Method for producing recombinant AAV particles

Abstract

Herein is reported a method for lysing mammalian cells producing recombinant AAV particles, the method comprising the steps of contacting a mammalian cell culture broth with an alkyl polyglucoside detergent, preferably Triton CG 110, and thereby lysing 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) for culturing the mammalian cells producing recombinant AAV particles.

Description

[0001] The present invention belongs to the field of gene therapy. More specifically, a method for releasing recombinant AAV particles from producer cells is reported herein, wherein the cells are lysed using Triton CG 110. Background Art

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

[0003] Today, most in vivo therapies rely on DNA delivery using recombinant adeno-associated virus (rAAV) vectors. AAV is a small, naturally occurring, non-pathogenic parvovirus that consists of an icosahedral capsid without an envelope. It contains a linear single-stranded DNA genome of approximately 4.7 kb. The genome of the wild-type AAV vector carries two genes, rep and cap, which are flanked by inverted terminal repeats (ITRs). ITRs are in cis and are necessary for viral replication and packaging. The rep gene encodes four different proteins, whose expression is driven by two alternative promoters, P5 and P19. In addition, different forms are generated by alternative splicing. Rep proteins have multiple functions, such as, for example, DNA binding, endonuclease and helicase activity. They play a role in gene regulation, site-specific integration, excision, replication and packaging. The cap gene encodes three capsid proteins and an assembly activation protein. Differential expression of these proteins is achieved by using alternative splicing and selective start codons, and expression is driven by a single promoter P40 located in the coding region of the rep gene.

[0004] In engineered therapeutic rAAV vectors, the viral genes are replaced with a transgene expression cassette that still flanks the viral ITRs but encodes the gene of interest under the control of a selected promoter. Unlike wild-type viruses, engineered rAAV vectors do not integrate site-specifically into the host genome but remain primarily episomal in the nucleus of transduced cells.

[0005] AAV itself does not have the ability to replicate, but requires the function of auxiliary genes. These are provided in nature by co-infected helper viruses, such as adenovirus or herpes simplex virus. For example, five adenovirus genes (i.e., E1A, E1B, E2A, E4, and VA) are known to be essential for AAV replication. Compared with other auxiliary genes that encode proteins, VA is a small RNA gene.

[0006] To produce rAAV vectors, DNA carrying the transgenic gene flanked by ITRs is introduced into a packaging host cell line that also contains rep and cap genes and required accessory genes. There are a variety of methods to introduce these three sets of DNA elements into cells and many methods to combine them on different DNA plasmids (see, e.g., Robert, MA et al. Biotechnol. J. 12 (2017) 1600193).

[0007] Two general production methods have been widely used. In the triple transfection method, a plasmid containing rep / cap and a plasmid containing rAAV transgenes are transiently co-transfected with an adenoviral helper plasmid carrying the desired adenoviral helper gene. This method can be performed using CHO or HEK cells. Alternatively, rep / cap and viral helper genes can be combined on a larger plasmid (double transfection method). The second method covers infection of insect cells (Sf9) with two baculoviruses, one baculovirus carrying the rAAV genome and the other baculovirus carrying rep and cap. In this system, the baculovirus plasmid itself provides auxiliary functions. Similarly, herpes simplex virus is used in combination with HEK293 cells or BHK cells. Recently, Mietzsch et al. (Hum. Gene Ther. 25 (2014) 212-222; Hum. Gene Ther. Methods 28 (2017) 15-22) stably integrated rep and cap into the genome of engineered Sf9 cells. For these cells, a single baculovirus carrying the rAAV transgene is sufficient to produce rAAV vectors. Clark et al. (Hum. Gene Ther. 6 (1995) 1329-1341) generated a HeLa cell line with the rep / cap gene and the rAAV transgene integrated into its genome. The rAAV vector production was induced by transfecting the cells with wild-type adenovirus, and a mixed stock of rAAV vector and adenovirus was produced.

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

[0009] Mafalda Moleirinho et al. reported the purification of clinical-grade oncolytic adenovirus using polysorbate 20 as an alternative to cell lysis (Curr. Gene Ther. 18 (2018) 1-9). WO 2022 / 003565 reported detergents and methods for purifying biotherapeutics. Summary of the invention

[0010] The present invention is based, at least in part, on the discovery that the recovery rate, i.e., the yield (both capsid-based and genome-based) of recombinant AAV particles in AAV affinity chromatography is affected by / depends on the detergent used to lyse the cells producing the recombinant AAV particles prior to AAV affinity chromatography.

[0011] The present invention is further based, at least in part, on the discovery that the ratio of intact to empty recombinant AAV particles obtained in AAV affinity chromatography is affected by / depends on the detergent used to lyse the recombinant AAV particle-producing cells prior to AAV affinity chromatography.

[0012] It has been found that by using an alkyl polyglucoside detergent for lysing cells producing recombinant AAV particles prior to the AAV affinity chromatography step, both the yield of recombinant AAV particles (capsid-based and genome-based) and the ratio of intact recombinant AAV particles to empty recombinant AAV particles can be increased in AAV affinity chromatography.

[0013] The prior art does not propose the use of alkyl polyglucoside (APG) for lysing AAV-producing cells, nor does it propose to solve the problem of providing a method to increase the yield of recombinant AAV particles and the ratio of complete recombinant AAV particles to empty recombinant AAV particles.

[0014] Therefore, the present invention encompasses the following embodiments:

[0015] 1. A method for lysing mammalian cells for producing recombinant AAV particles, the method comprising the steps of:

[0016] - contacting a mammalian cell culture broth with an alkyl polyglucoside detergent,

[0017] and thereby lysing mammalian cells producing recombinant AAV particles,

[0018] The mammalian cell culture broth comprises cultured mammalian cells producing recombinant AAV particles and culture medium (spent culture medium) used for culturing mammalian cells producing recombinant AAV particles.

[0019] 2. A method for releasing recombinant AAV particles from mammalian cells producing recombinant AAV particles, the method comprising the steps of:

[0020] - contacting a mammalian cell culture broth with an alkyl polyglucoside detergent,

[0021] and thereby lysing mammalian cells producing recombinant AAV particles,

[0022] The mammalian cell culture broth comprises cultured mammalian cells producing recombinant AAV particles and culture medium (spent culture medium) used for culturing mammalian cells producing recombinant AAV particles.

[0023] 3. A method for purifying recombinant AAV particles, the method comprising the following steps:

[0024] - contacting a mammalian cell culture broth with an alkyl polyglucoside detergent,

[0025] - remove cell debris from the mixture, and

[0026] - Purification of recombinant AAV particles using AAV affinity chromatography,

[0027] and thereby purifying the recombinant AAV particles,

[0028] The mammalian cell culture broth comprises cultured mammalian cells producing recombinant AAV particles and culture medium (spent culture medium) used for culturing mammalian cells producing recombinant AAV particles.

[0029] 4. A method for purifying recombinant AAV particles, the method comprising the following steps:

[0030] - releasing recombinant AAV particles from production mammalian cells by contacting a corresponding mammalian cell culture broth with an alkyl polyglucoside detergent, and

[0031] - Purification of the recombinant AAV particles using AAV affinity chromatography

[0032] Thus purifying the recombinant AAV particles,

[0033] The mammalian cell culture broth comprises cultured mammalian cells producing recombinant AAV particles and culture medium (spent culture medium) used for culturing mammalian cells producing recombinant AAV particles.

[0034] 5. A method for producing a recombinant AAV particle comprising a nucleic acid encoding a protein of interest or transcribed as a transcript of interest, wherein the method comprises the following steps:

[0035] (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding accessory proteins;

[0036] (ii) providing a plasmid comprising a nucleic acid encoding a protein of interest or transcribed as a transcript of interest, the transcript being inserted between the AAV ITRs;

[0037] (iii) contacting one or more mammalian cells with the provided plasmid and performing one of the following: further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture; or providing a physical means such as an electric current to introduce the nucleic acid into the cells;

[0038] (iv) culturing the transfected cells;

[0039] (v) harvesting the cultured cells and medium to produce a mammalian cell culture broth;

[0040] (vi) lysing the cells by contacting the mammalian cell culture broth with an alkyl polyglucoside detergent to produce a mammalian cell culture broth lysate; and

[0041] (vii) optionally isolating recombinant AAV particles from the culture broth lysate using AAV affinity chromatography;

[0042] Recombinant AAV particles are thereby produced that contain nucleic acid encoding a protein of interest or that is transcribed into a transcript of interest.

[0043] 6. A method for producing a recombinant AAV particle comprising a nucleic acid encoding a protein of interest or transcribed as a transcript of interest, wherein the method comprises the steps of:

[0044] (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding accessory proteins;

[0045] (ii) providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript;

[0046] (iii) or

[0047] (a) generating stably transfected cells by contacting one or more mammalian cells with the plasmid provided in (i), by one of the following operations: further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture, or providing a physical means such as an electric current to introduce nucleic acid into the cells; selecting a first stably transfected cell; contacting the selected first stably transfected cell with the plasmid provided in (ii) and performing one of the following operations: further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture, or providing a physical means such as an electric current to introduce nucleic acid into the cells; or

[0048] (b) generating transiently transfected cells by contacting one or more mammalian cells with the plasmids provided by (i) and (ii), and performing one of the following operations: further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture, or providing a physical means such as an electric current to introduce the nucleic acid into the cells;

[0049] Thereby, transfected cells are generated;

[0050] (iv) culturing the transfected cells of (iii);

[0051] (v) harvesting the cultured cells and medium to produce a mammalian cell culture broth;

[0052] (vi) lysing the cells by contacting the mammalian cell culture broth with an alkyl polyglucoside detergent to produce a mammalian cell culture broth lysate; and

[0053] (vii) optionally isolating recombinant AAV particles from mammalian cell culture broth lysate using AAV affinity chromatography;

[0054] Recombinant AAV particles are thereby produced that contain nucleic acid encoding a protein of interest or that is transcribed into a transcript of interest.

[0055] 7. A method for purifying recombinant AAV particles, the method comprising the steps of:

[0056] a) harvesting the cultured mammalian cells producing recombinant AAV particles and the cell culture supernatant containing rAAV particles to produce a mammalian cell culture broth;

[0057] b) optionally concentrating the harvest produced in step (a) to produce a concentrated mammalian cell culture broth;

[0058] c) lysing 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 contacting the culture broth with an alkyl polyglucoside detergent to produce a mammalian cell culture broth lysate;

[0059] d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate;

[0060] 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;

[0061] f) subjecting the nucleic acid-reduced lysate obtained in step (d) or the clarified lysate or the diluted clarified lysate produced in step (e) to AAV affinity column chromatography to produce a column eluate comprising 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;

[0062] Recombinant AAV particles are thus purified.

[0063] 8. The method according to embodiment 7, further comprising the following steps:

[0064] g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprising rAAV particles, 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;

[0065] h) optionally subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprising rAAV particles, 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; and

[0066] i) filtering the second column eluate or the diluted second column eluate produced in step (g), or filtering the third column eluate or the concentrated third column eluate produced in step (h),

[0067] And thereby purifying the recombinant AAV particles.

[0068] 9. The method according to embodiment 7, further comprising the following steps:

[0069] g) subjecting the column eluate or the diluted column eluate produced in step (f) to cation exchange column chromatography to produce a second column eluate comprising 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;

[0070] h) subjecting the column eluate or the diluted column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprising 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,

[0071] And thereby purifying the recombinant AAV particles.

[0072] 10. The method according to embodiment 7, further comprising the following steps:

[0073] g) subjecting the column eluate or the diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprising 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;

[0074] h) subjecting the column eluate or the diluted column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprising 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,

[0075] And thereby purifying the recombinant AAV particles.

[0076] 11. Use of an alkyl polyglucoside detergent for lysing mammalian cells producing recombinant AAV particles, so as to increase the yield of the obtained recombinant AAV particles in a subsequent AAV affinity chromatography method.

[0077] 12. Use of an alkyl polyglucoside detergent for lysing mammalian cells producing recombinant AAV particles to increase the ratio of intact recombinant AAV particles to empty recombinant AAV particles obtained (in the eluate fraction) in a subsequent AAV affinity chromatography.

[0078] 13. Use of an alkyl polyglucoside detergent for increasing the yield of recombinant AAV particles, wherein mammalian cells producing AAV particles are lysed with the alkyl polyglucoside detergent, and wherein the recombinant AAV particles are obtained in a subsequent AAV affinity chromatography.

[0079] 14. Use of an alkyl polyglucoside detergent for increasing the yield of recombinant AAV particles, wherein mammalian cells producing recombinant AAV particles are lysed with the alkyl polyglucoside detergent and wherein the yield is determined after a subsequent AAV affinity chromatography step.

[0080] 15. Use of an alkyl polyglucoside detergent for increasing the ratio of intact recombinant AAV particles to empty recombinant AAV particles obtained (in the eluate fraction) in a subsequent AAV affinity chromatography, wherein mammalian cells producing recombinant AAV particles are lysed with the alkyl polyglucoside detergent prior to the AAV affinity chromatography.

[0081] 16. The method and use according to any one of the preceding embodiments, wherein the alkyl polyglucoside detergent is a mixture of 58.0 (w / v)% to 62.0 (w / v)% D-pyranose oligodecyloctyl glycoside and 38.0 (w / v)% to 42.0 (w / v)% water.

[0082] 17. The method and use according to any one of the preceding embodiments, wherein the alkyl polyglucoside detergent has CAS number 68515-73-1.

[0083] 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.

[0084] 19. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent is incubating with the alkyl polyglucoside detergent for a defined time and at a defined concentration of the alkyl polyglucoside detergent.

[0085] 20. The method and use of any one of the preceding embodiments, wherein the alkyl polyglucoside detergent is in solution.

