacting an alkyl polyglucoside surfactant is at a pH value of 6.5 to 8.0, and the pH value is adjusted to 7.0 to 7.5 after being in a contact state with or contacting respectively.

[0071] 55. The method and use according to any one of the preceding embodiments, wherein being in a contact state with or contacting an alkyl polyglucoside surfactant is at a pH value of 7.0 to 7.5, and the pH value is adjusted to about pH 7.5 after being in a contact state with or contacting respectively.

[0072] 56. The method and use according to any one of embodiments 20 to 55, wherein a solution containing an alkyl polyglucoside surfactant has a pH value of 7.2 to 7.8.

[0073] 57. The method and use according to any one of embodiments 20 to 56, wherein a solution containing an alkyl polyglucoside surfactant has a pH value of 7.3 to 7.7.

[0074] 58. The method and use according to any one of embodiments 20 to 57, wherein a solution containing an alkyl polyglucoside surfactant has a pH value of 7.4 to 7.6.

[0075] 59. The method and use according to any one of embodiments 20 to 58, wherein the solution containing an alkyl polyglucoside surfactant preferably has a pH value of about 7.5.

[0076] 60. The method and use according to any one of the preceding embodiments, wherein a mammalian cell culture broth is brought into contact with an alkyl polyglucoside surfactant and a nuclease.

[0077] 61. The method and use according to any one of the preceding embodiments, wherein a mammalian cell culture broth is brought into contact with an alkyl polyglucoside surfactant and DNase I.

[0078] 62. The method and use according to any one of the preceding embodiments, wherein a mammalian cell culture broth is brought into contact with preferably an alkyl polyglucoside surfactant and benzonase.

[0079] 63. The method and use according to any one of embodiments 60 to 62, wherein a nuclease of 25 U / mL to 100 U / mL is added.

[0080] 64. The method and use according to any one of embodiments 60 to 63, wherein preferably 50 U / mL of each nuclease is added.

[0081] 65. The method and use according to any one of the preceding embodiments, wherein the recombinant AAV particle-producing cells are obtained by transfection with PEI.

[0082] 66. The method and use according to any one of the preceding embodiments, wherein the affinity chromatography is in a chromatographic material comprising a crosslinked poly(styrene-divinylbenzene) matrix to which an affinity ligand is covalently conjugated.

[0083] 67. The method and use according to any one of the preceding embodiments, wherein the affinity chromatography is in a chromatography material comprising a crosslinked poly(styrene-divinylbenzene) matrix to which an affinity ligand is covalently conjugated.

[0084] 68. The method and use according to embodiment 67, wherein the affinity ligand is a single domain antibody fragment (VHH).

[0085] 69. The method and use according to any one of embodiments 67 to 68, wherein the affinity ligand specifically binds to AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 and synthetic serotypes based thereon.

[0086] 70. The method and use according to any one of the preceding claims, wherein the affinity chromatography is in a chromatography material comprising a crosslinked poly(styrene-divinylbenzene) matrix to which a single domain antibody fragment (VHH) that specifically binds to AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 and synthetic serotypes based thereon is covalently conjugated.

[0087] In addition to the various embodiments described and claimed, the subject matter of the present disclosure also encompasses other embodiments having other combinations of the features disclosed and claimed herein. Accordingly, the specific features presented herein may be combined with each other in other ways within the scope of the subject matter of the present disclosure so that the subject matter of the present disclosure includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the subject matter of the present disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject matter of the present disclosure to the embodiments disclosed.

DETAILED DESCRIPTION OF THE INVENTION

[0088] Detailed Description of the Invention The present invention is at least partially based on the finding that the recovery rate of recombinant AAV particles in AAV affinity chromatography, i.e., the yield (both capsid-based yield and genome-based yield), is affected by / dependent on the surfactant used to lyse the cells that produced the recombinant AAV particles prior to AAV affinity chromatography.

[0089] The present invention is further at least partially based on the finding that the ratio of full recombinant AAV particles to empty recombinant AAV particles obtained by AAV affinity chromatography is affected by / dependent on the surfactant used to lyse the cells that produced the recombinant AAV particles prior to AAV affinity chromatography.

[0090] It has been found that by using an alkyl polyglucoside surfactant to lyse the cells that produced the recombinant AAV particles prior to the AAV affinity chromatography step, both the yield (based on capsid and genome) and the ratio of full recombinant AAV particles to empty recombinant AAV particles in AAV affinity chromatography can be increased.

[0091] Definitions Useful methods and techniques for carrying out the present invention are described, for example, in Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I - III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987).

[0092] By using recombinant DNA technology, it becomes possible to produce derivatives of nucleic acids. Such derivatives can be modified at individual or several nucleotide positions, for example, by substitution, alteration, exchange, deletion or insertion. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications can be easily carried out by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0093] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so on. Similarly, the terms "a" (or "an"), "one or more", and "at least one" may also be used synonymously herein. It should also be noted that the terms "comprising", "including", and "having" may be used synonymously.

[0094] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes plural such cells and their equivalents known to those skilled in the art, and so on. Similarly, the terms "a" (or "an"), "one or more", and "at least one" may also be used synonymously herein. It should also be noted that the terms "comprising", "including", and "having" may be used synonymously.

[0095] The term "AAV helper function" refers to AAV gene products that function in trans for the production of AAV replication and packaging and AAV-derived coding sequences (proteins) that can be expressed to provide AAV particles. Thus, the AAV helper function includes AAV open reading frames (ORFs) including rep and cap, as well as others such as AAP for specific AAV serotypes. The rep gene expression products have been shown to have many functions including, among other things, recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap gene expression products (capsids) supply the necessary packaging functions. The AAV helper function is used to complement the trans AAV functions missing from the AAV vector genome.

[0096] The term "about" means within ±20% of the numerical value that follows. In certain embodiments, the term "about" means within ±10% of the numerical value that follows. In certain embodiments, the term "about" means within ±5% of the numerical value that follows.

[0097] As used herein, the terms "comprise(s) / include(s)", "having / has", "capable of", "containing", and variations thereof 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" includes the term "consisting of". The present disclosure also contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether explicitly recited or not.

[0098] As used herein, the term "culturing" refers to the process of maintaining cells in a culture medium under conditions in which the cells are transfected and produce AAV particles.

[0099] As used herein, the term "culture broth" means the contents of the bioreactor at the end of the culture. The "culture broth" includes mammalian cells, dead and live, mammalian cell debris, culture medium, recombinant products produced by the mammalian cells, and other metabolites produced by the mammalian cells.

[0100] The terms "empty particle" and "empty recombinant AAV particle" can be used interchangeably and refer to an AAV particle that has an AAV protein shell but lacks a nucleic acid that encodes a protein or is transcribed into a transcript of interest adjacent to the AAV ITR, i.e., all or part of the vector. Thus, an empty particle does not function to transfer a nucleic acid that encodes a protein or is transcribed into a transcript of interest to a target cell.

[0101] The term "endogenous" refers to that which occurs naturally within a cell; that which is naturally produced by a cell; similarly, an endogenous locus / cell endogenous locus is a locus that occurs naturally within a cell.

[0102] As used herein, the term "exogenous" indicates that a nucleotide sequence is not derived from a particular cell and is introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation with a viral vector. Thus, an exogenous nucleotide sequence is an artificial sequence, and this artifact can arise, for example, from a combination of subsequences of different origins (e.g., a combination of a recombinase recognition sequence having an SV40 promoter and a coding sequence of a green fluorescent protein is an artificial nucleic acid), or from a partial deletion of a sequence (e.g., a sequence encoding only the extracellular domain of a membrane-bound receptor or cDNA) or a mutation of a nucleobase. The term "endogenous" means a nucleotide sequence derived from a cell. An "exogenous" nucleotide sequence can have an "endogenous" counterpart that has the same base composition but whose sequence is becoming an "exogenous" sequence, for example, by introduction into a cell via recombinant DNA technology.

[0103] As used herein, the term "fed-batch cell culture medium" means a culture medium in which cells and medium are initially supplied to a culture bioreactor, and additional culture nutrients are supplied continuously or individually incrementally to the culture medium during the culture process, and cells and / or products are periodically harvested, or not, before the end of the culture.

[0104] The terms "complete particle" and "complete recombinant AAV particle" can be used interchangeably and refer to an AAV particle having an AAV protein shell and encapsidating internally a nucleic acid that encodes a protein or is transcribed into a transcription product of interest adjacent to the AAV ITR, i.e., a vector. Thus, a complete particle can transfer an encapsidated nucleic acid that encodes a protein or is transcribed into a transcription product of interest to a target cell.

[0105] The terms "percentage of full to empty" and "ratio of full recombinant AAV particles to empty recombinant AAV particles" can be used interchangeably and represent the mathematical ratio of the number of full recombinant AAV particles to the total number of recombinant AAV particles (full and empty) in a recombinant AAV particle-containing sample or preparation of recombinant AAV particles. Since the number of full recombinant AAV particles can be at most the same as the total number of recombinant AAV particles, the ratio can be at most 1. Generally, the ratio is less than 1 and is expressed as a percentage. The number of full recombinant AAV particles is determined by determining the number of recombinant AAV particle-encapsidated nucleic acids in the sample or preparation. This can be done by PCR, particularly digital droplet PCR (ddPCR). The total number of recombinant AAV particles is determined by determining the number of capsid proteins in the sample or preparation. This can be done by ELISA, particularly a capsid protein-specific ELISA.

[0106] The term "nucleic acid encoding AAV packaging protein" generally refers to one or more nucleic acid molecules containing nucleotide sequences that provide AAV functions deleted from an AAV vector and are used to produce transduction-competent recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are commonly used to provide expression of the AAV rep and / or cap genes to complement the defective AAV functions required for AAV replication. However, the nucleic acid construct lacks AAV ITRs and cannot replicate or be packaged. Nucleic acids encoding AAV packaging proteins can be in the form of plasmids, phages, transposons, cosmids, viruses, or particles. Many nucleic acid constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM 29+45 that encode both the rep gene product and the cap gene product. See, for example, Samulski et al., J. Virol. 63 (1989) 3822-3828; and McCarty et al., J. Virol. 65 (1991) 2936-2945. Several plasmids encoding the rep and / or cap gene products have been described (e.g., U.S. Patent No. 5,139,941 and U.S. Patent No. 6,376,237). Any one of these nucleic acids encoding AAV packaging proteins can contain a DNA element or nucleic acid according to the present invention.

[0107] The term "nucleic acid encoding helper protein" generally refers to one or more nucleic acid molecules containing nucleotide sequences that encode proteins and / or RNA molecules that provide adenovirus helper function(s). A plasmid having a nucleic acid encoding a helper protein can be transfected into a suitable cell, and as a result, the plasmid can assist in the production of AAV particles in the cell. Any one of these nucleic acids encoding helper proteins can contain a DNA element or nucleic acid according to the present invention. Infectious virus particles existing in nature, such as adenovirus, herpesvirus, or vaccinia virus particles, are specifically excluded from this term.

