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.

What is claimed is: 
     
         1 . A viral vector comprising:
 (a) a first nucleic acid sequence encoding a first engineered nuclease;   (b) a first promoter operably linked to said first nucleic acid sequence, wherein said first promoter is positioned 5′ upstream of said first nucleic acid sequence and drives expression of said first engineered nuclease in a target cell; and   (c) a first vector recognition sequence which is recognized and cleaved by said first engineered nuclease.   
     
     
         2 . The viral vector of  claim 1 , wherein said viral vector further comprises a first polyA sequence positioned 3′ downstream of said first nucleic acid sequence. 
     
     
         3 . The viral vector of  claim 1  or  claim 2 , wherein cleavage of said first vector recognition sequence by said first engineered nuclease in said target cell causes said viral vector to have a lower persistence time in said target cell when compared to a viral vector which does not comprise a vector recognition sequence cleaved by said first engineered nuclease but which is otherwise identical. 
     
     
         4 . The viral vector of any one of  claims 1-3 , wherein said first vector recognition sequence is identical to a first chromosomal recognition sequence present in the genome of said target cell. 
     
     
         5 . The viral vector of any one of  claims 1-4 , wherein said first vector recognition sequence is a sub-optimal recognition sequence which is recognized and cleaved by said first engineered nuclease. 
     
     
         6 . The viral vector of any one of  claims 1-5 , wherein said viral vector 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 . The viral vector of  claim 6 , wherein said transgene sequence is positioned 5′ upstream of said first promoter. 
     
     
         8 . The viral vector of  claim 6 , wherein said transgene sequence is positioned 3′ downstream of said first nucleic acid sequence. 
     
     
         9 . The viral vector of any one of  claims 6-8 , wherein said first chromosomal recognition sequence is positioned within said region of interest in the genome of said target cell. 
     
     
         10 . The viral vector of any one of  claims 1-5 , wherein said viral vector further comprises a corrected gene sequence, wherein said corrected gene sequence does not comprise said first 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 viral vector of  claim 10 , wherein said mutated gene sequence differs from said corrected gene sequence by at least one nucleotide and comprises said first chromosomal recognition sequence. 
     
     
         12 . The viral vector of  claim 10  or  claim 11 , wherein said corrected gene sequence is positioned 5′ upstream of said first promoter. 
     
     
         13 . The viral vector of  claim 10  or  claim 11 , wherein said corrected gene sequence is positioned 3′ downstream of said first nucleic acid sequence. 
     
     
         14 . The viral vector of any one of  claims 1-5 , wherein said viral vector further comprises a second nucleic acid sequence encoding a second engineered nuclease. 
     
     
         15 . The viral vector of  claim 14 , wherein said viral vector further comprises a second promoter operably linked to said second nucleic acid sequence, wherein said second promoter is positioned 5′ upstream of said second nucleic acid sequence and drives expression of said second engineered nuclease in said target cell. 
     
     
         16 . The viral vector of  claim 14  or  claim 15 , wherein said second nucleic acid sequence is positioned 5′ upstream of said first promoter. 
     
     
         17 . The viral vector of  claim 14  or  claim 15 , wherein said second nucleic acid sequence is positioned 3′ downstream of said first nucleic acid sequence. 
     
     
         18 . The viral vector of any one of  claims 14-17 , wherein said viral vector further comprises a second polyA sequence positioned 3′ downstream of said second nucleic acid sequence. 
     
     
         19 . The viral vector of any one of  claims 14-18 , wherein said second engineered nuclease recognizes and cleaves a second chromosomal recognition sequence present in the genome of said target cell. 
     
     
         20 . The viral vector of  claim 19 , wherein said first chromosomal recognition sequence and said second chromosomal recognition sequence are positioned on the same chromosome. 
     
     
         21 . The viral vector of  claim 19  or  claim 20 , wherein said first chromosomal recognition sequence and said second chromosomal recognition sequence flank a region of interest in the genome of said target cell. 
     
