AAV production method

Abstract

Provided herein are methods for producing an AAV. In some embodiments, the method comprises transfecting a packaging cell with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection. In particular embodiments, the method comprises transfecting a packaging cell with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection, wherein a first round of transfection comprises transfecting the packaging cell with at least a first nucleic acid of the plurality of nucleic acids encoding the AAV, and a second round of transfection comprises transfecting the packaging cell with at least a second nucleic acid of the plurality of nucleic acids encoding the AAV.

Description

Atty. Docket No. 01277-0058-00PCTAAV PRODUCTION METHODCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 717,606, filed November 7, 2024, the entire contents of which are incorporated by reference herein for all purposes.FIELD OF THE INVENTION

[0002] The present disclosure provides compositions and methods related to producing an adeno-associated virus (AAV), such as methods comprising transfecting a packaging cell with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection.INTRODUCTION

[0003] Adeno-associated viruses (AAVs), such as recombinant AAVs, are typically produced in permissive (packaging) host cell cultures. Standard transfection methods for AAV production include introducing into packaging cells plasmids encoding a heterologous nucleic acid (such as a nucleic acid molecule of interest), AAV replication (“rep”) and capsid (“cap”) genes, and adenoviral accessory functions in a single round of transfection.

[0004] Commercial-scale methods of producing AAVs, such as recombinant AAVs, for a variety of gene therapy and vaccine approaches have been described. However, there remains a need for efficient, scalable methods for production of AAVs, including solutions for improving AAV titers and reducing AAV production costs.SUMMARY

[0005] The present disclosure provides improved, scalable methods of producing AAVs, such as for therapeutic use in a subject, including improved solutions for enhancing AAV production efficiency. The following exemplary embodiments are provided.

[0006] Embodiment 1 is a method of producing adeno-associated virus (AAV), the method comprising transfecting a packaging cell with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection, wherein:Atty. Docket No. 01277-0058-00PCT a) a first round of transfection comprises transfecting the packaging cell with at least a first nucleic acid of the plurality of nucleic acids encoding the AAV; and b) a second round of transfection comprises transfecting the packaging cell with at least a second nucleic acid of the plurality of nucleic acids encoding the AAV.

[0007] Embodiment 2 is the method of embodiment 1, wherein a) the packaging cell is not transfected with the second nucleic acid of the plurality of nucleic acids encoding the AAV in the first round of transfection; and / or b) the packaging cell is not transfected with the first nucleic acid of the plurality of nucleic acids encoding the AAV in the second round of transfection.

[0008] Embodiment 3 is the method of any one of the preceding embodiments, wherein: a) the first round of transfection comprises transfecting the packaging cell with a first set of the plurality of nucleic acids encoding the AAV; and b) the second round of transfection comprises transfecting the packaging cell with a second set of the plurality of nucleic acids encoding the AAV; wherein the first set and the second set of nucleic acids of the plurality of nucleic acids encoding the AAV are not the same.

[0009] Embodiment 4 is the method of the immediately preceding embodiment, wherein the first set of nucleic acids of the plurality of nucleic acids encoding the AAV comprises or consists of 1-6. 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, 5, or 6 nucleic acids; the second set of nucleic acids of the plurality of nucleic acids encoding the AAV comprises or consists of 1-6, 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, 5, or 6 nucleic acids; and the first set and the second set of nucleic acids of the plurality of nucleic acids encoding the AAV are not the same.

[0010] Embodiment 5 is the method of embodiment 1, wherein the plurality of nucleic acids encoding the AAV is the same in the first round of transfection and in the second round of transfection, and wherein a ratio of at least one of the plurality of nucleic acids transfected in the first round of transfection is different from a ratio of the at least one of the plurality of nucleic acids transfected in the second round of transfection.

[0011] Embodiment 6 is the method of any one of the preceding embodiments, wherein the plurality of nucleic acids encoding the AAV comprises one or more of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein; (iii) at least one nucleic acid encoding an AAV capsid protein; and (iv) at least one nucleic acid encoding an adenovirus packaging helper gene.Atty. Docket No. 01277-0058-00PCT

[0012] Embodiment 7 is the method of any one of the preceding embodiments, wherein the plurality of nucleic acids is sufficient for production of the AAV when the packaging cell is cultured under conditions suitable for production of the AAV.

[0013] Embodiment 8 is the method of any one of the preceding embodiments, wherein the first round of transfection comprises dual transfection, triple transfection, or quad transfection.

[0014] Embodiment 9 is the method of any one of the preceding embodiments, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV capsid protein.

[0015] Embodiment 10 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV replication protein.

[0016] Embodiment 11 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the gene of interest.

[0017] Embodiment 12 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; andAtty. Docket No. 01277-0058-00PCT b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein.

[0018] Embodiment 13 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with a nucleic acid encoding the adenovirus packaging helper gene.

[0019] Embodiment 14 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; and (ii) at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein.

[0020] Embodiment 15 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; and (ii) at least one nucleic acid encoding the adenovirus packaging helper gene.

[0021] Embodiment 16 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; andAtty. Docket No. 01277-0058-00PCT b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein; and (ii) at least one nucleic acid encoding the adenovirus packaging helper gene.

[0022] Embodiment 17 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with one of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; or (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; b) the second round of transfection comprises or consists of transfecting the packaging cell with any one of nucleic acids that was not transfected into the packaging cell in step (a); and c) a third round of transfection comprises or consists of transfecting the packaging cell with the nucleic acid that was not transfected into the packaging cell in step (a) or step (b).

[0023] Embodiment 18 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with one of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; or (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with the two nucleic acids that were not transfected into the packaging cell in step (a).

[0024] Embodiment 19 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with any two of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with the nucleic acid that was not transfected into the packaging cell in step (a).

[0025] Embodiment 20 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with any one or two of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; andAtty. Docket No. 01277-0058-00PCT b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding the adenovirus packaging helper gene.

[0026] Embodiment 21 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene; and

[0027] wherein the packaging cell is transfected with at least one nucleic acid encoding the AAV capsid protein during the second round of transfection and / or during an additional round of transfection following the second round of transfection.

[0028] Embodiment 22 is the method of any one of embodiments 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene; and

[0029] wherein the packaging cell is transfected with at least one nucleic acid encoding the AAV capsid protein during the second round of transfection and / or during an additional round of transfection following the second round of transfection.Atty. Docket No. 01277-0058-00PCT

[0030] Embodiment 23 is the method of any one of the preceding embodiments, further comprising a plurality of additional rounds of transfection, optionally after the second or the third round of transfection.

[0031] Embodiment 24 is the method of the immediately preceding embodiment, wherein each of the plurality of additional rounds of transfection comprises transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein; (iii) at least one nucleic acid encoding an AAV capsid protein; and (iv) at least one nucleic acid encoding an adenovirus packaging helper gene.

[0032] Embodiment 25 is the method of embodiment 23 or embodiment 24, wherein the plurality of additional rounds of transfection comprises 1-100 additional rounds of transfection, such as 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2,2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 additional rounds of transfection.

[0033] Embodiment 26 is the method of any one of the preceding embodiments, comprising a number of rounds of transfection sufficient to attain a desired level of expression of a gene of interest.

[0034] Embodiment 27 is the method of the immediately preceding embodiment, wherein the number of rounds of transfection comprises 1-100 rounds of transfection, such as 1-90, 1-80, 1- 70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2- 20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 rounds of transfection.

[0035] Embodiment 28 is the method of any one of the preceding embodiments, wherein an amount of elapsed time between any two consecutive rounds of transfection is independently selected from 3-48, 3-47, 3-46, 3-45. 3-44, 3-43, 3-42, 3-41, 3-40. 3-39, 3-38, 3-37, 3-36, 3-35.3-34, 3-33, 3-32, 3-31, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-36, 4-35, 4-34, 4- 33, 4-32, 4-31. 4-30. 4-29, 4-28, 4-27, 4-26. 4-25. 4-24, 4-23, 4-22, 4-21. 4-20. 4-19, 4-18, 4-17,4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,Atty. Docket No. 01277-0058-00PCT14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0036] Embodiment 29 is the method of any one of the preceding embodiments, wherein an amount of elapsed time between the first round of transfection and the second round of transfection is 3-48, 3-47, 3-46. 3-45, 3-44, 3-43, 3-42, 3-41. 3-40, 3-39, 3-38, 3-37, 3-36. 3-35,3-34, 3-33, 3-32, 3-31, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-22, 3-21, 3-20, 3-19, 3- 18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-36, 4-35, 4-34, 4- 33, 4-32, 4-31, 4-30. 4-29, 4-28, 4-27, 4-26, 4-25. 4-24, 4-23, 4-22, 4-21, 4-20. 4-19, 4-18, 4-17,4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25. 26. 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0037] Embodiment 30 is the method of any one of the preceding embodiments, wherein a concentration of a nucleic acid encoding an AAV is the same in each round of transfection wherein the nucleic acid encoding the AAV is transfected into the packaging cell.

[0038] Embodiment 31 is the method of any one of embodiments 1-29, wherein a concentration of a nucleic acid encoding an AAV is different in at least two rounds of transfection wherein the nucleic acid encoding the AAV is transfected into the packaging cell.

[0039] Embodiment 32 is the method of any one of the preceding embodiments, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is the same in each round of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell.

[0040] Embodiment 33 is the method of any one of embodiments 1-30, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in at least two rounds of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell.

[0041] Embodiment 34 is the method of any one of embodiments 1-30, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in each round of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell.

[0042] Embodiment 35 is the method of any one of embodiments 1-30. wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in at least two rounds of transfection, and wherein both the first nucleic acid encoding an AAV andAtty. Docket No. 01277-0058-00PCT the second nucleic acid encoding an AAV are transfected into the packaging cell in each of the at least two rounds of transfection.

[0043] Embodiment 36 is the method of any one of embodiments 1-30, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in each round of transfection, and wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell in each of the at least two rounds of transfection.

[0044] Embodiment 37 is the method of any one of the preceding embodiments, wherein the AAV is or is derived from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, or Myo AAV.

[0045] Embodiment 38 is the method of any one of the preceding embodiments, wherein the AAV is a chimeric AAV.

[0046] Embodiment 39 is the method of any one of the preceding embodiments, further comprising harvesting the AAV from a cell culture comprising the packaging cell.

[0047] Embodiment 40 is the method of any one of the preceding embodiments, wherein, after the two or more rounds of transfection, the AAV is harvested from the cell culture comprising the packaging cell every 1-6 days, every 1-5 days, every 1-4 days, every 1-3 days, every 2-6 days, every 2-5 days, or every 2-4 days.

[0048] Embodiment 41 is the method of any one of the preceding embodiments, wherein the AAV is harvested from the cell culture comprising the packaging cell 1-6 days, 1-5 days, 1-4 days, 1-3 days, 2-6 days, 2-5 days, or 2-4 days after the two or more rounds of transfection.

[0049] Embodiment 42 is the method of the immediately preceding embodiment, wherein harvesting the AAV from the cell culture comprising the packaging cell comprises harvesting the AAV from a supernatant of the cell culture.

[0050] Embodiment 43 is the method of embodiment 41 or embodiment 42, wherein harvesting the AAV from the cell culture comprising the packaging cell comprises isolating the AAV from the cell culture.

[0051] Embodiment 44 is the method of the immediately preceding embodiment, wherein the isolating comprises chemical lysis, mechanical lysis, depth filtration, microfiltration, ultrafiltration, alternating tangential flow filtration, tangential flow depth filtration, batch chromatography, acoustic wave separation, and / or centrifugation.Atty. Docket No. 01277-0058-00PCT

[0052] Embodiment 45 is the method of embodiment 43 or embodiment 44, wherein the isolating further comprises concentrating the AAV using a method selected from chromatography, column-based chromatography, membrane-based chromatography, filtration, and / or precipitation.

[0053] Embodiment 46 is the method of any one of the preceding embodiments, wherein the packaging cell comprises one or more nucleic acids encoding one or more helper genes, and / or the packaging cells are infected with one or more helper viruses.

[0054] Embodiment 47 is the method of the immediately preceding embodiment, wherein the one or more helper genes comprise at least one nucleic acid encoding an adenovirus packaging helper gene, at least one nucleic acid encoding an AAV replication protein sufficient for packaging, and / or at least one nucleic acid encoding an AAV capsid protein sufficient for packaging.

[0055] Embodiment 48 is the method of the immediately preceding embodiment, wherein the packaging cells are stably transformed with the at least one nucleic acid encoding an adenovirus packaging helper gene, the at least one nucleic acid encoding an AAV replication protein sufficient for packaging, and / or the at least one nucleic acid encoding an AAV capsid protein sufficient for packaging.

[0056] Embodiment 49 is the method of any one of the preceding embodiments, wherein the packaging cell comprises(a) a nucleic acid encoding E2A, a nucleic acid encoding E4, and a nucleic acid encoding VA genes;(b) a nucleic acid encoding an AAV replication protein;(c) a nucleic acid encoding an AAV capsid protein; and / or(d) a nucleic acid encoding a gene of interest, optionally comprising an inverted terminal repeat.

