Self-limiting viral vectors encoding nucleases

Patent metadata

Field	Value
Publication number	US-10662440-B2
Application number	US-201615737918-A
Country	US
Kind code	B2
Filing date	Jun 20, 2016
Priority date	Jun 19, 2015
Publication date	May 26, 2020
Grant date	May 26, 2020

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.

First claim

What is claimed is:

1. A viral vector comprising:
(a) a first nucleic acid sequence encoding a first engineered nuclease and comprising, from 5′ to 3′, a first exon, an intron, and a second exon;
(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, wherein said first vector recognition sequence is positioned within said intron of said first nucleic acid sequence.

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 , 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 claim 1 , 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 claim 1 , 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 claim 1 , 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 a first chromosomal recognition sequence is positioned within said region of interest in the genome of said target cell.

8. The viral vector of claim 1 , 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.

9. The viral vector of claim 8 , wherein said mutated gene sequence differs from said corrected gene sequence by at least one nucleotide and comprises a first chromosomal recognition sequence.

10. The viral vector of claim 1 , wherein said viral vector is an adeno-associated virus (AAV) vector, a retroviral vector, a lentiviral vector, or an adenoviral vector.

11. The viral vector of claim 1 , wherein said viral vector is an AAV vector comprising a 5′ inverted terminal repeat and a 3′ inverted terminal repeat.

12. The viral vector of claim 11 , wherein said AAV vector is a single-stranded AAV vector or a self-complementary AAV vector.

13. The viral vector of claim 1 , wherein said first promoter is a tissue-specific promoter, a species-specific promoter, or an inducible promoter.

14. The viral vector of claim 1 , wherein said engineered nuclease is an engineered meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a compact transcription activator-like effector nuclease (Compact TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas).

15. The viral vector of claim 1 , wherein said engineered nuclease is an engineered meganuclease.

16. The viral vector of claim 1 , wherein said first promoter comprises one or more binding sites for a transcription repressor that binds to and silences said first promoter.

17. The viral vector of claim 16 , wherein said transcription repressor is a Tet repressor, a Lac repressor, a Cre repressor, or a Lambda repressor.

18. A recombinant DNA construct encoding said viral vector of claim 16 , wherein said recombinant DNA construct further comprises a nucleic acid sequence encoding said transcription repressor.

19. The viral vector of claim 1 , 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.

20. A recombinant DNA construct encoding said viral vector of claim 1 .

21. An in vitro method for producing a viral vector, said method comprising:
(a) transforming a packaging cell with said recombinant DNA construct of claim 20 ;
(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 viral vector.

Assignees

• Prec Biosciences Inc
• Jantz Derek
• Smith James Jefferson
• Nicholson Michael G

Inventors

• Jantz Derek
• Smith James Jefferson
• Nicholson Michael G

Classifications

• C12N2750/14143

  viral genome or elements thereof as genetic vector · CPC title

• C12N2830/42

  being an intron or intervening sequence for splicing and/or stability of RNA · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N2830/008

  cell type or tissue specific enhancer/promoter combination · CPC title