Self-limiting viral vectors encoding nucleases

Abstract

Disclosed herein are viral vectors for use in recombinant molecular biology techniques. In particular, the present disclosure relates to self-limiting viral vectors comprising genes encoding site-specific endonucleases as well as recognition sequences for site-specific endonucleases such that expression of the endonuclease in a cell cleaves the viral vector and limits its persistence time. In some embodiments, the viral vectors disclosed herein also carry directives to delete, insert, or change a target sequence.

1 . An adeno-associated viral (AAV) vector comprising a polynucleotide, wherein said polynucleotide comprises:
 (a) a 5′ inverted terminal repeat (ITR);   (b) a nucleic acid sequence encoding an engineered nuclease and an intron that prevents expression of said engineered nuclease in a packaging cell;   (c) a promoter operably linked to said nucleic acid sequence, wherein said first promoter is positioned 5′ upstream of said nucleic acid sequence and drives expression of said engineered nuclease in a target cell;   (d) a vector recognition sequence which is recognized and cleaved by said engineered nuclease, wherein said vector recognition sequence is identical to a chromosomal recognition sequence present in the genome of said target cell, or wherein said vector recognition sequence is a sub-optimal recognition sequence which is recognized and cleaved by said engineered nuclease; and   (e) a 3′ ITR;   
       wherein said polynucleotide does not comprise more than one promoter. 
     
     
         2 . The AAV of  claim 1 , wherein said polynucleotide further comprises a polyA sequence positioned 3′ downstream of said nucleic acid sequence. 
     
     
         3 . The AAV of  claim 1 , wherein cleavage of said vector recognition sequence by said engineered nuclease in said target cell causes said AAV vector to have a lower persistence time in said target cell when compared to an AAV vector which does not comprise a vector recognition sequence cleaved by said engineered nuclease but which is otherwise identical. 
     
     
         4 - 5 . (canceled) 
     
     
         6 . The AAV of  claim 1 , wherein said polynucleotide further comprises a transgene sequence, wherein said transgene sequence is flanked by sequences homologous to sequences flanking a region of interest in the genome of said target cell. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The AAV of  claim 6 , wherein said chromosomal recognition sequence is positioned within said region of interest in the genome of said target cell. 
     
     
         10 . The AAV of  claim 1 , wherein said polynucleotide further comprises a corrected gene sequence, wherein said corrected gene sequence does not comprise said vector recognition sequence, and wherein said corrected gene sequence corresponds to a mutated gene sequence present in the genome of said target cell. 
     
     
         11 . The AAV of  claim 10 , wherein said mutated gene sequence differs from said corrected gene sequence by at least one nucleotide and comprises said chromosomal recognition sequence. 
     
     
         12 - 43 . (canceled) 
     
     
         44 . The AAV of  claim 1 , wherein said promoter is a tissue-specific promoter, a species-specific promoter, or an inducible promoter. 
     
     
         45 . The AAV of  claim 1 , wherein said engineered nuclease is an engineered meganuclease, a zinc finger nuclease (ZFN), a TALEN, a compact TALEN, or a CRISPR/Cas. 
     
     
         46 . The AAV of  claim 1 , wherein said engineered nuclease is an engineered meganuclease. 
     
     
         47 . The AAV of  claim 1 , wherein said promoter comprises one or more binding sites for a transcription repressor that binds to and silences said promoter. 
     
     
         48 . The AAV of  claim 47 , wherein said transcription repressor is a Tet repressor, a Lac repressor, a Cre repressor, or a Lambda repressor. 
     
     
         49 . The AAV of  claim 1 , wherein said promoter is an inducible promoter, and wherein said polynucleotide further comprises a nucleic acid sequence encoding a ligand-inducible transcription factor which regulates activation of said first promoter. 
     
     
         50 . A recombinant DNA construct encoding said AAV of  claim 1 . 
     
     
         51 . A recombinant DNA construct encoding said AAV of  claim 47 , wherein said recombinant DNA construct further comprises a nucleic acid sequence encoding said transcription repressor. 
     
     
         52 . (canceled) 
     
     
         53 . A method for producing an AAV, said method comprising:
 (a) transforming a packaging cell with said recombinant DNA construct of  claim 50 ;   (b) transforming said packaging cell with a second recombinant DNA construct comprising a cap gene and a rep gene; and   (c) transforming said packaging cell with a third recombinant DNA construct comprising adenoviral helper components;   wherein said packaging cell produces said AAV.   
     
     
         54 - 68 . (canceled)