[0057] Embodiment 50 is the method of any one of embodiments 6-49, wherein the gene of interest encodes at least one protein or RNA of interest.

[0058] Embodiment 51 is the method of any one of embodiments 6-50. wherein the gene of interest encodes at least one protein or RNA of therapeutic interest.

[0059] Embodiment 52 is the method of any one of embodiments 6-51, wherein the gene of interest encodes a chimeric antigen receptor.Atty. Docket No. 01277-0058-00PCT

[0060] Embodiment 53 is the method of any one of embodiments 6-52, wherein the one or more helper genes is expressed under a constitutive promoter, an activatable promoter, or an inducible promoter.

[0061] Embodiment 54 is the method of any one of embodiments 46-53, wherein the one or more helper viruses comprises an adenovirus, a baculovirus, or a herpes simplex virus.

[0062] Embodiment 55 is the method of the immediately preceding embodiment, wherein the adenovirus is a wild-type adenovirus.

[0063] Embodiment 56 is the method of any one of the preceding embodiments, wherein a titer of the AAV at harvest is higher than that of a control.

[0064] Embodiment 57 is the method of the immediately preceding embodiment, wherein the control comprises the same AAV harvested from a cell culture comprising only a single round of transfection, or comprising at least two rounds of transfection, wherein each round of transfection comprises transfecting the packaging cell with an identical set of nucleic acids encoding the AAV.

[0065] Embodiment 58 is the method of embodiment 56 or embodiment 57, wherein the titer of the AAV at harvest is at least 1.1-fold, at least 1.15-fold, at least 1.2-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, or at least 2-fold greater than that of the control.

[0066] Embodiment 59 is the method of any one of embodiments 56-58, wherein the titer of the AAV at harvest is at least 10%. at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% greater, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% greater than that of the control.

[0067] Embodiment 60 is the method of any one of embodiments 39-59, wherein the cell culture comprising the packaging cell comprises a HEK cell culture medium.

[0068] Embodiment 61 is the method of any one of embodiments 39-60, wherein the cell culture comprising the packaging cell comprises glutamine and / or a shear protectant.

[0069] Embodiment 62 is the method of any one of embodiments 39-61, wherein the cell culture comprising the packaging cell is serum-free.

[0070] Embodiment 63 is the method of any one of embodiments 39-62, wherein the cell culture comprising the packaging cell is a suspension culture.

[0071] Embodiment 64 is the method of any one of embodiments 39-63, wherein the cell culture comprising the packaging cell is in a bioreactor.Atty. Docket No. 01277-0058-00PCT

[0072] Embodiment 65 is the method of any one of embodiments 39-62, wherein the cell culture comprising the packaging cell is a batch-fed cell culture.

[0073] Embodiment 66 is the method of any one of embodiments 39-62, wherein the cell culture comprising the packaging cell is a continuous cell culture.

[0074] Embodiment 67 is the method of any one of embodiments 39-62, wherein the cell culture comprising the packaging cell is a perfusion cell culture.

[0075] Embodiment 68 is the method of any one of the preceding embodiments, wherein the packaging cell is a mammalian cell.

[0076] Embodiment 69 is the method of the immediately preceding embodiment, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) cell, Madin-Darby canine kidney (MDCK) cell, or Vero cell.

[0077] Embodiment 70 is the method of embodiment 68 or embodiment 69, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0078] Embodiment 71 is the method of embodiment 68 or embodiment 69, wherein the mammalian cell is a human cell.

[0079] Embodiment 72 is the method of the immediately preceding embodiment, wherein the human cell is a HEK293 cell.

[0080] Embodiment 73 is the method of any one of embodiments 1-67, wherein the packaging cell is an insect cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 illustrates an exemplary method of the disclosed embodiments wherein packaging cells are transfected with pHelper, pRep / Cap, and pTransgene plasmids in a first round of transfection, and are transfected with a pCap plasmid in a second round of transfection after (such as approximately 24 hours after) the first round of transfection.

[0082] FIG. 2 shows experimental results of the method illustrated in FIG. 1 on AAV titer and encapsidation.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0083] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with such embodiments, it will be understood thatAtty. Docket No. 01277-0058-00PCT they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims.

[0084] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids, reference to “a cell” includes a plurality of cells, and the like.

[0085] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.

[0086] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0087] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls.I. Definitions

[0088] As used herein, “or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.

[0089] As used herein, “adeno-associated virus” or “AAV” refers to an adeno-associated virus vector, including any AAV serotype or variant, including but not limited to an AAV1, AAV2,Atty. Docket No. 01277-0058-00PCTAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO (see, e.g., SEQ ID NO: 81 of US 9,790.472), AAVrh74 (see, e.g., SEQ ID NO: 1 of US 2015 / 0111955), AAV9, AAV9P also known as AAVMYO (see, e.g., Weinmann et al., Nature Communications, 2020, 11:5432), AAV 11, AAV 12, and Myo- AAV (as described, for example, in Tabebordbar et al., 2021, Cell, 184:1-20 (e.g., MyoAAV 1A, 2A, 3A, 4A, 4C, or 4E)), and chimeras thereof (such as those listed in Viney et. Al, J Virol. 2021, 95(7): e02023-20), wherein the number following AAV indicates the AAV serotype. The term “AAV” can also refer to any known AAV (vector) system. In some embodiments, the AAV vector is a single- stranded AAV (ssAAV). In some embodiments, the AAV vector is a double- stranded AAV (dsAAV). Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV6 vector, etc. See, e.g., McCarty et al.. Gene Ther. 2001; 8:1248-54, Naso et al., BioDrugs. 2017; 31:317-334, and references cited therein for detailed discussion of various AAV vectors. Structurally, AAVs are small (~25 nm), singlestranded DNA, non-enveloped viruses with an icosahedral capsid. As used herein, “AAV” can refer to naturally occurring or engineered AAV serotypes and recombinant AAVs (rAAVs) and variants that can differ in the composition and structure of their capsid protein, and can have varying tropism, i.e., ability to transduce different cell types. When combined with active promoters, this tropism defines the site of gene expression, e.g., in a host.

[0090] As used herein, “recombinant AAV” refers to an AAV with a capsid having packaged therein a heterologous nucleic acid molecule comprising an expression cassette for a desired product, such as a gene product. Such an expression cassette may contain an AAV 5’ and / or 3’ inverted terminal repeat sequence flanking a nucleic acid of interest, such as a gene sequence, in which the nucleic acid of interest is operably linked to expression control sequences. An expression cassette is thus useful for effecting the expression of the desired product (e.g., protein or RNA) in an intended target cell. Expression cassettes of use herein are known and available in the art or can be readily constructed from components that are available in the art. A “heterologous nucleic acid” as used herein is a nucleic acid derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a nucleic acid introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous nucleic acid. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linkedAtty. Docket No. 01277-0058-00PCT is a heterologous promoter. The term “heterologous nucleic acid” as used herein includes coding as well as non-coding nucleotide sequences.

[0091] As used herein, “isolate” or “isolating” refers to separation of a biological component (such as an AAV) from some or all other components of a mixture (such as cell culture media, whole cells, cellular materials, and / or cell lysate). A biological component (such as an AAV) need not be completely separated from other components of the mixture to be “isolated,” but may be separated from at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%. at least 96%, at least 97%, at least 98%, or at least 99% of other components of the mixture.

[0092] As used herein, a “packaging cell” refers to a cell wherein an AAV may be “packaged.” “Packaging” refers to a series of intracellular events that result in the assembly of the capsid proteins and encapsidation of the vector genome to form an AAV particle, thereby “packaging” the vector (such as a vector comprising a nucleic acid molecule of interest) in a delivery vehicle (the AAV particle). Unless context dictates otherwise, as used herein, the terms “cell line” or “cell culture” refer to a packaging cell line or packaging cell culture. The term “packaging cell” as used herein can also mean a “producer cell” as known in the art and as described herein. A producer cell may comprise (e.g., be transfected with) the AAV genome to be produced. In some embodiments, a producer cell may be generated by stably integrating AAV genes (such as along with an ITR-flanked therapeutic gene of interest) into a cell line (such as CHO, HEK293, or SF9 cells). AAV production may then be triggered by the addition of a helper virus to provide functional genes for AAV replication.

[0093] As used herein, a cell has been “transfected,” e.g., with at least one nucleic acid encoding an AAV, when such nucleic acids have been introduced inside the cell. In some embodiments, a nucleic acid encoding an AAV as described herein is a plasmid (such as a recombinant plasmid). In some embodiments, each of a plurality of nucleic acids encoding an AAV is a separate nucleic acid (such as a separate plasmid). In some embodiments, a single nucleic acid (such as a single plasmid) may comprise two or more (or each) of the plurality of nucleic acids encoding an AAV. A cell may be transfected, e.g., with one or more (such as one, two, three, or four) recombinant plasmids or other nucleic acids through any process known in the art, including but not limited to electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex. The term “transfection” as used herein encompasses any means of introducing one or more (such as one, two, three, or four) nucleic acids inside a cell, such as, but not limited to,Atty. Docket No. 01277-0058-00PCT transduction or infection with a DNA or RNA virus or viral vector. In some embodiments, a transfected nucleic acid may be introduced into a chromosome or mini-chromosome in the cell.

[0094] As used herein, a “concentration of a nucleic acid encoding an AAV” refers to an amount of the nucleic acid encoding the AAV, such as an amount of the nucleic acid encoding the AAV added to a packaging cell culture during a transfection procedure. A concentration of a nucleic acid encoding an AAV may be provided in any suitable units as known in the art, e.g., picograms per milliliter, nanograms per milliliter, micrograms per milliliter, milligrams per milliliter, picomoles, nanomoles, micromoles, or millimoles. A concentration may also be provided as an amount (such as picograms, nanograms, micrograms, milligrams, picomoles, nanomoles, micromoles, or millimoles) of the nucleic acid encoding the AAV added to a packaging cell culture during a transfection procedure per the total number of packaging cells in a packaging cell culture. By way of an illustrative, non-limiting example, a concentration provided in this format may be 1 pg of the nucleic acid encoding the AAV per 1 x 106cells.

[0095] As used herein a “ratio,” such as a “ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV,” refers to a relationship in quantity, such as a proportion, of the first nucleic acid to the second nucleic acid. A ratio herein can also be used to refer to a relationship in quantity, such as a proportion, of a first nucleic acid of a plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids. By way of an illustrative, nonlimiting example, a ratio of a first nucleic acid and a second nucleic acid, or a ration of a first nucleic acid of a plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids, may be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:10, 1:20, etc., as suitable.II. Exemplary Methods of Producing an AAVA. Overview

[0096] Various factors may limit AAV production using current cell culture methods. Standard transfection methods for AAV production include introducing into packaging cells plasmids encoding a heterologous nucleic acid (such as a nucleic acid molecule of interest), AAV rep and cap genes, and adenoviral accessory functions in a single round of transfection. Recent modifications of standard methods include transfecting packaging cells with these plasmids together in two rounds of transfection. However, AAV production efficiencies, such asAtty. Docket No. 01277-0058-00PCT production of recombinant AAVs for use in therapeutic applications, remain limited, and improved methods are needed.

[0097] Accordingly, the present disclosure provides improved methods for AAV production using packaging cell cultures, such as methods comprising transfecting one or more plasmids into packaging cell cultures in at least two rounds of transfection. In the disclosed methods, at least two rounds of transfection (such as at least first and second rounds of transfection) differ from one another. For example, in some embodiments, one round of transfection comprises one or more nucleic acids encoding an AAV that is not present in another round of transfection. In some embodiments, a first round of transfection comprises one or more nucleic acids encoding an AAV that is not present in a second round of transfection. In some embodiments, a second round of transfection comprises one or more nucleic acids encoding an AAV that is not present in a first round of transfection. In another example, in some embodiments, a concentration of at least one nucleic acid encoding an AAV is different (such as higher or lower) in one round of transfection (such as a first round of transfection) as compared to another round of transfection (such as a second round of transfection). By way of another example, in some embodiments, a ratio of at least one nucleic acid encoding an AAV to at least a second nucleic acid encoding the AAV is different (e.g., higher or lower) in one round of transfection (such as a first round of transfection) as compared to another round of transfection (such as a second round of transfection). In methods comprising more than two rounds of transfection (such as 3, 4, 5, 6. or more rounds of transfection), at least two of the more than two rounds of transfection differ from one another, such as described above and elsewhere herein. The improved methods disclosed herein can, e.g., increase an AAV titer and / or increase a percentage of full capsid produced (i.e„ increase a full capsid to empty capsid ratio) as compared to standard transfection methods.