[0108] As used herein, the term "operably linked" means the juxtaposition of two or more components in a relationship that enables them to function in the desired manner. For example, when a promoter and / or enhancer serves to regulate the transcription of a coding sequence / open reading frame / gene, the promoter and / or enhancer is operably linked to the coding sequence / open reading frame / gene. In certain embodiments, operably linked DNA sequences are contiguous. In certain embodiments, for example, when it is necessary to join the coding regions of two proteins such as a secretion leader and a polypeptide, these sequences are contiguous and are in the same reading frame. In certain embodiments, an operably linked promoter is located upstream of the coding sequence / open reading frame / gene and can be adjacent to the coding sequence. In certain embodiments, for example, with respect to an enhancer sequence that regulates the expression of a coding sequence / open reading frame / gene, the two components may not be adjacent but can be operably linked. When the enhancer increases the transcription of the coding sequence / open reading frame / gene, the enhancer is operably linked to the coding sequence / open reading frame / gene. An operably linked enhancer can be located upstream, within, or downstream of the coding sequence / open reading frame / gene, and can be located at a considerable distance from the promoter of the coding sequence / open reading frame / gene.

[0109] The term "packaging protein" refers to non-AAV-derived viral and / or cellular functions upon which AAV depends for its replication. Thus, this term encompasses the proteins and RNAs necessary for AAV replication, including those involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products, and activation of AAV capsid assembly. Viral-based accessory functions can be derived from any of a number of known helper viruses, such as adenovirus, herpesviruses (other than herpes simplex virus type I), and vaccinia virus.

[0110] As used herein, "AAV packaging protein" refers to AAV-derived sequences that function in trans to produce AAV replication. Thus, AAV packaging proteins are encoded by the major AAV open reading frames (ORFs), rep and cap. The rep proteins have been shown to have a number of functions, including, among others, recognition, binding, and nicking of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap (capsid) proteins provide the necessary packaging functions. AAV packaging proteins are used herein to complement the trans AAV functions missing from an AAV vector.

[0111] As used herein, the term "recombinant cell" means a cell after final genetic modification, e.g., a cell that expresses a polypeptide of interest or produces rAAV particles of interest and can be used for production of the polypeptide of interest or rAAV particles of interest at any scale. For example, a "mammalian cell comprising an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE) such that the coding sequence of a polypeptide of interest has been introduced into the genome of the host cell is a "recombinant cell." This cell can still perform an RMCE reaction, but doing so is not the purpose.

[0112] A "recombinant AAV vector" is obtained from the wild-type genome of a virus (e.g., AAV) by using molecular biological methods to remove the wild-type genome from the virus and replace it with a non-native nucleic acid, such as a nucleic acid transcribed into a transcript or a nucleic acid encoding a protein. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the recombinant AAV vector. A "recombinant" AAV vector is distinguished from the wild-type viral AAV genome because all or part of the viral genome has been replaced with a sequence that is non-native (i.e., heterologous) with respect to the viral genomic nucleic acid. Thus, the incorporation of the non-native sequence defines the viral vector (e.g., AAV) as a "recombinant" vector, which can be referred to as an "rAAV vector" in the case of AAV.

[0113] Recombinant vectors (e.g., AAV) are packaged for subsequent infection (transduction) of cells ex vivo, in vitro, or in vivo and can be referred to herein as "particles." When the recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle can also be referred to as an "rAAV." Such particles contain proteins that encapsulate or package the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, capsid proteins such as AAV VP1, VP2, and VP3.

[0114] As used herein, the term "serotype" is a distinction based on serologically distinct AAV capsids. Serological specificity is determined based on the lack of cross-reactivity between antibodies to one AAV compared to other AAVs. Such differences in cross-reactivity are usually due to differences in the capsid protein sequence / epitope (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). AAV variants, including capsid variants, may have at least one nucleotide or amino acid residue that differs compared to a reference AAV or other AAV serotype, even though they may not be serologically distinguishable from the reference AAV or other AAV serotypes.

[0115] Under the conventional definition, a serotype means that the virus of interest is tested against sera specific for all existing and characterized serotypes for neutralizing activity and no antibodies are found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid variants are generated, there may or may not be serological differences from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) has no serological differences, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not yet been performed on variant viruses with capsid sequence modifications to determine whether they are other serotypes according to the conventional definition of serotypes. Thus, for convenience and to avoid repetition, the term "serotype" is used herein in a broad sense to refer to both serologically distinct viruses (e.g., AAV) and serologically distinct viruses (e.g., AAV) that can be within a subgroup or variant of a given serotype.

[0116] The term "transgene" is used herein to conveniently refer to a nucleic acid that is intended or introduced into a cell or organism. A transgene includes any nucleic acid, e.g., a gene that is transcribed into a transcript or encodes a polypeptide or protein.

[0117] The term "Triton CG 110" refers to an alkyl polyglucoside surfactant. Triton CG 110 has the CAS number 68515-73-1. Triton CG 110 is a mixture of 58.0 - 62.0 (w / v)% D-glucopyranose, oligomeric, decyloctyl glucoside and 38.0 - 42.0 (w / v)% water.

[0118] "Vector" refers to the portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated, either directly or in single-stranded or RNA form, to form viral (e.g., AAV) particles. When using a recombinant plasmid to construct or produce recombinant viral particles, the viral particles do not contain the "plasmid" portion that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called the "plasmid backbone," which is important for plasmid cloning and amplification, processes necessary for growth and recombinant virus production, but is not itself packaged or encapsulated in viral (e.g., AAV) particles. Thus, "vector" refers to the nucleic acid packaged or encapsulated by viral particles (e.g., AAV).

[0119] Recombinant cell Generally, for the efficient and large-scale production of recombinant AAV particles (rAAV particles), cells that express and, if possible, secrete the rAAV particles. Such cells are referred to as "recombinant cells" or "recombinant production cells."

[0120] For the generation of "recombinant production cells," appropriate mammalian cells are transfected with the nucleic acid sequences necessary to produce the rAAV particles, including the necessary AAV helper functions.

[0121] Expression of a coding sequence, i.e., an open reading frame, requires additional regulatory elements such as a promoter and a polyadenylation signal (sequence). Thus, the open reading frame is operably linked to such additional regulatory elements for transcription. This can be achieved by incorporating it into a so-called expression cassette. The minimal control elements required for an expression cassette to function in mammalian cells are a promoter functional in the mammalian cells, located upstream, i.e., on the 5'-side, of the open reading frame, and a polyadenylation signal (sequence) functional in the mammalian cells, located downstream, i.e., on the 3'-side, of the open reading frame. Further, a terminator sequence may be present on the 3'-side of the polyadenylation signal (sequence). For expression, the promoter, open reading frame / coding region and polyadenylation signal sequence must be arranged in an operably linked form.

[0122] Similarly, a nucleic acid transcribed into a non-protein coding RNA is called an "RNA gene". For the expression of an RNA gene, additional regulatory elements such as a promoter and a transcription termination signal or a polyadenylation signal (sequence) are also required. The nature and localization of such elements depend on the RNA polymerase intended to drive the expression of the RNA gene. Thus, an RNA gene is also usually incorporated into an expression cassette.

[0123] For AAV particles composed of different (monomeric) capsid polypeptides and single-stranded DNA molecules and further requiring other adenovirus helper functions for production and encapsidation, a number of expression cassettes with different open reading frames / coding sequences contained therein are required. In this case, at least one expression cassette for each of the transgene, the different polypeptides forming the capsid of the AAV vector, and VA RNA is required for the necessary helper functions. Thus, individual expression cassettes for each of the helper functions E1A, E1B, E2A, E4orf6, VA RNA, rep, and cap genes are required. HEK293 cells constitutively express the E1A and E1B helper functions.

[0124] In certain embodiments of all aspects and embodiments of the present invention, each expression cassette comprises, in the 5' to 3' direction, a promoter, an open reading frame / coding sequence or RNA gene, and a polyadenylation signal sequence, and / or a terminator sequence. In certain embodiments, the open reading frame encodes a polypeptide and the expression cassette comprises a polyadenylation signal sequence with or without an additional terminator sequence. In certain embodiments, the expression cassette comprises an RNA gene, the promoter is a type 2 Pol III promoter, and a polyadenylation signal sequence or polyU terminator is present. See, for example, Song et al. Biochemical and Biophysical Research Communications 323 (2004) 573-578. In certain embodiments, the expression cassette comprises an RNA gene and the promoter is a type 2 Pol III promoter and a polyU terminator sequence.

[0125] In certain embodiments of all aspects and embodiments of the present invention, the open reading frame encodes a polypeptide, the promoter is a human CMV promoter with or without intron A, the polyadenylation signal sequence is the bGH (bovine growth hormone) poly A signal sequence, and the terminator is hGT (human gastrin terminator).

[0126] In certain embodiments of all aspects and embodiments of the present invention, the promoter is a human CMV promoter having intron A, the polyadenylation signal sequence is the bGH polyadenylation signal sequence, the terminator is hGT, and except for the expression cassette of the RNA gene and the expression cassette of the selection marker, for the selection marker, the promoter is the SV40 promoter, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence, the terminator is absent, for the RNA gene, the promoter is the wild-type type 2 polymerase III promoter, and the terminator is a polymerase II or III terminator.

[0127] Adeno-associated virus (AAV) For a general review of the helper functions of AAV and adenovirus or herpesvirus, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32(1987)243-306. The AAV genome is described in Srivastava et al., J. Virol., 45(1983)555-564. U.S. Patent No. 4,797,368 describes design considerations for constructing recombinant AAV vectors (see also International Publication No. 93 / 24641). Additional references describing AAV vectors are West et al., Virol. 160(1987)38-47; Kotin, Hum. Gene Ther. 5(1994)793-801; and Muzyczka J. Clin. Invest. 94(1994)1351. Construction of recombinant AAV vectors is described in U.S. Patent No. 5,173,414; Lebkowski et al., Mol. Cell. Biol. 8(1988)3988-3996; Tratschin et al., Mol. Cell. Biol. 5(1985)3251-3260; Tratschin et al., Mol. Cell. Biol., 4(1994)2072-2081; Hermonat and Muzyczka Proc. Natl. Acad. Sci. USA 81(1984)6466-6470; Samulski et al. J. Virol. 63(1989)3822-3828.

[0128] Adeno-associated virus (AAV) is a replication-defective parvovirus. It can replicate only in cells that are provided with specific viral functions by a co-infecting helper virus such as adenovirus, herpesvirus, and in some cases poxviruses such as vaccinia. Nevertheless, AAV can replicate in substantially any cell line of human, simian, or rodent origin if appropriate helper virus functions are present.

[0129] In the absence of helper virus genes, AAV establishes a latent period in its host cell. Its genome integrates into a specific site on chromosome 19 [(Chr)19(q13.4)], called the adeno-associated virus integration site 1 (AAVS1). For certain serotypes such as AAV-2, other integration sites have been found, such as on chromosome 5 [(Chr)5(p13.3)] called AAVS2 and on chromosome 3 [(Chr)3(p24.3)] called AAVS3.

[0130] AAV is classified into different serotypes. These are assigned based on parameters such as hemagglutination, tumorigenicity, and DNA sequence homology. To date, more than 10 different serotypes and more than 100 sequences corresponding to different clades of AAV have been identified.

[0131] The type and symmetry of the capsid protein determine the tissue tropism of each AAV. For example, AAV-2, AAV-4, and AAV-5 are specific to the retina, AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are specific to the brain, AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are specific to heart tissue, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are specific to the liver, and AAV-1, AAV-2, AAV-5, and AAV-9 are specific to the lung.