     
         22 . The viral vector of  claim 19 , wherein said first chromosomal recognition sequence and said second chromosomal recognition sequence are positioned on different chromosomes. 
     
     
         23 . The viral vector of any one of  claims 1-22 , wherein said first vector recognition sequence is positioned 5′ upstream of said first promoter. 
     
     
         24 . The viral vector of any one of  claims 1-22 , wherein said first vector recognition sequence is positioned 3′ downstream of said first promoter and 5′ upstream of said first nucleic acid sequence. 
     
     
         25 . The viral vector of any one of  claims 1-22 , wherein said first vector recognition sequence is positioned 3′ downstream of said first nucleic acid sequence. 
     
     
         26 . The viral vector of any one of  claims 1-22 , wherein said first nucleic acid sequence comprises, from 5′ to 3′, a first exon, an intron, and a second exon. 
     
     
         27 . The viral vector of  claim 26 , wherein said first vector recognition sequence is positioned within said intron of said first nucleic acid sequence. 
     
     
         28 . The viral vector of any one of  claims 2-22 , wherein said first vector recognition sequence is positioned 3′ downstream of said first nucleic acid sequence and 5′ upstream of said first polyA sequence. 
     
     
         29 . The viral vector of any one of  claims 2-22 , wherein said first vector recognition sequence is positioned 3′ downstream of said first polyA sequence. 
     
     
         30 . The viral vector of any one of  claims 6-9 , wherein said first vector recognition sequence is positioned 5′ upstream of said transgene sequence. 
     
     
         31 . The viral vector of any one of  claims 6-9 , wherein said first vector recognition sequence is positioned 3′ downstream of said transgene sequence. 
     
     
         32 . The viral vector of any one of  claims 10-13 , wherein said first vector recognition sequence is positioned 5′ upstream of said corrected gene sequence. 
     
     
         33 . The viral vector of any one of  claims 10-13 , wherein said first vector recognition sequence is positioned 3′ downstream of said corrected gene sequence. 
     
     
         34 . The viral vector of any one of  claims 14-22 , wherein said first vector recognition sequence is positioned 5′ upstream of said second nucleic acid sequence. 
     
     
         35 . The viral vector of any one of  claims 15-22 , wherein said first vector recognition sequence is positioned 3′ downstream of said second promoter and 5′ upstream of said second nucleic acid sequence. 
     
     
         36 . The viral vector of any one of  claims 14-22 , wherein said first vector recognition sequence is positioned 3′ downstream of said second nucleic acid sequence. 
     
     
         37 . The viral vector of any one of  claims 14-22 , wherein said second nucleic acid sequence comprises, from 5′ to 3′, a first exon, an intron, and a second exon. 
     
     
         38 . The viral vector of  claim 37 , wherein said first vector recognition sequence is positioned within said intron of said second nucleic acid sequence. 
     
     
         39 . The viral vector of any one of  claims 18-22 , wherein said first vector recognition sequence is positioned 3′ downstream of said second nucleic acid sequence and 5′ upstream of said second polyA sequence. 
     
     
         40 . The viral vector of any one of  claims 18-22 , wherein said first vector recognition sequence is positioned 3′ downstream of said second polyA sequence. 
     
     
         41 . The viral vector of any one of  claims 1-40 , wherein said viral vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviral vector. 
     
     
         42 . The viral vector of any one of  claims 1-41 , wherein said viral vector is an AAV vector comprising a 5′ inverted terminal repeat and a 3′ inverted terminal repeat. 
     
     
         43 . The viral vector of  claim 42 , wherein said AAV vector is a single-stranded AAV vector or a self-complementary AAV vector. 
     
     
         44 . The viral vector of any one of  claims 1-43 , wherein said first promoter is a tissue-specific promoter, a species-specific promoter, or an inducible promoter. 
     
     
         45 . The viral vector of any one of  claims 1-44 , wherein said engineered nuclease is an engineered meganuclease, a zinc finger nuclease (ZFN), a TALEN, a compact TALEN, or a CRISPR/Cas. 
     
