Production of oversized adeno-associated vectors
Provided herein are methods for producing an adeno-associated virus (AAV) particle containing an oversized recombinant AAV genome (e.g., greater than 4.7 kb). In some aspects, the invention provides AAV particles and AAV vectors comprising oversized rAAV genomes. Producer cell lines to produce AAV particles comprising oversized genomes are also provided.
1. A method for producing an adeno-associated virus (AAV) particle comprising an oversized recombinant AAV genome, the method comprising
a) culturing an AAV producer cell line under conditions to generate rAAV particles, wherein the AAV producer cell line comprises
i) nucleic acid encoding AAV rep and cap genes, and
ii) a rAAV genome, wherein the rAAV genome is 5.4 kb or greater;
b) providing AAV helper functions; and
c) collecting the rAAV particles comprising oversized rAAV genomes;
wherein the nucleic acid encoding AAV rep and cap genes and/or the oversized rAAV genome are stably integrated into the genome of the producer cell line.
2. The method of claim 1 , wherein the rAAV genome comprises one or more AAV inverted terminal repeats (ITRs) and a heterologous transgene.
3. The method of claim 1 , wherein the rAAV genome is between 5.4 kb and 9.4 kb.
4. The method of claim 1 , wherein the AAV particles collected in step c) comprise rAAV genomes between 5.4 kb and 9.4 kb.
5. The method of claim 2 , wherein the heterologous transgene encodes Factor VIII, dystrophin, dysferlin or cystic fibrosis transmembrane conductance regulator (CFTR).
6. The method of claim 2 , wherein the heterologous transgene is operably linked to a promoter.
7. The method of claim 6 , wherein the promoter is the mouse transthyretin (mTTR) promoter.
8. The method of claim 1 , wherein the rAAV genome comprises an intron.
9. The method of claim 1 , wherein the rAAV genome comprises a polyadenylation signal.
10. The method of claim 1 , wherein the rAAV particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, a goat AAV, AAV1/AAV2 chimeric, bovine AAV, or mouse AAV capsid rAAV2/HBoV1 serotype capsid.
11. The method of claim 2 , wherein the AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, a goat AAV, bovine AAV, or mouse AAV serotype ITRs.
12. The method of claim 10 , wherein the AAV particles comprise AAV2 ITRs and AAVrh8R or AAV8 capsid.
13. The method of claim 1 , wherein the AAV helper functions are provided by adenovirus, HSV or baculovirus.
14. The method of claim 13 , wherein the rAAV particles are collected from between about 48 hours and about 96 hours after the provision of helper functions.
15. The method of claim 1 , further comprising purification of the rAAV particles.
16. A rAAV particle comprising an oversized rAAV genome produced by a method for producing the AAV particle comprising an oversized recombinant AAV genome, the method comprising
a) culturing an AAV producer cell line under conditions to generate rAAV particles, wherein the AAV producer cell line comprises
i) nucleic acid encoding AAV rep and cap genes, and
ii) a rAAV genome, wherein the rAAV genome is 5.4 kb or greater;
b) providing AAV helper functions; and
c) collecting the rAAV particles comprising oversized rAAV genomes;
wherein the nucleic acid encoding AAV rep and cap genes and/or the oversized rAAV genome are stably integrated into the genome of the producer cell line.
17. A composition comprising rAAV particles wherein at least about 45% of the rAAV particle encapsidate an rAAV genome 5.4 kb or greater
wherein the AAV particles are produced by a method for producing the AAV particle comprising an oversized recombinant AAV genome, the method comprising
a) culturing an AAV producer cell line under conditions to generate rAAV particles, wherein the AAV producer cell line comprises
i) nucleic acid encoding AAV rep and cap genes, and
ii) a rAAV genome, wherein the rAAV genome is 5.4 kb or greater;
b) providing AAV helper functions; and
c) collecting the rAAV particles comprising oversized rAAV genomes;
wherein the nucleic acid encoding AAV rep and cap genes and/or the oversized rAAV genome are stably integrated into the genome of the producer cell line.
18. A cell line for producing an adeno-associated virus (AAV) particle comprising an oversized recombinant AAV genome, the cell line comprising
a) nucleic acid encoding AAV rep and cap genes, and
b) a rAAV genome, wherein the rAAV genome is 5.4 kb or greater;
wherein the nucleic acid encoding AAV rep and cap genes and/or the oversized rAAV genome are stably integrated into the genome of the producer cell line.
19. An adeno-associated virus (AAV) particle comprising a rAAV genome encapsidated by an AAV capsid, wherein the rAAV genome is 6 kb or greater.
20. A rAAV vector comprising a rAAV genome, wherein the rAAV genome comprises 5′ to 3′ an AAV2 ITR, a mTTR promoter, a synthetic intron, a transgene encoding human FVIII, a synthetic polyadenylation sequence, and an AAV2 ITR, wherein the rAAV genome is 6 kb or greater.
21. The rAAV vector of claim 20 , wherein the synthetic polyadenylation sequence is a bovine growth hormone synthetic polyadenylation sequence.
22. A method for treatment of a disease or disorder in an individual comprising administering an effective amount of an AAV particle comprising an oversized AAV genome, wherein the oversized AAV genome comprises a transgene suitable for treating the disease or disorder, and wherein the oversized AAV genome is 6 kb or greater, and wherein the method results in treatment of the disease or disorder in the individual.