[0132] Pseudotyping refers to the process that involves cross-packaging of the AAV genome between various serotypes, i.e., the genome is packaged with capsid proteins of different origins.

[0133] The wild-type AAV genome has a size of about 4.7 kb. The AAV genome further contains two overlapping genes called rep and cap, which include multiple open reading frames (see, e.g., Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral. 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The Rep proteins encoded by the open reading frames provide four proteins of different sizes called Rep78, Rep68, Rep52, and Rep40. These are involved in the replication, rescue, and integration of AAV. The open reading frame encoding the Cap proteins provides four proteins called VP1, VP2, VP3, and AAP. VP1, VP2, and VP3 are part of the proteinaceous capsid of the AAV particle. The combined rep and cap open reading frames are flanked at their 5' and 3' ends by the so-called inverted terminal repeats (ITRs). For replication, AAV requires, in addition to the Rep and Cap proteins, the products of the adenoviral genes E1A, E1B, E4orf6, E2A, and VA or the corresponding factors of another helper virus.

[0134] For example, in the case of AAV of serotype 2 (AAV-2), the ITRs each have a length of 145 nucleotides and flank a coding sequence region of about 4470 nucleotides. Of the 145 nucleotides of the ITR, 125 nucleotides have a palindromic sequence and can form a T-shaped hairpin structure. This structure has the function of a primer during viral replication. The remaining 20 unpaired nucleotides are shown as the D sequence.

[0135] The AAV genome has three transcription promoters, P5, P19, and P40, for the expression of the rep and cap genes (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571).

[0136] The ITR array must be present cis to the coding region. The ITR provides a functional origin of replication (ori), signals necessary for integration into the genome of the target cell, and efficient excision and rescue from the host cell chromosome or recombinant plasmid. The ITR further contains origin - like elements such as the Rep protein binding site (RBS) and the terminal resolution site (TRS). It has been found that the ITR itself can have the function of a transcriptional promoter in an AAV vector (Flotte et al., J. Biol. Chem. 268 (1993) 3781 - 3790; Flotte et al., Proc. Natl. Acad. Sci. USA 93 (1993) 10163 - 10167).

[0137] Trans - complementation of the rep gene product and the cap gene product is required for replication and encapsidation of the viral single - stranded DNA genome, respectively.

[0138] The rep locus contains two internal promoters called P5 and P19. It contains open reading frames for four proteins. Promoter P5 is operably linked to a nucleic acid sequence that provides a non - splicing type 4.2 kb mRNA encoding the Rep protein Rep78 (a chromatin nickase for arresting the cell cycle) and a splicing type 3.9 kb mRNA encoding the Rep protein Rep68 (a site - specific endonuclease). Promoter P19 is operably linked to a nucleic acid sequence that provides a non - splicing type mRNA encoding the Rep protein Rep52 and a splicing type 3.3 kb mRNA encoding the Rep protein Rep40 (a DNA helicase for accumulation and packaging).

[0139] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV double-stranded DNA replication, while the two smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0140] The larger Rep proteins, Rep68 and Rep78, can specifically bind to the hairpin conformation of the AAV ITR. They exhibit certain enzymatic activities required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 may be sufficient for infectious particle formation (Holscher, C., et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0141] All Rep proteins, mainly Rep78 and Rep68, are thought to exhibit regulatory activities such as induction and repression of AAV genes and inhibitory effects on cell growth (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0142] Overexpression of recombinant Rep78 results in a phenotype associated with reduced cell growth due to induction of DNA damage. This causes the host cell to arrest in the S phase, thereby promoting latent infection by the virus (Berthet, C., et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0143] Tratschin et al. reported that the P5 promoter is negatively auto-regulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, very low expression has been reported for certain cell lines after stable integration of AAV (see, for example, Mendelson et al., Virol. 166 (1988) 154-165).

[0144] The cap locus contains one promoter called P40. Promoter P40 is operably linked to a nucleic acid sequence that provides a 2.6 kb mRNA encoding the Cap proteins VP1 (87 kDa, unspliced mRNA transcript), VP2 (72 kDa from the spliced mRNA transcript), and VP3 (61 kDa from the alternative start codon) by alternative splicing and alternative start codon usage. VP1-VP3 constitute the components of the viral capsid. The capsid has the function of binding to cell surface receptors and enabling intracellular transport of the virus. VP3 accounts for approximately 90% of the total viral particle protein. Nevertheless, all three proteins are essential for efficient capsid production.

[0145] Inactivation of all three capsid proteins VP1-VP3 has been reported to prevent the accumulation of single-stranded progeny AAV DNA. Mutations at the VP1 amino terminus ("lipid negative" or "Inf negative") still allow the assembly of single-stranded DNA into viral particles, thereby significantly reducing the infectivity titer.

[0146] The AAP open reading frame encodes the assembly activation protein (AAP). This has a size of approximately 22 kDa and transports the native VP proteins to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0147] Individual AAV particles contain only single-stranded DNA molecules. This can be either the "plus" or "minus" strand. AAV virus particles containing the DNA molecule are infectious. Inside the infected cell, the parental infecting single strand is converted to double-stranded and then amplified. Amplification results in a large pool of double-stranded DNA molecules from which single strands are displaced and packaged into the capsid.

[0148] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. Transgenes introduced into target cells using AAV vectors are thought to be expressed for long periods. One drawback of using AAV vectors is the limitation on the size of the transgene that can be introduced into cells.

[0149] Viral vectors such as parvovirus particles including AAV serotypes and their variants provide a means for delivery of nucleic acids to cells ex vivo, in vitro and in vivo that encode proteins such that the cells express the encoded proteins. AAV is a virus useful as a gene therapy vector because it can penetrate cells and introduce nucleic acid / genetic material so that the nucleic acid / genetic material can be stably maintained within the cells. Further, these viruses can introduce nucleic acid / genetic material into specific sites, for example. Since AAV is not associated with pathogenic diseases in humans, AAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV etiology or disease.

[0150] AAV particles used as vehicles for effective gene delivery have several desirable features for such applications, including tropism for both dividing and non-dividing cells. Even in the early clinical experience with these vectors, no persistent toxicity was shown and the immune response was minimal or undetectable. AAV is 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, airway, brain, joints, and hematopoietic stem cells.

[0151] Recombinant AAV particles typically do not contain viral genes associated with the etiology. Such vectors typically have one or more of the wild-type AAV genes that are wholly or partially deleted, such as, for example, the rep and / or cap genes, but retain at least one functional adjacent ITR sequence as required for rescue, replication, and packaging of the AAV particles of the recombinant vector. For example, only the essential parts of the vector, such as the ITR element and the LTR element respectively, are included. Thus, the AAV vector genome will contain sequences that are cis-required for replication and packaging (e.g., functional ITR sequences).

[0152] Recombinant AAV vectors, as well as methods and uses thereof, include any viral strain or serotype. By way of non-limiting example, recombinant AAV vectors can be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, 2i8, AAV rh74 or AAV 7m8. Such vectors can be based on the same strain or serotype (or subgroup or variant), or can be different from one another. By way of non-limiting example, a recombinant AAV vector based on one serotype genome can be identical to one or more of the capsid proteins that package the vector. Further, the recombinant AAV vector genome can be based on an AAV (e.g., AAV2) serotype genome that is different from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based on AAV2, but at least one of the three capsid proteins can be, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, AAV 7m8 or a variant thereof. AAV variants include variants and chimeras of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV 7m8 capsids.

[0153] In certain embodiments of all aspects and embodiments of the present invention, the rAAV particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74, and AAV 7m8, and variants thereof (e.g., capsid variants such as amino acid insertions, additions, substitutions and deletions), as described, for example, in International Publication No. WO 2013 / 158879, International Publication No. WO 2015 / 013313 and U.S. Patent Application Publication No. 2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0154] In certain embodiments of all aspects and embodiments of the present invention, the rAAV particles comprise a capsid sequence having at least 70% sequence identity with the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV Rh74, or AAV 7m8 capsid sequence.

[0155] In certain embodiments of all aspects and embodiments of the present invention, the rAAV particles comprise an ITR sequence having at least 70% sequence identity with the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequence.

[0156] Recombinant particles (e.g., rAAV particles) can be incorporated into a pharmaceutical composition. Such pharmaceutical compositions are useful, inter alia, for in vivo or ex vivo administration and delivery to a subject. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical that can be administered without inducing an immune response harmful to the individual receiving the composition itself and without undue toxicity.

[0157] Protocols for the production of adenoviral vectors are described in U.S. Patent No. 5,998,205; U.S. Patent No. 6,228,646; U.S. Patent No. 6,093,699; U.S. Patent No. 6,100,242; International Publication No. 94 / 17810 and International Publication No. 94 / 2374, which are hereby incorporated by reference in their entirety.

[0158] Recombinant AAV particles (rAAV particles) Various methods known in the art for generating rAAV particles. For example, transfection using an AAV plasmid and an AAV helper sequence in conjunction with co-infection with one AAV helper virus (e.g., adenovirus, herpes virus, or vaccinia virus), or transfection with a recombinant AAV plasmid, an AAV helper plasmid, and a helper function plasmid. Non-limiting methods for generating rAAV particles are described, for example, in U.S. Patent No. 6,001,650, U.S. Patent No. 6,004,797, International Publication No. 2017 / 096039, and International Publication No. 2018 / 226887. After recombinant rAAV particle production (i.e., particle production in a cell culture system), the rAAV particles can be obtained and purified from host cells and cell culture supernatants.

[0159] Production of recombinant AAV particles requires the expression of Rep protein and Cap protein, helper proteins E1A, E1B, E2A, and E4orf6, and adenovirus VA RNA in a single mammalian cell. The helper proteins E1A, E1B, E2A, and E4orf6 can be expressed using any promoter shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), particularly the CMV IE promoter. Thus, any promoter can be used.

[0160] Generally, to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of the plasmids contains a transgene flanked by two cis-acting AAV ITRs. The defective AAV elements required for replication of the progeny recombinant genome and subsequent packaging, namely the open reading frames of the Rep and Cap proteins, are contained in trans on a second plasmid. Overexpression of the Rep protein results in an inhibitory effect on cell growth (Li, J., et al., J. Virol. 71 (1997) 5236-5243). Furthermore, a third plasmid containing helper virus genes, namely E1, E4orf6, E2A, and VA from adenovirus, is required for AAV replication.

[0161] To reduce the number of plasmids required, the Rep, Cap, and adenovirus helper genes may be combined on a single plasmid.

[0162] Alternatively, the host cell may already stably express the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone designated 293 was generated in 1977 by integrating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, F. L., et al., J. Gen. Virol. 36 (1977) 59-74). The HEK293 cell line contains base pairs 1 to 4344 of the adenovirus serotype 5 genome. This includes the E1A and E1B genes as well as the adenovirus packaging signal (Louis, N., et al., Virology 233 (1997) 423-429).