[0086] 21. The method and use of any one of the preceding embodiments, wherein the mammalian cell culture broth is combined with a solution comprising an alkyl polyglucoside detergent at 2.5% to 20% (2.5% (v / v) to 20% (v / v)) of its volume.

[0087] 22. The method and use of any one of the preceding embodiments, wherein the mammalian cell culture broth is combined with a solution comprising an alkyl polyglucoside detergent at 5% to 10% by volume (5% (v / v) to 10% (v / v)).

[0088] 23. The method and use of any one of embodiments 20 to 22, wherein the solution comprising the alkyl polyglucoside detergent comprises a concentration of 5% to 20% alkyl polyglucoside detergent.

[0089] 24. The method and use of any one of embodiments 20 to 22, wherein the solution comprising the alkyl polyglucoside detergent comprises a concentration of 7.5% to 15% alkyl polyglucoside detergent.

[0090] 25. The method and use of any one of embodiments 20 to 22, wherein the solution comprising the alkyl polyglucoside detergent preferably comprises the alkyl polyglucoside detergent at a concentration of about 10%.

[0091] 26. The method and use according to any one of the preceding embodiments, wherein the contacting or contacting with the alkyl polyglucoside cleaner, respectively, is continued for 30 minutes to 90 minutes.

[0092] 27. The method and use according to any one of the preceding embodiments, wherein the contacting or contacting with the alkyl polyglucoside cleaner, respectively, is continued for 45 minutes to 75 minutes.

[0093] 28. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside cleaner, respectively, preferably lasts for about 60 minutes.

[0094] 29. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent, respectively, is performed at a temperature of 25°C to 45°C.

[0095] 30. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent, respectively, is carried out at a temperature of 28°C to 42°C.

[0096] 31. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent, respectively, is carried out at a temperature of 32°C to 40°C.

[0097] 32. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent, respectively, is preferably performed at a temperature of about 37°C.

[0098] 33. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent, respectively, is performed with stirring or shaking.

[0099] 34. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside cleaning agent, respectively, is performed without aeration.

[0100] 35. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with an alkyl polyglucoside cleaner, respectively, is performed without pH adjustment during contacting or contacting with an alkyl polyglucoside cleaner.

[0101] 36. The method and use according to any one of the preceding embodiments, wherein after contacting with the alkyl polyglucoside cleaner or contacting with the alkyl polyglucoside cleaner, respectively, diatomaceous earth is added and the mixture is incubated for 5 to 20 minutes.

[0102] 37. The method and use according to any one of the preceding embodiments, wherein after contacting with or with the alkyl polyglucoside detergent or diatomaceous earth, respectively, the mixture is centrifuged and / or sterile filtered.

[0103] 38. The method and use of any one of embodiments 20 to 37, wherein the solution comprising the alkyl polyglucoside detergent comprises a buffer salt.

[0104] 39. The method and use of any one of embodiments 20 to 38, wherein the solution comprising the alkyl polyglucoside cleaning agent comprises TRIS.

[0105] 40. The method and use of any one of embodiments 20 to 39, wherein the solution comprising the alkyl polyglucoside detergent comprises a buffer salt at a concentration of 100 mM to 1000 mM.

[0106] 41. The method and use of any one of embodiments 20 to 40, wherein the solution comprising the alkyl polyglucoside detergent comprises a buffer salt at a concentration of 250 mM to 750 mM.

[0107] 42. The method and use of any one of embodiments 20 to 41, wherein the solution comprising the alkyl polyglucoside detergent comprises a buffer salt at a concentration of 400 mM to 600 mM.

[0108] 43. The method and use of any one of embodiments 20 to 42, wherein the solution comprising the alkyl polyglucoside detergent comprises a buffer salt at a concentration of about 500 mM.

[0109] 44. The method and use of any one of embodiments 20 to 43, wherein the solution comprising the alkyl polyglucoside detergent comprises an ion modifier salt.

[0110] 45. The method and use according to any one of embodiments 20 to 44, wherein the solution comprising the alkyl polyglucoside detergent preferably comprises magnesium (II) chloride as the ion modifier salt.

[0111] 46. ​​The method and use of any one of embodiments 20 to 45, wherein the solution comprising the alkyl polyglucoside detergent comprises the ion modifier salt at a concentration of 5 mM to 200 mM.

[0112] 47. The method and use of any one of embodiments 20 to 46, wherein the solution comprising the alkyl polyglucoside detergent comprises the ion modifier salt at a concentration of 7.5 mM to 150 mM.

[0113] 48. The method and use of any one of embodiments 20 to 47, wherein the solution comprising the alkyl polyglucoside detergent comprises the ion modifier salt at a concentration of 10 mM to 50 mM.

[0114] 49. The method and use according to any one of embodiments 20 to 48, wherein the solution comprising the alkyl polyglucoside detergent preferably comprises the ion modifier salt at a concentration of 10 mM to 40 mM.

[0115] 50. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent, respectively, is performed at a pH value of pH 6.5 to 9.0.

[0116] 51. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent, respectively, is performed at a pH value of pH 6.5 to 8.0.

[0117] 52. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with the alkyl polyglucoside detergent, respectively, is performed at a pH value of pH 7.0 to 7.5.

[0118] 53. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with an alkyl polyglucoside cleaner, respectively, is performed at a pH value of pH 6.5 to 9.0, and the pH value is adjusted to a pH of 7.0 to 7.5 after contacting or contacting with an alkyl polyglucoside cleaner, respectively.

[0119] 54. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with an alkyl polyglucoside cleaner, respectively, is performed at a pH value of pH 6.5 to 8.0, and the pH value is adjusted to a pH of 7.0 to 7.5 after contacting or contacting with an alkyl polyglucoside cleaner, respectively.

[0120] 55. The method and use according to any one of the preceding embodiments, wherein contacting or contacting with an alkyl polyglucoside cleaner, respectively, is carried out at a pH value of pH 7.0 to 7.5, and the pH value is adjusted to a pH of about 7.5 after contacting or contacting with an alkyl polyglucoside cleaner, respectively.

[0121] 56. The method and use of any one of embodiments 20 to 55, wherein the solution comprising the alkyl polyglucoside cleaning agent has a pH value of pH 7.2 to 7.8.

[0122] 57. The method and use of any one of embodiments 20 to 56, wherein the solution comprising the alkyl polyglucoside cleaning agent has a pH of 7.3 to 7.7.

[0123] 58. The method and use of any one of embodiments 20 to 57, wherein the solution comprising the alkyl polyglucoside cleaning agent has a pH of 7.4 to 7.6.

[0124] 59. The method and use according to any one of embodiments 20 to 58, wherein the solution comprising the alkyl polyglucoside cleaning agent preferably has a pH of about 7.5.

[0125] 60. The method and use of any one of the preceding embodiments, wherein the mammalian cell culture broth is contacted with an alkyl polyglucoside detergent and a nuclease.

[0126] 61. The method and use of any one of the preceding embodiments, wherein the mammalian cell culture broth is contacted with an alkyl polyglucoside detergent and DNase I.

[0127] 62. The method and use according to any one of the preceding embodiments, wherein preferably the mammalian cell culture broth is contacted with an alkyl polyglucoside detergent and Benzonase.

[0128] 63. The method and use according to any one of embodiments 60 to 62, wherein 25 U / mL to 100 U / mL of nuclease is added.

[0129] 64. The method and use according to any one of embodiments 60 to 63, wherein preferably 50 U / mL of each nuclease is added.

[0130] 65. The method and use according to any one of the preceding embodiments, wherein the cells producing recombinant AAV particles have been obtained by transfection with PEI.

[0131] 66. The method and use according to any one of the preceding embodiments, wherein affinity chromatography is performed on a chromatography material comprising a cross-linked poly(styrene-divinylbenzene) matrix to which the affinity ligand is covalently conjugated.

[0132] 67. The method and use according to any one of the preceding embodiments, wherein affinity chromatography is performed on a chromatography material comprising a cross-linked poly(styrene-divinylbenzene) matrix to which the affinity ligand is covalently conjugated.

[0133] 68. The method and use according to embodiment 67, wherein the affinity ligand is a single domain antibody fragment (VHH).

[0134] 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.

[0135] 70. The method and use according to any of the preceding claims, wherein the affinity chromatography is performed on a chromatographic material comprising a cross-linked poly(styrene-divinylbenzene) matrix to which are covalently conjugated single domain antibody fragments (VHH) that specifically bind to AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 and synthetic serotypes based thereon.

[0136] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. For purposes of illustration and description, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed. DETAILED DESCRIPTION

[0137] The present invention is based, at least in part, on the discovery that the recovery rate, i.e., the yield (both capsid-based and genome-based) of recombinant AAV particles in AAV affinity chromatography is affected by / depends on the detergent used to lyse the cells producing the recombinant AAV particles prior to AAV affinity chromatography.

[0138] The present invention is further based, at least in part, on the discovery that the ratio of intact to empty recombinant AAV particles obtained in AAV affinity chromatography is affected by / depends on the detergent used to lyse the recombinant AAV particle-producing cells prior to AAV affinity chromatography.

[0139] It has been found that by using an alkyl polyglucoside detergent for lysing cells producing recombinant AAV particles prior to the AAV affinity chromatography step, both the yield of recombinant AAV particles (capsid-based and genome-based) and the ratio of intact recombinant AAV particles to empty recombinant AAV particles can be increased in AAV affinity chromatography.

[0140] definition

[0141] Methods and techniques that can be used to practice the present invention are described in, for example, Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND and Hames, BD, eds., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, JD et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, Second Edition, Alan R. Liss, Inc., NY (1987).

[0142] Derivatives of nucleic acids can be generated using recombinant DNA technology. Such derivatives can be modified, for example, by substitution, change, exchange, deletion or insertion at one or more nucleotide positions. Modification or derivatization can be performed, for example, by means of site-directed mutagenesis. Such modifications can be easily performed 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, BD and Higgins, SG, Nucleic acid hybridization-apractical approach (1985) IRL Press, Oxford, England).

[0143] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Likewise, the terms "a", "one or more", and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising", "including", and "having" may be used interchangeably.

[0144] It must be noted that, as used herein and in 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 forth. Likewise, the terms "a," "one or more," and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" may be used interchangeably.

[0145] The term "AAV helper function" refers to AAV-derived coding sequences (proteins) that can be expressed to provide AAV gene products and AAV particles, which in turn act in trans to carry out productive AAV replication and packaging. Thus, AAV helper functions include AAV open reading frames (ORFs), including rep and cap, and others, such as AAP for certain AAV serotypes. The rep gene expression product has been shown to have many functions, including: recognition, binding and cleavage of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap gene expression product (capsid) provides the necessary packaging function. AAV helper functions are used to supplement trans-AAV functions that are missing in the AAV vector genome.

[0146] The term "about" means a range of + / -20% of the value that follows. In certain embodiments, the term "about" means a range of + / -10% of the value that follows. In certain embodiments, the term "about" means a range of + / -5% of the value that follows.

[0147] As used herein, the terms "comprises," "including," "having," "having," "may," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. The term "comprising" also encompasses the term "including..." The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements set forth herein, whether or not explicitly set forth.

[0148] As used herein, the term "culturing" refers to the step of maintaining cells in a culture medium under conditions where the cells are transfected and produce AAV particles.

[0149] As used herein, the term "culture broth" refers to the contents of a bioreactor at the end of a culture."Culture broth" contains dead and live mammalian cells, mammalian cell debris, culture medium, recombinant products produced by mammalian cells, and other metabolites produced by mammalian cells.

[0150] The terms "empty particles" and "empty recombinant AAV particles" used interchangeably refer to AAV particles (i.e., vectors) that have an AAV protein capsid but lack, in whole or in part, nucleic acids encoding proteins or transcribed into target transcripts flanked by AAV ITRs. Therefore, empty particles cannot function to transfer nucleic acids encoding proteins or transcribed into target transcripts into target cells.

[0151] The term "endogenous" means that something occurs naturally within a cell; is naturally produced by the cell; similarly, an "endogenous locus / cell endogenous locus" is a locus that occurs naturally in a cell.

[0152] As used herein, the term "exogenous" refers to a nucleotide sequence that is not derived from a specific cell, but is introduced into the cell by a DNA delivery method (e.g., by transfection, electroporation, or transduction by a viral vector). Therefore, an exogenous nucleotide sequence is an artificial sequence, wherein the artificiality can be derived from, for example, a combination of subsequences from different sources (e.g., a combination of a recombinase recognition sequence with an SV40 promoter and a coding sequence for a green fluorescent protein is an artificial nucleic acid) or from a sequence (e.g., a sequence encoding only the extracellular domain of a membrane-bound receptor or a cDNA) or a partial deletion, or a nuclear base mutation. The term "endogenous" refers to a nucleotide sequence derived from a cell. An "exogenous" nucleotide sequence can have an "endogenous" counterpart with the same base composition, but wherein the sequence becomes an "exogenous" sequence, for example, by being introduced into a cell via recombinant DNA technology.

[0153] As used herein, the term "fed-batch cell culture" refers to a culture in which cells and culture medium are first supplied to a culture bioreactor, and additional culture nutrients are fed to the culture continuously or in discontinuous increments during the culture, with or without periodic cell and / or product harvesting prior to terminating the culture.

[0154] The terms "intact particle" and "intact recombinant AAV particle" used interchangeably refer to an AAV particle (i.e., a vector) having an AAV protein capsid and having packaged therein a nucleic acid encoding a protein or transcribed as a target transcript flanked by AAV ITRs. Thus, the intact particle is capable of transferring the packaged nucleic acid encoding a protein or transcribed as a target transcript into a target cell.

