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 . A recombinant viral vector comprising a single-stranded heterologous nucleotide sequence comprising a region which forms intrastrand base pairs such that expression of a coding region of the heterologous sequence is enhanced relative to a recombinant viral vector that lacks sufficient intrastrand base pairing to enhance said expression. 
     
     
         2 . The recombinant viral vector of  claim 1 , wherein the region which forms intrastrand base pairs is a coding region. 
     
     
         3 . The recombinant viral vector of  claim 1 , which is a recombinant parvovirus vector, comprising one or more inverted terminal repeat (ITR) sequences flanking said heterologous sequence. 
     
     
         4 . The recombinant viral vector of  claim 3 , which is a recombinant adeno-associated virus (rAAV) vector. 
     
     
         5 . The rAAV vector of  claim 4 , wherein said rAAV vector is capable of being packaged in an AAV particle. 
     
     
         6 . The rAAV vector of  claim 4 , wherein the sequence complexity of the heterologous sequence is about one-half of the heterologous sequence. 
     
     
         7 . The rAAV vector of  claim 6 , further comprising an ITR flanked on both sides by heterologous sequences. 
     
     
         8 . A host cell comprising the recombinant viral vector of  claim 1 . 
     
     
         9 . A host cell comprising the rAAV vector of  claim 5 . 
     
     
         10 . The host cell of  claim 9 , which is mammalian. 
     
     
         11 . A recombinant viral particle comprising the recombinant viral vector of  claim 1 . 
     
     
         12 . A recombinant AAV particle comprising the rAAV vector of  claim 5 . 
     
     
         13 . A recombinant AAV particle comprising the rAAV vector of  claim 6 . 
     
     
         14 . A composition comprising the recombinant viral vector of  claim 1 . 
     
     
         15 . A composition comprising the rAAV vector of  claim 5 . 
     
     
         16 . The composition of  claim 15 , further comprising a pharmaceutically acceptable excipient. 
     
     
         17 . A library comprising a population of recombinant viral vectors according to  claim 1 . 
     
     
         18 . A library comprising a population of rAAV vectors according to  claim 5 . 
     
     
         19 . A method for preparing an rAAV according to  claim 6 , wherein the method comprises incubating a host cell under conditions that allow AAV replication and encapsidation, wherein said host cell comprises:
 (a) an rAAV vector comprising a heterologous nucleotide sequence and one or more AAV inverted terminal repeat (ITR) sequences flanking said heterologous sequence, wherein the vector is less than about 2.5 kb, and   (b) AAV rep function, AAV cap function, and helper virus function for AAV.   
     
     
         20 . The method of  claim 19  wherein rep and cap functions are provided by a rep-cap cassette that is stably integrated in the host cell genome. 
     
     
         21 . The method of  claim 19  wherein rep and cap functions are provided by a plasmid. 
     
     
         22 . The method of  claim 19  wherein the rAAV vector is provided in a plasmid. 
     
     
         23 . The method of  claim 19 , wherein the rAAV vector is stably integrated into the host cell genome. 
     
     
         24 . The method of  claim 19  wherein helper functions are provided by adenovirus infection. 
     
     
         25 . A population of rAAV particles produced according to the method of  claim 19 . 
     
     
         26 . A population of rAAV vectors produced according to the method of  claim 19 . 
     
     
         27 . A method for introducing a polynucleotide into a host cell, comprising contacting the cell with a recombinant viral vector according to  claim 1  under conditions that allow uptake of the vector, whereby the vector is introduced into the cell. 
     
     
         28 . A method for introducing a polynucleotide into a host cell, comprising contacting the cell with an rAAV vector according to  claim 4  under conditions that allow uptake of the rAAV vector, whereby the rAAV vector is introduced into the cell. 
     
     
         29 . A method for expressing a polynucleotide in a host cell, said host cell containing a recombinant viral vector of  claim 1 , comprising subjecting the cell to conditions which allow expression of the coding region of the heterolgous sequence of the viral vector, whereby a coding region of the heterolgous sequence is expressed. 
     
     
         30 . A method for expressing a polynucleotide in a host cell, said host cell containing an rAAV vector of  claim 4 , comprising subjecting the cell to conditions which allow expression of a coding region of the heterolgous sequence of the rAAV vector, whereby a coding region of the heterolgous sequence is expressed. 
     
     
         31 . A method of introducing an rAAV vector into a host cell comprising contacting the host cell with an rAAV of  claim 12  under conditions that allow infection, whereby the rAAV vector is introduced into the cell. 
     
     
         32 . A method for constructing a library comprising: (a) contacting a population of host cells with recombinant viral vectors according to  claim 1  under conditions that allow introduction of said vectors into the cells; and (b) selecting cells that contain a recombinant viral vector. 
     
     
         33 . A method for constructing a library comprising: (a) contacting a population of host cells with rAAV vectors according to  claim 4  under conditions that allow introduction of said vectors into the cells; and (b) selecting cells that contain an rAAV vector. 
     
     
         34 . A method for constructing a library comprising: (a) contacting a population of host cells with an rAAV particle according to  claim 12  under conditions that allow infection; and (b) selecting cells containing the rAAV vector. 
     
     
         35 . A method of identifying a phenotype associated with expression of a coding region of a recombinant viral vector of  claim 1  comprising:
 (a) contacting a host cell with a recombinant viral vector according to  claim 1  under conditions that allow uptake of the vector; 
 (b) assaying the cell for expression of the heterologous coding region of the vector; and 
 (c) comparing a phenotype of the cell expressing the heterologous coding region with a phenotype of a cell that lacks the vector, 
 wherein a phenotypic difference indicates that the phenotype of the cell expressing the heterologous sequence is a phenotype associated with expression of the coding region. 
 
     
     
         36 . A method of identifying a phenotype associated with expression of a coding region of an rAAV vector of  claim 4  comprising:
 (a) contacting a host cell with an rAAV vector according to  claim 4  under conditions that allow uptake of the rAAV vector; 
 (b) assaying the cell for expression of the heterologous coding region of the rAAV vector; and 
 (c) comparing a phenotype of the cell expressing the heterologous coding region with a phenotype of a cell that lacks the rAAV vector, 
 wherein a phenotypic difference indicates that the phenotype of the cell expressing the heterologous sequence is a phenotype associated with expression of the coding region. 
 
     
     
         37 . A method of identifying a phenotype associated with expression of a coding region of a recombinant viral vector of  claim 1 , comprising subjecting host cells containing the recombinant viral vector to conditions that allow expression, wherein a phenotypic difference between said host cells and host cells which do not contain the recombinant viral vector indicates a phenotype associated with expression of the coding region. 
     
     
         38 . A method of identifying a phenotype associated with expression of a coding region of an rAAV vector of  claim 4 , comprising subjecting host cells containing the rAAV vector to conditions that allow expression, wherein a phenotypic difference between said host cells and host cells which do not contain the rAAV vector indicates a phenotype associated with expression of the coding region.