[0098] In some embodiments of the disclosed methods, the plurality of nucleic acids encoding an AAV encodes all of the nucleic acids necessary for producing an AAV, such as under conditions suitable for AAV production known in the art and described herein. In some embodiments, a packaging cell is transfected, in two rounds of transfection, with a plurality of nucleic acids encoding an AAV, wherein the plurality of nucleic acids encoding the AAV together encode all of the nucleic acids necessary for producing an AAV. In some embodiments, a packaging cell is transfected, in more than two rounds of transfection (such as 3, 4, 5. 6, or more rounds of transfection, such as described elsewhere herein), with a plurality of nucleic acids encoding an AAV. wherein the plurality of nucleic acids encoding the AAV together encode all of the nucleicAtty. Docket No. 01277-0058-00PCT acids necessary for producing an AAV. In some embodiments, the plurality of nucleic acids encoding an AAV that together encode all of the nucleic acids necessary for producing an AAV comprise each of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication (“rep”) protein; (iii) at least one nucleic acid encoding an AAV capsid (“cap”) protein; and (iv) at least one nucleic acid encoding an adenovirus packaging helper gene. A “gene of interest” herein may also be referred to as a “transgene,” a “nucleotide sequence of interest,” or a “nucleic acid molecule of interest.” In some embodiments, a gene of interest encodes at least one protein or RNA of interest. In some embodiments, a gene of interest encodes at least one protein or RNA of therapeutic interest. In particular embodiments, a gene of interest encodes a chimeric antigen receptor.

[0099] In some embodiments, the method comprises transfecting a packaging cell with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection, wherein (a) a first round of transfection comprises transfecting the packaging cell with at least a first nucleic acid of the plurality of nucleic acids encoding the AAV; and (b) a second round of transfection comprises transfecting the packaging cell with at least a second nucleic acid of the plurality of nucleic acids encoding the AAV. In some embodiments, the packaging cell is not transfected with the second nucleic acid of the plurality of nucleic acids encoding the AAV in the first round of transfection; and / or the packaging cell is not transfected with the first nucleic acid of the plurality of nucleic acids encoding the AAV in the second round of transfection.

[0100] In some embodiments, the packaging cell is not transfected with the second nucleic acid of the plurality of nucleic acids encoding the AAV in the first round of transfection. In some embodiments, the packaging cell is not transfected with the first nucleic acid of the plurality of nucleic acids encoding the AAV in the second round of transfection. In some embodiments, the packaging cell is not transfected with the second nucleic acid of the plurality of nucleic acids encoding the AAV in the first round of transfection, and the packaging cell is not transfected with the first nucleic acid of the plurality of nucleic acids encoding the AAV in the second round of transfection.

[0101] In some embodiments, the packaging cell is not transfected with the second nucleic acid of the plurality of nucleic acids encoding the AAV in the first round of transfection, and the packaging cell is transfected with the first nucleic acid of the plurality of nucleic acids encoding the AAV in the second round of transfection.Atty. Docket No. 01277-0058-00PCT

[0102] In some embodiments, the packaging cell is transfected with the second nucleic acid of the plurality of nucleic acids encoding the AAV in the first round of transfection, and the packaging cell is not transfected with the first nucleic acid of the plurality of nucleic acids encoding the AAV in the second round of transfection.

[0103] In some embodiments, the plurality of nucleic acids encoding an AAV encodes, e.g., AAV rep and / or cap genes, a nucleotide sequence of interest (e.g., a nucleic acid encoding a protein or RNA of interest, or a noncoding sequence of interest), one or more helper functions, an additional element (such as one or more elements that can enhance viral second-strand DNA synthesis, one or more aspects of viral assembly and production, and / or AAV-mediated transgene expression (See, e.g., Ma W et al.. Hum Gene Ther. 2011, 22(5):633-40; Khan N et al. Cancer Med. 2020, 9(9):3188-3201), or any combination thereof. In some embodiments, a nucleic acid encoding an AAV is a plasmid (such as a recombinant plasmid). In some embodiments, each of a plurality of nucleic acids encoding an AAV are separate nucleic acids (such as separate plasmids). In some embodiments, a single nucleic acid (such as a single plasmid) may comprise two or more (or each) of the plurality of nucleic acids encoding an AAV. For example, in some embodiments, a nucleic acid (such as a plasmid) encodes both an AAV replication gene and an AAV capsid gene. In another example, in some embodiments, a nucleic acid encodes an AAV capsid gene and does not encode an AAV replication gene.

[0104] In some embodiments, the at least first nucleic acid of the plurality of nucleic acids encoding an AAV encodes AAV rep and / or cap genes. In some embodiments, the at least second nucleic acid of the plurality of nucleic acids encoding an AAV encodes AAV rep and / or cap genes. In some embodiments, the at least first nucleic acid of the plurality of nucleic acids encoding an AAV encodes a nucleotide sequence of interest (e.g., a nucleic acid encoding a protein or RNA of interest, or a noncoding sequence of interest). In some embodiments, the at least second nucleic acid of the plurality of nucleic acids encoding an AAV encodes a nucleotide sequence of interest (e.g., a nucleic acid encoding a protein or RNA of interest, or a noncoding sequence of interest). In some embodiments, the at least first nucleic acid of the plurality of nucleic acids encoding an AAV encodes one or more helper functions. In some embodiments, the at least second nucleic acid of the plurality of nucleic acids encoding an AAV encodes one or more helper functions. In some embodiments, the at least first nucleic acid of the plurality of nucleic acids encoding an AAV encodes an additional element, such as one or more elements that can enhance viral second-strand DNA synthesis, one or more aspects of viral assembly andAtty. Docket No. 01277-0058-00PCT production, and / or AAV-mediated transgene expression. In some embodiments, the at least second nucleic acid of the plurality of nucleic acids encoding an AAV encodes an additional element, such as one or more elements that can enhance viral second-strand DNA synthesis, one or more aspects of viral assembly and production, and / or AAV-mediated transgene expression. In some embodiments, the at least first nucleic acid of the plurality of nucleic acids encoding an AAV comprises a final component for production of the AAV in the packaging cell. In some embodiments, the at least second nucleic acid of the plurality of nucleic acids encoding an AAV comprises a final component for production of the AAV in the packaging cell. In such embodiments, a packaging cell has been transfected and / or infected and / or co-infected with other components needed to produce the AAV, and transfection of the at least one nucleic acid encoding the AAV triggers production of the AAV.

[0105] In some embodiments of the disclosed methods, the first round of transfection comprises transfecting the packaging cell with a first set of the plurality of nucleic acids encoding the AAV, and the second round of transfection comprises transfecting the packaging cell with a second set of the plurality of nucleic acids encoding the AAV, wherein the first set and the second set of nucleic acids of the plurality of nucleic acids encoding the AAV are not the same. For example, in some embodiments, at least one nucleic acid of the first set and at least one nucleic acid of the second set are not the same. In some embodiments, the first set of the plurality of nucleic acids encoding the AAV comprises at least one nucleic acid encoding the AAV that is not present in the second set of the plurality of nucleic acids encoding the AAV. In some embodiments, the second set of the plurality of nucleic acids encoding the AAV comprises at least one nucleic acid encoding the AAV that is not present in the first set of nucleic acids encoding the AAV. For example, in a particular embodiment, the first set of the plurality of nucleic acids encoding the AAV comprises a nucleic acid encoding an AAV replication gene and a nucleic acid encoding an AAV capsid gene (and optionally additional nucleic acids encoding the AAV), and the second set of the plurality of nucleic acids encoding the AAV comprises a nucleic acid encoding an AAV capsid gene (and optionally additional nucleic acids encoding the AAV), but does not comprise a nucleic acid encoding an AAV replication gene.

[0106] In some embodiments, the first set of the plurality of nucleic acids encoding the AAV comprises at least one nucleic acid encoding the AAV that shares less than 95%. less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less thanAtty. Docket No. 01277-0058-00PCT25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0% sequence identity with at least one nucleic acid of the second set of the plurality of nucleic acids encoding the AAV. In some embodiments, the second set of the plurality of nucleic acids encoding the AAV comprises at least one nucleic acid encoding the AAV that shares less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%. less than 4%, less than 3%, less than 2%, less than 1%, or 0% sequence identity with at least one nucleic acid of the first set of the plurality of nucleic acids encoding the AAV. In some embodiments, a ratio of at least one of the plurality of nucleic acids transfected in one round of transfection (such as a first round of transfection) is different from a ratio of the at least one of the plurality of nucleic acids transfected in another round of transfection (such as a second round of transfection). In some embodiments, the ratio is a ratio of the at least one of the plurality of nucleic acids to at least a second nucleic acid of the plurality of nucleic acids. In some embodiments, the ratio is a ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids. In some embodiments, a ratio of the at least one of the plurality of nucleic acids to the at least second nucleic acid of the plurality of nucleic acids in one round of transfection (such as a first round of transfection) is 1:1.25 to 1:100, such as 1: 1.5. 1:2, 1:3. 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10, 1:15. 1:20. 1:30. 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, and the ratio is different in another round of transfection (such as a second round of transfection). In some embodiments, a ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids in one round of transfection (such as a first round of transfection) is 1:1.25 to 1:100, such as 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, and the ratio is different in another round of transfection (such as a second round of transfection).

[0107] In some embodiments, the ratio of the at least one of the plurality of nucleic acids to the at least second nucleic acid of the plurality of nucleic acids is between 0.1 and 100 times higher, such as between 0.1 and 75, between 0.1 and 50, between 0.1 and 40, between 0.1 and 30, between 0.1 and 25, between 0.1 and 20, between 0.1 and 15, between 0.1 and 10, between 0.1 and 9. between 0.1 and 8, between 0.1 and 7, between 0.1 and 6. between 0.1 and 5, between 0.1 and 4, between 0.1 and 3, between 0.1 and 2.5, between 0.1 and 2, between 0.1 and 1.5, between 0.1 and 1, between 0.1 and 0.5, between 1 and 10, between 1 and 7.5, between 1 and 5, betweenAtty. Docket No. 01277-0058-00PCT1 and 4, between 1 and 3, between 1 and 2, or between 1 and 1.5 times higher in one round of transfection (such as in a first round of transfection) as compared to the ratio of the at least one of the plurality of nucleic acids to the at least second nucleic acid of the plurality of nucleic acids in another round of transfection (such as in a second round of transfection). In some embodiments, the ratio of the at least one of the plurality of nucleic acids to the at least second nucleic acid of the plurality of nucleic acids is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times higher in one round of transfection (such as in a first round of transfection) as compared to the ratio of the at least one of the plurality of nucleic acids to the at least second nucleic acid of the plurality of nucleic acids in another round of transfection (such as in a second round of transfection). In some embodiments, the ratio of the at least one of the plurality of nucleic acids to the at least second nucleic acid of the plurality of nucleic acids is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9. at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 times higher in one round of transfection (such as in a first round of transfection) as compared to the ratio of the at least one of the plurality of nucleic acids to the at least second nucleic acid of the plurality of nucleic acids in another round of transfection (such as in a second round of transfection).

[0108] In some embodiments, the ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids is between 0.1 and 100 times higher, such as between 0.1 and 75, between 0.1 and 50, between 0.1 and 40, between 0.1 and 30, between 0.1 and 25, between 0.1 and 20, between 0.1 and 15, between 0.1 and 10, between 0.1 and 9. between 0.1 and 8, between 0.1 and 7, between 0.1 and 6. between 0.1 and 5, between 0.1 and 4, between 0.1 and 3, between 0.1 and 2.5, between 0.1 and 2, between 0.1 and 1.5, between 0.1 and 1, between 0.1 and 0.5, between 1 and 10, between 1 and 7.5, between 1 and 5, between 1 and 4, between 1 and 3, between 1 and 2, or between 1 and 1.5 times higher in one round of transfection (such as in a first round of transfection) as compared to the ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids in another round of transfection (such as in a second round of transfection). In some embodiments, the ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5. 6, 7, 8, 9, 10, 15,Atty. Docket No. 01277-0058-00PCT20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 times higher in one round of transfection (such as in a first round of transfection) as compared to the ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids in another round of transfection (such as in a second round of transfection). In some embodiments, the ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20. at least 25, at least 30, at least 40, at least 50. at least 60, at least 70, at least 80, at least 90, or at least 100 times higher in one round of transfection (such as in a first round of transfection) as compared to the ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids in another round of transfection (such as in a second round of transfection).

[0109] In some embodiments, the plurality of nucleic acids encoding the AAV is the same in a first round of transfection and in a second round of transfection, and a ratio of at least one of the plurality of nucleic acids transfected in the first round of transfection is different from a ratio of the at least one of the plurality of nucleic acids transfected in the second round of transfection. In some such embodiments, the ratio is a ratio of the at least one of the plurality of nucleic acids to at least a second nucleic of the plurality of nucleic acids. In some such embodiments, the ratio is a ratio of the at least one of the plurality of nucleic acids to the remaining nucleic acids of the plurality of nucleic acids.