[0163] When using HEK293 cells, the missing E2A, E4orf6, and VA genes can be introduced by co-infection with adenovirus or by co-transfection with E2A, E4orf6, and VA expression plasmids (see, for example, Samulski, R. J., et al., J. Virol. 63 (1989) 3822-3828; Allen, J. M., et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K., et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, T. R., et al., Gene Ther. 2 (1995) 29-37; Conway, J. E., et al., J. Virol. 71 (1997) 8780-8789; Chiorini, J. A., et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, F. K., et al., J. Virol. 70 (1996) 3227-3234; Salvetti, A., et al., Hum. Gene Ther. 9 (1998) 695-706; Xiao, X., et al., J. Virol. 72 (1998) 2224-2232; Grimm, D., et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X., et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors can be used (see, for example, Conway, J. E., et al., J. Virol. 71 (1997) 8780-8789; Johnston, K. M., et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, A. J., et al., Gene Ther. 2 (1995) 481-485; Fisher, J. K., et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, K. M., et al., Hum. Gene Ther. 8 (1997) 359-370).

[0164] To restrict transgene activity to specific tissues, i.e., to restrict the integration site, the transgene can be operably linked to an inducible promoter or a tissue-specific promoter (see, e.g., Yang, Y., et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0165] E1A, E1B, E2 and E4 The coding sequences of E1A and E1B (open reading frames) can be derived from human adenoviruses, such as, in particular, human adenovirus serotype 2 or serotype 5. Exemplary sequences of human Ad5 (adenovirus serotype 5) can be found in GenBank entry X02996, AC_000008, and exemplary sequences of human Ad2 are found in GenBank entry AC_000007. Nucleotides 505-3522 contain the nucleic acid sequences encoding E1A and E1B of human adenovirus serotype 5. The plasmid pSTK146 reported in European Patent No. 1230354, as well as the plasmids pGS119 and pGS122 reported in International Publication No. 2007 / 056994, can also be used as sources of the E1A and E1B open reading frames.

[0166] E1A is the first viral helper gene expressed after adenovirus DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins based on the same E1A mRNA by alternative splicing. The expression of 12S and 13S proteins leads to the activation of other viral functions E1B, E2, E3 and E4. Furthermore, the expression of 12S and 13S proteins pushes the cell into the S phase of the cell cycle. When only E1A-derived proteins are expressed, the cells die (apoptosis).

[0167] E1B is the second viral helper gene that is expressed. It is activated by E1A-derived proteins 12S and 13S. The mRNA derived from the E1B gene can be spliced in two different ways, resulting in a first 55 kDa transcript and a second 19 kDa transcript. The E1B 55 kDa protein is involved in the regulation of the cell cycle, the prevention of the transport of cellular mRNA in the late stages of infection, and the prevention of E1A-induced apoptosis. The E1B 19 kDa protein is involved in the prevention of E1A-induced apoptosis in cells.

[0168] The E2 gene encodes different proteins. The E2A transcript encodes a single-stranded binding protein (SSBP) that is essential for AAV replication.

[0169] Similarly, the E4 gene encodes several proteins. The 34 kDa protein (E4orf6) derived from the E4 gene, together with the E1B 55 kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

[0170] Adenovirus VA RNA gene Viral-associated RNA (VA RNA) is a non-coding RNA of adenovirus (Ad) that regulates translation. The adenovirus genome contains two independent copies: VAI (VA RNAI) and VAII (VA RNAII). Both are transcribed by RNA polymerase III from the type 2 polymerase III promoter (see, for example, Machitani, M., et al., J. Contr. Rel. 154 (2011) 285-289). For recombinant production, the adenovirus VA RNA gene can be driven by any promoter.

[0171] The structure, function, and evolution of adenovirus-related RNAs using a systems biology approach were investigated by Ma, Y. and Mathews, M. B. (J. Virol. 70 (1996) 5083-5099). They provided alignments and consensus VA RNA sequences based on 47 known human adenovirus serotypes. The disclosure is incorporated herein by reference in its entirety.

[0172] VA RNAs, VAI, and VAII consist of 157-160 nucleotides (nt).

[0173] Depending on the serotype, adenoviruses contain one or two VA RNA genes. VA RNAI is thought to play a dominant proviral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, V. K. and Conn, G. L., Virus Res. 212 (2016) 39-52).

[0174] VA RNA is not essential but plays an important role in efficient virus growth by overcoming the cellular antiviral machinery. That is, VA RNA is not essential for virus growth, but VA RNA-deleted adenoviruses may not be able to grow during the early stages of vector production where there are only a few copies of the viral genome per cell, perhaps because viral genes other than VA RNA that block the cellular antiviral machinery are not sufficiently expressed (see Maekawa, A., et al. Nature Sci. Rep. 3 (2013) 1136).

[0175] Maekawa, A., et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenovirus vectors lacking viral-related RNA genes that disrupt the cellular RNAi machinery, where HEK293 cells constitutively and highly expressing flippase recombinase were infected to obtain VA RNA-deleted adenoviruses by FLP recombinase-mediated excision of the VA RNA locus.

[0176] Human adenovirus 2 VA RNAI corresponds to nucleotides 10586 to 10810 of the GenBank entry AC_000007 sequence. Human adenovirus 5 VA RNAI corresponds to nucleotides 10579 to 10820 of the GenBank entry AC_000008 sequence.

[0177] Method for producing rAAV particles Carter et al. showed that the entire rep and cap open reading frames within the wild-type AAV genome can be deleted and replaced with a transgene (Carter, B. J., "Handbook of Parvoviruses", ed. by P. Tijssen, CRC Press, pp. 155-168 (1990)). Furthermore, it has been reported that the ITRs must be maintained to retain the functions of replication, rescue, packaging, and integration of the transgene into the genome of the target cell.

[0178] When cells containing each viral helper gene are transduced by an AAV vector, or vice versa, or when cells containing the integrated AAV provirus are transduced by an appropriate helper virus, the AAV provirus is activated and enters the lytic infection cycle again (Clark, K. R., et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, R. J., Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0179] Producer cells contain the rep and cap gene sequences, as well as a transgene cassette adjacent to the ITR sequences on one or more plasmids maintained via drug selection. Production of rAAV particles in these cell lines generally occurs after their infection by the necessary helper functions. Thus, the cells are infected with either a replication-competent AdV (usually wild-type Ad5) or a plasmid containing the respective helper genes to supply the helper virus proteins and initiate rAAV particle production. Packaging cell lines differ from producer cell lines in that they contain only the rep and cap genes.

[0180] The method according to the invention comprises the step of transducing mammalian cells with a nucleic acid (e.g., a plasmid) containing all the elements necessary for the production of recombinant AAV particles. Thus, since the plasmid encodes viral packaging proteins and / or helper proteins, the cells can produce recombinant virus particles containing a nucleic acid encoding the protein of interest or containing sequences transcribed into the transcript of interest.

[0181] The present invention provides a recombinant AAV virus particle production platform that includes features distinguishing it from current "industry standard" recombinant AAV particle production processes that include a lysis step according to the present invention.

[0182] More generally, cells that are transfected or transduced with DNA for the recombinant production of AAV particles can be referred to as "recombinant cells." Such cells are any mammalian cells that are used as recipients of nucleic acids (plasmids) encoding packaging proteins such as AAV packaging proteins, nucleic acids (plasmids) encoding helper proteins, and nucleic acids (plasmids) that encode a protein or are transcribed into a transcription product of interest, i.e., a transgene placed between two AAV ITRs. This term includes the progeny of the originally transfected or transduced cells. It is understood that the progeny of a single parent cell may not necessarily be identical in form or genomic or total nucleic acid complement to the original parent due to natural, accidental, or intentional mutations.

[0183] A number of suitable cell growth media are commercially available for maintaining cell viability or providing cell growth and / or proliferation. Examples of such media include serum-free eukaryotic growth media such as media for maintaining viability or providing growth of mammalian (e.g., human) cells. Non-limiting examples include Ham’s F12 or F_{12}K media (Sigma-Aldrich), FreeStyle (FS) F17 media (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. Such media can be supplemented with vitamins and / or trace minerals and / or salts and / or amino acids, such as essential amino acids for mammalian (e.g., human) cells.

[0184] Accordingly, provided herein is a method for producing recombinant AAV vectors or AAV particles comprising recombinant AAV vectors that contain nucleic acids encoding a protein or transcribed into a transcription product of interest using the lysis step according to the invention.

[0185] For this purpose, three plasmids are co-transfected into mammalian cells. The transgene plasmid encodes an expression cassette cloned between AAV ITRs, but the rep and cap genes are provided in trans by co-transfecting a second packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also called the helper plasmid, contains minimal helper virus factors, generally the adenovirus E2A, E4 and VA genes, but lacks AAV ITRs.

[0186] One aspect of the invention is a method for producing a recombinant AAV vector comprising a nucleic acid encoding a protein or transcribed into a transcript of interest, or an AAV particle comprising the recombinant AAV vector, the method comprising: (i) providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) providing a plasmid comprising a nucleic acid encoding a protein of interest or transcribed into a transcript of interest intervening between AAV ITRs; (iii) contacting one or more mammalian cells with the provided plasmids, further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture; or providing physical means such as an electric current to introduce the nucleic acid into the cells; (iv) culturing the transfected cells; (v) recovering the cultured cells and culture medium to produce a mammalian cell culture broth; (vi) lysing the cells by contacting the mammalian cell culture broth with an alkyl polyglucoside surfactant to produce a mammalian cell culture broth lysate; (vii) optionally isolating recombinant AAV particles from the culture broth lysate using AAV affinity chromatography. Thereby, recombinant AAV particles containing a nucleic acid encoding a protein of interest or transcribed into a transcript of interest are produced.

[0187] One aspect of the present invention is a method for producing a recombinant AAV vector containing a nucleic acid encoding a protein of interest or transcribed into a transcript of interest or an AAV particle containing the recombinant AAV vector, the method comprising: (i) providing one or more plasmids containing a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (ii) providing a plasmid containing a nucleic acid encoding a protein of interest or transcribed into a transcript of interest; (iii) (a) contacting one or more mammalian cells with the plasmid provided in (i), further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture, or providing physical means such as an electric current to introduce the nucleic acid into the cells to generate stably transfected cells; selecting the first stably transfected cells; contacting the selected first stably transfected cells with the plasmid provided in (ii), further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cells; or (b) contacting one or more mammalian cells with the plasmids provided in (i) and (ii), further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture or providing physical means such as an electric current to introduce the nucleic acid into the cells to generate transiently transfected cells wherein either step results in the generation of transfected cells; (iv) culturing the transfected cells of (iii); (v) A step of recovering cultured cells and a culture medium to produce a mammalian cell culture broth; (vi) A step of lysing cells by bringing a mammalian cell culture broth into contact with an alkyl polyglucoside surfactant to produce a mammalian cell culture broth lysate; (vii) Optionally, a step of isolating recombinant AAV particles from a mammalian cell culture broth lysate using AAV affinity chromatography, and thereby producing recombinant AAV particles containing a nucleic acid encoding a protein of interest or transcribed into a transcript of interest.

[0188] The introduction of nucleic acid (plasmid) into cells can be performed by a plurality of methods.

[0189] A variety of methods for DNA transfer into mammalian cells have been reported in the art. All of these are useful in the method according to the present invention. In certain embodiments of all aspects and embodiments, electroporation, nucleofection or microinjection for nucleic acid transfer / transfection is used. In certain embodiments of all aspects and embodiments, inorganic substances (such as calcium phosphate / DNA coprecipitation, etc.), cationic polymers (such as polyethyleneimine, DEAE-dextran, etc.) or cationic lipids (lipofection) are used for nucleic acid transfer / transfection. Calcium phosphate and polyethyleneimine are the most commonly used reagents for transfection for nucleic acid transfer on a larger scale (see, for example, Baldi et al., Biotechnol. Lett. 29 (2007) 677-684), and polyethyleneimine is preferred.

