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. Also provided is a hybrid virus linked via a polycation conjugate to an AAV rep gene to form a single particle. These trans-infection particles 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 and viruses to produce large quantities of recombinant AAV.
Claims39
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 sequences flanked by the adenovirus sequences of (a);
- and
- (c) a selected transgene operatively linked to sequences which regulate its expression in a target cell, said gene and regulatory sequences flanked by the AAV sequences of (b);
- wherein the adenovirus sequences comprise a functional deletion in the adenovirus E1 gene, the adenovirus E3 gene, and the adenovirus E4 gene, and
- wherein the hybrid virus is provided with sufficient adenovirus sequences to permit packaging into a capsid and infection of a target cell.
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 sequences flanked by the adenovirus sequences of (a);
- and
- (c) a selected transgene operatively linked to sequences which regulate its expression in a target cell, said gene and regulatory sequences flanked by the AAV sequences of (b);
- wherein the adenovirus sequences comprise a functional deletion in the adenovirus E1 gene, the adenovirus E2 gene, the adenovirus E3 gene, and the adenovirus E4 gene, intermediate gene IX, intermediate gene IXa, and late genes L1 through L5;
- and
- wherein the hybrid virus is provided with sufficient adenovirus sequences to permit packaging into a capsid and infection of a target cell.
A recombinant hybrid vector 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 sequences flanked by the adenovirus sequences of (a);
- and
- (c) a selected transgene operatively linked to sequences which regulate its expression in a target cell, said gene and regulatory sequences flanked by the AAV sequences of (b);
- and
- wherein the adenovirus sequences comprise a functional deletion in the adenovirus E1 gene, the adenovirus E3 gene, and the adenovirus E4 gene.
A method for producing a recombinant adeno-associated virus in a cell comprising the step of culturing a cell containing
- (a) a recombinant, replication-defective, hybrid virus comprising:
- (i) adenovirus sequences comprising the adenovirus 5′ and 3′ cis-elements necessary for replication and virion encapsidation,
- (ii) adeno-associated virus (AAV) sequences comprising the 5′ and 3′ inverted terminal repeat (ITRs) of an AAV, said AAV sequences flanked by the adenovirus sequences of (i);
- and
- (iii) a selected transgene operatively linked to sequences which regulate its expression in a target cell, said gene and regulatory sequences flanked by the AAV sequences of (ii),
- wherein the hybrid virus is provided with sufficient adenovirus sequences to permit packaging into a capsid and infection of a target cell;
- (b) an optional helper virus;
- (c) a rep gene under the control of sequences which regulate expression of said rep;
- and
- (d) a cap gene under the control of sequences which regulate expression of said cap.
A method for producing a recombinant hybrid adenovirus comprising the step of culturing a host cell containing
- (a) a hybrid vector comprising:
- (i) adenovirus sequences comprising the adenovirus 5′ and 3′ cis-elements necessary for replication and virion encapsidation;
- and
- (ii) adeno-associated virus (AAV) sequences comprising the 5′ and 3′ inverted terminal repeat (ITRs) of an AAV, said AAV sequences flanked by the adenovirus sequences of (ii);
- and
- (iii) a selected transgene operatively linked to sequences which regulate its expression in a target cell, said gene and regulatory sequences flanked by the AAV sequences of (ii);
- and
- (b) an optional helper adenovirus, wherein the host cell and/or the helper virus provide the adenovirus sequences necessary to package the hybrid vector and generate a recombinant hybrid adenovirus.
Dependencies are inferred from the claim text.