[0110] In some embodiments, a concentration of at least one nucleic acid of the plurality of nucleic acids transfected in one round of transfection (such as in a first round of transfection) is different from a concentration of the at least one nucleic acid of the plurality of nucleic acids transfected in another round of transfection (such as in a second round of transfection). In some embodiments, a concentration of two or more (such as 2, 3, 4, 5, or 6) nucleic acids of the plurality of nucleic acids transfected in one round of transfection (such as in a first round of transfection) is different from a concentration of the two or more (such as 2, 3, 4, 5, or 6) nucleic acids of the plurality of nucleic acids transfected in another round of transfection (such as in a second round of transfection). In some embodiments, the concentration of the at least one nucleic acid is between 0.01 and 100 times higher, such as between 0.01 and 75, between 0.01 and 50, between 0.01 and 40, between 0.01 and 30, between 0.01 and 25, between 0.01 and 20, between 0.01 and 15, between 0.01 and 10, between 0.01 and 9, between 0.01 and 8, between 0.01 and 7,Atty. Docket No. 01277-0058-00PCT between 0.01 and 6, between 0.01 and 5, between 0.01 and 4, between 0.01 and 3, between 0.01 and 2.5, between 0.01 and 2, between 0.01 and 1.5, between 0.01 and 1, between 0.01 and 0.5, between 1 and 10, between 1 and 7.5, between 1 and 5, between 1 and 4, between 1 and 3, between 1 and 2, or between 1 and 1.5 times higher in one round of transfection (such as in a first round of transfection) as compared to the concentration of the at least one nucleic acid in another round of transfection (such as in a second round of transfection). In some embodiments, the concentration of the at least one nucleic acid is 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 15, 20. 25. 30. 40, 50, 60, 70, 80, 90, or 100 times higher in one round of transfection (such as in a first round of transfection) as compared to the concentration of the at least one nucleic acid in another round of transfection (such as in a second round of transfection). In some embodiments, the concentration of the at least one nucleic acid is at least 0.01, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 times higher in one round of transfection (such as in a first round of transfection) as compared to the concentration of the at least one nucleic acid in another round of transfection (such as in a second round of transfection).

[0111] In some embodiments, the first set of nucleic acids of the plurality of nucleic acids encoding the AAV comprises or consists of 1-6, 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, 5, or 6 nucleic acids. In some embodiments, the second set of nucleic acids of the plurality of nucleic acids encoding the AAV comprises or consists of 1-6, 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, 5, or 6 nucleic acids. In particular embodiments, the first set and the second set of nucleic acids of the plurality of nucleic acids encoding the AAV are not the same. In other embodiments, the first set and the second set of nucleic acids of the plurality of nucleic acids encoding the AAV are the same. In some such embodiments, however, a ratio or concentration of at least one nucleic acid in the first set of nucleic acids is different from a ratio or concentration of the at least one nucleic acid in the second set of nucleic acids.

[0112] In some embodiments of the disclosed methods, the plurality of nucleic acids encoding the AAV comprises one or more of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein; (iii) at least one nucleic acid encoding an AAV capsid protein; and (iv) at least one nucleic acid encoding an adenovirus packaging helper gene.Atty. Docket No. 01277-0058-00PCT

[0113] In some embodiments, the plurality of nucleic acids is sufficient for production of the AAV when the packaging cell is cultured under conditions suitable for production of the AAV. In some embodiments, the first round of transfection comprises dual transfection, triple transfection, or quad transfection, such as described elsewhere herein.

[0114] In particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV capsid protein.

[0115] In other particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest;(ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV replication protein.

[0116] In particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and(iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the gene of interest.

[0117] In some particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest;(ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein.

[0118] In particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and(iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the secondAtty. Docket No. 01277-0058-00PCT round of transfection comprises or consists of transfecting the packaging cell with a nucleic acid encoding the adenovirus packaging helper gene.

[0119] In other particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest;(ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; and (ii) at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein.

[0120] In particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and(iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; and (ii) at least one nucleic acid encoding the adenovirus packaging helper gene.

[0121] In some particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein; and (ii) at least one nucleic acid encoding the adenovirus packaging helper gene.

[0122] In particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with one of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; or (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; (b) the second round of transfection comprises or consists of transfecting the packaging cell with any one of nucleic acids that was not transfected into the packaging cell in step (a); and (c) a third round of transfection comprises or consists of transfecting the packaging cell with the nucleic acid that was not transfected into the packaging cell in step (a) or step (b).Atty. Docket No. 01277-0058-00PCT

[0123] In some particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with one of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; or (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with the two nucleic acids that were not transfected into the packaging cell in step (a).

[0124] In some particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with any two of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with the nucleic acid that was not transfected into the packaging cell in step (a).

[0125] In some particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with any one or two of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding the adenovirus packaging helper gene.

[0126] In some particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest;(ii) at least one nucleic acid encoding an AAV replication protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene. In some embodiments, the packaging cell is transfected with at least one nucleic acid encoding the AAV capsid protein during the second round of transfection and / or during an additional round of transfection following the second round of transfection.Atty. Docket No. 01277-0058-00PCT

[0127] In some particular embodiments, (a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene; and (b) the second round of transfection comprises or consists of transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene. In some embodiments, the packaging cell is transfected with at least one nucleic acid encoding the AAV capsid protein during the second round of transfection and / or during an additional round of transfection following the second round of transfection.

[0128] Some embodiments of the disclosed methods further comprise a plurality of additional rounds of transfection, optionally after the second or the third round of transfection. In some embodiments, each of the plurality of additional rounds of transfection comprises transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein; (iii) at least one nucleic acid encoding an AAV capsid protein; and (iv) at least one nucleic acid encoding an adenovirus packaging helper gene.

[0129] In some embodiments, the plurality of additional rounds of transfection comprises 1-100 additional rounds of transfection, such as 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1- 9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 additional rounds of transfection, optionally after the second or the third round of transfection. In some embodiments, the plurality of additional rounds of transfection comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional rounds of transfection, optionally after the second or the third round of transfection.

[0130] In some embodiments, the number of rounds of transfection is sufficient to attain a desired level of expression of a gene of interest. In some embodiments, the number of rounds of transfection comprises 1-100 rounds of transfection, such as 1-90. 1-80, 1-70, 1-60, 1-50, 1-40. 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-9, 2-Atty. Docket No. 01277-0058-00PCT8, 2-7, 2-6, 2-5, 2-4, 2-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 rounds of transfection.

[0131] In some embodiments, an amount of elapsed time between any two consecutive rounds of transfection is independently selected from 3-48, 3-47, 3-46, 3-45, 3-44, 3-43, 3-42, 3-41, 3-40, 3-39, 3-38, 3-37. 3-36, 3-35, 3-34, 3-33, 3-32. 3-31, 3-30, 3-29, 3-28, 3-27. 3-26, 3-25, 3-24, 3- 23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6,3-5, 3-4, 4-36, 4-35, 4-34, 4-33, 4-32, 4-31, 4-30, 4-29, 4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22,4-21, 4-20, 4-19. 4-18, 4-17, 4-16, 4-15, 4-14. 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours. In certain embodiments, an amount of elapsed time between the first round of transfection and the second round of transfection is 3-48, 3-47, 3-46, 3-45, 3-44, 3-43, 3-42, 3-41, 3-40, 3-39, 3-38, 3-37, 3-36, 3-35,3-34, 3-33, 3-32, 3-31, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-22, 3-21, 3-20, 3-19, 3- 18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-36, 4-35, 4-34, 4-33, 4-32, 4-31, 4-30, 4-29, 4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17,4-16, 4-15, 4-14. 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 3, 4, 5, 6, 7, 8, 9. 10. 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0132] In some embodiments, a concentration of a nucleic acid encoding an AAV is the same in each round of transfection wherein the nucleic acid encoding the AAV is transfected into the packaging cell. In other embodiments, a concentration of a nucleic acid encoding an AAV is different in at least two rounds of transfection wherein the nucleic acid encoding the AAV is transfected into the packaging cell.

[0133] In some embodiments, a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is the same in each round of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell.

[0134] In other embodiments, a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in at least two rounds of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell.Atty. Docket No. 01277-0058-00PCT

[0135] In certain embodiments, a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in each round of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell. In some embodiments, a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in at least two rounds of transfection, and both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell in each of the at least two rounds of transfection. In some embodiments, a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in each round of transfection, and both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell in each round of transfection.

[0136] Some embodiments of the present disclosure further comprise harvesting the AAV from the cell culture. In some embodiments of the disclosed methods, a titer of an AAV at harvest is higher than that of a control, such as a control comprising the same AAV harvested from a cell culture comprising only a single round of transfection, or comprising at least two rounds of transfection, wherein each round of transfection comprises transfecting the packaging cell with an identical set of nucleic acids encoding the AAV. In some embodiments of the disclosed methods, the titer of the AAV at harvest is at least 1.1-fold, at least 1.15-fold, at least 1.2-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, or at least 2-fold greater than that of the control. In some embodiments, the titer of the AAV at harvest is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% greater, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% greater than that of the control.B. Packaging Cells

[0137] Embodiments of the disclosed methods comprise transfecting a packaging cell with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection. In particular embodiments, the method comprises transfecting a packaging cell with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection, wherein a first round of transfection comprises transfecting the packaging cell with at least a first nucleic acid of the plurality of nucleic acids encoding the AAV, and a second round of transfection comprises transfecting the packaging cell with at least a second nucleic acid of the plurality of nucleic acidsAtty. Docket No. 01277-0058-00PCT encoding the AAV. In some embodiments, the method comprises culturing packaging cells in the presence of an AAV under conditions suitable for production of the AAV

[0138] A variety of suitable cells and cell lines have been described for use in AAV production. For example, a packaging cell useful in the present disclosure can be a eukaryotic cell, a fungal cell, an insect cell, a prokaryotic cell (e.g.. bacterial or archaeal cell), or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, and include the progeny of the original cell if such cell has been transformed by the nucleic acid. In some embodiments, the cell is a eukaryotic cell, such as a 293T cell (such as a HEK293T cell). Exemplary mammalian cells include without limitation, Chinese hamster ovary (CHO) cells, CHO-K1 cells, CHO-derived cells, human embryonic kidney (HEK) cells (such as HEK293 cells, which express functional adenoviral El, and HEK-derived cells such as HEK293T or HEK293F), Madin-Darby canine kidney (MDCK) cells, Vero cells, EB66 cells, chicken embryo cells, RK cells, RAF cells, PK15 cells. MRC-5 cells, A549 cells, WEHI cells, 3T3 cells, 10T1 / 2 cells, BHK cells, COS 1 cells, COS 7 cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, WI38 cells, HeLa cells, Saos cells, C2C12 cells, L cells, HT1080 cells, HepG2 cells, NS-1 cells, and primary fibroblast, hepatocyte, and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. In certain embodiments, the cells are suspension-adapted cells. The selection of the mammalian species providing the cells is not a limitation of this invention; nor is the type of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

[0139] In other embodiments, the cell is a fungal cell, such as a yeast cell, such as a yeast cell of species Saccharomyces (such as Saccharomyces cerevisiae). In other embodiments, the cell is an insect cell (e.g., for use in baculovirus-based AAV production systems), such as an SF-9 cell. A “recombinant packaging cell” (also referred to as a “genetically modified packaging cell”) is a packaging cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a bacterial packaging cell is a genetically modified bacterial packaging cell by virtue of introduction of an exogenous nucleic acid (e.g., a plasmid or recombinant expression vector) into a suitable bacterial packaging cell, and a eukaryotic packaging cell is a genetically modified eukaryotic packaging cell (e.g., a mammalian cell), by virtue of introduction of an exogenous nucleic acid into a suitable eukaryotic packaging cell.

[0140] In particular, non-limiting embodiments, the packaging cells are Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells (such as HEK293 cells), Madin-DarbyAtty. Docket No. 01277-0058-00PCT canine kidney (MDCK) cells, or Vero cells. In one particular embodiment, the packaging cells are CHO cells. In another particular embodiment, the packaging cells are HEK293 cells.

[0141] In certain embodiments, the disclosed methods comprise culturing an AAV packaging cell in media that allows production of the AAV. Suitable media known in the art may be used for AAV production including, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and custom formulations such as those described in U.S. Pat. No. 6,566,118, and Sf-900 II SFM media as described in U.S. Pat. No. 6,723.551. AAV production culture media may be supplemented with serum or serum-derived recombinant proteins, e.g., at a level of 0.5%-20% (v / v or w / v). Alternatively, as is known in the art, AAV vectors may be produced in serum-free conditions which may also be referred to as media with no animal-derived products. Commercial or custom media designed to support production of A A Vs may also be supplemented with one or more cell culture components know in the art, including without limitation, glucose, vitamins, amino acids, and / or growth factors, in order to increase the titer or yield of AAV in production cultures.

[0142] In some embodiments, the cell culture comprises a culture medium suitable for culturing HEK293 cells, such as a chemically defined HEK293 cell culture medium. In a particular embodiment, the cell culture comprises glutamine and / or a shear protectant. In some embodiments, the cell culture is serum-free. Exemplary commercially available cell culture media include, but are not limited to, BalanCD media (e.g., BalanCD HEK293 medium or BalanCD CHO medium; Fujifilm and Irvine Scientific), Viral Production Media (e.g., Gibco LV-MAX™ Production Medium), and F17 medium (Invitrogen).C. Cell Culture Systems

[0143] Methods of culturing an AAV packaging cell to produce an AAV are known in the art, and exemplary methods are discussed herein. In certain embodiments, the disclosed methods comprise culturing an AAV packaging cell in media that allows production of the AAV. Cell culture procedures for both large and small-scale production of AAVs are encompassed by the present disclosure.

