Methods for generating high titer helper-free preparations of released recombinant AAV vectors

• US 6,566,118 B1
• Filed: 03/15/2000
• Issued: 05/20/2003
• Est. Priority Date: 09/05/1997
• Status: Expired due to Term

• Current Assignee: Targeted Genetics Corporation (Innoviva, Inc.)
• Original Assignee: Targeted Genetics Corporation (Innoviva, Inc.)
• Inventors: Aranha, Ian L., Takeya, Ryan K., Atkinson, Edward M.
• Primary Examiner(s): Housel, James
• Assistant Examiner(s): Hill, Myron G.
• Application Number:
  US09/526,333
• Time in Patent Office: 1,161 Days
• Field of Search: 435/235.1, 435/239, 435/320.1
• US Class Current: 435/239
• CPC Class Codes: C07C 2/32 as complexes, e.g. acetyl-acetonates complexes of salts of acids of halogen C07C2/20
• CPC Class Codes: C07C 2531/22 Organic complexes
• CPC Class Codes: C07D 207/32 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 207/34 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• CPC Class Codes: C07D 207/50 Nitrogen atoms
• CPC Class Codes: C07D 295/30 non-acylated
• CPC Class Codes: C07D 401/14 containing three or more hetero rings
• CPC Class Codes: C07D 409/14 containing three or more hetero rings
• CPC Class Codes: C07F 15/045 without a metal-carbon linkage
• CPC Class Codes: C07F 7/0812 comprising a heterocyclic ring
• CPC Class Codes: C07F 9/572 Five-membered rings
• CPC Class Codes: C07F 9/58 Pyridine rings
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14151 Methods of production or purification of viral material
• CPC Class Codes: C12N 2750/14152 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

This invention provides methods and compositions for producing high titer, substantially purified preparations of recombinant adeno-associated virus (AAV) that can be used as vectors for gene delivery. At the onset of vector production, AAV producer cells of this invention typically comprise one or more AAV packaging genes, an AAV vector comprising a heterologous (i.e. non-AAV) transgene of interest, and a helper virus such as an adenovirus. The AAV vector preparations produced are generally replication incompetent but are capable of mediating delivery of a transgene of interest (such as a therapeutic gene) to any of a wide variety of tissues and cells. The AAV vector preparations produced according to this invention are also substantially free of helper virus as well as helper viral and cellular proteins and other contaminants. The invention described herein provides methods of producing rAAV particles by culturing producer cells under conditions, such as temperature and pH, that promote release of virus. Also provided is a quantitative, high-throughput assay useful in the assessment of viral infectivity and replication, as well as in the screening of agent that affect viral infectivity and/or replication.

Claims

1. A method of generating a population of recombinant adeno-associated virus (rAAV) particles, comprising the following steps:

a) incubating producer cells in a cell culture medium under conditions comprising a condition that promotes release of rAAV particles into the medium without lysing the cells, wherein the producer cells comprise;

(i) one or more packaging genes, wherein each said AAV packaging gene encodes an AAV replication or encapsidation protein;

(ii) a recombinant AAV (rAAV) vector that comprises a heterologous non-AAV polynucleotide flanked by at least one AAV inverted terminal repeat (ITR); and

(iii) helper virus function for AAV, whereby rAAV virus particles are released from the producer cells into the cell culture medium, and

b) harvesting the rAAV Particles from the cell culture medium without lysing the cells.

2. The method of claim 1, wherein the condition that promotes release of virus particles is pH.

3. The method of claim 2, wherein the pH is about 7.4 to about 8.0.

4. The method of claim 3, wherein the pH is about 8.0.

5. The method of claim 1, wherein the condition that promotes virus release is incubating cells under sublethal osmotic stress.

6. The method of claim 5, wherein said condition that promotes virus release is incubating producer cells in culture medium that has an osmolality of about 333 mOsm.

7. The method of claim 6, wherein the initial pH of the culture medium is about 8.00.

8. The method of claim 7, wherein the pH of the culture medium is monitored during culture and maintained at about 8.00.

9. The method of claim 8, wherein the pH is adjusted using a sodium base.

10. The method of claim 6, wherein the osmolality of the cell culture medium is adjusted using an ionic salt.

11. The method of claim 10, wherein the ionic salt is NaCl.

12. The method of claim 6, wherein the initial osmolality of the cell culture medium is about 300 mOsm.

13. The method of claim 12, wherein the pH is about 8.00.

14. The method of claim 12, wherein the osmolality of the culture medium is monitored during culture and maintained at about 200 to about 400 mOsm using an ionic salt.

15. The method of claim 14, wherein the ionic salt is NaCl.

16. The method of claim 1, wherein the condition that promotes virus release is temperature.

17. The method of claim 16, wherein the temperature is about 37°

C. to about 40°

C.

18. The method of claim 17, wherein the temperature is about 39°

C.

19. The method of claim 8, wherein the temperature is about 39°

C.

20. The method of claim 19, wherein the osmolality is about 300 to about 350 mOsm.

21. The method of claim 12, wherein the temperature is about 39°

C.

22. The method of claim 1, wherein the condition that promotes release of virus particles is conductivity.

23. The method of claim 22, wherein the conductivity is at least about 10 mS.

24. The method of claim 22, wherein the conductivity is about 10 mS.

25. The method of claim 22, wherein the conductivity is about 15 mS.

26. The method of claim 22, wherein the conductivity is adjusted using a sodium salt.

28. The method of claim 8, wherein producer cells are cultured for about 48 to about 96 hours after introduction of helper virus function.

29. The method of claim 18, wherein producer cells are cultured for about 48 to about 96 hours after introduction of helper virus function.

30. The method of claim 11, wherein helper virus function is provided by helper virus.

31. A method of generating a population of rAAV particles according to claim 30, wherein said helper virus is an adenovirus.

32. The method of claim 1, further comprising the steps of:

c) chromatographing the AAV producer cell culture medium on a positively-charged anion exchange resin; and

d) purifying the chromatographic fractions containing rAAV particles of step c) by cation exchange chromatography or tangential flow filtration to generate a purified population of rAAV vector particles.

33. The method of claim 32, wherein step (d) is cation exchange chromatography.

34. The method of claim 1, further comprising the steps of

c) chromatographing the AAV producer cell culture medium on a negatively-charged cation exchange resin;

d) purifying the chromatographic fractions containing rAAV particles of step c) by anion exchange chromatography; and

e) purifying the chromatographic fractions containing rAAV particles of step d) by cation exchange chromatography to generate a purified population of rAAV vector particles.

35. The method of claim 34, wherein the chromatography of step e) is performed using heparin sulfate.

36. A method of generating a population of rAAV particles according to claim 1, wherein said rAAV vector comprises a heterologous non-AAV polynucleotide flanked by two AAV inverted terminal repeats (ITRs).

37. A method of generating a population of rAAV particles according to claim 1, wherein said AAV producer cell comprises at least one AAV packaging gene that is stably integrated into the genome of said AAV producer cell.

38. A method of generating a population of rAAV particles according to claim 1, wherein said AAV producer cell comprises an AAV rep gene and an AAV cap gene.

39. A method of generating a population of rAAV particles according to claim 38, wherein said AAV rep gene and AAV cap gene are stably integrated into the genome of said AAV producer cell.

40. A method of generating a population of rAAV particles according to claim 1, wherein the producer cell is an attachment-dependent mammalian cell line.

41. A method of generating a population of rAAV particles according to claim 1, wherein the producer cell is a suspension-adapted mammalian cell line.

27. The method of 26, wherein the sodium salt is NaCl.