Recombinantly-modified adeno-associated virus (rAAV) having improved packaging efficiency
The present invention is directed to recombinantly-modified adeno-associated virus (rAAV) having improved packaging efficiency, pharmaceutical compositions comprising such rAAV, and methods for their production and use. The present invention is particularly directed to recombinantly-modified adeno-associated virus (rAAV) that have been further modified to comprise Cis-Elements, including replication origins, promoters and enhancers, that are capable of regulating the replication of an rAAV genome and that improve rAAV replication. Preferably, such Cis-Elements are provided within domains of the rAAV that precede and/or follow the 5′ and/or 3′ inverted terminal repeated sequences (ITR) of an rAAV. The invention particularly concerns the presence and the use of polynucleotide Cis-Elements that comprise actual or potential G-Quadruplex Sequences, polynucleotide Cis-Elements that comprise DNA sequences from wild-type AAV (wt AAV) and polynucleotide Cis-Elements that comprise DNA sequences from other viral genomes or from the human genome.
1. A recombinantly-modified adeno-associated virus (rAAV) that comprises a Cis-Element in one or more of its P1, P2, P3 or P4 Domains, wherein:
(1) said P1 Domain is 5′ to a 5′ ITR of said rAAV;
(2) said P2 Domain is 3′ to said 5′ ITR of said rAAV and 5′ to a transgene cassette of said rAAV;
(3) said P3 Domain is 3′ to said transgene cassette of said rAAV and 5′ to a 3′ ITR of said rAAV; and
(4) said P4 Domain is 3′ to said 3′ ITR of said rAAV;
wherein the Cis-Element comprises any of SEQ ID NOs: 45-74; and
wherein the presence of said Cis-Element causes rAAV-producing cells to produce said rAAV at a higher production titer than would be attained with such rAAV if lacking said Cis-Element.
2. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said rAAV comprises the Cis-Element in its P1 Domain.
3. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said rAAV comprises the Cis-Element in its P2 Domain.
4. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said rAAV comprises the Cis-Element in its P3 Domain.
5. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said rAAV comprises the Cis-Element in its P4 Domain.
6. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said rAAV comprises the Cis-Element in its P1 Domain and in one or more of its P2, P3 or P4 Domain.
7. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said rAAV comprises the Cis-Element in its P2 Domain and in one or more of its P3 or P4 Domain.
8. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said rAAV comprises the Cis-Element in its P3 Domain and its P4 Domain.
9. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said transgene cassette encodes a protein, or wherein said transgene cassette comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.
10. The recombinantly-modified adeno-associated virus (rAAV) of claim 1 , wherein said rAAV belongs to the rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAV10 serotype, or to a hybrid of said serotypes.
11. A pharmaceutical composition that comprises:
(A) the recombinantly-modified adeno-associated virus (rAAV) of claim 1 ; and
(B) a pharmaceutically acceptable carrier.
12. The pharmaceutical composition of claim 11 , wherein said rAAV belongs to the rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAV10 serotype, or to a hybrid of said serotypes.
13. A method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), wherein said method comprises:
(A) employing an rAAV that has been modified to comprise an added Cis-Element in one or more of its P1, P2, P3 or P4 Domains, wherein:
(1) said P1 Domain is 5′ to a 5′ ITR of said rAAV;
(2) said P2 Domain is 3′ to said 5′ ITR of said rAAV and 5′ to a transgene cassette of said rAAV;
(3) said P3 Domain is 3′ to said transgene cassette of said rAAV and 5′ to a 3′ ITR of said rAAV; and
(4) said P4 Domain is 3′ to said 3′ ITR of said rAAV;
wherein the Cis-Element comprises any of SEQ ID NOs: 45-74; and
(B) culturing cells that have been transfected with said rAAV, wherein said cells additionally contain an AAV helper function-providing polynucleotide and a non-AAV helper function-providing polynucleotide, and wherein said culturing is conducted in a culture medium under conditions sufficient to permit the production of rAAV;
wherein the presence of said Cis-Element in said rAAV causes said cells to produce said rAAV at an increased production titer relative to that which would be attained if said rAAV had lacked said Cis-Element.
14. The method of claim 13 , wherein said cells are human embryonic kidney cells or baby hamster kidney cells.
15. The method of claim 14 , wherein said cells are HEK293 human embryonic kidney cells.
16. The method of claim 14 , wherein said cells are BHK21 baby hamster kidney cells.
17. The method of claim 13 , wherein said cells are sf9 insect cells.