Viral vectors and methods for producing and using the same

Abstract

A recombinant hybrid virus, including: (a) a deleted adenovirus vector genome comprising the adenovirus 5′ and 3′ cis-elements for viral replication and encapsidation, and further comprising a deletion in an adenovirus genomic region selected from the group consisting of: (i) the polymerase region, wherein said deletion essentially prevents the expression of a functional polymerase protein from said deleted region and said hybrid virus does not otherwise express a functional polymerase protein, (ii) the preterminal protein region, wherein said deletion essentially prevents the expression of a functional preterminal protein from said deleted region, and said hybrid virus does not otherwise express a functional preterminal protein, and (iii) both the regions of (i) and (ii); and (b) a recombinant adeno-associated virus (AAV) vector genome flanked by the adenovirus vector genome sequences of (a), said recombinant AAV vector genome comprising (i) AAV 5′ and 3′ inverted terminal repeats, (ii) an AAV packaging sequence, and (iii) a heterologous nucleic acid sequence, wherein said heterologous nucleic acid sequence is flanked by the 5′ and 3′ AAV inverted terminal repeats of (i). Methods of making and using the recombinant hybrid virus are also disclosed.

1 - 36 . (canceled) 
     
     
         37 . A method of producing a recombinant hybrid adeno-associated virus (AAV) particle, comprising providing to a cell:
 (a) a recombinant hybrid virus; comprising:
 (i) a deleted adenovirus vector genome comprising the adenovirus 5′ and 3′ cis-elements for viral replication and encapsidation; a functional adenovirus genomic region selected from the group consisting of an adenovirus E1a region, E2a region, E4orf6 region, VA RNA region, and any combination of the foregoing; and further comprising a deletion in an adenovirus genomic region selected from the group consisting of:
 (1) the polymerase region, wherein said deletion essentially prevents the expression of a functional polymerase protein from said deleted region and said hybrid virus does not otherwise express a functional polymerase protein, 
 (2) the preterminal protein region, wherein said deletion essentially prevents the expression of a functional preterminal protein from said deleted region, and said hybrid virus does not otherwise express a functional preterminal protein, and 
 (3) both the regions of (1) and (2); and 
 
 (ii) a recombinant adeno-associated virus (AAV) vector genome flanked by the adenovirus vector genome sequences of (i), said recombinant AAV vector genome comprising:
 (4) AAV 5′ and 3′ inverted terminal repeats; 
 (5) an AAV packaging sequence; and 
 (6) a heterologous nucleic acid sequence, wherein said heterologous nucleic acid sequence is flanked by the 5′ and 3′ AAV inverted terminal repeats of (4), and further wherein the AAV vector genome does not encode the AAV Rep or AAV capsid proteins; 
 
   (b) AAV sequences sufficient for replication and packaging of the AAV vector genome; and   (c) AAV sequences sufficient to produce a functional AAV capsid, wherein (a), (b), and (c) are provided to the cell under conditions sufficient for replication of the AAV vector genome and packaging thereof in the AAV capsid such that AAV particles comprising the AAV vector genome encapsidated within the AAV capsid are produced in the cell essentially without producing contaminating adenovirus.   
     
     
         38 . The method of  claim 37 , further comprising collecting the recombinant AAV particle. 
     
     
         39 . (canceled) 
     
     
         40 . The method of  claim 37 , wherein each of adenovirus E1a, E2a, E4orf6, and VA RNA helper sequences are provided. 
     
     
         41 . The method of  claim 37 , wherein the cell is selected from the group consisting of a HeLa cell, a 293 cell, a muscle cell, and a liver cell. 
     
     
         42 . (canceled) 
     
     
         43 . The method of  claim 37 , wherein the yield of recombinant AAV particles is at least 5-fold greater than in the presence of the adenovirus polymerase and/or preterminal proteins. 
     
     
         44 . The method of  claim 37 , wherein sequences encoding an AAV Rep protein and/or sequences encoding the AAV capsid protein are stably expressed by the cell. 
     
     
         45 . The method of  claim 37 , wherein sequences encoding an AAV Rep protein and/or sequences encoding the AAV capsid protein are provided by a vector other than the recombinant hybrid virus. 
     