     
         46 . The viral vector of any one of  claims 1-45 , wherein said engineered nuclease is an engineered meganuclease. 
     
     
         47 . The viral vector of any one of  claims 1-46 , wherein said first promoter comprises one or more binding sites for a transcription repressor that binds to and silences said first promoter. 
     
     
         48 . The viral vector of  claim 47 , wherein said transcription repressor is a Tet repressor, a Lac repressor, a Cre repressor, or a Lambda repressor. 
     
     
         49 . The viral vector of any one of  claims 1-46 , wherein said first promoter is an inducible promoter, and wherein said viral vector 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 viral vector of any one of  claims 1-49 . 
     
     
         51 . A recombinant DNA construct encoding said viral vector of  claim 47  or  claim 48 , wherein said recombinant DNA construct further comprises a nucleic acid sequence encoding said transcription repressor. 
     
     
         52 . The recombinant DNA construct of  claim 51 , wherein said nucleic acid sequence encoding said transcription repressor is positioned outside of the coding sequence of said viral vector. 
     
     
         53 . A method for producing a viral vector, said method comprising transforming a packaging cell with said recombinant DNA construct of any one of  claims 50-52 , wherein said packaging cell produces said viral vector. 
     
     
         54 . The method of  claim 53 , wherein said packaging cell is transformed with said recombinant DNA construct of  claim 51  or  claim 52 . 
     
     
         55 . The method of  claim 54 , wherein said recombinant DNA construct further comprises a nucleic acid sequence encoding said transcription repressor. 
     
     
         56 . The method of  claim 55 , wherein said nucleic acid sequence encoding said transcription repressor is positioned outside of the coding sequence of said viral vector. 
     
     
         57 . The method of  claim 54 , wherein said packaging cell is further transformed with a second recombinant DNA construct comprising a nucleic acid sequence encoding said transcription repressor. 
     
     
         58 . The method of  claim 54 , wherein said packaging cell comprises in its genome a nucleic acid sequence encoding said transcription repressor, and wherein said packaging cell stably expresses said transcription repressor. 
     
     
         59 . The method of  claim 53 , wherein said first promoter of said recombinant DNA construct is a tissue-specific promoter that is inactive in said packaging cell. 
     
     
         60 . The method of  claim 53 , wherein said first promoter of said recombinant DNA construct is a species-specific promoter that is inactive in said packaging cell. 
     
     
         61 . The method of  claim 60 , wherein said first promoter is a mammalian promoter and said packaging cell is a microbial cell, an insect cell, or a plant cell. 
     
     
         62 . The method of  claim 53 , wherein said first promoter of said recombinant DNA construct is an inducible-promoter which is regulated by a ligand-inducible transcription factor, and wherein said recombinant DNA construct further comprises a nucleic acid sequence encoding said ligand-inducible transcription factor. 
     
     
         63 . The method of  claim 62 , wherein said nucleic acid sequence encoding said ligand-inducible transcription factor is positioned within the coding sequence of said viral vector. 
     
     
         64 . The method of any one of  claims 53-63 , wherein said packaging cell is an insect cell, and wherein said first nucleic acid sequence encoding said first engineered nuclease comprises an intron that prevents expression of said first engineered nuclease in said packaging cell. 
     
     
         65 . The method of  claim 64 , wherein said intron is a human growth hormone intron (SEQ ID NO: 2) or an SV40 large T antigen intron (SEQ ID NO: 3). 
     
     
         66 . The method of any one of  claims 53-65 , wherein said viral vector is an AAV vector, a retroviral vector, a lentiviral vector, or an adenoviral vector. 
     
     
         67 . The method of any one of  claims 53-66 , wherein said viral vector is an AAV vector. 
     
     
         68 . The method of  claim 67 , said method further comprising transforming said packaging cell with:
 (a) a second recombinant DNA construct comprising a cap gene and a rep gene; and   (b) a third recombinant DNA construct comprising adenoviral helper components; wherein said packaging cell produces said AAV vector.