[0155] The terms "intact particle / empty particle ratio" and "ratio of intact recombinant AAV particles to empty recombinant AAV particles", which are used interchangeably, refer to the mathematical ratio of the number of intact recombinant AAV particles to the total number of recombinant AAV particles (intact and empty) in a sample containing recombinant AAV particles or in a recombinant AAV particle preparation. Since the number of intact recombinant AAV particles can be at most the same as the total number of recombinant AAV particles, the ratio can be at most 1. In general, the ratio is less than 1 and is expressed as a percentage. The number of intact recombinant AAV particles is determined by determining the amount of nucleic acid packaged by the recombinant AAV particles in the sample or preparation. This can be done by PCR, in particular digital droplet PCR (ddPCR). The total number of recombinant AAV particles is determined by determining the amount of capsid protein in the sample or preparation. This can be done by ELISA, in particular by a capsid protein-specific ELISA.

[0156] "Nucleic acid encoding AAV packaging proteins" generally refers to one or more nucleic acid molecules, which include nucleotide sequences that provide AAV functions deleted from AAV vectors, and the nucleic acid molecules are used to produce transduction-active recombinant AAV particles. Nucleic acids encoding AAV packaging proteins are generally used to provide expression of AAV rep and / or cap genes to supplement the missing AAV functions required for AAV replication; however, the nucleic acid construct lacks AAV ITR and can neither replicate nor package itself. 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 pIM29+45, which encode both rep gene expression products and cap gene expression products. See, for example, Samulski et al., J. Virol. 63 (1989) 3822-3828; and McCarty et al., J. Virol. 65 (1991) 2936-2945. Many plasmids encoding rep and / or cap gene expression products have been described (e.g., US 5,139,941 and US 6,376,237). Any of these nucleic acids encoding AAV packaging proteins may contain a DNA element or nucleic acid according to the present invention.

[0157] The term "nucleic acid encoding an auxiliary protein" generally refers to one or more nucleic acid molecules that include nucleotide sequences encoding proteins and / or RNA molecules that provide adenoviral auxiliary functions. Plasmids with nucleic acids encoding auxiliary proteins can be transfected into suitable cells, where the plasmid is then able to support the production of AAV particles in the cells. Any of these nucleic acids encoding auxiliary proteins may comprise a DNA element or nucleic acid according to the present invention. The term explicitly excludes infectious viral particles as they occur in nature, such as adenovirus, herpes virus or vaccinia virus particles.

[0158] As used herein, the term "operably connected" refers to the juxtaposition of two or more components, wherein the relationship of these components allows them to play a role in an expected manner. For example, if a promoter and / or enhancer plays a role in regulating the transcription of a coding sequence / open reading frame / gene, the promoter and / or enhancer are operably connected to the coding sequence / open reading frame / gene. In certain embodiments, the DNA sequence of "operably connected" is adjacent. In certain embodiments, for example, when two protein coding regions (such as a secretory leader and a polypeptide) must be joined, these sequences are adjacent and in the same reading frame. In certain embodiments, the promoter that is operably connected is located upstream of the coding sequence / open reading frame / gene and can be adjacent to the coding sequence / open reading frame / gene. In certain embodiments, for example, about the enhancer sequence regulating the expression of the coding sequence / open reading frame / gene, these two components can be operably connected, but not adjacent. If an enhancer increases the transcription of the coding sequence / open reading frame / gene, the enhancer is operably connected to the coding sequence / open reading frame / gene. An operably linked enhancer may be located upstream, within, or downstream of a coding sequence / open reading frame / gene and may be located at a considerable distance from the promoter of the coding sequence / open reading frame / gene.

[0159] The term "packaging protein" refers to non-AAV derived viral and / or cellular functions on which AAV replication depends. Thus, the term embodies proteins and RNAs required for AAV replication, including those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, Cap expression product synthesis, and AAV capsid assembly. Virus-based helper functions can be derived from any of the known helper viruses, such as adenovirus, herpes virus (except herpes simplex virus type I) and vaccinia virus.

[0160] As used herein, "AAV packaging proteins" refer to AAV-derived sequences that act in trans to carry out productive AAV replication. Therefore, AAV packaging proteins are encoded by the major AAV open reading frames (ORFs), rep and cap. The rep protein has been shown to have many functions, including: recognition, binding and cleavage of the AAV origin of DNA replication; DNA helicase activity; and regulation of transcription from AAV (or other heterologous) promoters. The cap (capsid) protein provides the necessary packaging function. AAV packaging proteins are used herein to supplement the trans-AAV functions missing in AAV vectors.

[0161] As used herein, the term "recombinant cell" refers to a cell that has been genetically modified, such as, for example, a cell that expresses a polypeptide of interest or produces rAAV particles of interest and can be used to produce the polypeptide of interest or rAAV particles of interest on any scale. For example, a "mammalian cell comprising an exogenous nucleotide sequence" that has undergone recombinase-mediated cassette exchange (RMCE) and has thus introduced the coding sequence of the polypeptide of interest into the host cell genome is a "recombinant cell". Although the cell is still capable of further RMCE reactions, it is not desirable to do so.

[0162] "Recombinant AAV vectors" are derived from the wild-type genome of a virus (such as AAV) by using molecular methods to remove the wild-type genome from the virus (e.g., AAV) and replace the wild-type genome with a non-native nucleic acid (such as a nucleic acid transcribed into a transcript or encoding a protein). For AAV, one or two inverted terminal repeat (ITR) sequences of the wild-type AAV genome are usually retained in the recombinant AAV vector. "Recombinant" AAV vectors are distinguished from the wild-type viral AAV genome because all or part of the viral genome has been replaced with a non-native (i.e., heterologous) sequence relative to the viral genomic nucleic acid. Therefore, the incorporation of non-native sequences defines a viral vector (e.g., AAV) as a "recombinant" vector, which in the case of AAV can be referred to as a "rAAV vector."

[0163] Recombinant vector (e.g., AAV) sequences can be packaged - referred to herein as "particles" - for subsequent cell infection (transduction) in vitro, in vitro, or in vivo. When the recombinant vector sequence is encapsulated or packaged into an AAV particle, the particle may also be referred to as "rAAV". Such particles include 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.

[0164] As used herein, the term "serotype" is a distinction based on serologically different AAV capsids. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to antibodies to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Although AAV variants, including capsid variants, may be serologically indistinguishable from reference AAV or other AAV serotypes, they differ in at least one nucleotide or amino acid residue compared to the reference AAV serotype or other AAV serotypes.

[0165] Under the traditional definition, serotype refers to the target virus that has been tested for serum, which has specificity for neutralizing activity to all existing and characterized serotypes, and no antibodies to neutralize the target virus are found. With the discovery of more naturally occurring virus isolates and / or the generation of capsid mutants, there may or may not be serological differences with any of the currently existing serotypes. Therefore, in the case where a new virus (such as AAV) does not have serological differences, the new virus (such as AAV) will be a subgroup or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not yet been performed on mutant viruses with modified capsid sequences to determine whether they have another serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid duplication, the term "serotype" refers broadly to serologically different viruses (such as AAV) and serologically no different viruses (such as AAV), which may be within a subgroup or variant of a given serotype.

[0166] The term "transgene" is used herein for convenience to refer to a nucleic acid that is intended to be or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a gene, that is transcribed into a transcript or encodes a polypeptide or protein.

[0167] The term "Triton CG 110" means an alkyl polyglucoside detergent. Triton CG 110 has CAS number 68515-73-1. Triton CG 110 is a mixture of 58.0 (w / v)% to 62.0 (w / v)% D-pyranose oligodecyloctyl glycoside and 38.0 (w / v)% to 42.0 (w / v)% water.

[0168] "Vector" refers to a portion of a recombinant plasmid sequence that is ultimately packaged or encapsulated directly or in the form of a single strand or RNA to form a virus (e.g., AAV) particle. In the case where a recombinant plasmid is used to construct or manufacture a recombinant virus particle, the virus particle does not include a "plasmid" portion that does not correspond to the vector sequence of the recombinant plasmid. This non-vector portion of the recombinant plasmid is called a "plasmid backbone," which is important for cloning and amplification of the plasmid, a process required for propagation and recombinant virus production, but it itself is not packaged or encapsulated into a virus (e.g., AAV) particle. Therefore, "vector" refers to a nucleic acid packaged or encapsulated by a virus particle (e.g., AAV).

[0169] Recombinant cells

[0170] Generally speaking, for efficient and large-scale production of recombinant AAV particles (rAAV particles), cells express and, if possible, also secrete the rAAV particles. Such cells are referred to as "recombinant cells" or "recombinant production cells."

[0171] To generate "recombinant producer cells," appropriate mammalian cells are transfected with the nucleic acid sequences required for production of the rAAV particles (including the desired AAV helper functions).

[0172] For expressing coding sequence, i.e. open reading frame, additional regulatory elements are needed, such as promoter and polyadenylation signal (sequence). Therefore, open reading frame is operably connected to the additional regulatory elements for transcription. This can be achieved by being integrated into the so-called expression cassette. The minimum regulatory elements required for the expression cassette to work in mammalian cells are the promoter working in the mammalian cell, the promoter is located at the upstream of the open reading frame, i.e. 5', and the polyadenylation signal (sequence) working in the mammalian cell, the polyadenylation signal is located at the downstream of the open reading frame, i.e. 3'. In addition, the terminator sequence may be present in the 3' of the polyadenylation signal (sequence). For expression, promoter, open reading frame / coding region and polyadenylation signal sequence must be arranged in an operably connected form.

[0173] Similarly, nucleic acids that are transcribed into RNA encoding non-proteins are referred to as "RNA genes". Also for the expression of RNA genes, additional regulatory elements are required, such as promoters and transcription termination signals or polyadenylation signals (sequences). The nature and positioning of such elements depend on the RNA polymerase intended to drive RNA gene expression. Therefore, RNA genes are also typically integrated into expression cassettes.

[0174] In the case of AAV particles, which consist of different (monomeric) capsid polypeptides and single-stranded DNA molecules and also require other adenoviral helper functions for production and packaging, a number of expression cassettes are required, which contain different open reading frames / coding sequences. In this case, at least an expression cassette is required for each of the transgene, the different polypeptides forming the capsid of the AAV vector, the required helper functions and the VA RNA. Thus, a separate expression cassette is required for each of the helper functions E1A, E1B, E2A, E4orf6, VA RNA, rep and cap genes. HEK293 cells constitutively express the E1A and E1B helper functions.

[0175] In certain embodiments of all aspects and embodiments of the invention, each of the expression cassettes comprises a promoter, an open reading frame / coding sequence or an RNA gene and a polyadenylation signal sequence and / or a terminator sequence in the 5' to 3' direction. 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 there is a polyadenylation signal sequence or a polyU terminator. See, e.g., Song et al. Biochemical and Biophysical Research Communications 323 (2004) 573–578. In certain embodiments, the expression cassette comprises an RNA gene, the promoter is a type 2 Pol III promoter and a polyU terminator sequence.

[0176] In certain embodiments of all aspects and embodiments of the 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 a bGH (bovine growth hormone) polyA signal sequence, and the terminator is hGT (human gastrin terminator).

[0177] In certain embodiments of all aspects and embodiments of the invention, the promoter is the human CMV promoter with intron A, the polyadenylation signal sequence is the bGH polyadenylation signal sequence and the terminator is hGT, except for the expression cassette for the RNA gene and the expression cassette for the selection marker, wherein for the selection marker, the promoter is the SV40 promoter and the polyadenylation signal sequence is the SV40 polyadenylation signal sequence and the terminator is not present, and wherein 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.

[0178] Adeno-associated virus (AAV)

[0179] For a general review of AAV and adenovirus or herpes helper functions, see Berns and Bohensky, Advances in Virus Research, Academic Press., 32 (1987) 243-306. The genome of AAV is described in Srivastava et al., J. Virol., 45 (1983) 555-564. Design considerations for constructing recombinant AAV vectors are described in US 4,797,368 (see also WO 93 / 24641). Other references describing AAV vectors are West et al., Virol. 160 (1987) 38-47; Kotin, Hum. Gene Ther. 5 (1994) 793-801; and Muzyczka J. Clin. Invest. 94 (1994) 1351. The construction of recombinant AAV vectors is described in US 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

[0180] Adeno-associated virus (AAV) is a replication-defective parvovirus. It can only replicate in cells where certain viral functions are provided by a co-infecting helper virus, such as adenovirus, herpes virus, and in some cases poxvirus, such as vaccinia. Nevertheless, AAV can replicate in almost any cell line of human, simian or rodent origin, provided that appropriate helper virus functions are present.

[0181] If helper viral genes are not present, AAV establishes latency in its host cells. Its genome integrates into a specific site in chromosome 19 [(Chr)19(q13.4)], called adeno-associated virus integration site 1 (AAVS1). For specific serotypes, such as AAV-2, other integration sites have been found, such as, for example, on chromosome 5 [(Chr)5(p13.3)], called AAVS2, and on chromosome 3 [(Chr)3(p24.3)], called AAVS3.

[0182] AAV is divided 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 a hundred sequences corresponding to different branches of AAV have been identified.

[0183] The capsid protein type and symmetry determine the tissue tropism of the corresponding AAV. For example, AAV-2, AAV-4, and AAV-5 are specific for the retina, AAV-2, AAV-5, AAV-8, AAV-9, and AAVrh-10 are specific for the brain, AAV-1, AAV-2, AAV-6, AAV-8, and AAV-9 are specific for cardiac tissue, AAV-1, AAV-2, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, and AAV-10 are specific for the liver, and AAV-1, AAV-2, AAV-5, and AAV-9 are specific for the lung.

[0184] Pseudotyping refers to a process that involves cross-packaging of the AAV genome between different serotypes, i.e., the genome is packaged with capsid proteins of different origin.

