Hybrid adenovirus-AAV virus and methods of use thereof
AssigneeThe Trustees of the University of Pennsylvania
InventorsJames M. Wilson, William M. Kelley, Krishna J. Fisher
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Abstract
The present invention provides a hybrid vector construct which comprises a portion of an adenovirus, 5′ and 3′ ITR sequences from an AAV, and a selected transgene. Other hybrid vectors form a polycation conjugate and incorporate an AAV rep gene in a single particle. These hybrid virus vectors are characterized by high titer transgene delivery to a host cell and the ability to stably integrate the transgene into the host cell chromosome. Also disclosed is the use of the hybrid vectors to produce large quantities of recombinant AAV.
Claims21
A recombinant hybrid virus comprising:
- (a) adenovirus sequences comprising the adenovirus 5′ and 3′ cis-elements necessary for replication and virion encapsidation;
- (b) adeno-associated virus (AAV) sequences comprising the 5′ and 3′ inverted terminal repeats (ITRs) of an AAV, said AAV ITRs flanked by the adenovirus sequences of (a), and a mutant AAV rep gene which encodes sufficient rep protein functions for efficient replication of the hybrid virus;
- (c) a selected transgene operably linked to sequences which regulate its expression in a target cell, said transgene and regulatory sequences flanked by the AAV sequences of (b);
- wherein the hybrid virus is provided with sufficient adenovirus sequences to permit packaging into a capsid and infection of a target cell.
A method of detecting a recombinant adeno-associated virus (AAV) in cells containing a hybrid virus in which the rAAV was produced,
- (a) passaging cells which have been infected with a hybrid virus for several generations,
- said hybrid virus comprising adenovirus sequences comprising (i) the adenovirus 5′ and 3′ cis-elements necessary for replication and virion encapsidation and (ii) a minigene comprising AAV 5′ inverted terminal repeats (ITRs), a selected transgene operatively linked to sequences which regulate its expression in a target cell, and AAV 3′ ITRs, wherein said minigene is flanked by the adenovirus 5′ and 3′ cis-elements;
- (b) tagging the cells to detect the presence of transgene in the cells;
- and
- (c) separating the cells detected to contain the transgene in a fluorescence activated cell sorter, whereby the presence of said transgene indicated integration of the minigene into the cells.
A method of detecting contaminating hybrid virus in cells used for production of recombinant adeno-associated virus, said method comprising the steps of:
- (a) culturing cells containing a recombinant hybrid virus, the hybrid virus comprising:
- (i) adenovirus sequences comprising the adenovirus 5′ and 3′ cis-elements necessary for replication and virion encapsidation;
- (ii) a first minigene comprising AAV 5′ inverted terminal repeats (ITRs), a first transgene operatively linked to sequences which regulate its expression in a target cell, and AAV 3′ ITRs, wherein said minigene is located between the adenovirus 5′ and 3′ cis-elements;
- and
- (iii) a second minigene comprising a reporter transgene under the control of sequences which control expression of the reporter, said second minigene being located outside of the first minigene and between the adenovirus 5′ and 3′ cis-elements;
- and
- (b) detecting the presence of the reporter in the cell culture, whereby presence of the reporter indicates the presence of the hybrid virus.
A method of purifying recombinant adeno-associated virus (rAAV) from contaminating hybrid virus in cells used for production of recombinant adeno-associated virus, said method comprising the steps of:
- (a) culturing cells containing a recombinant hybrid virus, the hybrid virus comprising:
- (i) adenovirus sequences comprising the adenovirus 5′ and 3′ cis-elements necessary for replication and virion encapsidation;
- (ii) a first minigene comprising AAV 5′ inverted terminal repeats (ITRs), a first transgene operatively linked to sequences which regulate its expression in a target cell, and AAV 3′ ITRs, wherein said minigene is located between the adenovirus 5′ and 3′ cis-elements;
- and
- (iii) a second minigene comprising a reporter transgene under the control of sequences which control expression of the reporter, said second minigene being located outside of the first minigene and between the adenovirus 5′ and 3′ cis-elements;
- (b) detecting the presence of the reporter in the cell culture, whereby presence of the reporter indicates the presence of the hybrid virus;
- and
- (c) removing hybrid virus detected, thereby providing purified rAAV.
Dependencies are inferred from the claim text.