[0190] Improvement in the efficiency and reproducibility of transfection conditions using PEI as a growth and transfection reagent in serum-free suspension culture enables easy scale-up of AAV production using shake flasks, waves or stirred tank bioreactors.

[0191] In certain embodiments of all aspects and embodiments, the nucleic acid (plasmid) is provided as a composition in combination with polyethyleneimine (PEI), optionally in combination with cells. In certain embodiments, the composition comprises a plasmid / PEI mixture having the following plurality of components: (a) one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; (b) a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcript of interest; (c) a polyethyleneimine (PEI) solution. In certain embodiments, the plasmids are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0.01, and the mixture of components (a), (b) and (c) is optionally incubated for a period of about 10 seconds to about 4 hours.

[0192] In certain embodiments of all aspects and embodiments, the composition further comprises cells. In certain embodiments, the cells are in contact with the plasmid / PEI mixture of components (a), (b) and / or (c).

[0193] In certain embodiments of all aspects and embodiments, the composition further comprises free PEI, optionally in combination with cells. In certain embodiments, the cells are in contact with free PEI.

[0194] In certain embodiments of all aspects and embodiments, the cells are in contact with the mixture of components (a), (b) and / or (c) for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours. In a preferred embodiment, the cells are in contact with the mixture of components (a), (b) and / or (c), and optionally free PEI, for at least about 4 hours.

[0195] The composition may further comprise additional plasmid(s) and / or cells. Such plasmids and cells may be in contact with free PEI. In certain embodiments, the plasmid(s) and / or cells are in contact with free PEI for at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours.

[0196] The method according to the invention also comprises the step of transfecting cells. Thus, the method comprises providing one or more plasmids, preparing a solution comprising polyethyleneimine (PEI), and mixing the plasmid(s) with the PEI solution to produce a plasmid / PEI mixture. In certain embodiments, such a mixture is incubated for a period ranging from about 10 seconds to about 4 hours. In such a method, the cells are then contacted with the plasmid / PEI mixture to produce a plasmid / PEI cell culture, and then free PEI is added to the produced plasmid / PEI cell culture to produce a free PEI / plasmid / PEI cell culture, and then the produced free PEI / plasmid / PEI cell culture is incubated for at least about 4 hours, thereby producing transfected cells. In certain embodiments, the plasmid comprises one or more or all of the rep open reading frame, the cap open reading frame, the E1A, E1B, E2 and E4orf6 open reading frames, and a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0197] Furthermore, the method according to the present invention includes a step for producing transfected cells that produce recombinant AAV vectors or AAV particles, the method comprising providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; providing a plasmid comprising a nucleic acid encoding a protein or transcribed into a transcription product of interest; providing a solution comprising polyethyleneimine (PEI); mixing the aforementioned plasmid with the PEI solution to produce a plasmid / PEI mixture, wherein the plasmid is in a molar ratio range of about 1:0.01 to about 1:100 or in a molar ratio range of about 100:1 to about 1:0.01 (and optionally incubating the plasmid / PEI mixture for a period in the range of about 10 seconds to about 4 hours); contacting mammalian cells with the plasmid / PEI mixture to produce a plasmid / PEI cell culture; adding free PEI to the produced plasmid / PEI cell culture to produce a free PEI / plasmid / PEI cell culture; and incubating the free PEI / plasmid / PEI cell culture for at least about 4 hours, thereby producing transfected cells that produce recombinant AAV vectors or particles comprising a nucleic acid encoding a protein or transcribed into a transcription product of interest, whereby the mammalian cells are obtained by the method according to the present invention.

[0198] Furthermore, a method for producing a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcription product of interest, the method comprising: providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding a helper protein; providing a plasmid comprising a nucleic acid encoding a protein of interest or a nucleic acid transcribed into a transcription product of interest; providing a solution comprising polyethyleneimine (PEI); mixing the aforementioned plasmids with the PEI solution to produce a plasmid / PEI mixture, wherein the plasmid is in a molar ratio range of about 1:0.01 to about 1:100 or in a molar ratio range of about 100:1 to about 1:0.01 (and optionally incubating the plasmid / PEI mixture for a period ranging from about 10 seconds to about 4 hours); contacting mammalian cells with the plasmid / PEI mixture produced as described to produce a plasmid / PEI cell culture; adding free PEI to the plasmid / PEI cell culture produced as described to produce a free PEI / plasmid / PEI cell culture; incubating the produced plasmid / PEI cell culture or free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells; recovering the transfected cells and / or the culture medium produced from the produced transfected cells to produce a culture broth; lysing the cells by the method according to the invention and optionally isolating the recombinant AAV vector or particle from the culture broth lysate using an AAV affinity chromatography step; and thereby producing a recombinant AAV vector or particle comprising a nucleic acid encoding a protein or a nucleic acid transcribed into a transcription product of interest is provided.

[0199] In certain embodiments of all aspects and embodiments, PEI is added to the plasmid and / or cells at various times. In certain embodiments, free PEI is added to the cells before, simultaneously with, or after contacting the plasmid / PEI mixture with the cells.

[0200] In certain embodiments of all aspects and embodiments, the cells have a specific density and / or cell growth phase and / or viability when contacted with the plasmid / PEI mixture and / or free PEI. In a preferred embodiment, the cells have a density in the range of about 1×10^5 cells / mL to about 1×10^8 cells / mL when contacted with the plasmid / PEI mixture and / or free PEI. In certain embodiments, the viability of the cells when contacted with the plasmid / PEI mixture or free PEI is about 60% or more than 60%, or the cells are in the logarithmic growth phase when contacted with the plasmid / PEI mixture, or the viability of the cells when contacted with the plasmid / PEI mixture or free PEI is about 90% or more than 90%, or the cells are in the logarithmic growth phase when contacted with the plasmid / PEI mixture or free PEI.

[0201] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA is a branched short-chain fatty acid that inhibits histone deacetylase activity. Therefore, it is commonly added to mammalian cell cultures as an enhancer of recombinant protein production.

[0202] In certain embodiments of all aspects and embodiments, the encoded AAV packaging proteins include AAV rep and / or AAV cap. In certain embodiments of all aspects and embodiments, such AAV packaging proteins include AAV rep and / or AAV cap proteins of any AAV serotype.

[0203] In certain embodiments of all aspects and embodiments, the encoded helper proteins include adenovirus E1A and E1B, adenovirus E2 and / or E4, VA RNA, and / or non-AAV helper proteins.

[0204] In certain embodiments of all aspects and embodiments, the nucleic acid (plasmid) is used in a specific amount or ratio. In certain embodiments, the total amount of a plasmid containing a nucleic acid that encodes a protein or is transcribed into a transcription product of interest, and one or more plasmids containing a nucleic acid that encodes an AAV packaging protein and / or a nucleic acid that encodes a helper protein, ranges from about 0.1 μg to about 15 μg per mL of cells. In certain embodiments, the molar ratio of a plasmid containing a nucleic acid that encodes a protein or is transcribed into a transcription product of interest, and one or more plasmids containing a nucleic acid that encodes an AAV packaging protein and / or a nucleic acid that encodes a helper protein, is within the range of about 1:5 to about 1:1, or within the range of about 1:1 to about 5:1.

[0205] In certain embodiments of all aspects and embodiments, the first plasmid contains a nucleic acid that encodes an AAV packaging protein, and the second plasmid contains a nucleic acid that encodes a helper protein.

[0206] In certain embodiments of all aspects and embodiments, the molar ratio of a plasmid containing a nucleic acid that encodes a protein or is transcribed into a transcription product of interest, a first plasmid containing a nucleic acid that encodes an AAV packaging protein, and a second plasmid containing a nucleic acid that encodes a helper protein, is in the range of about 1 - 5:1:1, or 1:1 - 5:1, or 1:1:1 - 5 in co - transfection.

[0207] In certain embodiments of all aspects and embodiments, the cells are mammalian cells. In one preferred embodiment, the cells are HEK293 cells or CHO cells.

[0208] Cultivation can be carried out using conditions of about 37 °C, 95% humidity and 8% by volume CO2, which are commonly used for culturing eukaryotic cells. Cultivation can be performed in a serum-containing medium or a serum-free medium, and in adherent culture or suspension culture. Suspension culture can be carried out in any fermentation vessel such as, for example, a stirred tank reactor, a wave reactor, a rocking bioreactor, a shaker vessel or a spinner vessel, or a so-called roller bottle. Transfection can be carried out in high-throughput format and screening, respectively, for example in 96 or 384 well format.

[0209] The method according to the present invention can comprise AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles comprise any of AAV serotypes 1-12, AAV VP1, VP2 and / or VP3 capsid proteins, or modified or variant AAV VP1, VP2 and / or VP3 capsid proteins, or wild-type AAV VP1, VP2 and / or VP3 capsid proteins. In certain embodiments of all aspects and embodiments, the AAV particles comprise an AAV serotype or an AAV pseudotype, and the AAV pseudotype comprises an AAV capsid serotype different from the ITR serotype.

[0210] The methods according to the invention that provide or comprise AAV vectors or particles can also comprise other elements. Examples of such elements include, but are not limited to, introns, expression control elements, one or more adeno-associated virus (AAV) inverted terminal repeat sequences (ITRs) and / or filler / stuffer polynucleotide sequences. Such elements can be present within or adjacent to a nucleic acid encoding a protein or transcribed into a transcript of interest, or an expression control element can be operably linked to a nucleic acid encoding a protein or transcribed into a transcript of interest, or an AAV ITR can be adjacent to the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a transcript of interest, or a filler polynucleotide sequence can be adjacent to the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a transcript of interest.

[0211] Expression control elements include constitutive or regulatable control elements such as tissue-specific expression control elements or promoters.

[0212] The ITR can be of any of the AAV2, AAV6, AAV8, or AAV9 serotypes, or a combination thereof. The AAV particles can comprise any VP1, VP2, and / or VP3 capsid protein having at least 75% sequence identity to any of the VP1, VP2, and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74, or AAV 7m8, or can comprise a modified or variant VP1, VP2, and / or VP3 capsid protein selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74, and AAV 7m8 AAV serotypes.

[0213] After the production of the recombinant virus (e.g., AAV) particles described in this specification, if desired, the virus (e.g., rAAV) particles can be purified and / or isolated from host cells using various conventional methods. Such methods include column chromatography, CsCl gradient, iodixanol gradient, and the like.

[0214] For example, multiple column purification steps such as purification by anion exchange columns, affinity columns and / or cation exchange columns can be used. (See, for example, WO 02 / 12455 and US 2003 / 0207439 A1). Alternatively or additionally, iodixanol or CsCl gradient steps can be used (see, for example, US 2012 / 0135515 A1 and US 2013 / 0072548 A1). Further, when using infectious virus to express packaging and / or helper proteins, various methods can be used to inactivate residual virus. For example, adenovirus can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or more. Since AAV is heat stable while helper adenovirus is heat labile, this treatment effectively inactivates the helper virus.

[0215] The goal of the rAAV vector production and purification system is to implement strategies to minimize / control the generation of production-related impurities such as wild-type / pseudo-wild-type AAV species (wtAAV) and protein, nucleic acid, and vector-related impurities including AAV encapsidated residual DNA impurities.