[0144] AAV production cultures can be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. As is known in the art, AAV production cultures include attachment-dependentAtty. Docket No. 01277-0058-00PCT cultures that can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. AAV vector production cultures may also include suspension-adapted host cells such as HEK.293, HeLa, and SF-9 cells that can be cultured in a variety of attachment-independent vessels including, for example, spinner flasks, stirred tank bioreactors, batch bioreactors, fed batch bioreactors, continuous culture bioreactors (e.g., perfusion bioreactors), and disposable systems such as the Wave bag system. In some embodiments, cells are initially ‘bulked up’ in tissue culture flasks or bioreactors and subsequently grown in multi-layered culture vessels or larger bioreactors (e.g., greater than 50 L). In such embodiments, media into which the cells are inoculated generally comprises an antifoam agent prior to the inoculation. Suitable conditions for culturing cells are known, and further, numerous suspension culture systems are known in the art for production of AAV (such as rAAV) particles (See, e.g., U.S. Pat. Nos. 6,995,006 and 9,783,826; U.S. Pat. Appl. Pub. No. 20120122155; Tissue Culture, Academic Press, Kruse and Paterson, editors (1973); and R. I. Freshney, Culture of animal cells: A manual of basic technique, fourth edition, Wiley-Liss Inc., 2000, ISBN 0-471-34889-9, each of which is incorporated herein by reference in its entirety).

[0145] In some embodiments, a cell culture disclosed herein is a suspension culture. In some embodiments, a cell culture is a suspension culture comprising HEK293 cells. In some embodiments, a cell culture is a suspension culture comprising HEK293 cells adapted for growth in suspension culture. In some embodiments, a suspension cell culture comprises a serum-free medium, an animal-component free medium, and / or a chemically defined medium. In some embodiments, suspension-adapted cells are cultured in a shaker flask, a spinner flask, a cellbag, or a bioreactor.

[0146] In some embodiments, a cell culture comprises cells attached to a substrate (e.g., microcarriers) that are themselves in suspension in a medium. In some such embodiments, the cells are HEK293 cells.

[0147] In some embodiments, a cell culture is an adherent culture. In some embodiments, a cell culture disclosed herein is an adherent culture comprising HEK293 cells. In some embodiments, an adherent cell culture comprises a serum-free medium, an animal-component free medium, and / or or a chemically defined medium.

[0148] In some embodiments, a cell culture of the disclosed methods comprises a high-density cell culture. In some embodiments, the cell culture has a total cell density of at least about 2xl06Atty. Docket No. 01277-0058-00PCT vc / mL, at least about 2xl07vc / mL, or at least about 2xl08vc / mL. In some embodiments, the cell culture has a total cell density of between IxlO6and 2xl07vc / mL, such as between IxlO6and IxlO7vc / mL, IxlO6and 75xl06vc / mL, IxlO6and 50xl06vc / mL, or IxlO6and 25xl06vc / mL. In some embodiments, more than about 50% of the cells of the cell culture are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells), CHO cells, Vero cells, or SF-9 cells. In particular embodiments, the cells are HEK293 cells. In other particular embodiments, the cells are HEK293 cells adapted for growth in suspension culture.

[0149] It will be understood by the skilled person that the conditions used in the methods disclosed herein will be dependent upon the host cell used. Typical conditions, for example the culture medium or temperature to be used, are well known in the art. In one embodiment, culturing is performed by incubating a mammalian packaging cell line (e.g., HEK293 or CHO cells) under humidified conditions. In a particular embodiment, the humidified conditions comprise incubating the transfected cells at about 37°C and about 5% CO2.

[0150] In some embodiments of the methods disclosed herein large volumes of cell culture can be present (e.g., during the commercial manufacturing processes). In some embodiments the methods disclosed herein are suitable for the processing of a large volume of cell culture comprising AAV (such as rAAV) particles. In particular embodiments, the cell culture is a suspension culture, a batch fed culture, a continuous cell culture, or a perfusion cell culture. In some embodiments, the methods disclosed herein are scalable, and thus can be carried out in any desired volume of culture medium, e.g., from 10 ml (e.g., in shaker flasks) to 0.5 L, 1 L, 5 L, 10 L, 20 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 500 L, 1,000 L, 5,000 L, 10,000 L, or more (e.g. in bioreactors such as wave bioreactor systems and stirred tanks). In some embodiments, the cell culture is cultured (such as in a bioreactor) in a volume of at least 0.01 L, at least 0.1 L, at least 0.2 L, at least 0.3 L, at least 0.4 L, at least 0.5 L, at least 0.6 L, at least 0.7 L, at least 0.8 L, at least 0.9 L, at least 1 L, at least 1.25 L, at least 1.5 L, at least 1.75 L, at least 2 L, at least 3 L, at least 4 L, at least 5 L, at least 6 L, at least 7 L, at least 8 L, at least 9 L, at least 10 L, at least 15 L, at least 20 L, at least 25 L, at least 50 L, at least 75 L, at least 100 L, at least 200 L, at least 300 L, at least 400 L, at least 500 L, or at least 1,000 L. In some embodiments, the cell culture is cultured (such as in a bioreactor) in a volume of 0.1-1,000 L, such as 1-1,000 L, 20-1,000 L, 50-1,000 L, 75-1,000 L, 100-1,000 L, 500-1,000 L, 1-900 L, 1-800 L, 1-700 L, 1- 600 L, 1-500 L, 1-400 L, 1-300 L, 1-200 L, 0.1-90 L, 0.1-80 L, 0.1-70 L, 0.1-60 L, 0.1-50 L, 0.1-Atty. Docket No. 0I277-0058-00PCT40 L, 0.1-30 L, 0.1-25 L, 0.1-20 L, 0.1-15 L, 0.1-10 L, 0.1-9 L, 0.1-8 L, 0.1-7 L, 0.1-6 L, 0.1-5 L, 0.1-4 L, 0.1-3 L, 0.1-2 L, 0.1-1 L, 0.1-0.75 L, 0.1-0.5 L, 0.1-0.25 L, 90-100 L, 80-100 L, 70- 100 L, 60-100 L, 50-100 L, 40-100 L, 30-100 L, 25-100 L, 20-100 L, 15-100 L, 10-100 L, 5-100 L, 1-100 L, 1-90 L, 1-80 L, 1-70 L, 1-60 L, 1-50 L, 1-40 L, 1-30 L, 1-20 L, 1-10 L, 1-5 L, 5-100 L, 5-90 L, 5-80 L, 5-70 L. 5-60 L, 5-50 L, 5-40 L, 50-30 L, 5-20 L, 5-10 L. 10-100 L, 10-90 L, 10-80 L, 10-70 L, 10-60 L, 10-50 L, 10-40 L, 10-30 L, 10-20 L, 20-100 L, 20-90 L, 20-80 L, 20- 70 L, 20-60 L, 20-50 L, 20-40 L, or 20-30 L.

[0151] In some embodiments, the cell culture is in a bioreactor. In some embodiments, the cell culture is cultured at a pH of between about 6 and about 8, such as at a pH between about 6.1 and 7.9, 6.2 and 7.8, 6.3 and 7.7, 6.4 and 7.6, 6.5 and 7.5, 6.6 and 7.4, 6.7 and 7.4, 6.8 and 7.4, 6.8 and 7.3, or 6.8 and 7.2. In some embodiments, the cell culture is cultured with a dissolved oxygen (DO) setpoint of about 20-60%, such as about 25-60%, about 30-60%, about 35-55%, about 40-55%, about 45-55%, about 20-55%, about 20-50%. about 20-45%, about 20-35%, or about 20-25%. In some embodiments, the cell culture is cultured at an agitation rate that imparts a power input per volume (P / V) of about 15-30 W / m3, such as about 15-25, 15-20, 20-30, or 25- 35 W / m3.D. Transfection

[0152] In some embodiments of the disclosed methods, a packaging cell is transfected with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection. The preparation of a packaging cell suitable for use in the disclosed methods involves techniques such as assembly of selected DNA sequences. This assembly may be accomplished utilizing conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., including polymerase chain reaction, synthetic methods, and any other suitable methods which provide the desired nucleotide sequence.

[0153] A cell (such as an AAV packaging cell) has been transfected by an exogenous nucleic acid, e.g.. a recombinant expression vector (such as a recombinant plasmid), when such a nucleic acid (e.g., DNA) has been introduced inside the cell. The presence of an exogenous nucleic acid can result in permanent or transient genetic change. The transfected exogenous nucleic acid may or may not be integrated (covalently linked) into the genome of the cell. A cell wherein an exogenous nucleic acid has been integrated into the genome of the cell is “stably transformed.”Atty. Docket No. 01277-0058-00PCT

[0154] On its own, AAV does not possess the ability to efficiently replicate its genetic material, and thus typically requires the presence of a helper virus. Three helper viruses are commonly used in AAV production: adenovirus (Ad), herpes simplex virus (HSV), and baculovirus (Bac). Accordingly, in some embodiments, the packaging cells are infected with one or more helper viruses, such as an adenovirus (such as a wild-type adenovirus), a baculovirus, or a herpes simplex virus. Ad and HSV helper AAV production methods use mammalian cell lines while the Bac system uses insect cells. Unlike the HSV and Bac systems, the Ad-based system genes required for helper function have been identified and can be expressed via a plasmid, thus eliminating the need for actual Ad to be produced. An exemplary “helper free” system can use multiple plasmids (such as one, two, three, or four plasmids). In some examples, a helper free system uses three plasmids, e.g., one containing the Ad E2A, E4, and VA RNA helper genes, a second expressing the AAV rep and cap genes, and third, a nucleic acid of interest (such as described elsewhere herein), e.g.. flanked by AAV inverted terminal repeats (ITRs).Accordingly, an AAV production system useful in the disclosed methods may be helper free. In some embodiments, the one or more helper functions, the AAV Rep protein, the AAV capsid protein, and / or the nucleic acid molecule of interest, is expressed under an activatable or inducible promoter. In certain embodiments, the nucleic acid molecule of interest encodes a chimeric antigen receptor (CAR) or T cell receptor (TCR), such as are known in the art and / or described elsewhere herein.

[0155] Genetic material can be introduced into cells using any of a variety of means. By way of illustration, such techniques include, for example, transfection with viral, bacteria, or yeast- derived plasmids. Transfection methods have been described in the art and include, for example, calcium phosphate co-precipitation, direct micro-injection into cultured cells, electroporation, liposome mediated gene transfer, lipid-mediated transfection or nucleic acid delivery using high- velocity microprojectiles. Other suitable transfection media include strontium phosphate, polycationic polymers, e.g., Superfect™ (Qiagen), liposomes, and cationic polymers such as polyethylenimine (PEI). In the disclosed embodiments, the term transfection encompasses any means of introducing a nucleic acid inside a cell, including, but not limited to, transduction or infection with a DNA or RNA virus or viral vector.

[0156] Any of these techniques can be used to introduce one or more exogenous nucleic acids, such as vector constructs, into suitable host cells. Generally, the exogenous nucleic acid traverses the host cell plasma membrane in order to be exposed to the cell's transcription and replicationAtty. Docket No. 01277-0058-00PCT machinery. The resulting cell can be transiently transfected with the exogenous nucleic acid molecule, i.e., the exogenous DNA will not be integrated into the genome of a transfected cell, but rather can exist episomally. Alternatively, the resulting cell can be stably transfected, i.e., the nucleic acid molecule becomes covalently linked with the host cell genome or is maintained and replicated as an episomal unit that can be passed on to progeny cells (e.g., is capable of extra- chromosomal replication at a sufficient rate).

[0157] In some embodiments, transfecting packaging cells with an AAV (such as with at least one nucleic acid encoding the AAV) comprises dual transfection, triple transfection, quad transfection, induction of a stable cell line, or co-infection with one or more helper viruses. In some embodiments of dual transfection, triple transfection, or quad transfection, the packaging cells are HEK293 cells or derivatives thereof. In some embodiments, HEK, CHO, or SF9 cells are used as stable cell lines from which AAV is induced. Stable cell lines may be engineered by introducing either the AAV rep and cap genes (packaging cell lines) and / or the AAV genome to be produced (producer cells). An AAV can be produced from packaging cell lines upon transfection of the AAV construct and coinfection with a helper virus (such as an Ad virus) or upon infection with a recombinant hybrid helper (such as an Ad / AAV). For producer cells containing both AAV rep / cap and AAV genome, production can follow a single step of infection with a helper virus. In some embodiments, co-infection with one or more helper viruses may comprise co-infection with a BAcv, Ad5, or HSV helper virus in SF9 cells. HEK293 cells, or BHK cells, respectively. Such transfection methods are known in the art, such as described in Clement and Grieger, Mol Ther Methods Clin Dev. 2016 Mar 16;3: 16002, which is incorporated by reference herein in its entirety.