[0185] The wild-type AAV genome size is about 4.7 kb. The AAV genome further comprises two overlapping genes, called rep and cap, which contain multiple open reading frames (see, for example, Srivastava et al., J. Viral., 45 (1983) 555-564; Hermonat et al., J. Viral. 51 (1984) 329-339; Tratschin et al., J. Virol., 51 (1984) 611-619). The open reading frame encoding the Rep protein provides four proteins of different sizes, which are called Rep78, Rep68, Rep52 and Rep40. These are involved in the replication, rescue and integration of AAV. The open reading frame encoding the Cap protein provides four proteins, called VP1, VP2, VP3 and AAP. VP1, VP2 and VP3 are part of the protein capsid of the AAV particle. The combined rep and cap open reading frames are flanked by so-called inverted terminal repeats (ITRs) at their 5' and 3' ends. 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.

[0186] For example, in the case of AAV of serotype 2 (AAV-2), each ITR has a length of 145 nucleotides and is flanked by 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 functions as a primer during viral replication. The remaining 20 non-paired nucleotides are represented as D sequences.

[0187] The AAV genome contains three transcription promoters P5, P19 and P40 (Laughlin et al., Proc. Natl. Acad. Sci. USA 76 (1979) 5567-5571) for the expression of rep and cap genes.

[0188] ITR sequence must be present in the coding region in cis. ITR provides a functional origin of replication (ori), is integrated into the target cell genome required signal, and effectively excised and rescued from the host cell chromosome or recombinant plasmid. ITR further comprises an origin of replication-like element, such as Rep protein binding site (RBS) and terminal resolution site (TRS). It has been found that ITR itself can have the function of a transcription 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).

[0189] The trans organization of the rep and cap gene products is required for the replication and encapsidation of the viral single-stranded DNA genome, respectively.

[0190] The rep locus comprises two internal promoters, called P5 and P19. It comprises open reading frames for four kinds of proteins. Promoter P5 is operably connected to a nucleotide sequence providing a non-spliced ​​4.2kb mRNA encoding Rep protein Rep78 (chromatin nickase that blocks the cell cycle) and a spliced ​​3.9kb mRNA encoding Rep protein Rep68 (site-specific endonuclease). Promoter P19 is operably connected to a nucleotide sequence providing a non-spliced ​​mRNA encoding Rep protein Rep52 and a spliced ​​3.3kb mRNA encoding Rep protein Rep40 (DNA helicase for accumulation and packaging).

[0191] The two larger Rep proteins, Rep78 and Rep68, are essential for AAV duplex DNA replication, whereas the smaller Rep proteins, Rep52 and Rep40, appear to be essential for progeny single-stranded DNA accumulation (Chejanovsky & Carter, Virology 173 (1989) 120-128).

[0192] The larger Rep proteins Rep68 and Rep78 can specifically bind to the hairpin conformation of AAV ITR. They exhibit defined enzymatic activity, which is required for resolving replication at the AAV termini. Expression of Rep78 or Rep68 may be sufficient for the formation of infectious particles (Holscher, C. et al. J. Virol. 68 (1994) 7169-7177 and 69 (1995) 6880-6885).

[0193] All Rep proteins, primarily Rep78 and Rep68, are believed to exhibit regulatory activities such as induction and repression of AAV genes and inhibition of cell growth (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894; Labow et al., Mol. Cell. Biol., 7 (1987) 1320-1325; Khleif et al., Virology, 181 (1991) 738-741).

[0194] Recombinant overexpression of Rep78 produces a phenotype of reduced cell growth caused by inducing DNA damage, thereby arresting host cells in the S phase, thereby promoting latent infection of the virus (Berthet, C. et al., Proc. Natl. Acad. Sci. USA 102 (2005) 13634-13639).

[0195] Tratschin et al. reported that the P5 promoter is negatively autoregulated by Rep78 or Rep68 (Tratschin et al., Mol. Cell. Biol. 6 (1986) 2884-2894). Due to the toxic effects of Rep protein expression, it has been reported that after stable integration of AAV, expression for some cell lines is only very low (see, e.g., Mendelson et al., Virol. 166 (1988) 154-165).

[0196] The cap locus contains a promoter, called P40. The promoter P40 is operably linked to a nucleic acid sequence that provides 2.6 kb mRNA, which encodes the Cap proteins VP1 (87 kDa, non-spliced ​​mRNA transcripts), VP2 (72 kDa, from spliced ​​mRNA transcripts) and VP3 (61 kDa, from alternative start codons) by alternative splicing and using alternative start codons. VP1 to VP3 constitute the components of the viral capsid. The capsid has the function of binding to cell surface receptors and allowing intracellular transport of the virus. VP3 accounts for about 90% of the total protein of the viral particle. However, all three proteins are essential for effective capsid production.

[0197] Inactivation of all three capsid proteins VP1 to VP3 reportedly prevents the accumulation of single-stranded progeny AAV DNA. Mutations at the amino terminus of VP1 (“Lip negative” or “Inf negative”) still allow single-stranded DNA to assemble into viral particles, thereby greatly reducing infectious titers.

[0198] The AAP open reading frame encodes the assembly activation protein (AAP). It is about 22 kDa in size and transports native VP protein to the nucleolar region for capsid assembly. This open reading frame is located upstream of the VP3 protein coding sequence.

[0199] A single AAV particle contains only one single-stranded DNA molecule. This can be either the "plus" strand or the "minus" strand. AAV virus particles containing DNA molecules are infectious. Within infected cells, the parental infectious single strand is converted to a double strand and subsequently amplified. Amplification produces a large number of double-stranded DNA molecules, which displace the single strand and are packaged into capsids.

[0200] Adeno-associated virus (AAV) vectors can transduce both dividing and quiescent cells. It can be assumed that transgenes introduced into target cells using AAV vectors will be expressed long term. One disadvantage of using AAV vectors is that the size of the transgene that can be introduced into cells is limited.

[0201] Viral vectors such as parvovirus particles, including AAV serotypes and variants thereof, provide a way to deliver nucleic acids to protein-encoding cells in vitro, in vitro, and in vivo, so that the cells express the encoded protein. AAV is a virus that can be used as a gene therapy vector because they can penetrate cells and introduce nucleic acids / genetic material so that nucleic acids / genetic material can be stably maintained in cells. In addition, for example, these viruses can introduce nucleic acids / genetic material into specific sites. Since AAV is not related to human pathogenic diseases, AAV vectors are able to deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV pathogenesis or disease.

[0202] AAV particles used as vehicles for efficient gene delivery have many characteristics required for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also showed no persistent toxicity and little or no detectable immune response. AAV is known to infect a variety of cell types in vivo and in vitro by receptor-mediated endocytosis or by transcytosis. These vector systems have been tested in humans targeting retinal epithelium, liver, skeletal muscle, airways, brain, joints, and hematopoietic stem cells.

[0203] Recombinant AAV particles generally do not include viral genes associated with pathogenesis. Such vectors generally have one or more of the wild-type AAV genes that are completely or partially deleted, such as rep and / or cap genes, but retain at least one functional flanking ITR sequence, which is necessary for rescuing, replicating, and packaging the recombinant vector into AAV particles. For example, only the essential parts of the vector are included, such as ITR and LTR elements, respectively. Therefore, the AAV vector genome will include cis sequences (e.g., functional ITR sequences) required for replication and packaging.

[0204] Recombinant AAV vectors and methods and uses thereof include any viral strain or serotype. As a non-limiting example, the recombinant AAV vector can be based on any AAV genome, such as, for example, 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 different from each other. As a non-limiting example, a recombinant AAV vector based on a serotype genome can be the same as one or more of the capsid proteins that package the vector. In addition, 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, and at least one of the three capsid proteins can be 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, AAV rh74 and AAV 7m8 capsids.

[0205] In certain embodiments of all aspects and embodiments of the 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), for example, as shown in WO 2013 / 158879, WO 2015 / 013313 and US2013 / 0059732 (disclosing LK01, LK02, LK03, etc.).

[0206] In certain embodiments of all aspects and embodiments of the invention, the rAAV particle comprises a capsid sequence having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAV Rh74, or AAV 7m8 capsid sequence.

[0207] In certain embodiments of all aspects and embodiments of the invention, the rAAV particle comprises an ITR sequence having 70% or greater sequence identity to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 ITR sequence.

[0208] Recombinant particles (e.g., rAAV particles) can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are particularly useful for in vivo or ex vivo administration and delivery to a subject. In certain embodiments, the pharmaceutical composition contains a pharmaceutical carrier or excipient. Such excipients include any medicament that does not itself induce an immune response that is harmful to the individual receiving the composition and can be administered without excessive toxicity.

[0209] Protocols for generating adenoviral vectors have been described in US 5,998,205; US 6,228,646; US 6,093,699; US 6,100,242; WO 94 / 17810 and WO 94 / 23744, the entire contents of which are incorporated herein by reference.

[0210] Recombinant AAV particles (rAAV particles)

[0211] Different methods for generating rAAV particles are known in the art. For example, transfection using AAV plasmids and AAV auxiliary sequences and co-infection with an AAV helper virus (e.g., adenovirus, herpes virus or vaccinia virus), or transfection using recombinant AAV plasmids, AAV helper plasmids and auxiliary function plasmids. Non-limiting methods for generating rAAV particles are described in, for example, US 6,001,650, US 6,004,797, WO 2017 / 096039 and WO 2018 / 226887. After recombinant rAAV particle production (i.e., particle generation in a cell culture system), rAAV particles can be obtained from host cells and cell culture supernatants and purified.

[0212] For the production of recombinant AAV particles, it is necessary to express the Rep and Cap proteins, the accessory proteins E1A, E1B, E2A and E4orf6, and the adenovirus VA RNA in a single mammalian cell. The accessory proteins E1A, E1B, E2A and E4orf6 can be expressed using any promoter, as shown by Matsushita et al. (Gene Ther. 5 (1998) 938-945), in particular the CMV IE promoter. Therefore, any promoter can be used.

[0213] In general, in order to produce recombinant AAV particles, different complementary plasmids are co-transfected into host cells. One of these plasmids contains a transgene sandwiched between two cis-acting AAV ITRs. The deleted AAV elements (i.e., open reading frames for Rep and Cap proteins) required for offspring recombinant genome replication and subsequent packaging are contained in the second plasmid in trans. Overexpression of Rep protein causes an inhibitory effect on cell growth (Li, J. et al., J. Virol. 71 (1997) 5236-5243). In addition, AAV replication requires a third plasmid containing genes of helper viruses (i.e., E1, E4orf6, E2A and VA from adenovirus).

[0214] To reduce the number of plasmids required, Rep, Cap, and adenoviral helper genes can be combined on a single plasmid.

[0215] Alternatively, the host cell may have stably expressed the E1 gene product. Such cells are HEK293 cells. The human embryonic kidney clone, denoted 293, was generated as early as 1977 by integrating adenovirus DNA into human embryonic kidney cells (HEK cells) (Graham, FL 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 covers E1A and E1B genes and adenovirus packaging signals (Louis, N. et al., Virology 233 (1997) 423-429).

[0216] When HEK293 cells are used, the deleted E2A, E4orf6 and VA genes can be introduced by co-infection with adenovirus or by co-transfection with plasmids expressing E2A, E4orf6 and VA (see, e.g., Samulski, RJ et al., J. Virol. 63 (1989) 3822-3828; Allen, JM et al., J. Virol. 71 (1997) 6816-6822; Tamayose, K. et al., Hum. Gene Ther. 7 (1996) 507-513; Flotte, TR et al., Gene Ther. 2 (1995) 29-37; Conway, JE et al., J. Virol. 71 (1997) 8780-8789; Chiorini, JA et al., Hum. Gene Ther. 6 (1995) 1531-1541; Ferrari, FK et al., J. Virol. 70 (1996) 3227-3234; Salvetti, A. et al., Hum. Gene Ther. 9 (1998) 695-706; Xiao, X. et al., J. Virol. 72 (1998) 2224-2232; Grimm, D. et al., Hum. Gene Ther. 9 (1998) 2745-2760; Zhang, X. et al., Hum. Gene Ther. 10 (1999) 2527-2537). Alternatively, adenovirus / AAV or herpes simplex virus / AAV hybrid vectors may be used (see, e.g., Conway, JE et al., J. Virol. 71 (1997) 8780-8789; Johnston, KM et al., Hum. Gene Ther. 8 (1997) 359-370; Thrasher, AJ et al., Gene Ther. 2 (1995) 481-485; Fisher, JK et al., Hum. Gene Ther. 7 (1996) 2079-2087; Johnston, KM et al., Hum. Gene Ther. 8 (1997) 359-370).

[0217] To restrict transgene activity to specific tissues, ie, to limit integration sites, the transgene can be operably linked to an inducible or tissue-specific promoter (see, eg, Yang, Y. et al. Hum. Gene. Ther. 6 (1995) 1203-1213).

[0218] E1A, E1B, E2 and E4

[0219] The coding sequences (open reading frames) of E1A and E1B may be derived from human adenoviruses, such as, for example, in particular human adenovirus serotype 2 or serotype 5. Exemplary sequences of human Ad5 (adenovirus serotype 5) are found in GenBank entries X02996, AC_000008, and the sequence of an exemplary human Ad2 is found in GenBank entry AC_000007. Nucleotides 505 to 3522 comprise the nucleic acid sequence encoding E1A and E1B of human adenovirus serotype 5. Plasmid pSTK146 reported in EP 1 230 354 and plasmids pGS119 and pGS122 reported in WO2007 / 056994 may also be used as sources of E1A and E1B open reading frames.