[0216] Considering that rAAV particles represent only a very small fraction of the biomass, rAAV particles need to be purified to a level of purity that can be used as a clinical human gene therapy product (see, for example, reports from Smith P.H., et al., Mo. Therapy 7(2003)8348; Chadeuf G., et al, Mo. Therapy 12(2005)744; CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0217] As a first step, typically, the cultured cells that produce rAAV particles are recovered, optionally in combination with the harvested cell culture supernatant (medium) in which the cells (suspension or adherent) that produce rAAV particles were cultured. The recovered cells and optionally the cell culture supernatant can be used as is, lysed, or concentrated as needed. Further, if infection is used to express helper functions, residual helper virus can be inactivated. For example, adenovirus can be inactivated by heating to a temperature of about 60 °C for, for example, 20 minutes or more, which inactivates only the helper virus since AAV is heat stable while the helper adenovirus is heat labile.

[0218] The cells in the harvested culture broth are lysed by the method according to the invention to release rAAV particles. During or after cell lysis, a nuclease, such as benzonase, is added to degrade contaminating DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtration or centrifugation, giving a clarified cell lysate. In certain examples, the lysate is filtered through a filter with a pore diameter in microns (for example, a filter with a pore diameter of 0.1 - 10.0 μm, for example, a filter with a pore diameter of 0.45 μm and / or 0.2 μm) to produce a clarified lysate.

[0219] The lysate (optionally clarified) contains AAV particles (including rAAV vectors and empty capsids) as well as soluble cell components from the host cells that may include production / process-related impurities, such as inter alia cellular proteins, lipids and / or nucleic acids, as well as cell culture medium components. The optionally clarified lysate is then subjected to a purification step to purify the AAV particles (including rAAV vectors) from the impurities using chromatography. The clarified lysate can be diluted or concentrated with a suitable buffer prior to the first chromatography step.

[0220] After cell lysis, any clarification, and any dilution or concentration, the rAAV particles can be purified using a plurality of subsequent consecutive chromatography steps.

[0221] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0222] If the first chromatography step is affinity chromatography, the second chromatography step can be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, the rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography and / or cation exchange chromatography and / or size exclusion chromatography, in any order or sequence or combination.

[0223] For example, the removal of empty capsids from full capsids during downstream processing is based on their different isoelectric points (pI) in anion exchange chromatography. The average pI calculated across all serotypes is 5.9 for full capsids and 6.3 for empty capsids (Venkatakrishnan, B., et al., J. Virol. 87 (2013) 4974 - 4984).

[0224] In certain embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by anion exchange chromatography, followed by purification by size exclusion chromatography (SEC).

[0225] In certain embodiments of all aspects and embodiments, rAAV particle purification is by affinity chromatography, followed by purification by size exclusion chromatography (SEC), followed by purification by anion exchange chromatography.

[0226] Cation exchange chromatography functions to separate AAV particles from cell components and other components present in the clarified lysate and / or column eluate from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins that can bind to rAAV particles over a wide pH range include, but are not limited to, any sulfonic acid-based resin indicated by the presence of a 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). Further examples include Capto S, Capto S ImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and the commercially available DOWEX®, AMBERLITE®, and AMBERLYST® resin families available from Aldrich Chemical Company (Milliwaukee, WI, USA). Examples of weak cation exchange resins include, but are not limited to, any carboxylic acid-based resin. Exemplary cation exchange resins include carboxymethyl (CM), phospho (based on phosphate functional groups), sulfomethyl (S) and sulfopropyl (SP) resins.

[0227] Anion exchange chromatography functions to separate AAV particles from proteins, cellular components, and other components present in the clarified lysate and / or column eluate from affinity chromatography, cation exchange chromatography, or size exclusion chromatography. The amount of empty capsids in the eluate can also be reduced and thereby controlled using anion exchange chromatography. For example, an anion exchange column bound with rAAV particles can be washed with a solution containing a moderate concentration (e.g., about 100 - 125 mM, e.g., 110 - 115 mM) of NaCl, and a portion of the empty capsids can be eluted in the flow-through without substantially eluting the rAAV particles. Subsequently, the rAAV particles bound to the anion exchange column can be eluted using a solution containing a higher concentration (e.g., about 130 - 300 mM NaCl) of NaCl to produce a column eluate having a reduced or depleted amount of empty capsids and a proportionally increased amount of rAAV particles containing the rAAV vector.

[0228] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins. Examples of strong anion exchange resins include, but are not limited to, those generally based on quaternized nitrogen atoms, including quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (a strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (a strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (a weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (powerful anion exchangers available from GE healthcare, Marlborough, MA, USA); DEAE Sepharose (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q Sepharose (a powerful anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Further exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE) and quaternary aminoethyl (QAE).

[0229] The manufacturing process for purifying recombinant AAV particles intended as a product for treating human diseases should achieve the following objectives: 1) consistent particle purity, potency, and safety; 2) scalability of the manufacturing process; 3) acceptable manufacturing costs.

[0230] An exemplary process for purifying recombinant AAV particles is reported in International Publication No. WO 2019 / 006390.

[0231] The methods for purifying and producing recombinant adeno-associated virus particles (rAAV particles) outlined below are scalable to large scales. For example, up to suspension cultures with volumes of 5, 10, 10-20, 20-50, 50-100, 100-200, 200-500 liters or more. The methods for purifying and producing recombinant adeno-associated virus particles are applicable to a wide variety of AAV serotypes / capsid variants.

[0232] In certain embodiments of all aspects and embodiments, the purification of rAAV particles a) recovering a cell culture supernatant containing cultured recombinant AAV particle-producing mammalian cells and rAAV particles to produce a mammalian cell culture broth; b) optionally, concentrating the recovery produced in step (a) to produce a concentrated mammalian cell culture broth; [[ID=!7]] c) lysing the mammalian cells contained in the mammalian cell culture broth produced in step (a) or the concentrated mammalian cell culture broth produced in step (b) by bringing the broth into contact with an alkyl polyglucoside surfactant to produce a lysate of the mammalian cell culture broth; d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate; e) optionally, filtering the nucleic acid-reduced lysate produced in step (d) to produce a clarified lysate, and optionally, diluting the clarified lysate to produce a diluted clarified lysate. f) To produce a column eluate containing recombinant AAV particles, subjecting the nucleic acid-reduced lysate obtained in step (d), or the clarified lysate or diluted clarified lysate produced in step (e) to AAV affinity chromatography, thereby separating rAAV particles from protein impurities or other production / process-related impurities, and optionally concentrating the column eluate to produce a concentrated column eluate, including the step of Thereby, purifying the recombinant AAV particles.

[0233] In certain embodiments, steps (a)-(f) are maintained, and the following steps: g) To produce a second column eluate containing rAAV particles, subjecting the column eluate or concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC), thereby separating rAAV particles from protein impurities or other production / process-related impurities, and optionally diluting the second column eluate to produce a diluted second column eluate, including the step of h) Optionally, to produce a third column eluate containing rAAV particles, subjecting the second column eluate or diluted second column eluate produced in step (g) to anion exchange chromatography, thereby separating rAAV particles from protein impurities or other production / process-related impurities, and optionally diluting the third column eluate to produce a diluted third column eluate; and i) Combined with the step of filtering the second column eluate or diluted second column eluate produced in step (g), or filtering the third column eluate or concentrated third column eluate produced in step (h), Thereby, purifying the recombinant AAV particles.

[0234] In certain embodiments, steps (a)-(f) are maintained, and the following steps: g) a step of subjecting the column eluate or diluted column eluate produced in step (f) to cation exchange column chromatography to produce a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process-related impurities, and optionally diluting the column eluate to produce a diluted second column eluate; h) a step of subjecting the column eluate or diluted column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or production / process-related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate, combined with the step; Thereby, recombinant AAV particles are purified.

[0235] In certain embodiments, steps (a)-(g) are maintained, and the following steps: g) a step of subjecting the column eluate or diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or production / process-related impurities, and optionally concentrating the second column eluate to produce a concentrated second column eluate; h) a step of subjecting the column eluate or diluted column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate containing rAAV particles, thereby separating the rAAV particles from protein impurities or other production / process-related impurities, and optionally concentrating the third column eluate to produce a concentrated third column eluate, combined with the step; Thereby, recombinant AAV particles are purified.

[0236] In certain embodiments of all aspects and embodiments, the concentration in step (b) and / or step (f) and / or step (g) and / or step (h) is by ultrafiltration / diafiltration, such as tangential flow filtration (TFF).

[0237] In certain embodiments of all aspects and embodiments, the concentration in step (b) reduces the volume of the recovered cells and cell culture supernatant by about 2 to 20-fold.

[0238] In certain embodiments of all aspects and embodiments, the concentration in step (f) and / or step (g) and / or step (h) reduces the volume of the column eluate by about 5 to 20-fold.

[0239] In certain embodiments of all aspects and embodiments, step (d) includes treating with a nuclease, thereby reducing contaminating nucleic acids. Non-limiting examples of nucleases include benzonase.

[0240] In certain embodiments of all aspects and embodiments, filtering the clarified lysate or diluted clarified lysate of step (e) is by a filter. Non-limiting examples of the filter have a pore size of about 0.1 micron to 10.0 microns (including both ends).

[0241] In certain embodiments of all aspects and embodiments, the dilution of the clarified lysate of step (e) is by a buffered phosphate, acetate, or Tris aqueous solution. Non-limiting examples of the solution pH are about pH 4.0 to pH 7.4 (including both ends). Non-limiting examples of the pH of the Tris solution are greater than pH 7.5, such as about pH 8.0 to pH 9.0 (including both ends).

[0242] In certain embodiments of all aspects and embodiments, the dilution of the second column eluate of step (g) or the third column eluate of step (h) is by a buffered phosphoric acid, acetic acid, or Tris aqueous solution. Non-limiting examples of the solution pH are from about pH 4.0 to pH 7.4 (including both ends). Non-limiting examples of the pH of the Tris solution are greater than pH 7.5, such as from about pH 8.0 to pH 9.0 (including both ends).

[0243] In certain embodiments of all aspects and embodiments, the rAAV particles obtained from step (i) are formulated with a surfactant to produce an rAAV particle formulation.

[0244] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography of step (g) and / or (h) includes polyethylene glycol (PEG)-regulated column chromatography.

[0245] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography of step (g) and / or (h) is washed with a PEG solution prior to elution of the rAAV particles from the column.

[0246] In certain embodiments of all aspects and embodiments, PEG has an average molecular weight in the range of about 1,000 g / mol to 80,000 g / mol (including both ends).

[0247] In certain embodiments of all aspects and embodiments, PEG is at a concentration of about 4% to about 10% (w / v) (including both ends).

[0248] In certain embodiments of all aspects and embodiments, the anion exchange column of step (g) and / or (h) is washed with an aqueous surfactant solution prior to elution of the rAAV particles from the column.

[0249] In certain embodiments of all aspects and embodiments, the cation exchange column of step (g) and / or step (h) is washed with a surfactant solution prior to elution of the rAAV particles from the column.

[0250] In certain embodiments of all aspects and embodiments, the PEG solution and / or surfactant solution comprises an aqueous Tris-HCl / NaCl buffer, an aqueous phosphate / NaCl buffer, or an aqueous acetate / NaCl buffer.