[0158] For example, in some embodiments, “dual transfection” (also known as “2-plasmid transfection”) is performed using two plasmid vectors, typically wherein the first plasmid vector comprises a heterologous nucleotide sequence (such as a nucleic acid molecule of interest) flanked by ITRs and the second plasmid vector comprises the AAV rep and AAV cap gene sequences and adenovirus helper functions sequences, e.g., the Ad5 genes (VA RNAs, E2A, and E4OEF6). HEK293 cells constitutively express Ela / b, the fourth Ad function for AAV replication. In some embodiments, the second plasmid vector comprises from 5' to 3' an AAV rep coding region, an AAV cap coding region and a nucleotide sequence comprising a promoter region, e.g., an AAV p5 promoter region.Atty. Docket No. 01277-0058-00PCT

[0159] In other embodiments, “triple transfection” (also known as “3-plasmid transfection”) is performed using three plasmid vectors, such that the packaging cells are triple-transfected with at least one vector encoding a heterologous nucleic acid (such as a nucleic acid molecule of interest), at least one vector encoding AAV rep and cap genes, and at least one vector encoding adenoviral accessory functions, such as those described above for dual transfection. Most commonly, dual and triple transfections are performed using calcium phosphate (CaPCrf) plasmid precipitation in HEK293 cells.

[0160] In yet other embodiments, “quad transfection” (also known as “quadruple plasmid transfection” or “4-plasmid transfection”) is performed using four plasmid vectors, such that the packaging cells are quadruple-transfected with at least one vector encoding a heterologous nucleic acid (such as a nucleic acid molecule of interest), at least one vector encoding AAV rep and cap genes, at least one vector encoding adenoviral accessory functions, and at least one vector encoding an additional element, such as one or more elements that can enhance viral second-strand DNA synthesis, one or more aspects of viral assembly and production, and / or AAV-mediated transgene expression. See, e.g., Ma W et al., Hum Gene Ther. 2011, 22(5):633- 40; Khan N et al. Cancer Med. 2020, 9(9):3188-3201).

[0161] In some embodiments, the packaging cells comprise one or more nucleic acids encoding one or more helper genes, and / or the packaging cells are infected with one or more helper viruses. In some embodiments, the one or more helper genes comprise at least one nucleic acid encoding an adenovirus packaging helper gene, at least one nucleic acid encoding an AAV replication protein sufficient for packaging, and / or at least one nucleic acid encoding an AAV capsid protein sufficient for packaging. In a particular embodiment, the packaging cells are stably transformed with the at least one nucleic acid encoding an adenovirus packaging helper gene, the at least one nucleic acid encoding an AAV replication protein sufficient for packaging, and / or the at least one nucleic acid encoding an AAV capsid protein sufficient for packaging. In another particular embodiment, the packaging cells comprise (a) a nucleic acid encoding E2A, a nucleic acid encoding E4, and a nucleic acid encoding VA genes; (b) a nucleic acid encoding an AAV replication protein; (c) a nucleic acid encoding an AAV capsid protein; and / or (d) a nucleic acid encoding a gene of interest, optionally comprising an inverted terminal repeat.

[0162] In some embodiments of the disclosed methods, the AAV (such as an AAV5, AAV6, or AAV8) comprises an AAV capsid protein and a nucleic acid molecule that comprises an AAV 5’ inverted terminal repeat (ITR), a nucleic acid molecule of interest to be packaged into the AAVAtty. Docket No. 01277-0058-00PCT capsid, and a 3’ ITR. In such embodiments, the packaging cell can thus comprise (i) the at least one nucleic acid of interest to be packaged into the AAV capsid, (ii) a nucleic acid molecule encoding the AAV capsid protein under control of one or more sequences that direct its expression in the packaging cell, (iii) a nucleic acid molecule encoding an AAV Rep protein that expresses the AAV Rep protein in the cell to permit packaging of the nucleic acid of interest into the AAV capsid, and / or (iv) one or more helper functions required for packaging the nucleic acid molecule of interest into the AAV capsid. In certain embodiments, the nucleic acid molecule of interest encodes a chimeric antigen receptor (CAR) or T cell receptor (TCR)).

[0163] In some embodiments, the one or more helper genes is expressed under, for example, a constitutive promoter, an activatable promoter, or an inducible promoter. An exemplary promoter useful in the present disclosure can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / “ON” state), an activatable promoter, an inducible promoter (i.e., a promoter whose state, active / “ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein), a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc., e.g., a tissue specific promoter, a cell type specific promoter, etc.), or a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process).

[0164] Suitable promoters can be derived from viruses (viral promoters), or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a Rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology. 20: 497-500; 2002), an enhanced U6 promoter (e.g.. Xia et al., Nucleic Acids Res. Sep 1(31): 17; 2003), a human Hl promoter (Hl), and the like.

[0165] Selection of suitably transfected packaging cells may be conducted using any technique in the art. For example, the nucleic acid(s) introduced to the cell may be introduced simultaneously with or operably linked to one or more detectable or selectable markers as is known in the art. For example, a drug resistance gene can be used as a selectable marker. DrugAtty. Docket No. 01277-0058-00PCT resistant cells can then be picked and grown, and then tested for expression of a desired sequence, such as a packaging gene product, or a product of the heterologous nucleic acid, as appropriate. Testing for acquisition, localization, and / or maintenance of an introduced nucleic acid can be performed by means known in the art, for example, using DNA hybridization-based techniques (such as Southern blotting and other procedures as known in the art), Northern analysis of RNA extracted from the genetically altered cells, or by indirect immunofluorescence for the corresponding gene product.E. Harvesting AAV from a Packaging Cell Culture

[0166] In some embodiments, an AAV (such as an AAV5, AAV6, or AAV8) is harvested from AAV packaging cells and / or from the cell culture media. In some embodiments, after transfecting the cell culture with at least one nucleic acid encoding the AAV, the AAV is harvested from the cell culture every 1-6 days, every 1-5 days, every 1-4 days, every 1-3 days, every 2-6 days, every 2-5 days, or every 2-4 days. In particular embodiments, the AAV is harvested from the cell culture 1-6 days, 1 -5 days, 1-4 days, 1-3 days, 2-6 days, 2-5 days, or 2-4 days after transfecting the cell culture with the at least one nucleic acid encoding the AAV.

[0167] The AAV harvesting may comprise cell disruption (such as cell lysis) or may substantially not comprise cell disruption (such as in embodiments wherein harvesting the AAV from the cell culture comprises harvesting the AAV from cell culture media (e.g., from a supernatant of the cell culture)). Thus, A A Vs of the invention may be harvested from AAV production cultures by lysis of the packaging cells of the production culture or by harvest of the media (“spent” media) from the production culture, provided the cells are cultured under conditions known in the art to cause release of AAV particles into the media from intact cells (such as described more fully in U.S. Pat. No. 6,566,118). Suitable methods of lysing cells are also known in the art and include for example multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and / or proteases.

[0168] In some embodiments, harvesting the AAV from the cell culture comprises isolating the AAV from the cell culture. Methods of isolating an AAV from a cell culture can include, but are not limited to, chemical lysis, mechanical lysis, depth filtration, microfiltration, ultrafiltration, alternating tangential flow filtration, tangential flow depth filtration, batch chromatography, acoustic wave separation, and / or centrifugation. In some embodiments, isolating the AAV from the cell culture further comprises concentrating the AAV using a method selected fromAtty. Docket No. 01277-0058-00PCT chromatography, column-based chromatography, membrane-based chromatography, filtration, and / or precipitation. Accordingly, an AAV of the disclosed methods can be an AAV packaging cell culture harvest without further processing, or can be treated, e.g., prior to loading on a column using one or more of a clarifying treatment (such as filtration and / or centrifugation), one or more nucleases and / or proteases (to digest contaminating nucleic acids and / or proteins), an additionally chromatography step (such as affinity chromatography), a concentrating step, and the like.

[0169] The AAV particles produced according to a method disclosed herein can be isolated using methods known in the art. In some embodiments, methods of isolating AAV particles produced according to a method disclosed herein comprises downstream processing such as, for example, harvest of a cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, sterile filtration, or any combination thereof. In some embodiments, downstream processing includes at least 2, at least 3, at least 4, at least 5, or at least 6 of: harvest of a cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and sterile filtration.

[0170] At harvest, an AAV of the present disclosure may contain one or more of the following: packaging cell materials (such as packaging cell proteins and / or DNA); plasmid DNA; helper virus; helper virus proteins; helper virus DNA; and media components including, for example, serum proteins, amino acids, transferrins, and other low molecular weight proteins. In some embodiments, prior to separating the AAV from the other components of the AAV (such as by use of a column purification method), the AAV is clarified to remove packaging cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters including, for example, a grade DOHC Millipore Millistak+HC Pod Filter, a grade A1HC Millipore Millistak+HC Pod Filter, and / or a 0.2 pm Filter Opticap XL10 Millipore Express SHC Hydrophilic Membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any suitable filter (such as any suitable cellulose acetate filter of 0.2 pm or greater pore size) knownAtty. Docket No. 0I277-0058-00PCT in the art. Still other suitable depth filters, e.g., in the range of about 0.045 pm to about 0.2 pm, or other filtration techniques may be used.

[0171] The AAV starting preparation can be treated with a nuclease, or a combination of nucleases, to digest any contaminating high molecular weight nucleic acid present in the production culture. Suitable nucleases include but are not limited to a DNAse, e.g., Benzonase® digestion performed under standard conditions known in the art. For example, a final concentration of 1 unit / mL to 2.5 units / mL of Benzonase® is used at a temperature ranging from ambient temperature to 37°C for a period of 30 minutes to several hours, or about 2 hours. In another example, a turbonuclease is used. However, one of skill in the art may utilize another suitable nuclease, or a mixture of nucleases. Exonucleases may also be used to remove contaminating nucleic acids. Such nucleases may be selected to degrade single stranded DNA and / or double- stranded DNA, and RNA. Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases.

[0172] AAVs produced using a disclosed method may be isolated or purified prior to loading onto a column chromatography resin (e.g., an anion exchange column or a cation exchange column) using one or more of the following steps: tangential flow filtration for concentrating the AAV particles, heat inactivation of helper virus, AAV capture by hydrophobic interaction chromatography, affinity capture chromatography to remove production system contaminants, buffer exchange by size exclusion chromatography (SEC), and / or nanofiltration. These steps may be used alone, in various combinations, or in different orders. In some embodiments, the method comprises all the steps in the order.

[0173] In some embodiments, a nuclease (e.g., Benzonase®)-treated mixture is concentrated via tangential flow filtration. Large scale concentration of viruses using tangential flow filtration ultrafiltration has been described by R. Paul et al., Human Gene Therapy, 4:609-615 (1993). Tangential flow filtration concentration of the AAV enables a technically manageable volume of the preparation to be subjected to the methods of the present disclosure and allows for more reasonable sizing of a solid support (such as beads, such as in a column). In some embodiments, the AAV is concentrated between at least two-fold or at least ten-fold. In some embodiments, the AAV is concentrated between at least ten-fold and at least twenty-fold, such as at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold. In some embodiments, the AAV is concentrated between at least twenty-fold and at least fifty-fold. One of ordinary skill inAtty. Docket No. 01277-0058-00PCT the art will also recognize that tangential flow filtration can also be used at any step in the disclosed methods where it is desirable to exchange buffers before performing the next step in the method.

[0174] In one embodiment, AAVs produced using a disclosed method have been separated from contaminants (e.g., packaging cell, viral, and other nucleic acid or proteinaceous materials which are present in the production culture or are by-products thereof) present from the production system. In some embodiments, the AAVs separated from contaminants contain less than about 10% contamination from non- AAV viral and cellular proteinaceous and nucleic acid materials, or less than about 5% contaminants, or less than about 1% contaminating viral and cellular proteinaceous and nucleic acid materials. Thus, in some embodiments, the AAV (e.g., in some examples, prior to loading onto a column, such as a cation or anion exchange column, for a further separation step) is about 95% to about 99% free of contaminants.

[0175] In some embodiments, affinity capture chromatography may be used to separate AAVs from production system contaminants. This affinity capture can be performed, e.g., using an antibody-capture affinity resin. In one embodiment, a solid support is a cross-linked poly(styrene-divinylbenzene) having an average particle size of about 50 pm and having an AAV-specific antibody. An example of one such commercially available affinity resin is POROS™ high performance affinity resin commercially available from Thermo Fisher Scientific. The resin contains ligands created by a proprietary technology based on camelid- derived single-domain antibody fragments coupled to the resin via carbonyldiimidazole (CDI). The ligand is a 13-kDa single-domain fragment that comprises the 3 CDRs that form the antigen binding domain and is efficiently produced by the yeast Saccharomyces cerevisiae in a production process free of animal components. Other suitable affinity resins may be selected or designed which contain an AAV-specific antibody (such as an AAV5, AAV6, or AAV8 specific antibody), or other immunoglobulin construct which is an AAV-specific ligand. Such solid supports may be any suitable polymeric matrix material, e.g., agarose, sepharose, sephadex, amongst others.

[0176] In some embodiments, an AAV (such as an AAV that has undergone one or more processing steps as described herein) is diluted, such as in a buffer, e.g., prior to loading onto a chromatography column for further purification.

