Closed-ended, linear, duplex adenoassociated virus dna, and uses thereof

Abstract

Closed-ended, linear, duplex (CELiD) DNA molecules, recombinant AAV (rAAV), particles comprising CELiD DNA, methods of making such molecules and particles, and therapeutic applications of such particles.

What is claimed is: 
     
         1 . A method of producing a recombinant AAV (rAAV) particle comprising heterologous DNA, the method comprising culturing a cell comprising:
 i) closed ended, linear duplex (CELiD) DNA comprising the heterologous DNA; and,   ii) one or more nucleic acid molecules encoding one or more AAV capsid (Cap) proteins;    under conditions suitable for formation of AAV particles containing the CELiD DNA.   
     
     
         2 . The method of  claim 1 , wherein the one or more nucleic acid molecules encoding one or more AAV capsid (Cap) proteins is/are stably inserted into the genome of the cell. 
     
     
         3 . The method of  claim 1 , wherein the cultured cell comprises one or more polynucleotide molecules encoding one or more AAV Rep proteins, and wherein the culture conditions comprise a culture environment suitable for replication of the CELiD DNA, expression of the one or more Cap proteins, and expression of the one or more Rep proteins. 
     
     
         4 . The method of  claim 1 , wherein the cell is an invertebrate cell or a mammalian cell. 
     
     
         5 . The method of  claim 1 , wherein the (CELiD) DNA comprising the heterologous DNA is flanked by inverted terminal repeats (ITRs), at least one of which comprises an AAV Rep protein binding site and an AAV terminal resolution site (trs) site. 
     
     
         6 . The method of  claim 1 , wherein the heterologous DNA encodes a protein or a therapeutic RNA. 
     
     
         7 . The method of  claim 6 , wherein therapeutic RNA comprises a siRNA, a RNAi, a shRNA, a miRNA, an aptamer, or a ribozyme. 
     
     
         8 . A recombinant AAV (rAAV) particle produced by the method of  claim 1 , wherein the particle comprises closed ended, linear duplex (CELiD)-derived DNA comprising heterologous DNA and wherein DNA from a source other than an AAV or the heterologous DNA in the CELiD-derived DNA makes up less than 10% of the total amount of DNA in the particle, and wherein the heterologous DNA encodes a protein or a therapeutic RNA. 
     
     
         9 . A kit comprising the rAAV particle of  claim 8 . 
     
     
         10 . A method of protecting an individual against a disease, comprising administering the rAAV particle of  claim 8  to the individual, wherein the rAAV particle comprises heterologous DNA encoding a therapeutic molecule that protects the individual against the disease. 
     
     
         11 . A method of treating an individual for an illness, comprising administering the rAAV particle of  claim 8  to the individual, wherein the rAAV particle comprises heterologous DNA encoding a therapeutic molecule that treats the illness. 
     
     
         12 . A method of eliciting an immune response in an individual, comprising administering the rAAV particle of  claim 8  to the individual, wherein the rAAV particle comprises heterologous DNA encoding an immunogenic protein. 
     
     
         13 . A recombinant AAV (rAAV) particle comprising closed ended, linear duplex (CELiD)-derived DNA comprising heterologous DNA wherein DNA from a source other than an AAV or the heterologous DNA in the CELiD-derived DNA makes up less than 10% of the total amount of DNA in the particle, and wherein the heterologous DNA encodes a protein or a therapeutic RNA. 
     
     
         14 . The rAAV particle of  claim 13 , wherein the rAAV particle is produced using a method comprising culturing a cell comprising:
 i) closed ended, linear duplex (CELiD) DNA comprising the heterologous DNA; and,   ii) one or more nucleic acid molecules encoding one or more AAV capsid (Cap) proteins;    under conditions suitable for formation of AAV particles containing the CELiD-derived DNA.   
     
     
         15 . The rAAV particle of  claim 14 , wherein the one or more nucleic acid molecules encoding one or more AAV capsid (Cap) proteins is/are stably inserted into the genome of the cell. 
     
     
         16 . The rAAV particle of  claim 14 , wherein the cultured cell comprises one or more polynucleotide molecules encoding one or more AAV Rep proteins, and wherein the culture conditions comprise a culture environment suitable for replication of the CELiD DNA, expression of the one or more Cap proteins, and expression of the one or more Rep proteins. 
     
     
         17 . The rAAV particle of  claim 14 , wherein the cell is an invertebrate cell or a mammalian cell. 
     
     
         18 . The rAAV particle of  claim 13 , wherein the heterologous DNA encodes a therapeutic RNA. 
     
     
         19 . The rAAV particle of  claim 18 , wherein the therapeutic RNA comprises a siRNA, a RNAi, a shRNA, a miRNA, an aptamer, or a ribozyme. 
     
     
         20 . A method of producing closed ended, linear duplex (CELiD) DNA, comprising heterologous DNA, the method comprising:
 a) culturing a cell comprising:
 i) one or more polynucleotide sequences encoding one or more adeno-associated virus (AAV) Rep proteins; and, 
 ii) a nucleic acid molecule comprising heterologous DNA flanked by a pair of inverted terminal repeats (ITRs), wherein at least one ITR comprises an AAV Rep protein binding site and a AAV terminal resolution site (trs), and wherein at least one ITR can be used as an origin of replication; 
    under conditions suitable for replication of the nucleic acid molecule, thereby producing new CELiD DNA molecules; and   b) isolating the newly produced CELiD DNA molecules from the cell.   
     
     
         21 . The method of  claim 20 , wherein the nucleic acid molecule comprises CELiD DNA. 
     
     
         22 . A method of protecting an individual against a disease, comprising administering the rAAV particle of  claim 13  to the individual, wherein the rAAV particle comprises heterologous DNA encoding a therapeutic molecule that protects the individual against the disease. 
     
     
         23 . A method of treating an individual for an illness, comprising administering the rAAV particle of  claim 13  to the individual, wherein the rAAV particle comprises heterologous DNA encoding a therapeutic molecule that treats the illness. 
     
     
         24 . A method of eliciting an immune response in an individual, comprising administering the rAAV particle of  claim 13  to the individual, wherein the rAAV particle comprises heterologous DNA encoding an immunogenic protein.