[0251] In certain embodiments of all aspects and embodiments, the NaCl concentration in the buffer or solution is in the range of about 20 to 300 mM NaCl (including both ends) or about 50 to 250 mM NaCl (including both ends).

[0252] In certain embodiments of all aspects and embodiments, the surfactant comprises a cationic or anionic surfactant.

[0253] In certain embodiments of all aspects and embodiments, the surfactant comprises a 12-carbon chain surfactant.

[0254] In certain embodiments of all aspects and embodiments, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or sarcosyl.

[0255] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the anion exchange column of step (f), (g) and / or (h) with an aqueous Tris-HCl / NaCl buffer.

[0256] In certain embodiments of all aspects and embodiments, the Tris-HCl / NaCl buffer comprises 100 to 400 mM NaCl (including both ends), optionally at a pH in the range of about pH 7.5 to about pH 9.0 (including both ends).

[0257] In certain embodiments of all aspects and embodiments, the anion exchange column of step (g) and / or (h) is washed with an aqueous Tris-HCl / NaCl buffer.

[0258] In certain embodiments of all aspects and embodiments, the NaCl concentration in the aqueous Tris-HCl / NaCl buffer is in the range of about 75 to 125 mM (including both ends).

[0259] In certain embodiments of all aspects and embodiments, the aqueous Tris-HCl / NaCl buffer has a pH in the range of about pH 7.5 to about pH 9.0 (including both ends).

[0260] In certain embodiments of all aspects and embodiments, the anion exchange column in step (g) and / or (h) is washed one or more times to reduce the amount of empty capsids in the second or third column eluate.

[0261] In certain embodiments of all aspects and embodiments, anion exchange column washing removes empty capsids from the column before and / or instead of rAAV particle elution, thereby reducing the amount of empty capsids in the second or third column eluate.

[0262] In certain embodiments of all aspects and embodiments, anion exchange column washing removes at least about 50% of the total empty capsids from the column before and / or instead of rAAV particle elution, thereby reducing the amount of empty capsids in the second or third column eluate by about 50%.

[0263] In certain embodiments of all aspects and embodiments, the NaCl concentration in the aqueous Tris-HCl / NaCl buffer is in the range of about 110 to 120 mM (including both ends).

[0264] In certain embodiments of all aspects and embodiments, the ratio and / or amount of eluted rAAV particles and empty capsids is controlled by the washing buffer.

[0265] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the cation exchange column of step (g) and / or (h) in an aqueous phosphate / NaCl buffer or an aqueous acetate / NaCl buffer. Non-limiting NaCl concentrations in the buffer range from about 125 to 500 mM NaCl (including both ends). Non-limiting examples of buffer pH are from about pH 5.5 to about pH 7.5 (including both ends).

[0266] In certain embodiments of all aspects and embodiments, the anion exchange column of step (g) and / or (h) contains a quaternary ammonium functional group such as quaternized polyethyleneimine.

[0267] In certain embodiments of all aspects and embodiments, the size exclusion column (SEC) has a separation / fractionation range (molecular weight) from about 10,000 g / mol to about 600,000 g / mol (including both ends).

[0268] In certain embodiments of all aspects and embodiments, the cation exchange column of step (g) or / and (h) contains a functional group such as sulfonic acid or sulfopropyl.

[0269] In certain embodiments of all aspects and embodiments, the AAV affinity column contains a protein or ligand that binds to the AAV capsid protein. Non-limiting examples of proteins include antibodies that bind to the AAV capsid protein. More specific non-limiting examples include single-chain camelid antibodies (Camelid) that bind to the AAV capsid protein.

[0270] In certain embodiments of all aspects and embodiments, the method excludes the step of cesium chloride gradient ultracentrifugation.

[0271] In certain embodiments of all aspects and embodiments, the method produces rAAV particles with a higher purity than rAAV particles produced or purified by single AAV affinity column purification.

[0272] In certain embodiments of all aspects and embodiments, steps (c) and (d) are carried out substantially simultaneously.

[0273] In certain embodiments of all aspects and embodiments, the NaCl concentration is adjusted to be in the range of about 100 - 400 mM NaCl (including both ends), or in the range of about 140 - 300 mM NaCl (including both ends) after step (c) but before step (f).

[0274] In certain embodiments of all aspects and embodiments, the cells are suspension-growing cells or adherent-growing cells.

[0275] In certain embodiments of all aspects and embodiments, the cells are mammalian cells. Non-limiting examples include HEK cells such as HEK-293 cells, and CHO cells such as CHO-K1 cells.

[0276] Methods for determining the infectivity 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 assaying empty capsids and rAAV particles packaged with a transgene are known (see, for example, Grimm et al., Gene Therapy 6 (1999) 1322 - 1330; Sommer et al., Malec. Ther. 7 (2003) 122 - 128).

[0277] To determine the presence or amount of disassembled / denatured capsids, purified rAAV particles are subjected to SDS-polyacrylamide gel electrophoresis consisting of any gel capable of separating the three capsid proteins, e.g., a gradient gel, and then the gel is run until the sample is separated and can be blotted onto a nylon or nitrocellulose membrane. An anti-AAV capsid antibody is then used as the primary antibody that binds to the denatured capsid protein (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody that binds to the primary antibody includes means for detecting the primary antibody. The binding between the primary and secondary antibodies is detected semi-quantitatively to determine the amount of capsid. Another method is analytical HPLC using an SEC column or an analytical ultracentrifuge.

[0278] Description of specific embodiments of the present invention The present invention is at least partially based on the finding that the recovery rate of recombinant AAV particles in AAV affinity chromatography, i.e., the yield (both capsid-based yield and genome-based yield), is affected by / dependent on the surfactant used to lyse the cells that produced the recombinant AAV particles prior to AAV affinity chromatography.

[0279] The present invention is further at least partially based on the finding that the ratio of full recombinant AAV particles to empty recombinant AAV particles obtained by AAV affinity chromatography is affected by / dependent on the surfactant used to lyse the cells that produced the recombinant AAV particles prior to AAV affinity chromatography.

[0280] It has been found that by using an alkyl polyglucoside surfactant to lyse the cells that produced the recombinant AAV particles prior to the AAV affinity chromatography step, both the yield (based on capsid and genome) and the ratio of full recombinant AAV particles to empty recombinant AAV particles in AAV affinity chromatography can be increased.

[0281] The method according to the present invention is illustrated below using Triton CG 110 as an example of an alkyl polyglucoside surfactant. This is presented merely as an illustration of the concept of the present invention and should not be construed as limiting. Similarly, any other alkyl polyglucoside surfactant can be used. The true scope of the present invention is set forth in the appended claims.

[0282] More specifically, an increase in the absolute AAV particle recovery rate in AAV affinity chromatography above 5% can be achieved by using Triton CG 110 as an example of an alkyl polyglucoside surfactant, as compared to the current “gold standard” Triton X-100 (a p-tert octylphenol derivative having a polyethylene glycol side chain), i.e., an absolute recovery rate of 76% when lysing cells using Triton CG 110 can be achieved as compared to an absolute recovery rate of 72% when lysing cells using Triton X-100.

[0283] Since this method is intended for large-scale production of recombinant AAV particles, even a slight relative increase in the AAV particle recovery rate results in a large absolute increase in the recovery rate, thereby providing an advantage.

[0284] Simultaneously with the increase in the absolute AAV particle recovery rate, the ratio of full-length recombinant AAV particles to empty recombinant AAV particles in the AAV affinity chromatography eluate increases as compared to the ratio in the lysate applied to the AAV affinity chromatography material.

[0285] More specifically, by using Triton CG 110 as an example of an alkyl polyglucoside surfactant in AAV affinity chromatography, an increase in genomic recovery rate of more than 60% is achieved compared to the current "gold standard" Triton X-100. That is, an absolute genomic yield of 4.7×1E11 viral genomes / mL (vg / mL) can be obtained when cells are lysed using Triton CG 110, compared to an absolute genomic yield of 2.8×1E11 viral genomes / mL (vg / mL) when cells are lysed using Triton X-100. Also, compared to Triton X-100 lysed culture broth, an increase of more than 50% in the total viral particle yield in the eluate of AAV affinity chromatography of Triton CG 110 lysed culture broth is achieved. That is, an absolute particle yield of 1.2×1E13 viral particles / mL (vp / mL) can be achieved when cells are lysed using Triton CG 110, compared to an absolute particle yield of 0.77×1E13 viral particles / mL (vp / mL) when cells are lysed using Triton X-100.

[0286] Since the increase in genomic yield is higher than the total viral particle yield, an increase in the ratio of full recombinant AAV particles to empty recombinant AAV particles is obtained.

[0287] Each data is shown in the following table. TIFF2025524619000001.tif89161

[0288] Therefore, the present invention is at least partially based on the finding that when recombinant AAV particle-producing cells are lysed using Triton CG 110 as an example of an alkyl polyglucoside surfactant, the recovery rate of full recombinant AAV particles increases.

[0289] Combinations of different final concentrations of Triton CG 110 and dissolution times (incubation times in the presence of Triton CG 110) were tested in small-scale experiments using aliquots of the same culture broth obtained 72 hours after triple PEI-mediated transfection.

[0290] It was found that an incubation time of approximately 60 minutes at a final concentration of 1% (v / v) of Triton CG 110 provided the best overall results. This can be further improved by incubation with diatomaceous earth for approximately 20 minutes after incubation with the alkyl polyglucoside surfactant Triton CG 110. These are preferred embodiments of the present invention.

[0291] Each data is shown in the following table. TIFF2025524619000002.tif116144

[0292] Plasmids required for the production of recombinant AAV particles, namely, plasmids containing a transgene (transgene plasmid), plasmids encoding AAV packaging proteins (rep / cap plasmids), and plasmids containing the necessary AAV helper functions not yet present in HEK293 cells (helper plasmids), were transfected into mammalian HEK293 cells.

[0293] More specifically, to produce recombinant AAV particles, pre-cultured HEK293 cells were cultured in a wave bioreactor (10 L working volume) in a batch process using F17 medium with an initial cell density of approximately 1E6 cells / ml.

[0294] PEI (polyethyleneimine)-mediated transfection with three plasmids (helper plasmid, transgene plasmid, rep / cap plasmid) was performed several hours after seeding of the bioreactor. The total DNA amount was calculated based on the cell density after seeding (1 μg DNA / E6 cells). The molar ratio of the three plasmids was 1:1:1. The transfection reagent was used in an amount of 2 μg / 1 μg plasmid.

[0295] Cultivation was carried out at a temperature of 37 °C, a humidity of 70%, pCO2 of 5% with a stirrer speed of 30 - 35 rpm without oxygen and pH control. Aeration was performed using air at a flow rate of approximately 500 ml / min. Cultivation was terminated 72 hours after transfection.

[0296] In another process, HEK cells can be cultured in a fed-batch process for 4 days with a starting cell density of approximately 15×1E5 cells / ml with feed and glucose addition.

[0297] PEI (polyethyleneimine)-mediated transfection by triple transfection with three plasmids (helper plasmid, transgene plasmid, rep / cap plasmid) can be performed 24 hours after seeding. First, , 20% v / v of fresh medium is added to the culture. Subsequently, a transfection mix is prepared and added to the culture. The total DNA concentration is 3 μg / mL and the molar ratio of the three plasmids is 1:1:1. The transfection reagent is used at a concentration of 2.5 μg / 1 μg plasmid and free PEI is added at a concentration of 1.5 μg / ml of culture volume.