[0177] An AAV packaging cell culture of the present disclosure may yield a mixture of AAV full capsids, AAV empty capsids, and / or AAV partially empty capsids.Atty. Docket No. 01277-0058-00PCTIII. Additional Features of Certain Disclosed MethodsA. Adeno-associated Viruses

[0178] In embodiments of the present disclosure, an AAV is produced using an AAV packaging cell culture, wherein the packaging cells are transfected with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection. Adeno-associated virus (AAV), a member of the Parvovirus family, is a small, non-enveloped vims. AAV particles comprise an AAV capsid composed of 60 capsid protein subunits that are each made up of VP1, VP2, and VP3 proteins. The VP1, VP2, and VP3 proteins are present in a predicted ratio of about 1:1:10 and have icosahedral symmetry. The AAV capsid encloses a small, single-stranded DNA (ssDNA) genome of about 4.8 kilobases (kb). The ssDNA AAV genome includes two open reading frames, Rep and Cap. flanked by two 145-base inverted terminal repeats (ITRs). These ITRs base pair to allow for synthesis of the complementary DNA strand. Rep and Cap are translated to produce multiple distinct proteins (Rep78, Rep68, Rep52, Rep40, which play important roles in the AAV life cycle; and the VP1. VP2, and VP3 capsid proteins).

[0179] In some embodiments, the AAV is a recombinant or engineered AAV. In some embodiments, the AAV is a pseudotyped AAV. Use of a recombinant or engineered AAV enables insertion, deletion, or substitution of target DNA sequences into the genomes of mammalian cells. AAV comprises a protein capsid surrounding and protecting a single-stranded DNA genome of approximately 4.8 kilobases (kb). Naso et al., BioDrugs. 2017; 31(4): 317-334. AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Its single- stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression gives rise to the viral capsid proteins (VP; VP1 / VP2 / VP3), which form the outer capsid shell that protects the viral genome, and is involved in cell binding and internalization. The viral capsid is comprised of approximately 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1:1:10 (VP1:VP2:VP3). The aapAtty. Docket No. 01277-0058-00PCT gene encodes the assembly-activating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly, but may be nonessential in certain AAV serotypes.

[0180] Recombinant AAV (rAAV), which lacks viral DNA, is a protein-based nanoparticle engineered to traverse the cell membrane, where it can ultimately traffic and deliver a DNA cargo (comprised within the viral capsid) into the nucleus of a cell. Naso et al., BioDrugs. 2017; 31(4): 317-334. In the absence of Rep proteins, ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of a transduced cell. Because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time as the cell undergoes repeated rounds of replication. This will eventually result in the loss of the transgene and transgene expression, with the rate of transgene loss dependent on the turnover rate of the transduced cell. These characteristics make rAAV appealing for certain gene therapy applications. Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in U.S. Pat. Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; 9,284,357; 9,409,953; 9.169,299; 9,193,956; 9,458,517; and 9,587,282; US patent application publication nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024;2017 / 0051257; International patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335; WO 2003 / 052051, WO 2005 / 033321. WO 03 / 042397, WO 2006 / 068888, WO 2006 / 110689, W02009 / 104964, WO 2010 / 127097, and WO 2015 / 191508, and U.S. Appl. Publ. No. 20150023924.

[0181] In some embodiments, the AAV that is produced using the disclosed methods is, or is derived from, an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhlO, AAVrh74, AAV9, AAV9P. AAV10. AAV11. AAV12, or Myo-AAV, or novel chimeras thereof. In particular embodiments, the AAV is AAV5, AAV6, or AAV8. Methods disclosed herein can be used in the production of rAAV particles comprising a capsid protein from any AAV capsid serotype. In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO. AAV.rh20, AAV.rh39. AAV.Rh74. AAV.RHM4-1. AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7,Atty. Docket No. 01277-0058-00PCTAAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid protein.

[0182] In some embodiments, the AAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV 5, AAV6, or AAV8. In some embodiments, the AAV particles have an AAV capsid serotype of AAV5. In some embodiments, the AAV particles have an AAV capsid serotype of AAV6. In some embodiments, the AAV particles have an AAV capsid serotype of AAV8.

[0183] AAV6 has been shown to have enhanced capsid-associated tropism in tissues, such as the lungs, cardiac muscle, and skeletal muscle (Halbert et al., J Virol. 2001, 75(14):6615-24; Rengo et al., Circulation. 2009, 119(l):89-98; Bortolanza et al. Mol Ther. 2011, 19(11):2055-64). Like other AAVs, AAV6 can transduce non-dividing cells (Halbert et al.. J Virol. 2001, 75 ( 14):6615- 24). AAV5 exhibits enhanced capsid-associated tropism in the central nervous system, lung, photoreceptor cells, and retinal pigment epithelium. Similarly, AAV8 exhibits enhanced capsid- associated tropism in the central nervous system, photoreceptor cells, and retinal pigment epithelium, as well as the heart, liver, and skeletal muscle. AAV5 and AAV8 vectors are also currently being explored for gene therapy applications in hemophilia B and hemophilia A, respectively. See, e.g.. Issa et al., Cells. 2023, 12(5), 785.

[0184] In some embodiments, the AAV (such as the AAV5, AAV6, or AAV8) is less than 5 kb from ITR to ITR in size, inclusive of both ITRs. In particular embodiments, the AAV (such as the AAV5, AAV6, or AAV8) is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV (such as the AAV5, AAV6, or AAV8) is less than 4.85 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV (such as the AAV5, AAV6. or AAV8) is less than 4.8 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV (such as the AAV5, AAV6, or AAV8) is less than 4.75 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV (such as theAtty. Docket No. 01277-0058-00PCTAAV5, AAV6, or AAV8) is less than 4.7 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the AAV (such as the AAV5, AAV6, or AAV8) is 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3.9-4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4.9 kb, 3.9-4.85 kb, 4.2- 4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the AAV (such as the AAV5, AAV6, or AAV8) is 4.4-4.85 kb from ITR to ITR in size, inclusive of both ITRs.

[0185] In some embodiments, the AAV (such as the AAV5, AAV6, or AAV8) comprises a nucleic acid molecule (e.g., enclosed within the fully assembled AAV capsid) that encodes at least one protein or RNA of interest, such as at least one protein or RNA of therapeutic interest. In any of the embodiments described herein, the nucleic acid of interest to be packaged into the AAV capsid may encode a chimeric antigen receptor (CAR) or T cell receptor (TCR)). In certain embodiments, the CAR comprises an antigen-binding domain and an intracellular signaling region comprising an intracellular signaling domain. In certain embodiments, the antigen-binding domain is or comprises an antibody or an antibody fragment thereof, which optionally is a single chain fragment. In certain embodiments, the fragment comprises an scFv. The intracellular signaling domain can comprise a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and / or a signaling domain comprising an immunoreceptor tyrosine-based activation motif. In certain embodiments, the intracellular signaling domain is or comprises an intracellular signaling domain of a CD3 chain, optionally a CD3-zeta chain, or a signaling portion thereof. The CAR can comprise a transmembrane domain disposed between the extracellular domain and the intracellular signaling region, which can further comprise a costimulatory signaling domain. The costimulatory signaling domain can comprise an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof, such as an intracellular signaling domain of a CD28, a 4- IBB or an ICOS or a signaling portion thereof. In certain embodiments, the costimulatory signaling domain is between the transmembrane domain and the intracellular signaling domain. In certain embodiments, the TCR comprises an alpha chain containing a variable alpha (Va) region and a beta chain containing a variable beta (V ) region, wherein the TCR is capable of binding to or recognizing a peptide epitope in the context of an MHC molecule, such as HLA-A2.Atty. Docket No. 01277-0058-00PCT

[0186] A nucleic acid molecule of interest can be operably linked to one or more regulatory elements, such as a promoter, such as a tissue-specific promoter. The term “operably linked” means that the nucleic acid molecule of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. AAVs produced using the disclosed methods can include any AAV comprising a tissue-specific promoter in facilitating administration of gene therapy, which can include any known gene editing system in the art. A promoter as described herein can also be “cell specific,” meaning that the particular promoter selected for the AAV can direct expression of the selected transgene / nucleotide sequence of interest in a particular cell or cell type.

[0187] An exemplary promoter useful in the present disclosure can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / “ON” state), an activatable promoter, an inducible promoter (i.e., a promoter whose state, active / “ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein), a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc., e.g., a tissue specific promoter, a cell type specific promoter, etc.), or a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process).

[0188] Suitable promoters can be derived from viruses (viral promoters), or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a Rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi etal., Nature Biotechnology. 20: 497-500; 2002), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. Sep 1(31): 17; 2003), a human Hl promoter (Hl), and the like.

[0189] Nucleic acids of interest useful herein can also include other regulatory elements, i.e., transcriptional and translational control sequences, such as enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., site- directed modifying polypeptide, or Cas9 polypeptide) and / or regulate translation of an encodedAtty. Docket No. 01277-0058-00PCT polypeptide. Exemplary regulatory sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology. Vol. 185, Academic Press, San Diego, CA (1990).

[0190] All patents, patent applications, websites, other publications or documents, accession numbers and the like cited herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number, if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated.EXAMPLESExample 1: Transfection of packaging cells with a plurality of nucleic acids encoding an AAV in two rounds of transfection

[0191] The workflow described in this example is illustrated in FIG. 1. This Example shows the transfection of a packaging cell with nucleic acids encoding an AAV6 in two rounds of transfection. The method is compared to a control, wherein the packaging cells were transfected with nucleic acids encoding an AAV6 in a single round of transfection. HEK.293 cells were inoculated into an Eppendorf 1 L bioreactor containing a chemically defined HEK293 cell culture medium. 72 hours after inoculation, cell cultures were transfected (using a triple transient transfection method) with a plurality of nucleic acids encoding an AAV. In the triple transfection, three separate plasmids encoding (1) a heterologous nucleotide sequence (encoding a gene of interest) flanked by ITRs, (2) the AAV rep and AAV cap gene sequences, and (3) adenovirus helper function sequences (e.g., the Ad5 genes: VA RNAs, E2A, and E4OEF6), respectively, were mixed and incubated with a cationic polymer to form complexes, and were then introduced to the HEK293 cells in the bioreactor. HEK293 cells constitutively expressAtty. Docket No. 01277-0058-00PCTEla / b, the fourth adenovirus function for AAV replication. Twenty-four hours post transfection, cells were transfected again with a plasmid encoding the AAV cap gene sequence. A control culture was not transfected after 24 hours with a plasmid encoding the AAV cap gene sequence.

[0192] Following transfection of the HEK293 cells and culturing of the transfected cells for 72 hours to produce the AAV, the HEK293 packaging cell cultures were lysed, and the lysate subsequently clarification using depth filtration. Additional AAV6 concentration and purification methods included tangential flow filtration, affinity chromatography (Poros CaptureSelect AAVX), anion exchange chromatography (CIMmultus QA monolith), nanofiltration (Planova 35N, Asahi Kasei), additional tangential flow filtration, and 0.2um sterile-grade filtration. Column chromatography was performed on an AKTA Avantl50 FPLC (Cytiva, Massachusetts, USA). Viral genome titers were calculated using a qPCR method. Capsid titers were calculated by an ELISA assay. Percent full was determined as viral genome titer divided by capsid titer.

[0193] Transfection with a plasmid encoding the AAV cap gene sequence at 24 hours following the initial triple transfection (with pHelper, pRep / Cap, and pTransgene nucleic acids) increased overall AAV titer and percent full capsid production as compared to a control that was not transfected with the plasmid encoding the AAV cap gene sequence at 24 hours following the initial triple transfection. As shown in FIG. 2, transfection with a plasmid encoding the AAV cap gene sequence at 24 hours following the initial triple transfection yielded an average titer increase of 17.3% compared to the control, and an average full capsid increase of 13% compared to the control. Specifically, the average viral genome titer (vg / mL) was 4.15xlOnvg / mL in cell cultures transfected with the plasmid encoding the AAV cap gene sequence at 24 hours following the initial triple transfection, as compared to 3.49xlOnvg / mL under control conditions. The average percent yield of full capsids was 27.1% in cell cultures transfected with the plasmid encoding the AAV cap gene sequence at 24 hours following the initial triple transfection, as compared to 23.8% under control conditions.

Claims

Atty. Docket No. 01277-0058-00PCTWhat is claimed is:

1. A method of producing adeno-associated virus (AAV), the method comprising transfecting a packaging cell with a plurality of nucleic acids encoding an AAV in two or more rounds of transfection, wherein: a) a first round of transfection comprises transfecting the packaging cell with at least a first nucleic acid of the plurality of nucleic acids encoding the AAV; and b) a second round of transfection comprises transfecting the packaging cell with at least a second nucleic acid of the plurality of nucleic acids encoding the AAV.

2. The method of claim 1, wherein a) the packaging cell is not transfected with the second nucleic acid of the plurality of nucleic acids encoding the AAV in the first round of transfection; and / or b) the packaging cell is not transfected with the first nucleic acid of the plurality of nucleic acids encoding the AAV in the second round of transfection.

3. The method of any one of the preceding claims, wherein: a) the first round of transfection comprises transfecting the packaging cell with a first set of the plurality of nucleic acids encoding the AAV; and b) the second round of transfection comprises transfecting the packaging cell with a second set of the plurality of nucleic acids encoding the AAV ; wherein the first set and the second set of nucleic acids of the plurality of nucleic acids encoding the AAV are not the same.

