Metabolically activated recombinant viral vectors and methods for their preparation and use

Abstract

Recombinant viral vectors, especially parvovirus vectors such as adeno-associated virus (AAV) vectors, capable of enhanced expression of heterologous sequences, and methods for their construction and use, are provided. The vectors have a structure, or are capable of rapidly adopting a structure, which involves intrastrand base pairing of at least one region in a heterologous sequence.

1 - 38 . (canceled) 
     
     
         39 : A composition comprising a purified recombinant adeno-associated virus (rAAV) particle, wherein the rAAV particle comprises an rAAV genome comprising a heterologous nucleotide sequence comprising a coding region and one or more inverted terminal repeat (ITR) sequences flanking said heterologous sequence, wherein the total amount of unique sequence present in the heterologous sequence is about one-half of the heterologous sequence and wherein the heterologous sequence forms intrastrand base pairs along most or all of its length. 
     
     
         40 : The composition of  claim 39 , wherein the coding region encodes a protein of therapeutic interest. 
     
     
         41 : The composition of  claim 39 , wherein the coding region encodes an RNA of therapeutic interest. 
     
     
         42 : The composition of  claim 41 , wherein the RNA of therapeutic interest is an antisense RNA or a ribozyme. 
     
     
         43 : The composition of  claim 39 , wherein the heterologous sequence is: (i) a polynucleotide encoding a protein useful in gene therapy to relieve deficiencies caused by missing, defective or sub-optimal levels of a structural protein or enzyme; (ii) a polynucleotide that is transcribed into an anti-sense molecule; (iii) a polynucleotide that is transcribed into a decoy that binds a transcription or translation factor, (iv) a polynucleotide that encodes a cellular modulator; (v) a polynucleotide that can make a recipient cell susceptible to a specific drug; (vi) a polynucleotide for cancer therapy; or (vii) a polynucleotide that encodes an antigen or antibody. 
     
     
         44 : The composition of  claim 39 , wherein the coding region encodes the herpes virus thymidine kinase gene, an E1A tumor suppressor gene or a p53 tumor suppressor gene. 
     
     
         45 : The composition of  claim 39 , wherein the coding region is operably linked to a promoter sequence. 
     
     
         46 : The composition of  claim 45 , wherein the promoter sequence is (i) an SV40 late promoter, (ii) a baculovirus polyhedron enhancer/promoter element, (iii) a Herpes Simplex Virus thymidine kinase (HSV tk), (iv) an immediate early promoter from cytomegalovirus (CMV), (v) a retroviral promoter including LTR elements, or (vi) a heavy metal ion inducible promoters and a promoter from T7 phage. 
     
     
         47 : The composition of  claim 45 , wherein the coding sequence is further operably linked to an enhancer sequence. 
     
     
         48 : The composition of  claim 39 , wherein the coding region is operably linked to a ribosome binding site (RBS). 
     
     
         49 : The composition of  claim 39 , wherein the coding region is operably linked to a polyadenylation signal. 
     
     
         50 : The composition of  claim 39 , wherein the rAAV genome comprises an internal ITR that is approximately in the center of the genome. 
     
     
         51 : The composition of  claim 39 , wherein the rAAV particle comprises an rAAV2 capsid. 
     
     
         52 : The composition of  claim 39 , wherein the rAAV genome has a size up to about 5.2 kilobase. 
     
     
         53 : The composition of  claim 39 , further comprising a pharmaceutically acceptable excipient. 
     
     
         54 : A method of introducing an rAAV genome into a cell, comprising contacting the cell essentially in the absence of an AAV helper virus with a composition comprising a purified rAAV particle under conditions that allow uptake of the rAAV vector, whereby the rAAV vector is introduced into the cell, wherein the rAAV particle comprises an rAAV genome comprising a heterologous nucleotide sequence comprising a coding region and one or more inverted terminal repeat (ITR) sequences flanking said heterologous sequence, wherein the total amount of unique sequence present in the heterologous sequence is about one-half of the heterologous sequence and wherein the heterologous sequence forms intrastrand base pairs along most or all of its length. 
     
     
         55 : A method of expressing a polynucleotide coding sequence in a cell, comprising subjecting the cell to conditions which allow expression of the coding sequence, wherein the coding sequence is introduced into the cell by contacting the cell essentially in the absence of an AAV helper virus with a composition comprising a purified rAAV particle, wherein the rAAV particle comprises an rAAV genome comprising a heterologous nucleotide sequence comprising a coding region and one or more inverted terminal repeat (ITR) sequences flanking said heterologous sequence, wherein the total amount of unique sequence present in the heterologous sequence is about one-half of the heterologous sequence and wherein the heterologous sequence forms intrastrand base pairs along most or all of its length.