     
         46 . The method of  claim 45 , wherein the vector is selected from the group consisting of a plasmid, an adenovirus, an Epstein Barr virus, and a herpesvirus vector. 
     
     
         47 . The method of  claim 37 , wherein the AAV inverted terminal repeats and the AAV capsid are derived from different AAV serotypes. 
     
     
         48 . The method of  claim 37 , wherein the AAV capsid is an AAV-6 capsid. 
     
     
         49 . The method of  claim 37 , wherein the AAV inverted terminal repeats are AAV-2 inverted terminal repeats. 
     
     
         50 . A method of producing a recombinant hybrid adeno-associated virus (AAV) particle, comprising providing to a cell a hybrid virus particle produced by the method of  claim 37 , said recombinant hybrid virus particle expressing the adenovirus helper functions for AAV replication and packaging; wherein the cell:
 (i) expresses AAV rep sequences sufficient for replication and packaging of the AAV vector genome;   (ii) expresses AAV cap sequences sufficient to produce a functional AAV capsid; and   (iii) does not express sequences sufficient to produce a functional adenovirus E1a protein;   
       and further wherein the hybrid virus particle is provided under conditions sufficient for replication of the AAV vector genome and packaging thereof in the AAV capsid such that AAV particles comprising the AAV vector genome encapsidated within the AAV capsid are produced in the cell. 
     
     
         51 . A method of producing a recombinant hybrid adeno-associated virus (AAV) particle, comprising providing to a cell a recombinant hybrid AAV particle produced by the method of  claim 37 , the recombinant hybrid AAV particle expressing:
 (i) adenovirus helper functions for AAV replication and packaging except the hybrid virus particle does not express a functional adenovirus E1a gene product,   (ii) AAV rep sequences sufficient for replication and packaging of the AAV vector genome, and   (iii) AAV cap sequences sufficient to produce a functional AAV capsid,   
       wherein the cell expresses functional adenovirus E1a gene products; and further wherein the hybrid virus particle is provided to the cell under conditions sufficient for replication of the AAV vector genome and packaging thereof in the AAV capsid such that AAV particles comprising the AAV vector genome encapsidated within the AAV capsid are produced in the cell. 
     
     
         52 . A method of producing a recombinant hybrid adeno-associated virus (AAV) particle, comprising providing to a cell:
 (a) a recombinant hybrid AAV particle produced by the method of  claim 37 , the recombinant hybrid virus AAV particle expressing adenovirus helper functions for AAV replication and packaging except the hybrid virus particle does not express a functional adenovirus E1a gene product,   (b) a separate vector comprising inducible AAV rep sequences sufficient for replication and packaging of the AAV vector genome, and AAV cap sequences sufficient to produce a functional AAV capsid,   
       wherein:
 (i) the cell expresses a functional adenovirus E1a gene product; and 
 (ii) (a) and (b) are provided to the cell under conditions sufficient for replication of the AAV vector genome and packaging thereof in the AAV capsid such that AAV particles comprising the AAV vector genome encapsidated within the AAV capsid are produced in the cell. 
 
     
     
         53 . A method of producing a recombinant hybrid adeno-associated virus (AAV) particle, comprising providing to a cell:
 (a) a recombinant hybrid AAV particle produced by the method of  claim 37 , the recombinant hybrid virus AAV particle expressing adenovirus helper functions for AAV replication and packaging,   (b) a separate vector comprising AAV rep sequences sufficient for replication and packaging of the AAV vector genome, and AAV cap sequences sufficient to produce a functional AAV capsid,   
       wherein:
 (i) the cell does not express a functional adenovirus E1a gene product; and 
 (ii) (a) and (b) are provided to the cell under conditions sufficient for replication of the AAV vector genome and packaging thereof in the AAV capsid such that AAV particles comprising the AAV vector genome encapsidated within the AAV capsid are produced in the cell. 
 
     
     
         54 . The method of  claim 52 , wherein the separate vector is a plasmid vector. 
     
     
         55 . The method of  claim 52 , wherein the separate vector is an adenovirus vector. 
     
     
         56 - 142 . (canceled) 
     
     
         143 . The method of  claim 53 , wherein the separate vector is a plasmid vector. 
     
     
         144 . The method of  claim 53 , wherein the separate vector is an adenovirus vector.