4. The method of the immediately preceding claim, wherein the first set of nucleic acids of the plurality of nucleic acids encoding the AAV comprises or consists of 1-6, 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, 5, or 6 nucleic acids; the second set of nucleic acids of the plurality of nucleic acids encoding the AAV comprises or consists of 1-6, 1-5, 1-4, 1-3, 1-2, 1, 2, 3,4, 5, or 6 nucleic acids; and the first set and the second set of nucleic acids of the plurality of nucleic acids encoding the AAV are not the same.

5. The method of claim 1, wherein the plurality of nucleic acids encoding the AAV is the same in the first round of transfection and in the second round of transfection, andAtty. Docket No. 01277-0058-00PCT wherein a ratio of at least one of the plurality of nucleic acids transfected in the first round of transfection is different from a ratio of the at least one of the plurality of nucleic acids transfected in the second round of transfection.

6. The method of any one of the preceding claims, wherein the plurality of nucleic acids encoding the AAV comprises one or more of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein; (iii) at least one nucleic acid encoding an AAV capsid protein; and (iv) at least one nucleic acid encoding an adenovirus packaging helper gene.

7. The method of any one of the preceding claims, wherein the plurality of nucleic acids is sufficient for production of the AAV when the packaging cell is cultured under conditions suitable for production of the AAV.

8. The method of any one of the preceding claims, wherein the first round of transfection comprises dual transfection, triple transfection, or quad transfection.

9. The method of any one of the preceding claims, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV capsid protein.

10. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV replication protein.Atty. Docket No. 01277-0058-00PCT11. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the gene of interest.

12. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein.

13. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with a nucleic acid encoding the adenovirus packaging helper gene.

14. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; andAtty. Docket No. 01277-0058-00PCT b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; and (ii) at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein.

15. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; and (ii) at least one nucleic acid encoding the adenovirus packaging helper gene.

16. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the AAV replication protein and the AAV capsid protein; and (ii) at least one nucleic acid encoding the adenovirus packaging helper gene.

17. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with one of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; or (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; b) the second round of transfection comprises or consists of transfecting the packaging cell with any one of nucleic acids that was not transfected into the packaging cell in step (a); andAtty. Docket No. 01277-0058-00PCT c) a third round of transfection comprises or consists of transfecting the packaging cell with the nucleic acid that was not transfected into the packaging cell in step (a) or step (b).

18. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with one of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; or (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with the two nucleic acids that were not transfected into the packaging cell in step (a).

19. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with any two of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with the nucleic acid that was not transfected into the packaging cell in step (a).

20. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with any one or two of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein and an AAV capsid protein; and (iii) at least one nucleic acid encoding the adenovirus packaging helper gene.Atty. Docket No. 01277-0058-00PCT21. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein; and (iii) at least one nucleic acid encoding an adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene; and wherein the packaging cell is transfected with at least one nucleic acid encoding the AAV capsid protein during the second round of transfection and / or during an additional round of transfection following the second round of transfection.

22. The method of any one of claims 1-9, wherein a) the first round of transfection comprises or consists of transfecting the packaging cell with (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene; and b) the second round of transfection comprises or consists of transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding the gene of interest; (ii) at least one nucleic acid encoding the AAV replication protein; (iii) at least one nucleic acid encoding the AAV capsid protein; and (iv) at least one nucleic acid encoding the adenovirus packaging helper gene; and wherein the packaging cell is transfected with at least one nucleic acid encoding the AAV capsid protein during the second round of transfection and / or during an additional round of transfection following the second round of transfection.

23. The method of any one of the preceding claims, further comprising a plurality of additional rounds of transfection, optionally after the second or the third round of transfection.Atty. Docket No. 01277-0058-00PCT24. The method of the immediately preceding claim, wherein each of the plurality of additional rounds of transfection comprises transfecting the packaging cell with one or more of (i) at least one nucleic acid encoding a gene of interest; (ii) at least one nucleic acid encoding an AAV replication protein; (iii) at least one nucleic acid encoding an AAV capsid protein; and (iv) at least one nucleic acid encoding an adenovirus packaging helper gene.

25. The method of claim 23 or claim 24, wherein the plurality of additional rounds of transfection comprises 1-100 additional rounds of transfection, such as 1-90, 1-80, 1-70,1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30,2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30.

35.

40. 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 additional rounds of transfection.

26. The method of any one of the preceding claims, comprising a number of rounds of transfection sufficient to attain a desired level of expression of a gene of interest.

27. The method of the immediately preceding claim, wherein the number of rounds of transfection comprises 1-100 rounds of transfection, such as 1-90, 1-80, 1-70, 1-60, 1-50,1-40, 1-30, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15,2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 rounds of transfection.

28. The method of any one of the preceding claims, wherein an amount of elapsed time between any two consecutive rounds of transfection is independently selected from 3-48,3-47, 3-46, 3-45, 3-44, 3-43, 3-42, 3-41, 3-40, 3-39, 3-38, 3-37, 3-36, 3-35, 3-34, 3-33, 3- 32, 3-31, 3-30, 3-29. 3-28, 3-27, 3-26, 3-25, 3-24. 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16. 3-15. 3-14, 3-13, 3-12, 3-11. 3-10. 3-9, 3-8. 3-7, 3-6. 3-5, 3-4. 4-36, 4-35, 4-34,4-33, 4-32, 4-31, 4-30, 4-29, 4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15. 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 3, 4, 5, 6, 7, 8,Atty. Docket No. 01277-0058-00PCT9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43. 44, 45, 46, 47, or 48 hours.

29. The method of any one of the preceding claims, wherein an amount of elapsed time between the first round of transfection and the second round of transfection is 3-48, 3-47,3-46, 3-45, 3-44, 3-43, 3-42, 3-41, 3-40, 3-39, 3-38, 3-37, 3-36, 3-35, 3-34, 3-33, 3-32, 3- 31, 3-30, 3-29, 3-28, 3-27, 3-26, 3-25, 3-24, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14. 3-13. 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-36. 4-35, 4-34, 4-33,4-32, 4-31, 4-30, 4-29, 4-28, 4-27, 4-26, 4-25, 4-24, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14. 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

30. The method of any one of the preceding claims, wherein a concentration of a nucleic acid encoding an AAV is the same in each round of transfection wherein the nucleic acid encoding the AAV is transfected into the packaging cell.

31. The method of any one of claims 1-29, wherein a concentration of a nucleic acid encoding an AAV is different in at least two rounds of transfection wherein the nucleic acid encoding the AAV is transfected into the packaging cell.

32. The method of any one of the preceding claims, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is the same in each round of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell.

33. The method of any one of claims 1-30, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in at least two rounds of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell.Atty. Docket No. 01277-0058-00PCT34. The method of any one of claims 1-30, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in each round of transfection wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell.

35. The method of any one of claims 1-30, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in at least two rounds of transfection, and wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell in each of the at least two rounds of transfection.

36. The method of any one of claims 1-30, wherein a ratio of a first nucleic acid encoding an AAV and a second nucleic acid encoding an AAV is different in each round of transfection, and wherein both the first nucleic acid encoding an AAV and the second nucleic acid encoding an AAV are transfected into the packaging cell in each of the at least two rounds of transfection.

37. The method of any one of the preceding claims, wherein the AAV is or is derived from an AAV1, AAV2. AAV3, AAV4, AAV5. AAV6, AAV7, AAV8. AAV9, AAV10, AAV1 1, AAV 12, or Myo AAV.

38. The method of any one of the preceding claims, wherein the AAV is a chimeric AAV.

39. The method of any one of the preceding claims, further comprising harvesting the AAV from a cell culture comprising the packaging cell.

40. The method of any one of the preceding claims, wherein, after the two or more rounds of transfection, the AAV is harvested from the cell culture comprising the packaging cell every 1-6 days, every 1-5 days, every 1-4 days, every 1-3 days, every 2-6 days, every 2-5 days, or every 2-4 days.Atty. Docket No. 01277-0058-00PCT41. The method of any one of the preceding claims, wherein the AAV is harvested from the cell culture comprising the packaging cell 1-6 days, 1-5 days, 1-4 days, 1-3 days, 2-6 days, 2-5 days, or 2-4 days after the two or more rounds of transfection.

42. The method of the immediately preceding claim, wherein harvesting the AAV from the cell culture comprising the packaging cell comprises harvesting the AAV from a supernatant of the cell culture.

43. The method of claim 41 or claim 42, wherein harvesting the AAV from the cell culture comprising the packaging cell comprises isolating the AAV from the cell culture.

44. The method of the immediately preceding claim, wherein the isolating comprises chemical lysis, mechanical lysis, depth filtration, microfiltration, ultrafiltration, alternating tangential flow filtration, tangential flow depth filtration, batch chromatography, acoustic wave separation, and / or centrifugation.

45. The method of claim 43 or claim 44, wherein the isolating further comprises concentrating the AAV using a method selected from chromatography, column-based chromatography, membrane-based chromatography, filtration, and / or precipitation.

46. The method of any one of the preceding claims, wherein the packaging cell comprises one or more nucleic acids encoding one or more helper genes, and / or the packaging cells are infected with one or more helper viruses.

47. The method of the immediately preceding claim, wherein the one or more helper genes comprise at least one nucleic acid encoding an adenovirus packaging helper gene, at least one nucleic acid encoding an AAV replication protein sufficient for packaging, and / or at least one nucleic acid encoding an AAV capsid protein sufficient for packaging.

48. The method of the immediately preceding claim, wherein the packaging cells are stably transformed with the at least one nucleic acid encoding an adenovirus packaging helper gene, the at least one nucleic acid encoding an AAV replication protein sufficient forAtty. Docket No. 01277-0058-00PCT packaging, and / or the at least one nucleic acid encoding an AAV capsid protein sufficient for packaging.

49. The method of any one of the preceding claims, wherein the packaging cell comprises(a) a nucleic acid encoding E2A, a nucleic acid encoding E4, and a nucleic acid encoding VA genes;(b) a nucleic acid encoding an AAV replication protein;(c) a nucleic acid encoding an AAV capsid protein; and / or(d) a nucleic acid encoding a gene of interest, optionally comprising an inverted terminal repeat.

50. The method of any one of claims 6-49, wherein the gene of interest encodes at least one protein or RNA of interest.

51. The method of any one of claims 6-50, wherein the gene of interest encodes at least one protein or RNA of therapeutic interest.

52. The method of any one of claims 6-51, wherein the gene of interest encodes a chimeric antigen receptor.

53. The method of any one of claims 6-52, wherein the one or more helper genes is expressed under a constitutive promoter, an activatable promoter, or an inducible promoter.

54. The method of any one of claims 46-53, wherein the one or more helper viruses comprises an adenovirus, a baculovirus, or a herpes simplex virus.

55. The method of the immediately preceding claim, wherein the adenovirus is a wild-type adenovirus.

56. The method of any one of the preceding claims, wherein a titer of the AAV at harvest is higher than that of a control.Atty. Docket No. 01277-0058-00PCT57. The method of the immediately preceding claim, wherein the control comprises the same AAV harvested from a cell culture comprising only a single round of transfection, or comprising at least two rounds of transfection, wherein each round of transfection comprises transfecting the packaging cell with an identical set of nucleic acids encoding the AAV.

58. The method of claim 56 or claim 57, wherein the titer of the AAV at harvest is at least 1.1-fold, at least 1.15-fold, at least 1.2-fold, at least 1.25-fold, at least 1.5-fold, at least 1.75-fold, or at least 2-fold greater than that of the control.

59. The method of any one of claims 56-58, wherein the titer of the AAV at harvest is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% greater, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% greater than that of the control.

60. The method of any one of claims 39-59, wherein the cell culture comprising the packaging cell comprises a HEK cell culture medium.

61. The method of any one of claims 39-60. wherein the cell culture comprising the packaging cell comprises glutamine and / or a shear protectant.

62. The method of any one of claims 39-61, wherein the cell culture comprising the packaging cell is serum-free.

63. The method of any one of claims 39-62, wherein the cell culture comprising the packaging cell is a suspension culture.

64. The method of any one of claims 39-63, wherein the cell culture comprising the packaging cell is in a bioreactor.

65. The method of any one of claims 39-62, wherein the cell culture comprising the packaging cell is a batch-fed cell culture.Atty. Docket No. 01277-0058-00PCT66. The method of any one of claims 39-62, wherein the cell culture comprising the packaging cell is a continuous cell culture.

67. The method of any one of claims 39-62, wherein the cell culture comprising the packaging cell is a perfusion cell culture.

68. The method of any one of the preceding claims, wherein the packaging cell is a mammalian cell.

69. The method of the immediately preceding claim, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell, human embryonic kidney (HEK) cell, Madin-Darby canine kidney (MDCK) cell, or Vero cell.

70. The method of claim 68 or claim 69, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

71. The method of claim 68 or claim 69, wherein the mammalian cell is a human cell.

72. The method of the immediately preceding claim, wherein the human cell is a HEK293 cell.

73. The method of any one of claims 1-67, wherein the packaging cell is an insect cell.

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