[0298] A feed, for example the HEK FS feed supplement from Xell AG, is added as a bolus feed on day 2 of fermentation (24 hours after transfection). Glucose is added as a bolus feed on days 1 and 3 of fermentation. The pH value is adjusted by adding CO2 and 1M Na2CO3 within 0.05 pH units each. An antifoam solution is added as needed. During the process, the parameters temperature, pH value and pO2 are monitored and controlled. The fermentation process is stopped on day 4 (72 hours after triple transfection) by adding a lysis buffer. ***

[0299] All references mentioned in this specification are incorporated herein by reference. ***

[0300] The following examples are provided to assist in the understanding of the present invention, the true scope of which is set forth in the claims. It is understood that modifications can be made to the procedures described without departing from the spirit of the present invention.

Examples

[0301] Materials Cell line Commercially available HEK293 cells were used to produce AAV particles using transient transfection with three plasmids.

[0302] Culture materials The culture medium was prepared according to the supplier's instructions (HEK ViP NB powder medium, Xell AG). HEK FS feed (Xell AG) and F17 medium were purchased ready for use. The medium and feed were stored in the dark at 4°C and consumed according to the manufacturer's instructions. The corrective agents were stored at room temperature (glucose solution; sodium carbonate solution; antifoam agent solution).

[0303] Example 1 Culturing of HEK293 cells and production of recombinant AAV particles Generally, the culture method is adapted from standard protocols (e.g., see Lindl, T., "Zell-und Gewebekultur: Einführung in die Grundlagen sowie ausgewählte Methoden und Anwendungen", Spektrum Akademischer Verlag GmbH, Heidelberg / Berlin, 2002) and the operating instructions of each supplier.

[0304] Pre-culture HEK cells were thawed and grown in a shaking flask at 37°C, 70% humidity, 5% pCO2 and a shaking frequency of 120 rpm in culture medium for 2 - 3 weeks. The cells were split every 3 - 4 days and expanded to the volume required for seeding the production culture in the medium.

[0305] Production culture To produce recombinant AAV particles, pre-cultured HEK293 cells were cultured in a wave bioreactor (10 L working volume) in a batch process using F17 medium with an initial cell density of 10xE5 cells / ml.

[0306] PEI (polyethyleneimine)-mediated transfection with three plasmids (helper plasmid, transgene plasmid, rep / cap plasmid) was performed several hours after inoculation of the bioreactor. The total DNA amount was calculated based on the cell density after inoculation (1 μg DNA / E6 cells). The molar ratio of the three plasmids was 1:1:1. The transfection reagent was PEIpro (Polyplus) containing plasmid DNA in an amount of 2 μg / 1 μg.

[0307] The culture was carried out at a temperature of 37°C, 70% humidity, 5% pCO2, with a stirrer speed of 30 - 35 rpm without oxygen and pH control. Aeration was carried out using air at a flow rate of approximately 500 ml / min. The culture was terminated 72 hours after transfection.

[0308] Example 2 Lysis To release AAV particles into the cell culture broth, 10% (v / v) lysis buffer (500 mM TRIS, 20 mM MgCl2, pH 7.5) containing different concentrations of the test surfactant was added to the culture broth. Additionally, 50 U / ml of DNase I (bovine pancreas, Roche) and 37.5 mM of MgSO4 were added. Subsequently, the cell culture broth was incubated at 37 °C for different times with stirring, without aeration and pH control. After each incubation, the lysate was passed through a sterile filter.

[0309] Example 3 AAV Particle Purification For the affinity chromatography step, a column containing 10.5 mL of AAVX resin from Thermo Fisher was used with an AKTA Avant 25 chromatography system. The system was operated at a flow rate of approximately 300 cm / h. After equilibration with buffer A (1×PBS, pH 7.4, 0.001% Pluronic F-68), 200 mL of the lysed culture broth was applied to the column, followed by two washing steps each with the equilibration buffer and 0.5 M NaCl, pH 6.0. 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 the addition of 2 M Tris (pH 10).

[0310] TIFF2025524619000003.tif68136

[0311] Example 4 Analysis Method Enzyme-Linked Immunosorbent Assay (ELISA) for Total Titer Measurement For the measurement of AAV capsid titer, a kit from PROGEN (catalog number PRAAV8) was used according to the manufacturer's instructions.

[0312] Briefly, this assay is a sandwich ELISA that uses a recombinant AAV capsid-specific antibody as the capture antibody and a biotin-labeled detection antibody.

[0313] Wells of pre-coated multi-titer plates (MTPs) were incubated overnight at 4 °C with 100 μL each of standard, sample or control. The next day, the wells were washed three times with ASSB buffer (1×) as provided in the kit. Thereafter, 100 μL / well of a solution containing biotinylated detection antibody (diluted according to the manufacturer's instructions) was added and incubated for 2 hours at room temperature with shaking. Thereafter, the wells were washed three times with ASSB buffer (1×) as provided in the kit. In the next step, 100 μl of a solution containing horseradish peroxidase conjugated to streptavidin was added to each well and incubated for 30 minutes at room temperature with shaking. Thereafter, the wells were washed three times with ASSB buffer (1×) as provided in the kit. For the color reaction, 100 μL of a solution containing ABTS prepared according to the manufacturer's instructions was added to each well and incubated with shaking. The color intensity was determined using an MTP-ELISA-Reader Versa Max (Molecular Devices) at 405 nm with a reference wavelength of 490 nm until the extinction difference between the blank and the highest concentration standard reached approximately 1.5.

[0314] Each sample, standard and control was measured in duplicate.

[0315] The amount of capsid (capsid / mL) was calculated based on a standard curve determined by four-parameter fitting using the average of the standards, for example according to the Wiemer-Rodbard algorithm.

[0316] Digital droplet polymerase chain reaction (ddPCR) for genome titer determination Reagents for enzyme sample treatment: 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; approximately 20 mg / mL = 800 U / mL): 16 U / mL 4) Proteinase K Buffer (BioRad): 400 mM Tris-HCl, 20 mM EDTA, 2000 mM NaCl, 1% SDS, pH 8 5) Sodium dodecyl sulfate (SDS) solution: 10% (w / v) Enzyme sample treatment: - Mix 30 μL H2O, 5 μL DNase I buffer, 5 μL DNase I, and 10 μL sample - Incubate at 37 °C for 30 minutes - Heat to 75 °C for 15 minutes to obtain the incubated DNase I-Mix - Short cooling and centrifugation - Mix 42 μL H2O + 2 μL Proteinase K + 5 μL Proteinase K buffer + 1 μL 10% SDS solution, and add the incubated DNase I-Mix - Incubate at 50 °C for 60 minutes - Heat to 95 °C for 15 minutes - Cool to 4 °C

[0317] ddPCR: For viral genome titration, a duplex ddPCR assay was performed. Primers and probes were designed against the CMV promoter and polyA / 3’UTR sequences used. The PCR master mix was prepared according to the following table (Droplet Digital PCR Guide - Bio-Rad).

[0318] TIFF2025524619000004.tif67144

[0319] The prepared master mix was pipetted into a 96-well plate at 16.5 μL / well. Next, a dilution series of the pretreated samples was performed: 10 μL of the sample was transferred to 90 μL of water in a LoBind Tube together with LoRetention Tips and mixed thoroughly. Then, 5.5 μL of the sample was added to the master mix solution in the 96-well plate at 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 an additional 1 minute. Using an automated droplet generator that removed 20 μL of the PCR mixture from each well, up to 20,000 droplets per well were generated and transferred to another 96-well plate. After sealing the droplet plate at 180 °C, PCR was performed. Each condition is shown in the following table.

[0320] TIFF2025524619000005.tif39153

[0321] Using a droplet reader, the fluorescence signal was measured for each droplet. QuantaSoft software processed the reader data and the calculated copy number per 20 μL well for the target sequence. The initial sample titer can be determined using the following formula. TIFF2025524619000006.tif18128

Claims

1. A method for purifying recombinant AAV particles, comprising the following steps: - A step of releasing recombinant AAV particles from the producing mammalian cells by contacting each mammalian cell culture broth with an alkyl polyglucoside surfactant, - A step of purifying the recombinant AAV particles by AAV affinity chromatography and Includes, This allows for the purification of the recombinant AAV particles, The method wherein the mammalian cell culture broth comprises cultured recombinant AAV particle-producing mammalian cells and a culture medium used for culturing the recombinant AAV particle-producing mammalian cells.

2. Use of alkyl polyglucoside surfactants to increase the yield of recombinant AAV particles, wherein recombinant AAV particle-producing mammalian cells are lysed with the alkyl polyglucoside surfactant, and the yield is determined after a subsequent AAV affinity chromatography step.

3. The use of an alkyl polyglucoside surfactant to increase the ratio of completely recombinant AAV particles (in the eluate fraction) obtained by subsequent AAV affinity chromatography to empty recombinant AAV particles, wherein recombinant AAV particle-producing mammalian cells are dissolved with the alkyl polyglucoside surfactant prior to the AAV affinity chromatography.

4. The method and use according to any one of claims 1 to 3, wherein the alkyl polyglucoside surfactant is a mixture of 58.0 to 62.0 (w / v)% D-glucopyranose oligomer, decyloctyl glycoside, and 38.0 to 42.0 (w / v)% water.

5. The method and use according to any one of claims 1 to 3, wherein the alkyl polyglucoside surfactant has CAS number 68515-73-1.

6. The method and use according to any one of claims 1 to 3, wherein the alkyl polyglucoside surfactant is in a solution.

7. The method and use according to any one of claims 1 to 3, wherein the mammalian cell culture broth is combined with a solution containing an alkyl polyglucoside surfactant in an amount of 5% to 10% (5% (v / v) to 10% (v / v)) of its volume.

8. The method and use according to claim 6, wherein the solution containing the alkyl polyglucoside surfactant contains the alkyl polyglucoside surfactant at a concentration of about 10%.

9. The method and use according to any one of claims 1 to 3, wherein each alkyl polyglucoside surfactant is brought into contact with the others, or the contact is maintained for about 60 minutes.

10. The method and use according to any one of claims 1 to 3, wherein the alkyl polyglucoside surfactants are brought into contact with each other, or the contact is performed at a temperature of about 37°C.

11. The method and use according to any one of claims 1 to 3, wherein the alkyl polyglucoside surfactants are brought into contact with each other, or diatomaceous earth is added after contact, and the mixture is incubated for 5 to 20 minutes.

12. The method and use according to claim 6, wherein the solution containing the alkyl polyglucoside surfactant contains magnesium(II) chloride at a concentration of 10 mM to 40 mM.

13. The method and use according to claim 6, wherein the solution containing the alkyl polyglucoside surfactant has a pH value of about 7.

5.

14. The method and use according to any one of claims 1 to 3, wherein the mammalian cell culture broth is brought into contact with the alkyl polyglucoside surfactant and a nuclease selected from DNase I and benzonase at a concentration of 50 U / mL.

15. The method and use according to any one of claims 1 to 3, wherein the affinity chromatography is performed on a chromatographic material comprising a crosslinked poly(styrene-divinylbenzene) matrix on which single-domain antibody fragments (VHHs) that specifically bind to AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 and synthetic serotypes based thereon are covalently conjugated.