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.
Claims35
A recombinant, replication-defective, 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 hybrid virus is provided with sufficient adenovirus sequences to permit infection of a target cell and stable integration of the transgene into the target cell in the presence of a functional portion of an AAV rep gene.
A recombinant trans-infection particle comprising:
- (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 repeats (ITRs) of an adeno-associated virus, said AAV sequences flanked by the adenovirus sequences of (i);
- and
- (iii) a selected trans ene operatively linked to sequences which regulate its expression in a target cell, said transgene and regulatory sequences flanked by the AAV sequences of (ii),
- wherein the hybrid virus is provided with sufficient adenovirus sequences to permit infection of a target cell and stable integration of the transgene into the target cell in the presence of a functional portion of an AAV rep gene;
- (b) a polycation sequence conjugated to said hybrid virus;
- and
- (c) a plasmid comprising a functional portion of an AAV rep gene operatively linked to sequences which regulate its expression, said plasmid bound to said polycation sequence.
A composition which delivers and stably integrates a selected transgene into the chromosome of a target cell, said composition comprising
- (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 repeats (ITRs) of an adeno-associated virus, said AAV sequences flanked by the adenovirus sequences of (i);
- and
- (iii) a selected ransgene operatively linked to sequences which regulate its expression in a target cell, said transgene and regulatory sequences flanked by the AAV sequences of (ii), and
- (iv) a functional portion of an AAV rep gene under the control of sequences which regulate expression of said functional portion of the rep gene in the target cell;
- wherein the hybrid virus is provided with sufficient adenovirus sequences to permit infection of a target cell and stable integration of the transgene into the target cell in the presence of a functional portion of an AAV rep gene;
- and
- (b) a pharmaceutically acceptable carrier.
A composition which delivers stably integrates a selected transgene into the chromosome of a target cell comprising an effective amount of a recombinant trans-infection particle in a pharmaceutically acceptable carrier, said particle comprising:
- (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 repeats (ITRs) of an adeno-associated virus, said AAV sequences flanked by the adenovirus sequences of (i);
- and
- (iii) a selected ransgene operatively linked to sequences which regulate its expression in a target cell, said transgene and regulatory sequences flanked by the AAV sequences of (ii);
- wherein the hybrid virus is provided with sufficient adenovirus sequences to permit infection of a target cell and stable integration of the transgene into the target cell in the presence of a functional portion of an AAV rep gene;
- (b) a polycation sequence conjugated to said hybrid virus;
- (c) a plasmid comprising a functional portion of an AAV rep gene operatively linked to sequences which regulate its expression, said plasmid bound to said polycation sequence.
A method for producing high levels of a recombinant adeno-associated virus in a cultured cell comprising the step of culturing a cell containing
- (A) a recombinant, replication-defective, hybrid vector 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 repeats (ITRs) of an adeno-associated virus, 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 transgene and regulatory sequences flanked by the AAV sequences of (ii);
- and
- (B) an optional helper virus in the presence of a plasmid containing an AAV rep gene under the control of sequences which regulate expression of said rep gene so as to produce recombinant adeno-associated virus.
A mammalian host cell comprising a recombinant, replication-defective, hybrid vector 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 repeats (ITRs) of an adeno-associated virus, said AAV sequences flanked by the adenovirus sequences of (i);
- and
- (iii) a selected transgene operatively linked to regulatory sequences which direct its expression in a target cell, said transgene and regulatory sequences flanked by the AAV sequences of (ii).
A recombinant, replication-defective hybrid virus comprising:
- (a) adenovirus sequences comprising the adenovirus 5' and 3' cis-elements necessary for replication and virion encapsidation, said adenovirus sequences comprising a functional deletion in the E1, E2, E3, and E4 genes,
- (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 transgene and regulatory sequences flanked by the AAV sequences of (b),
- wherein the hybrid virus is provided with sufficient adenovirus sequences to permit infection of a target cell and stable integration of the transgene into the target cell in the presence of a functional portion of an AAV rep gene.
A hybrid adenovirus-adeno-associated virus vector encoded by SEQ ID NO:
- 1.
Dependencies are inferred from the claim text.