[0220] E1A is the first viral helper gene expressed after adenoviral DNA enters the cell nucleus. The E1A gene encodes 12S and 13S proteins, which are based on the same E1A mRNA by alternative splicing. The expression of 12S and 13S proteins causes the activation of other viral functions E1B, E2, E3 and E4. In addition, the expression of 12S and 13S proteins forces the cell to enter the S phase of the cell cycle. If only E1A-derived proteins are expressed, the cell will die (apoptosis).

[0221] E1B is the second viral helper gene 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 55kDa transcript and a second 19kDa transcript. The E1B 55kDa protein is involved in regulating the cell cycle, preventing mRNA trafficking in cells late in infection, and preventing E1A-induced apoptosis. The E1B 19kDa protein is involved in preventing E1A-induced apoptosis.

[0222] The E2 genes encode different proteins. The E2A transcript encodes the single-stranded binding protein (SSBP), which is essential for AAV replication.

[0223] Similarly, the E4 gene also encodes multiple proteins. The 34kDa protein (E4orf6) derived from the E4 gene, together with the E1B 55kDa protein, prevents the accumulation of cellular mRNA in the cytoplasm, but also promotes the transport of viral RNA from the nucleus to the cytoplasm.

[0224] Adenovirus VA RNA gene

[0225] Virus-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 from a type 2 polymerase III promoter by RNA polymerase III (see, e.g., 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.

[0226] Ma, Y. and Mathews, MB (J. Virol. 70 (1996) 5083-5099) used a phylogenetic approach to study the structure, function and evolution of adenovirus-associated RNA. They provided an alignment based on 47 known human adenovirus serotypes and a consensus VA RNA sequence. The disclosure is hereby incorporated by reference into this application in its entirety.

[0227] VA RNA, VAI and VAII consist of 157 to 160 nucleotides (nt).

[0228] Depending on the serotype, adenovirus contains one or two VA RNA genes. VA RNAI is thought to play a major proviral role, while VA RNAII can partially compensate for the absence of VA RNAI (Vachon, VK and Conn, GL, Virus Res. 212 (2016) 39-52).

[0229] VA RNA is not essential, but plays an important role in efficient viral growth by overcoming cellular antiviral mechanisms. That is, although VA RNA is not essential for viral growth, adenoviruses lacking VA RNA fail to grow during the initial steps of vector generation, where only a few copies of the viral genome are present per cell, probably because viral genes other than VARNA that block cellular antiviral mechanisms may not be adequately expressed (see Maekawa, A. et al. Nature Sci. Rep. 3 (2013) 1136).

[0230] Maekawa, A. et al. (Nature Sci. Rep. 3 (2013) 1136) reported the efficient production of adenoviral vectors lacking genes for virus-associated RNAs that disrupt the cellular RNAi machinery, in which HEK293 cells that constitutively and highly express flippase recombinase were infected to obtain VA RNA-deficient adenoviruses through FLP recombinase-mediated excision of the VA RNA locus.

[0231] Human adenovirus 2VA RNAI corresponds to nucleotides 10586-10810 of the sequence of GenBank entry AC_000007. Human adenovirus 5VA RNAI corresponds to nucleotides 10579-10820 of the sequence of GenBank entry AC_000008.

[0232] Methods for producing rAAV particles

[0233] Carter et al. have shown that the entire rep and cap open reading frames in the wild-type AAV genome can be deleted and replaced with transgenes (Carter, BJ, "Handbook of Parvoviruses", P. Tijssen ed., CRC Press, pp. 155-168 (1990)). In addition, it is reported that ITRs must be maintained to retain the functions of transgene replication, rescue, packaging and integration into the target cell genome.

[0234] When cells containing the corresponding viral helper genes are transduced with AAV vectors, or vice versa, when cells containing integrated AAV provirus are transduced with appropriate helper virus, the AAV provirus is activated and enters the lytic infection cycle again (Clark, KR et al., Hum. Gene Ther. 6 (1995) 1329-1341; Samulski, RJ, Curr. Opin. Genet. Dev. 3 (1993) 74-80).

[0235] The production cells contain rep and cap gene sequences, and a transgenic cassette flanked by ITR sequences on one or more plasmids retained by drug selection. The production of rAAV particles in these cell lines generally occurs after they have been infected with the required helper functions. Therefore, the cells are infected with a replication-competent AdV (usually wild-type Ad5) or a plasmid containing the corresponding helper genes to provide helper viral proteins and initiate rAAV particle production. The packaging cell line is different from the production cell line because it only contains the rep and cap genes.

[0236] The method according to the invention comprises the step of transducing mammalian cells with a nucleic acid (e.g., a plasmid) comprising all the elements required for producing recombinant AAV particles. Thus, when the plasmid encodes viral packaging proteins and / or auxiliary proteins, the cells can produce recombinant viral particles comprising a nucleic acid encoding a protein of interest or a nucleic acid comprising a sequence that is transcribed into a transcript of interest.

[0237] The present invention provides a recombinant AAV viral particle production platform that includes features that distinguish it from the current "industry standard" recombinant AAV particle production process, which features include a lysis step according to the present invention.

[0238] More generally, cells transfected or transduced with DNA for recombinant production of AAV particles can be referred to as "recombinant cells". Such cells may be any mammalian cell that has been used as a receptor for nucleic acids (plasmids) encoding packaging proteins (such as AAV packaging proteins), nucleic acids (plasmids) encoding auxiliary proteins, and nucleic acids (plasmids) encoding proteins or transcribed into target transcripts (i.e., transgenes placed between two AAVITRs). The term includes the descendants of the original cells that have been transduced or transfected. It should be understood that the descendants of a single parent cell may not necessarily be exactly the same as the original parent in morphology or genome or total nucleic acid complement due to natural, accidental or deliberate mutations.

[0239] Many cell growth mediums suitable for maintaining cell viability or providing cell growth and / or proliferation are commercially available. The example of such culture medium includes serum-free eukaryotic growth medium, such as for maintaining mammalian (e.g., human) cell viability or providing mammalian (e.g., human) cell growth medium. Non-limiting examples include Ham's F12 or F12K culture medium (Sigma-Aldrich), FreeStyle (FS) F17 culture medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific) and mixtures thereof. Such culture medium 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.

[0240] Thus, provided herein is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector using the lysis step according to the present invention, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript.

[0241] For this purpose, three plasmids are co-transfected into mammalian cells. The transgenic plasmid encodes an expression cassette cloned between the AAV ITRs, while the rep and cap genes are provided in trans by co-transfection of a second packaging plasmid (rep / cap plasmid) to ensure AAV replication and packaging. The third plasmid, also called a helper plasmid, contains minimal helper viral factors (usually adenoviral E2A, EV and VA genes) but lacks the AAV ITRs.

[0242] One aspect of the present invention is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

[0243] (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding accessory proteins;

[0244] (ii) providing a plasmid comprising a nucleic acid encoding a protein of interest or transcribed as a transcript of interest, the transcript being inserted between the AAV ITRs;

[0245] (iii) contacting one or more mammalian cells with the provided plasmid and performing one of the following: further adding a transfection reagent, and optionally incubating the plasmid / transfection reagent / cell mixture; or providing a physical means such as an electric current to introduce the nucleic acid into the cells;

[0246] (iv) culturing the transfected cells;

[0247] (v) harvesting the cultured cells and medium to produce a mammalian cell culture broth;

[0248] (vi) lysing the cells by contacting the mammalian cell culture broth with an alkyl polyglucoside detergent to produce a mammalian cell culture broth lysate; and

[0249] (vii) optionally isolating recombinant AAV particles from the culture broth lysate using AAV affinity chromatography;

[0250] Recombinant AAV particles are thereby produced that contain nucleic acid encoding a protein of interest or that is transcribed into a transcript of interest.

[0251] One aspect of the present invention is a method for producing a recombinant AAV vector or an AAV particle comprising the recombinant AAV vector, wherein the recombinant AAV vector comprises a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising the following steps:

[0252] (i) providing one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding accessory proteins;

[0253] (ii) providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript;

[0254] (iii) or

[0255] (a) generating stably transfected cells by contacting one or more mammalian cells with the plasmid provided in (i), by one of the following operations: further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture, or providing a physical means such as an electric current to introduce nucleic acid into the cells; selecting a first stably transfected cell; contacting the selected first stably transfected cell with the plasmid provided in (ii) and performing one of the following operations: further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture, or providing a physical means such as an electric current to introduce nucleic acid into the cells; or

[0256] (b) generating transiently transfected cells by contacting one or more mammalian cells with the plasmids provided by (i) and (ii), and performing one of the following operations: further adding a transfection reagent and optionally incubating the plasmid / transfection reagent / cell mixture, or providing a physical means such as an electric current to introduce the nucleic acid into the cells;

[0257] Thereby, transfected cells are generated;

[0258] (iv) culturing the transfected cells of (iii);

[0259] (v) harvesting the cultured cells and medium to produce a mammalian cell culture broth;

[0260] (vi) lysing the cells by contacting the mammalian cell culture broth with an alkyl polyglucoside detergent to produce a mammalian cell culture broth lysate; and

[0261] (vii) optionally isolating recombinant AAV particles from mammalian cell culture broth lysate using AAV affinity chromatography;

[0262] Recombinant AAV particles are thereby produced that contain nucleic acid encoding a protein of interest or that is transcribed into a transcript of interest.

[0263] Introduction of nucleic acids (plasmids) into cells can be done in a variety of ways.

[0264] The various methods of transferring DNA to mammalian cells have been reported in the art. These can be used according to the method of the present invention. In certain embodiments of all aspects and embodiments, electroporation, nuclear transfection 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), cationic polymers (such as polyethyleneimine, DEAE-dextran) or cationic lipids (lipofection) for nucleic acid transfer / transfection are used. Calcium phosphate and polyethyleneimine are the most commonly used reagents in the transfection for larger-scale nucleic acid transfer (see, for example, Baldi et al., Biotechnol.Lett.29 (2007) 677-684), wherein polyethyleneimine is preferred.

[0265] Growing in serum-free suspension culture and using PEI as the transfection reagent improves the efficiency and reproducibility of transfection conditions, allowing real-time scale-up of AAV production using shake flasks, wave-type, or stirred tank bioreactors.

[0266] In certain embodiments of all aspects and embodiments, nucleic acid (plasmid) is provided in the form of a composition in combination with polyethyleneimine (PEI), optionally in combination with cells. In certain embodiments, the composition includes a plasmid / PEI mixture having multiple components: (a) one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding auxiliary proteins; (b) a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript; and (c) a polyethyleneimine (PEI) solution. In certain embodiments, the molar ratio of the plasmid ranges from about 1:0.01 to about 1:100, or a molar ratio ranges from 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.

[0267] In certain embodiments of all aspects and embodiments, the composition further comprises a cell. In certain embodiments, the cell is contacted with the plasmid / PEI mixture of components (a), (b) and / or (c).

[0268] In certain embodiments of all aspects and embodiments, the composition optionally combined with the cells further comprises free PEI. In certain embodiments, the cells are contacted with free PEI.

[0269] In certain embodiments of all aspects and embodiments, the cell has been contacted with a mixture of components (a), (b) and / or (c) for at least about 4 hours, or from about 4 hours to about 140 hours, or from about 4 hours to about 96 hours. In a preferred embodiment, the cell has been contacted with a mixture of components (a), (b) and / or (c) and optionally free PEI for at least about 4 hours.

[0270] Said composition can further comprise plasmid or / and cell.Such plasmid and cell can contact with free PEI.In certain embodiments, plasmid and / or cell have contacted with free PEI at least about 4 hours, or about 4 hours to about 140 hours, or about 4 hours to about 96 hours.

[0271] The method according to the present invention also includes the step of transfecting cells. Therefore, the method includes the following steps: providing one or more plasmids; providing a solution comprising polyethyleneimine (PEI); and mixing the plasmid 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 methods, the cells are then contacted with the plasmid / PEI mixture to produce a plasmid / PEI cell culture; free PEI is then added to the produced plasmid / PEI cell culture to produce free PEI / plasmid / PEI cell culture; the produced free PEI / plasmid / PEI cell culture is then incubated for at least about 4 hours, thereby producing transfected cells. In certain embodiments, the plasmid comprises one or more or all of a rep open reading frame, a cap open reading frame, an E1A, E1B, E2 and an E4orf6 open reading frame, and a nucleic acid encoding a protein or transcribed into a target transcript.

[0272] Further, the method according to the present invention includes a step for producing transfected cells (which produce recombinant AAV vectors or AAV particles), the method comprising one or more plasmids comprising nucleic acids encoding AAV packaging proteins and / or nucleic acids encoding auxiliary proteins; providing a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript; providing a solution comprising polyethyleneimine (PEI); mixing the above plasmid with the PEI solution, wherein the molar ratio of the plasmid ranges from about 1:0.01 to about 1:100, or the molar ratio ranges from about 100:1 to about 1:0.01, to produce a plasmid / PEI mixture (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 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 a recombinant AAV vector or particle comprising a nucleic acid encoding a protein or transcribed into a target transcript, wherein the mammalian cells have been obtained using the method according to the present invention.

