Methods and cell line useful for production of recombinant adeno-associated viruses
View Patent ↗Methods for efficient production of recombinant AAV employ a host cell which comprising AAV rep and cap genes stably integrated within the cell's chromosomes, wherein the AAV rep and cap genes are each operatively linked to regulatory sequences capable of directing the expression of the rep and cap gene products upon infection of the cell with a helper virus, a helper gene, and a helper gene product. A method for producing recombinant adeno-associated virus (rAAV) involves infecting such a host cell with a helper virus, gene or gene product and infecting the infected host cell with a recombinant hybrid virus or plasmid vector containing adenovirus cis-elements necessary for replication and virion encapsidation, AAV sequences comprising the 5′ and 3′ ITRs of an AAV, and a selected gene operatively linked to regulatory sequences directing its expression, which is flanked by the above-mentioned AAV sequences.
1. A method for producing recombinant AAV, said method comprising the steps of:
infecting a helper-containing host cell useful for production of recombinant adeno-associated virus (AAV), wherein said cell has stably integrated in its chromosome at least two copies of an AAV rep gene and AAV cap gene under the control of regulatory sequences comprising AAV P5 which is located in the native AAV position upstream of the AAV rep gene with
a recombinant hybrid virus, the recombinant hybrid virus comprising:
(1) a selected transgene operatively linked to regulatory sequences controlling the transgene's expression,
(2) AAV sequences comprising the 5′ and 3′ ITRs of an AAV, wherein the 5′ ITR flanks one side of the transgene and regulatory sequences of (1), and the 3′ ITR flanks the other side, and
(3) an adenovirus cis-element selected from the group consisting of cis-elements required for replication of adenovirus genomes or genes and cis-elements required for encapsidation of adenovirus virions, said cis-elements flanking each AAV sequence of (2);
whereby recombinant AAV is produced by the cell.
2. The method according to claim 1 , wherein the helper-containing host cell is infected about 24 hours after the helper is introduced to the host cell.
3. The method according to claim 1 , comprising the additional step of:
isolating from the hybrid virus-infected, helper-containing host cell a recombinant AAV comprising the transgene.
4. The method according to claim 3 , wherein the isolating step is performed 36 hours after the helper-containing host cell is infected with the hybrid virus.
5. The method according to claim 1 , wherein the recombinant AAV is produced at levels exceeding 1×10 3 genome copies per cell.
6. The method according to claim 3 , wherein the recombinant AAV is isolated in step (c) at a level exceeding 1×10 4 genome copies per cell.
7. The method according to claim 1 , wherein the recombinant AAV produced is essentially homogenous and is essentially free of replication-competent AAV.
8. The method according to claim 1 , wherein the host cell is a B-50 cell having ATCC Accession No. CRL 12401.
9. The method according to claim 1 , wherein the helper is a member selected from the group consisting of an adenovirus, a herpes virus, a recombinant adenovirus, a recombinant herpes virus, an adenovirus gene, a herpes virus gene, an adenovirus gene product, and a herpes virus gene product.
10. The method according to claim 9 , wherein the helper is a recombinant adenovirus comprising an adenovirus E1 gene.
11. The method according to claim 10 , wherein the recombinant adenovirus is replication-deficient.
12. The method according to claim 9 , wherein the helper comprises an adenovirus E1 gene.
13. The method according to claim 9 , wherein the recombinant herpes virus is replication-deficient.
14. The method according to claim 9 , wherein the helper comprises a recombinant virus selected from the group consisting of Herpes Simplex Type I, Herpes Simplex Type II, Cytomegalovirus and Vaccinia.