Self-complementary parvoviral vectors, and methods for making and using the same

Abstract

The teachings herein are generally directed to a method of enhancing the genetic stability of parvovirus vectors. The stability of conventional ss or dsAAV vector constructs can be enhanced, for example, to obtain a concurrent increase in vector titer and purity, as well as an improvement in vector safety, due at least in part to the elimination of stuffer DNA from the AAV vector. The method is broadly applicable to all gene transfer/therapy applications, such as those requiring delivery of foreign DNA containing recombinant gene expression cassettes. Such foreign DNA can range, for example, from about 0.2 up to about 5.2 kb in length. The enhanced vector constructs are highly flexible, user-friendly, and can be easily modified (via routine DNA cloning) and utilized (via standard AAV vector technology) by anyone skilled in the art.

1 - 53 . (canceled) 
     
     
         54 . A parvovirus vector template nucleic acid for creating a self-complementary, genetically stable, infectious recombinant, adeno-associated virus (rAAV) virion comprising:
 a) a 5′ inverted terminal repeat (ITR);   b) a 3′ ITR; and   c) a foreign DNA insert comprising one or more expression cassettes, each independently ranging in size from 0.2 kb to 2.2 kb, and adding up to a total size of no more than 2.4 kb in length between the 5′ITR and the 3′ ITR,   wherein: (i) the 3′ ITR serves as a primer for DNA replication, (ii) the 5′ and 3′ ITRs are from AAV, (iii) the 5′ and 3′ ITRs are not from the same AAV serotype, and (iv) at least one of the 5′ ITR and 3′ ITR does not contain a functional terminal resolution site (trs).   
     
     
         55 . The template nucleic acid of  claim 54 , wherein the expression cassette includes a promoter, a coding sequence and a termination signal. 
     
     
         56 . The template nucleic acid of  claim 54 , wherein the foreign DNA insert encodes a protein, an shRNA, or a micro-RNA. 
     
     
         57 . The template nucleic acid of  claim 54 , wherein the foreign DNA insert comprises more than one expression cassette. 
     
     
         58 . The template nucleic acid of  claim 54 , wherein the template nucleic acid is single-stranded. 
     
     
         59 . The template nucleic acid of  claim 54 , wherein the template nucleic acid is double-stranded. 
     
     
         60 . The template nucleic acid of  claim 54 , wherein the 5′ ITR or 3′ ITR is from AAV-2. 
     
     
         61 . The template nucleic acid of  claim 54 , wherein the 5′ ITR or 3′ ITR is from AAV-4. 
     
     
         62 . The template nucleic acid of  claim 61 , wherein the ITR from AAV-4 consists essentially of the trs-deleted BssHII fragment of SEQ ID NO: 1. 
     
     
         63 . The template nucleic acid of  claim 54 , wherein the 5′ ITR or 3′ ITR is from AAV-5. 
     
     
         64 . A plasmid comprising the template nucleic acid of  claim 54 . 
     
     
         65 . A method of producing a self-complementary, genetically stable, infectious recombinant, adeno-associated virus (rAAV) virion, said method comprising:
 a) introducing the nucleic acid of  claim 54  into a cell that permits viral replication;   b) introducing an AAV helper plasmid to provide rep and cap genes;   c) introducing an adenoviral helper plasmid to provide helper virus function; and   d) maintaining the cell under conditions sufficient to produce a stabilized, self-complementary double-stranded parvovirus vector, package the vector in a parvovirus capsid, and produce the parvovirus virion.   
     
     
         66 . A genetically stable viral genome, comprising:
 a) two inverted copies of a foreign DNA insert; and   b) first and second AAV ITR sequences that are not from the same AAV serotype, wherein the first or second ITR serves as a primer for DNA replication but does not contain a functional terminal resolution site, and wherein the first or the second ITR is between the two inverted copies of the foreign DNA insert.   
     
     
         67 . The genetically stable viral genome of  claim 66 , wherein the first or second ITR is from AAV-2. 
     
     
         68 . The genetically stable viral genome of  claim 66 , wherein the first or second ITR is from AAV-4. 
     
     
         69 . The genetically stable viral genome of  claim 68 , wherein the ITR from AAV-4 consists essentially of the trs-deleted BssHII fragment of SEQ ID NO: 1. 
     
     
         70 . The genetically stable viral genome of  claim 66 , wherein the first or second ITR is from AAV-5. 
     
     
         71 . The genetically stable viral genome of  claim 66 , wherein the foreign DNA insert encodes a protein, an shRNA, or a micro-RNA. 
     
     
         72 . A genetically stable, infectious recombinant, adeno-associated virus (rAAV) virion comprising:
 a) an AAV parvovirus capsid; and   b) the viral genome of  claim 66 .   
     
     
         73 . A composition comprising the genetically stable, infectious recombinant rAAV virion of  claim 72  in a pharmaceutically acceptable carrier. 
     
     
         74 . An isolated cell comprising the genetically stable, infectious recombinant rAAV virion of  claim 72 . 
     
     
         75 . A method for introducing a foreign DNA insert comprising one or more expression cassettes into at least one cell of a mammalian host, the method comprising:
 administering to the mammalian host an effective amount of the virion of  claim 72  so that the foreign DNA insert is introduced into at least one cell of the mammalian host.   
     
     
         76 . The method of  claim 75 , wherein the foreign DNA insert contains an expression cassette encoding Factor IX (FIX) to increase a level of FIX in blood of the mammalian host and induce clotting. 
     
     
         77 . A kit for use in producing an adeno-associated virus (rAAV) virion, said kit comprising:
 a) a plasmid comprising the template nucleic acid of  claim 54 ;   b) an AAV helper plasmid to provide rep and cap genes;   c) an adenoviral helper plasmid to provide helpervirus function; and   d) instructions for producing the adeno-associated virus (rAAV) virion.