[0273] Also provided is a method for producing a recombinant AAV vector or AAV particle comprising a nucleic acid encoding a protein or transcribed into a target transcript, the method comprising providing one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an auxiliary protein; providing a plasmid comprising a nucleic acid encoding a target protein or transcribed into a target transcript; providing a solution comprising polyethyleneimine (PEI); mixing the above plasmids with the PEI solution, wherein the molar ratio of the plasmids ranges from about 1:0.01 to about 1:100, or the molar ratio ranges from about 100:1 to about 1:0.01, to produce a plasmid / PEI mixture (and optionally incubating the plasmid / PEI mixture for a period of time ranging from about 10 seconds to about 4 hours); incubating mammalian cells with the above described The method of the present invention is as follows: contacting the produced plasmid / PEI mixture as described above to produce a plasmid / PEI cell culture; adding free PEI to the produced plasmid / PEI cell culture as described above to produce a free PEI / plasmid / PEI cell culture; incubating the produced plasmid / PEI cell culture or the free PEI / plasmid / PEI cell culture for at least about 4 hours to produce transfected cells; harvesting the produced transfected cells and / or harvesting the culture medium from the produced transfected cells to produce a culture broth; lysing the cells using the method according to the present invention, and optionally isolating the recombinant AAV vector or particle from the culture broth lysate using an AAV affinity chromatography step; thereby producing a recombinant AAV vector or particle comprising a nucleic acid encoding a protein or transcribed as a target transcript.

[0274] In certain embodiments of all aspects and embodiments, PEI is added to plasmid and / or cells at different time points. In certain embodiments, free PEI is added to cells before, simultaneously with or after contacting the plasmid / PEI mixture with cells.

[0275] In some embodiments of all aspects and embodiments, when contacting with plasmid / PEI mixture and / or when contacting with free PEI, cell is in specific density and / or cell growth phase and / or vigor.In a preferred embodiment, when contacting with plasmid / PEI mixture and / or when contacting with free PEI, the density of cell is in the range of about 1*10^5 individual cells / mL to about 1*10^8 individual cells / mL.In certain embodiments, when contacting with plasmid / PEI mixture or with free PEI, cell viability is about 60% or greater than 60%, or wherein when contacting with plasmid / PEI mixture, cell is in logarithmic growth phase, or when contacting with plasmid / PEI mixture or with free PEI, cell viability is about 90% or greater than 90%, or wherein when contacting with plasmid / PEI mixture or with free PEI, cell is in logarithmic growth phase.

[0276] In addition to PEI, valproic acid (VPA) can be used to improve transfection efficiency. VPA is a branched short-chain fatty acid and inhibits histone deacetylase activity. For this reason, it is often added to mammalian cell cultures as an enhancer for recombinant protein production.

[0277] 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.

[0278] In certain embodiments of all aspects and embodiments, the encoded accessory proteins include adenoviral E1A and E1B, adenoviral E2 and / or E4, VA RNA and / or non-AAV accessory proteins.

[0279] In certain embodiments of all aspects and embodiments, nucleic acids (plasmids) are used in specific amounts or ratios. In certain embodiments, the total amount of a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript and one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an auxiliary protein is in the range of about 0.1 μg to about 15 μg per mL of cells. In certain embodiments, the molar ratio of a plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript to one or more plasmids comprising a nucleic acid encoding an AAV packaging protein and / or a nucleic acid encoding an auxiliary protein is in the range of about 1:5 to about 1:1, or in the range of about 1:1 to about 5:1.

[0280] In certain embodiments of all aspects and embodiments, the first plasmid comprises a nucleic acid encoding an AAV packaging protein and the second plasmid comprises a nucleic acid encoding an accessory protein.

[0281] In certain embodiments of all aspects and embodiments, the molar ratio of the plasmid comprising a nucleic acid encoding a protein or transcribed into a target transcript to the first plasmid comprising a nucleic acid encoding an AAV packaging protein to the second plasmid comprising a nucleic acid encoding an auxiliary protein in the co-transfection is in the range of about 1-5:1:1 or 1:1-5:1 or 1:1:1-5.

[0282] In certain embodiments of all aspects and embodiments, the cell is a mammalian cell. In a preferred embodiment, the cell is a HEK293 cell or a CHO cell.

[0283] Approximately 37°C, 95% humidity, and 8% CO 2The culture can be carried out in serum-containing or serum-free medium, adherent culture or suspension culture. The suspension culture can be carried out in any fermentation vessel, such as, for example, in a stirred tank reactor, a wave reactor, a rocking bioreactor, a shaking container or a rotating container or a so-called roller bottle. The transfection can be carried out in a high-throughput format and screening, for example, in a 96- or 384-well format, respectively.

[0284] The methods according to the invention may include AAV particles of any serotype or variants thereof. In certain embodiments of all aspects and embodiments, the recombinant AAV particles comprise any one of the following: AAV serotypes 1 to 12, AAV VP1, VP2 and / or VP3 capsid proteins, or modified or variant AAV VP1, VP2 and / or VP3 capsid proteins, or wild-type AAV VP1, VP2 and / or VP3 capsid proteins. In certain embodiments of all aspects and embodiments, the AAV particles comprise an AAV serotype or an AAV pseudotype, wherein the AAV pseudotype comprises an AAV capsid serotype different from the ITR serotype.

[0285] The methods according to the invention that provide or include AAV vectors or particles may also include 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 repeats (ITRs) and / or filler / filler polynucleotide sequences. Such elements may be located within or flanking a nucleic acid encoding a protein or transcribed into a target transcript, or an expression control element may be operably linked to a nucleic acid encoding a protein or transcribed into a target transcript, or an AAV ITR may flank the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a target transcript, or a filler polynucleotide sequence may flank the 5' or 3' end of a nucleic acid encoding a protein or transcribed into a target transcript.

[0286] Expression control elements include constitutive or regulatable control elements, such as tissue-specific expression control elements or promoters.

[0287] The ITR may be any of AAV2 or AAV6 or AAV8 or AAV9 serotypes, or a combination thereof. The AAV particles may include any VP1, VP2 and / or VP3 capsid proteins having 75% or greater sequence identity with any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV10, AAV11, AAV-2i8, AAV rh74 or AAV 7m8 VP1, VP2 and / or VP3 capsid proteins, or 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.

[0288] After producing recombinant virus (e.g., AAV) particles as described herein, if desired, a variety of conventional methods can be used to purify and / or separate virus (e.g., rAAV) particles from host cells. Such methods include column chromatography, CsCl gradients, iodixanol gradients, etc.

[0289] For example, multiple column purification steps can be used, such as purification by anion exchange column, affinity column and / or cation exchange column. (See, for example, WO 02 / 12455 and US2003 / 0207439). Alternatively or additionally, iodixanol or CsCl gradient steps can be used (see, for example, US 2012 / 0135515; and US2013 / 0072548). In addition, if infectious viruses are used to express packaging proteins and / or auxiliary proteins, various methods can be used to inactivate residual viruses. For example, adenovirus can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or longer. This treatment effectively inactivates the helper virus because AAV is heat stable, while the helper adenovirus is heat unstable.

[0290] One goal of rAAV vector production and purification systems is to implement strategies to minimize / control the generation of production-related impurities, such as proteins, nucleic acids, and vector-related impurities, including wild-type / pseudo-wild-type AAV species (wtAAV) and residual DNA impurities encapsulated by AAV.

[0291] Considering that rAAV particles account for only a small part of the biomass, rAAV particles need to be purified to a purity level that can be used as a clinical human gene therapy product (see, for example, Smith PH et al., Mo. Therapy 7 (2003) 8348; Chadeuf G. et al., Mo. Therapy 12 (2005) 744; report from the CHMP gene therapy expert group meeting, European Medicines Agency EMEA / CHMP 2005, 183989 / 2004).

[0292] As an initial step, cultured cells producing rAAV particles are typically harvested and optionally combined with harvested cell culture supernatant (culture medium) in which cells (suspension cells or adherent cells) producing rAAV particles have been cultured. The harvested cells and optional cell culture supernatant can be used as is, lysed or concentrated as appropriate. In addition, if infection is used to express auxiliary functions, residual helper viruses can be inactivated. For example, adenoviruses can be inactivated by heating to a temperature of about 60° C. for, for example, 20 minutes or longer, which only inactivates the helper virus because AAV is heat stable, while helper adenoviruses are heat unstable.

[0293] By lysing the cells in the culture broth harvested according to the method of the present invention, to release rAAV particles. During cell lysis or after cell lysis, a nuclease (such as, for example, benzonase) is added to degrade the contaminated DNA. Typically, the resulting lysate is clarified to remove cell debris, for example, by filtering or centrifugation to obtain a clarified cell lysate. In a specific example, the lysate is filtered with a micron diameter pore size filter (such as a 0.1 μm to 10.0 μm pore size filter, for example, a 0.45 μm and / or pore size 0.2 μm filter) to produce a clarified lysate.

[0294] The lysate (optionally clarified) contains AAV particles (including rAAV vectors as well as empty capsids) and production / process-related impurities, such as soluble cellular components from host cells, which may include cell proteins, lipids and / or nucleic acids, and cell culture medium components, among others. The optionally clarified lysate is then subjected to a purification step to purify the AAV particles (including rAAV vectors) from the impurities using chromatography. Prior to the first chromatography step, the clarified lysate may be diluted or concentrated with an appropriate buffer.

[0295] Following cell lysis, optional clarification, and optional dilution or concentration, multiple subsequent and sequential chromatography steps may be used to purify the rAAV particles.

[0296] The first chromatography step is preferably an affinity chromatography step using an AAV affinity chromatography ligand.

[0297] If the first chromatography step is affinity chromatography, the second chromatography step may be anion exchange chromatography. Thus, in certain embodiments of all aspects and embodiments, rAAV particle purification is performed via affinity chromatography followed by purification via anion exchange chromatography or / and cation exchange chromatography or / and size exclusion chromatography in any order or sequence or combination.

[0298] For example, during downstream processing, empty capsids are removed from intact capsids based on their different isoelectric points (pi) in anion exchange chromatography. The average pI calculated for all serotypes is 5.9 for intact capsids and 6.3 for empty capsids (Venkatakrishnan, B. et al., J. Virol. 87 (2013) 4974-4984).

[0299] In certain embodiments of all aspects and embodiments, rAAV particle purification is performed via affinity chromatography, followed by purification via anion exchange chromatography, followed by purification via size exclusion chromatography (SEC).

[0300] In certain embodiments of all aspects and embodiments, rAAV particle purification is performed via affinity chromatography, followed by purification via size exclusion chromatography (SEC), followed by purification by anion exchange chromatography.

[0301] The role of cation exchange chromatography is to separate AAV particles from cells and other components present in the clarified lysate and / or column eluate from affinity chromatography or size exclusion chromatography. Examples of strong cation exchange resins capable of binding rAAV 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 ThermoFisher Scientific, Inc., Waltham, MA, USA). Additional examples include Capto S, Capto SImpAct, Capto S ImpRes (strong cation exchangers available from GE Healthcare, Marlborough, MA, USA), and commercial preparations of resins available from Aldrich Chemical Company (Milliwaukee, WI, USA). and Family. Weak cation exchange resins include, but are not limited to, any carboxylic acid-based resins. Exemplary cation exchange resins include carboxymethyl (CM) resins, phosphate resins (based on phosphate functional groups), methanesulfonate (S) resins, and sulfopropyl (SP) resins.

[0302] The effect of anion exchange chromatography is to separate AAV particles from proteins, cells and other components present in the lysate and / or column eluate of the clarification from affinity or cation exchange or size exclusion chromatography. Anion exchange chromatography can also be used to reduce and thus control the amount of empty capsids in the eluate. For example, the anion exchange column with the rAAV particles combined therewith can be washed with a solution of NaCl comprising a moderate concentration (e.g., about 100mM to 125mM, such as 110mM to 115mM), and a portion of empty capsids can be eluted when flowing through without eluting rAAV particles in large quantities. Subsequently, the rAAV particles bonded to the anion exchange column can be eluted using a solution comprising a higher concentration of NaCl (e.g., about 130mM to 300mM NaCl), thereby producing the column eluate of the rAAV particles comprising rAAV carriers with empty capsids reduced or depleted and proportionally increased amounts.

[0303] Exemplary anion exchange resins include, but are not limited to, those based on polyamine resins and other resins.Examples of strong anion exchange resins include those generally based on quaternized nitrogen atoms, including but not limited to quaternary ammonium salt resins such as trialkylbenzylammonium resins. Suitable exchange chromatography materials include, but are not limited to, MACRO PREP Q (strong anion exchanger available from BioRad, Hercules, CA, USA); UNOSPHERE Q (strong anion exchanger available from BioRad, Hercules, CA, USA); POROS 50HQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS XQ (strong anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS SOD (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); POROS 50PI (weak anion exchanger available from Applied Biosystems, Foster City, CA, USA); Capto Q, Capto XQ, Capto Q ImpRes, and SOURCE 30Q (strong anion exchangers available from GE Healthcare). Healthcare, Marlborough, MA, USA); DEAE SEPHAROSE (a weak anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA); Q SEPHAROSE (a strong anion exchanger available from Amersham Biosciences, Piscataway, NJ, USA). Additional exemplary anion exchange resins include aminoethyl (AE), diethylaminoethyl (DEAE), diethylaminopropyl (DEPE), and quaternary aminoethyl (QAE).

[0304] Manufacturing processes for purifying recombinant AAV particles intended as products for treating human disease should achieve the following goals: 1) consistent particle purity, potency, and safety; 2) manufacturing process scalability; and 3) acceptable manufacturing costs.

[0305] An exemplary process for purification of recombinant AAV particles is reported in WO 2019 / 006390.

[0306] The recombinant adeno-associated virus particle (rAAV particle) purification and production methods outlined below can be expanded to large scale. For example, to 5 liters, 10 liters, 10 liters to 20 liters, 20 liters to 50 liters, 50 liters to 100 liters, 100 liters to 200 liters, 200 liters to 500 liters or more liters of suspension culture volume. The recombinant adeno-associated virus particle purification and production methods are applicable to multiple AAV serotypes / capsid variants.

