Methods of unidirectional, site-specific integration into a genome, compositions and kits for practicing the same

• US 20050208021A1
• Filed: 12/03/2004
• Published: 09/22/2005
• Est. Priority Date: 06/04/2002
• Status: Active Grant

• Current Assignee: Poetic Genetics, LLC, Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Calos, Michele
• Granted Patent: US 8,420,395 B2
• Time in Patent Office: Days
• US Class Current: 424/93.200
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: A61K 48/0066 Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C12N 15/8209 Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/8217 Gene switch
• CPC Class Codes: C12N 15/8273 for drought, cold, salt resistance
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2800/90 Vectors containing a transposable element
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/38 being a stuffer
• CPC Class Codes: C12N 2830/55 from bacteria
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: Y02A 40/146 : Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The subject invention provides a unidirectional site-specific integration system for integrating a nucleic acid into the genome of a target cell. The provided systems include a (1) a mutant, unidirectional site specific integrase, which can be provided by an integrase vector encoding the mutant integrase and (2) a targeting vector that includes: (a) a nucleic acid to be integrated; and (b) a vector attachment site, where the targeting vector attachment site serves as a substrate for the mutant, unidirectional site-specific integrase. In using the subject systems for site-specific integration, the targeting vector and integrase are introduced into the target cell and the cell is maintained under conditions sufficient to provide for site-specific integration of the nucleic acid into the target cell genome via a recombination event mediated by the site-specific integrase. Also provided are kits that include the subject systems. The subjects systems, methods and kits find use in a variety of different applications, several representative ones of which are described in detail as well.

Claims

1. A method of producing a unidirectional, site-specific integrating vector system for integrating a nucleic acid into a target cell genome in a site-specific manner, said method comprising:

(a) identifying the following system elements;

(i) a genome attachment site;

(ii) a vector attachment site; and

(iii) a mutant unidirectional site-specific integrase that efficiently employs said target cell and vector attachment sites as substrates; and

(b) producing a site-specific integrating vector system that includes said identified system elements.

2. The method according to claim 1, wherein said identifying step (a) includes a variable integrase approach.

3. The method according to claim 1, wherein said identifying step (a) includes a variable attachment site approach.

4. The method according to claim 1, wherein at least one of said genome attachment site is identified by screening a desired integration region of said target cell genome for the presence of an integrase attachment consensus sequence.

5. The method according to claim 4, wherein said integrase attachment consensus sequence is a Φ

C31 integrase attachment consensus sequence.

6. The method according to claim 5, wherein said mutant unidirectional, site-specific integrase is identified by:

(a) producing at least one mutant integrase; and

(b) screening said mutant integrase for its ability to employ said identified genome attachment site as substrate.

7. The method according to claim 6, wherein said integrase is identified by producing and screening a plurality mutant integrases.

8. A unidirectional site-specific integrating vector system produced according to the method of claim 1.

9. The system according to claim 8, wherein said system comprises a single nucleic acid vector.

10. The system according to claim 9, wherein said nucleic acid vector is a viral vector.

11. A unidirectional site-specific integrating vector system, said system comprising:

(a) a targeting vector comprising a vector attachment site; and

(b) a mutant, unidirectional site-specific integrase that recognizes said vector attachment site as a substrate.

12. The system according to claim 11, wherein said targeting vector further comprises a coding sequence.

13. The system according to claim 12, wherein said coding sequence is present in an expression cassette.

14. A targeting vector comprising a vector attachment site that serves as a substrate for a mutant, unidirectional site-specific integrase.

15. The targeting vector according to claim 14, wherein said vector further includes a coding sequence.

16. The vector according to claim 15, wherein said coding sequence is present in an expression cassette.

17. The vector according to claim 14, wherein said vector further comprises an integrase coding sequence.

18. The vector according to claim 17, wherein said vector is a viral vector.

19. A method of site-specifically integrating a nucleic acid into a genome of a target cell of a multicellular organism, said method comprising:

introducing a targeting vector comprising said nucleic acid and a vector attachment site and a mutant, unidirectional site-specific integrase into a target cell and maintaining said target cell under conditions sufficient for said vector to integrate into said genome of said target cell by a recombination event mediated by said unidirectional site-specific integrase.

20. The method according to claim 19, wherein said target cell is present in vitro.

21. The method according to claim 19, wherein said target cell is present in vivo.

22. The method according to claim 19, wherein said nucleic acid comprises a coding sequence.

23. The method according to claim 19, wherein said coding sequence is present in an expression cassette.

24. A kit for use integrating a nucleic acid into a genome of a target cell of a multicellular organism, said kit comprising:

(a) a targeting vector comprising a vector attachment site; and

(b) a mutant, unidirectional site-specific integrase or nucleic acid encoding the same.