[0307] In certain embodiments of all aspects and embodiments, purification of rAAV particles comprises the following steps:

[0308] a) harvesting the cultured mammalian cells producing recombinant AAV particles and the cell culture supernatant containing rAAV particles to produce a mammalian cell culture broth;

[0309] b) optionally concentrating the harvest produced in step (a) to produce a concentrated mammalian cell culture broth;

[0310] c) lysing 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 contacting the culture broth with an alkyl polyglucoside detergent to produce a mammalian cell culture broth lysate;

[0311] d) treating the lysate produced in step (c) to reduce contaminating nucleic acids in the lysate, thereby producing a nucleic acid-reduced lysate;

[0312] 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;

[0313] f) subjecting the nucleic acid-reduced lysate obtained in step (d) or the clarified lysate or the diluted clarified lysate produced in step (e) to AAV affinity column chromatography to produce a column eluate comprising 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;

[0314] Recombinant AAV particles are thus purified.

[0315] In certain embodiments, steps (a) to (f) are retained and combined with the following steps:

[0316] g) subjecting the column eluate or the concentrated column eluate produced in step (f) to size exclusion column chromatography (SEC) to produce a second column eluate comprising rAAV particles, 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;

[0317] h) optionally subjecting the second column eluate or the diluted second column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprising rAAV particles, 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; and

[0318] i) filtering the second column eluate or the diluted second column eluate produced in step (g), or filtering the third column eluate or the concentrated third column eluate produced in step (h),

[0319] And thereby purifying the recombinant AAV particles.

[0320] In certain embodiments, steps (a) to (f) are retained and combined with the following steps:

[0321] g) subjecting the column eluate or the diluted column eluate produced in step (f) to cation exchange column chromatography to produce a second column eluate comprising 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;

[0322] h) subjecting the column eluate or the diluted column eluate produced in step (g) to anion exchange chromatography to produce a third column eluate comprising 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,

[0323] And thereby purifying the recombinant AAV particles.

[0324] In certain embodiments, steps (a) to (g) are retained and combined with the following steps:

[0325] g) subjecting the column eluate or the diluted column eluate produced in step (f) to anion exchange chromatography to produce a second column eluate comprising 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;

[0326] h) subjecting the column eluate or the diluted column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprising 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,

[0327] And thereby purifying the recombinant AAV particles.

[0328] In certain embodiments of all aspects and embodiments, the concentration of step (b) and / or step (f) and / or step (g) and / or step (h) is performed via ultrafiltration / diafiltration, such as by tangential flow filtration (TFF).

[0329] In certain embodiments of all aspects and embodiments, the concentration of step (b) reduces the volume of harvested cells and cell culture supernatant by about 2 to 20 fold.

[0330] In certain embodiments of all aspects and embodiments, the concentrating of step (f) and / or step (g) and / or step (h) reduces the volume of the column eluate by about 5 to 20 fold.

[0331] In certain embodiments of all aspects and embodiments, step (d) comprises treating with a nuclease, thereby reducing contaminating nucleic acids. Non-limiting examples of nucleases include benzonase.

[0332] In certain embodiments of all aspects and embodiments, the filtration of the clarified lysate or diluted clarified lysate of step (e) is performed via a filter. Non-limiting examples of filters are those having a pore size between about 0.1 microns and 10.0 microns (including end values).

[0333] In certain embodiments of all aspects and embodiments, the dilution of the clarified lysate of step (e) is performed with an aqueous buffered phosphate, acetate or Tris solution. Non-limiting examples of solution pH are between about pH 4.0 and pH 7.4, including end values. Non-limiting examples of Tris solution pH are greater than pH 7.5, such as between about pH 8.0 and pH 9.0, including end values.

[0334] In certain embodiments of all aspects and embodiments, the dilution of the second column eluate of step (g) or the dilution of the third column eluate of step (h) is carried out with an aqueous buffered phosphate, acetate or Tris solution. Non-limiting examples of solution pH are between about pH 4.0 and pH 7.4, including end values. Non-limiting examples of Tris solution pH are greater than pH 7.5, such as between about pH 8.0 and pH 9.0, including end values.

[0335] In certain embodiments of all aspects and embodiments, the rAAV particles produced in step (i) are formulated with a surfactant to produce a rAAV particle formulation.

[0336] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography of steps (g) and / or (h) comprises polyethylene glycol (PEG)-modulated column chromatography.

[0337] In certain embodiments of all aspects and embodiments, the anion exchange column chromatography of steps (g) and / or (h) is washed with a PEG solution prior to eluting the rAAV particles from the column.

[0338] In certain embodiments of all aspects and embodiments, the average molecular weight of PEG is in the range of about 1,000 g / mol to 80,000 g / mol, inclusive.

[0339] In certain embodiments of all aspects and embodiments, the concentration of PEG is about 4% (w / v) to about 10% (w / v), inclusive.

[0340] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (g) and / or (h) is washed with an aqueous surfactant solution prior to eluting the rAAV particles from the column.

[0341] 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 eluting the rAAV particles from the column.

[0342] In certain embodiments of all aspects and embodiments, the PEG solution and / or the surfactant solution comprises an aqueous Tris-HCl / NaCl buffer, an aqueous phosphate / NaCl buffer, or an aqueous acetate / NaCl buffer.

[0343] In certain embodiments of all aspects and embodiments, the NaCl concentration in the buffer or solution ranges between about 20 mM to 300 mM NaCl, inclusive, or between about 50 mM to 250 mM NaCl, inclusive.

[0344] In certain embodiments of all aspects and embodiments, the surfactant comprises a cationic or anionic surfactant.

[0345] In certain embodiments of all aspects and embodiments, the surfactant comprises a twelve carbon chain surfactant.

[0346] In certain embodiments of all aspects and embodiments, the surfactant comprises dodecyltrimethylammonium chloride (DTAC) or Sarkosyl.

[0347] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the anion exchange column of steps (f), (g) and / or (h) with aqueous Tris-HCl / NaCl buffer.

[0348] In certain embodiments of all aspects and embodiments, the Tris-HCl / NaCl buffer comprises 100 mM to 400 mM NaCl (inclusive), optionally at a pH in the range of about pH 7.5 to about pH 9.0 (inclusive).

[0349] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (g) and / or (h) is washed with aqueous Tris-HCl / NaCl buffer.

[0350] 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 mM to 125 mM, inclusive.

[0351] In certain embodiments of all aspects and embodiments, the aqueous Tris-HCl / NaCl buffer has a pH of about pH 7.5 to about pH 9.0, inclusive.

[0352] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (g) and / or (h) is washed one or more times to reduce the amount of empty capsids in the second or third column eluate.

[0353] In certain embodiments of all aspects and embodiments, washing of the anion exchange column removes empty capsids from the column prior to and / or in place of elution of the rAAV particles, thereby reducing the amount of empty capsids in the second or third column eluate.

[0354] In certain embodiments of all aspects and embodiments, washing the anion exchange column removes at least about 50% of the total empty capsids from the column prior to and / or in place of elution of the rAAV particles, thereby reducing the amount of empty capsids in the second or third column eluate by about 50%.

[0355] 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 mM to 120 mM, inclusive.

[0356] In certain embodiments of all aspects and embodiments, the ratio and / or amount of eluted rAAV particles and empty capsids is controlled by a wash buffer.

[0357] In certain embodiments of all aspects and embodiments, the rAAV particles are eluted from the cation exchange column of step (g) and / or (h) with an aqueous phosphate / NaCl buffer or an aqueous acetate / NaCl buffer. Non-limiting NaCl concentrations in the buffer range from about 125 mM to 500 mM NaCl, inclusive. Non-limiting examples of buffer pH range from about pH 5.5 to about pH 7.5, inclusive.

[0358] In certain embodiments of all aspects and embodiments, the anion exchange column of steps (g) and / or (h) comprises quaternary ammonium functional groups, such as quaternized polyethyleneimine.

[0359] In certain embodiments of all aspects and embodiments, the size exclusion column (SEC) has a separation / fractionation range (molecular weight) of about 10,000 g / mol to about 600,000 g / mol, inclusive.

[0360] In certain embodiments of all aspects and embodiments, the cation exchange column of steps (g) and / or (h) comprises a sulfonic acid or a functional group, such as a sulfopropyl group.

[0361] In certain embodiments of all aspects and embodiments, the AAV affinity column comprises a protein or ligand that binds to an AAV capsid protein. Non-limiting examples of proteins include antibodies that bind to an AAV capsid protein. More specific non-limiting examples include single-chain llama antibodies (Camelidae) that bind to an AAV capsid protein.

[0362] In certain embodiments of all aspects and embodiments, the method does not comprise the step of cesium chloride gradient ultracentrifugation.

[0363] In certain embodiments of all aspects and embodiments, the method produces rAAV particles having a higher purity than rAAV particles produced or purified by a single AAV affinity column purification.

[0364] In certain embodiments of all aspects and embodiments, steps (c) and (d) are performed substantially simultaneously.

[0365] In certain embodiments of all aspects and embodiments, after step (c) but before step (f), the NaCl concentration is adjusted to within the range of about 100 mM to 400 mM NaCl (inclusive), or within the range of about 140 mM to 300 mM NaCl (inclusive).

[0366] In certain embodiments of all aspects and embodiments, the cells are cells growing in suspension or adherently.

[0367] In certain embodiments of all aspects or embodiments, the cell is a mammalian cell. Non-limiting examples include HEK cells, such as HEK-293 cells, and CHO cells, such as CHO-K1 cells.

[0368] Methods for determining the infectious titer of rAAV particles containing a transgene are known in the art (see, e.g., Zhen et al., Hum. Gene Ther. 15 (2004) 709). Methods for determining empty capsids and rAAV particles with packaged transgenes are known (see, e.g., Grimm et al., Gene Therapy 6 (1999) 1322-1330; Sommer et al., Malec. Ther. 7 (2003) 122-128).

[0369] In order to determine the presence or amount of degraded / denatured capsids, purified rAAV particles can be subjected to SDS-polyacrylamide gel electrophoresis, which consists of any gel (e.g., gradient gel) capable of separating the three capsid proteins, and then running the gel until the sample is separated and blotted onto a nylon or nitrocellulose membrane. Anti-AAV capsid antibodies are then used as primary antibodies that bind to denatured capsid proteins (see, e.g., Wobus et al., J. Viral. 74 (2000) 9281-9293). The secondary antibody that binds the primary antibody contains a tool for detecting the primary antibody. Semi-quantitative detection of the binding between the primary and secondary antibodies determines the amount of capsids. Another method is to use an analytical HPLC or analytical ultracentrifuge using a SEC column. DETAILED DESCRIPTION

[0371] The present invention is based, at least in part, on the discovery that the recovery rate, i.e., the yield (both capsid-based and genome-based) of recombinant AAV particles in AAV affinity chromatography is affected by / depends on the detergent used to lyse the cells producing the recombinant AAV particles prior to AAV affinity chromatography.

[0372] The present invention is further based, at least in part, on the discovery that the ratio of intact to empty recombinant AAV particles obtained in AAV affinity chromatography is affected by / depends on the detergent used to lyse the recombinant AAV particle-producing cells prior to AAV affinity chromatography.

[0373] It has been found that by using an alkyl polyglucoside detergent for lysing cells producing recombinant AAV particles prior to the AAV affinity chromatography step, both the yield of recombinant AAV particles (capsid-based and genome-based) and the ratio of intact recombinant AAV particles to empty recombinant AAV particles can be increased in AAV affinity chromatography.

[0374] The method according to the present invention is illustrated below using Triton CG 110 as an example of an alkyl polyglucoside detergent. This is presented only as an illustration of the concept of the present invention and should not be construed as limiting. Likewise, any other alkyl polyglucoside detergent may be used. The true scope of the present invention is set forth in the appended claims.

[0375] In more detail, by using Triton CG 110 as an example of an alkyl polyglucoside detergent, an increase in absolute AAV particle recovery of greater than 5% can be achieved in AAV affinity chromatography compared to the current "gold standard" Triton X-100 (a p-tert-octylphenol derivative with polyethylene glycol side chains), i.e., an absolute recovery of 76% can be achieved when Triton CG 110 has been used to lyse the cells, compared to an absolute recovery of 72% when Triton X-100 has been used to lyse the cells.

[0376] Since this method is intended for large-scale production of recombinant AAV particles, even a small increase in the relative yield of AAV particle recovery would result in a large increase in absolute yield, thus providing an advantage.

[0377] Concurrently with the increase in absolute AAV particle recovery, the ratio of intact to empty recombinant AAV particles in the AAV affinity chromatography eluate was increased compared to the ratio in the lysate applied to the AAV affinity chromatography material.

[0378] In more detail, it has been found that by using Triton CG 110 as an example of an alkyl polyglucoside detergent, an increase in genome recovery of greater than 60% can be obtained in AAV affinity chromatography compared to the current "gold standard" Triton X-100, i.e., an absolute genome yield of 4.7 x 1E11 viral genomes per mL (vg / mL) when Triton CG 110 has been used to lyse the cells, compared to an absolute genome yield of 2.8 x 1E11 viral genomes per mL (vg / mL) when Triton X-100 has been used to lyse the cells. A greater than 50% increase in total viral particle yield was also obtained in the eluate of AAV affinity chromatography in Triton CG 110 lysis culture broth compared to Triton X-100 lysis culture broth, i.e., an absolute particle yield of 1.2 x 1E13 viral particles per mL (vg / mL) when Triton CG 110 had been used to lyse the cells, compared to an absolute particle yield of 0.77 x 1E13 viral particles per mL (vg / mL) when Triton X-100 had been used to lyse the cells.

[0379] Since the increase in genome yield was higher than the increase in total viral particle yield, an increase in the ratio of complete recombinant AAV particles to empty recombinant AAV particles was obtained.

[0380] The corresponding data are shown in the following table:

[0381]

[0382] Thus, the present invention is based, at least in part, on the discovery that the use of Triton CG 110 as an example of an alkyl polyglucoside detergent to lyse cells producing recombinant AAV particles results in increased recovery of intact recombinant AAV particles.

[0383] Different combinations of final Triton CG 110 concentration and lysis time (incubation time in the presence of Triton CG 110) have been tested in small-scale experiments using aliquots of the same culture broth obtained 72 hours after triple PEI-mediated transfection.

[0384] It has been found that an incubation time of about 60 minutes and a final concentration of 1% (v / v) Triton CG 110 provides the best overall results. This is further improved by incubating with diatomaceous earth for about 20 minutes after incubation with the alkyl polyglucoside detergent Triton CG 110. These are preferred embodiments of the present invention.

[0385] The corresponding data are shown in the table below.

[0386]

[0387]

[0388] Mammalian HEK293 cells have been transfected with: a plasmid required for production of recombinant AAV particles (i.e., a plasmid containing the transgene (transgene plasmid)), a plasmid encoding AAV packaging proteins (rep / cap plasmid), and a plasmid containing desired AAV helper functions not already contained in the HEK293 cells (helper plasmid).

[0389] In more detail, for the production of recombinant AAV particles, pre-cultured HEK293 cells were cultured in a batch process in a wave bioreactor (10 L working volume) using F17 medium with a starting cell density of approximately 1E6 cells / ml.

[0390] A few hours after inoculation of the bioreactor, PEI (polyethyleneimine)-mediated transfection was performed with three plasmids (helper plasmid, transgenic plasmid, rep / cap plasmid). 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 used in an amount of 2 μg / 1 μg plasmid.

[0391] Cultivation was performed at a temperature of 37°C, a humidity of 70%, a pCO2 of 5%, a stirrer speed of 30 to 35 rpm, without oxygen and pH control. Aeration was performed using air at a flow rate of about 500 ml / min. Cultivation was terminated 72 h after transfection.

[0392] In an alternative method, HEK cells can be cultured in a 4 day fed-batch process with feed and glucose added at a starting cell density of approximately 15 x 1E5 cells / ml.

[0393] PEI (polyethyleneimine) mediated transfection can be performed 24h after inoculation with a triple transfection of three plasmids (helper plasmid, transgenic plasmid, rep / cap plasmid). First, 20% v / v fresh medium is added to the culture. Subsequently, a transfection mixture 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 culture volume.

[0394] Feed (e.g. HEK FS feed supplement from Xell AG) was added as a bolus feed on the second day of fermentation (24 h after transfection). Glucose was added as a bolus feed on the first and third days of fermentation. CO was added separately 2 and 1M Na 2 CO 3 The pH value is adjusted in 0.05 pH units. If necessary, a defoamer solution is added. During this process, the parameters temperature, pH and pO 2 Monitoring and control were performed. The fermentation process was stopped on day 4 (72 h after triple transfection) by adding lysis buffer.

[0395] ***

[0396] All references mentioned herein are hereby incorporated by reference.

[0397] ***

[0398] The following examples are provided to aid the understanding of the present invention, the true scope of the invention being set forth in the appended claims.It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0399] Examples

[0400] Material

[0401] Cell lines

[0402] Commercially available HEK293 cells were used for the production of AAV particles using transient transfection with the three plasmids.

[0403] Culture materials

[0404] The culture medium (HEK ViP NB powder medium, Xell AG) was prepared according to the supplier's instructions. HEK FS feed (Xell AG) and F17 medium were used as purchased. The culture medium and feed were stored at 4°C in the dark and consumed according to the manufacturer's instructions. The calibrators were stored at room temperature (glucose solution; sodium carbonate solution; antifoam solution).

[0405] Example 1

[0406] HEK293 cell culture and production of recombinant AAV particles

[0407] In general, the culture methods were adapted from standard protocols (see, e.g., Lindl, T., "Zell- und Gewebekultur: Einführung in die Grundlagen sowie Methoden und Anwendungen", Spektrum Akademischer Verlag GmbH, Heidelberg / Berlin, 2002) and the operating instructions of the corresponding suppliers.

[0408] Pre-culture

[0409] HEK cells were thawed and propagated in culture medium in shake flasks at 37°C, 70% humidity, 5% pCO2 and 120 rpm shaking frequency for two to three weeks. Cells were split every three to four days and expanded in culture medium to the volume required for inoculation of production culture.

[0410] Production and cultivation

[0411] For production of recombinant AAV particles, pre-cultured HEK293 cells were cultured in a batch process in a wave bioreactor (10 L working volume) using F17 medium with a starting cell density of approximately 10 x E5 cells / ml.

[0412] A few hours after inoculation of the bioreactor, PEI (polyethyleneimine)-mediated transfection was performed with three plasmids (helper plasmid, transgenic plasmid, rep / cap plasmid). 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), and the amount was 2 μg / 1 μg plasmid DNA.

[0413] Cultivation was performed at a temperature of 37°C, a humidity of 70%, a pCO2 of 5%, a stirrer speed of 30 to 35 rpm, without oxygen and pH control. Aeration was performed using air at a flow rate of about 500 ml / min. Cultivation was terminated 72 h after transfection.

[0414] Example 2

[0415] Lysis

[0416] 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 tested detergents was added to the culture broth. In addition, 50 U / ml DNase I (bovine pancreas, Roche) and 37.5 mM MgSO4 were added. The cell culture broth was then incubated at 37°C for different times under stirring without aeration and pH control. After the corresponding incubation, the lysate was sterile filtered.

[0417] Example 3

[0418] AAV particle purification

[0419] For the affinity chromatography step, A column containing 10.5 mL AAVX resin obtained from Thermo Fisher was used on an Avant 25 chromatography system. The system was operated at a flow rate of approximately 300 cm / h. After equilibration with buffer A (1x PBS, pH 7.4, 0.001% Pluronic F-68), 200 mL of lysis culture broth was applied to the column, followed by 2 washing steps with equilibration buffer and 0.5 M NaCl (pH 6.0), respectively. The AAV particles were eluted with 0.1 M sodium citrate solution (pH 2.4). The pH of the eluate was adjusted to pH 7.5 by adding 2 M Tris (pH 10).

[0420] step Buffer Column volume [CV] balance 1x PBS, pH 7.4 3 Loading Lysate Washing I 1x PBS, pH 7.4 4 Washing II 0.5M NaCl pH 6.0 4 Washing III 1x PBS, pH 7.4 4 Elution 0.1M sodium citrate pH 2.4 3

[0421] Example 4

[0422] Analytical methods

[0423] Enzyme-linked immunosorbent assay (ELISA) for total titer determination

[0424] For AAV capsid titer determination, a kit from PROGEN (Cat. No. PRAAV8) was used according to the manufacturer's instructions.

[0425] Briefly, the assay is a sandwich ELISA using a recombinant AAV capsid-specific antibody and a biotin-labeled detection antibody as the capture antibody.

[0426] The wells of the pre-coated multi-titer plate (MTP) were incubated with 100 μL of standard, sample or control at 4 ° C overnight. The next day, the wells were washed three times with ASSB buffer (1x) as provided in the kit. Thereafter, 100 μL of a solution containing biotinylated detection antibody (diluted according to the manufacturer's instructions) was added to each well and incubated at room temperature for two hours under shaking. Then, the wells were washed three times with ASSB buffer (1x) 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 at room temperature under shaking for 30 minutes. Then, the wells were washed three times with ASSB buffer (1x) 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 under shaking. The color intensity was determined using a MTP-ELISA-Reader Versa Max (Molecular Devices) at 405 nm (reference wavelength 490 nm) until the extinction difference between the blank and the standard with the highest concentration reached approximately 1.5.

[0427] Each sample, standard, and control was measured in duplicate.

[0428] The average of the standards is used to calculate the amount of capsids (capsids / mL) based on a standard curve determined by a 4-parameter fit (eg, according to the Wiemer Rodbard algorithm).

[0429] Digital droplet polymerase chain reaction (ddPCR) for genomic titer determination

[0430] Reagents for enzymatic sample processing:

[0431] 1) DNase I buffer (NEB): 100 mM Tris-HCl, pH 7.6, 25 mM MgSO4, 5 mM CaCl2

[0432] 2) DNase I (NEB): 0.2 U / μL

[0433] 3) Proteinase K (NEB; approximately 20 mg / mL = 800 U / mL): 16 U / mL

[0434] 4) Proteinase K buffer (BioRad): 400 mM Tris-HCl, 20 mM EDTA, 2000 mM NaCl, 1% SDS, pH 8

[0435] 5) Sodium dodecyl sulfate (SDS) solution: 10% (w / v)

[0436] Enzymatic sample treatment:

[0437] -Mix 30μL H2O, 5μL DNase I buffer, 5μL DNase I, 10μL sample

[0438] - Incubate at 37°C for 30 minutes.

[0439] - Heat to 75°C for 15 minutes to obtain an incubated DNase I-mix

[0440] - Briefly cool and centrifuge

[0441] - 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

[0442] - Incubate at 50°C for 60 minutes

[0443] - Heat to 95°C for 15 minutes.

[0444] - Cool to 4°C

[0445] ddPCR:

[0446] For viral genome titration, a duplex ddPCR assay was performed. Primers and probes were designed for the CMV promoter used and for the polyA / 3'UTR sequence. PCR master mix was prepared according to the following table (Droplet Digital PCR Guide - Bio-Rad).

[0447] Components Volume per well [μL] Final concentration Supermix (2x) 11 1x 20 μM CMV forward primer 0.99 900nM 20 μM CMV reverse primer 0.99 900nM 20 μM CMV probe 0.275 250nM template 5.5 - water 0.99 - total 22

[0448] The prepared master mix was transferred into a 96-well plate, 16.5 μL per well. Then, the pre-treated sample was serially diluted: 10 μL of sample was transferred into 90 μL of water in a LoBind tube using LoRentention Tips and mixed thoroughly. Thereafter, 5.5 μL of sample was added to the master mix solution in the 96-well plate through several dilution steps. The plate was sealed at 180°C, vortexed at 2,200 rpm for 1 minute and centrifuged at 1,000 rpm for another 1 minute. 20 μL of PCR mixture was taken from each well using an automated droplet generator device, producing up to 20,000 droplets per well, and transferred to another 96-well plate.

[0449] The PCR run was performed after sealing the droplet plate at 180° C. The corresponding conditions are shown in the table below.

[0450] Cycle times transsexual annealing Final extension Finish 1 94°C, 10 min 39 94°C, 30 seconds 58°C, 1 min 1 98°C, 10 min 12℃, long term

[0451] In the droplet analyzer, the fluorescence signal of each droplet is measured. QuantaSoft software processes the analyzer data and calculates the copy number of each 20 μL well of the target sequence. The initial sample titer can be determined using the following formula:

[0452]

Claims

1. A method for purifying recombinant AAV particles, the method comprising the following steps: - releasing recombinant AAV particles from production mammalian cells by contacting a corresponding mammalian cell culture broth with an alkyl polyglucoside detergent, and - Purification of the recombinant AAV particles using AAV affinity chromatography Thus purifying the recombinant AAV particles, The mammalian cell culture broth comprises cultured mammalian cells producing recombinant AAV particles and a culture medium for culturing the mammalian cells producing recombinant AAV particles.

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

3. Use of an alkyl polyglucoside detergent for increasing the ratio of intact recombinant AAV particles to empty recombinant AAV particles obtained (in the eluate fraction) in a subsequent AAV affinity chromatography, wherein mammalian cells producing recombinant AAV particles are lysed with the alkyl polyglucoside detergent prior to the AAV affinity chromatography.

4. The method and use according to any one of the preceding claims, wherein the alkyl polyglucoside detergent is a mixture of 58.0 (w / v)% to 62.0 (w / v)% D-pyranose oligodecyloctyl glycoside and 38.0 (w / v)% to 42.0 (w / v)% water.

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

6. The method and use according to any one of the preceding claims, wherein the alkyl polyglucoside cleaning agent is in solution.

7. The method and use according to any one of the preceding claims, wherein the mammalian cell culture broth is combined with a solution comprising an alkyl polyglucoside detergent at 5% to 10% (5% (v / v) to 10% (v / v)) of its volume.

8. The method and use according to any one of claims 6 to 7, wherein the solution comprising the alkyl polyglucoside detergent comprises the alkyl polyglucoside detergent at a concentration of about 10%.

9. The method and use according to any one of the preceding claims, wherein said contacting with the alkyl polyglucoside cleaning agent or said contacting with the alkyl polyglucoside cleaning agent, respectively, is carried out for about 60 minutes.

10. The method and use according to any one of the preceding claims, wherein said contacting with an alkyl polyglucoside detergent or said contacting with an alkyl polyglucoside detergent, respectively, is carried out at a temperature of about 37°C.

11. The method and use according to any one of the preceding claims, wherein diatomaceous earth is added after said contacting with the alkyl polyglucoside detergent or said contacting with the alkyl polyglucoside detergent, respectively, and the mixture is incubated for 5 to 20 minutes.

12. The method and use according to any one of claims 6 to 11, wherein the solution comprising the alkyl polyglucoside detergent comprises magnesium (II) chloride at a concentration of 10 mM to 40 mM.

13. The method and use according to any one of claims 6 to 12, wherein the solution comprising the alkyl polyglucoside cleaning agent has a pH value of about 7.

5.

14. The method and use according to any one of the preceding claims, wherein the mammalian cell culture broth is contacted with the alkyl polyglucoside detergent and 50 U / mL of a nuclease selected from DNase I and Benzonase.

15. The method and use according to any of the preceding claims, wherein the affinity chromatography is performed on a chromatography material comprising a cross-linked poly(styrene-divinylbenzene) matrix to which are covalently conjugated 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.

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