Self-extinguishing recombinases, nucleic acids encoding them and methods of using the same

• US 6,852,530 B2
• Filed: 04/12/2001
• Issued: 02/08/2005
• Est. Priority Date: 04/12/2000
• Status: Expired due to Term

• Current Assignee: Dana-Farber Cancer Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Silver, Daniel P., Livingston, David M.
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): Sullivan, Daniel M.
• Application Number:
  US09/834,778
• Publication Number:
  US 20020062489A1
• Time in Patent Office: 1,398 Days
• Field of Search: 435/320.1, 435/325, 514/44, 800/8
• US Class Current: 435/325
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• 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 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00

Abstract

Nucleic acid molecules comprising at least a first signal site and a recombinase gene operably linked to an expression control sequence, such that upon entry into a cell, there is a first signal site and a second signal site positioned to mediate excision of a sufficient portion of either the recombinase gene or the expression control sequence to extinguish recombinase activity when the first and second signal sites are contacted with a recombinase, cells, transgenics and uses of the foregoing.

Claims

1. A nucleic acid molecule comprising at least a first signal sequence and a second signal sequence and a recombinase gene operably linked to an expression control sequence, said first and second signal sequences being positioned to mediate in inversion of a sufficient portion of either the recombinase gene or the expression control sequence to inactivate or decrease recombinase activity when the first and second signal sequences contacted with a recombinase, thereby decreasing or eliminating recombinase-mediated toxicity.

2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is included in a retioviral vector and said signal sequence is inserted to a retroviral long terminal repeat of said vector.

3. The nucleic acid molecule of claim 2, wherein said signal sequence is inserted into the U3 region of the 3′

retroviral long terminal repeat of said vector.

4. The nucleic acid molecule of claim 1, wherein said recombinase is selected from the group consisting of a cre recombinase and a Flp recombinase and the signal sequence is selected from the group consisting of lox sequences and FRT sequences.

5. The nucleic acid molecule of claim 1, wherein said signal sequences flank said recombinase gene or said expression control sequence of said recombinase gene.

6. An isolated cell comprising the nucleic acid molecule of claim 1.

7. The isolated cell of claim 6, further comprising a second nucleic acid molecule comprising a target gene and signal sequences recognized by said recombinase.

8. The isolated cell of claim 7, wherein said recombinase, when expressed in said cell, inverts a sequence in said second nucleic acid molecule that is located between said signal sequences in said second nucleic acid molecule, and the inversion results in modulation of expression of said target gene.

9. The isolated cell of claim 8, wherein said signal sequences in said second nucleic acid molecule are in inverted orientation with respect to one another.

10. The isolated cell of claim 9, wherein said signal sequences in said second nucleic acid molecule flank said target gene, so that expression of said recombinase results in inversion of said target gene, and inactivation of expression of said target gene.

11. The isolated cell of claim 9, wherein said signal sequences in said second nucleic acid molecule flank a positive regulatory element of said target gene, so that expression of said recombinase results in inversion of said positive regulatory element, and inactivation of expression of said target gene.

16. The isolated cell of claim 6, wherein said signal sequences in said nucleic acid molecule comprising said sequence encoding said recombinase flank said nucleic acid sequence encoding said recombinase.

17. The isolated cell of claim 6, wherein said signal sequences in said nucleic acid molecule comprising said sequence encoding said recombinase flank a positive regulatory element of said nucleic acid sequence encoding recombinase.

18. The isolated cell of claim 7, wherein said nucleic acid molecule comprising said sequence encoding said recombinase and said second nucleic molecule are present in the same vector.

20. The nucleic acid molecule of claim 1, wherein said signal sequences are in inverted orientation with respect to one another.

12. A isolated cell comprising two nucleic acid molecules, wherein the first nucleic acid molecule comprises a recombinase gene operably linked to an expression control sequence and signal sequences recognized by a recombinase and the second nucleic acid molecule comprises a target gene and signal sequences recognized by a recombinase, wherein said signal sequences in said second nucleic acid molecule flank a negative regulatory element of said target gene, so that expression of said recombinase results in excision of said negative regulatory element, and activation of expression of said target gene.

13. A isolated cell comprising two nucleic acid molecules, wherein the first nucleic acid molecule comprises a recombinase gene operably linked to an expression control sequence and signal sequences recognized by a recombinase and the second nucleic acid molecule comprises a target gene and signal sequences recognized by a recombinase, wherein said signal sequences in said second nucleic acid molecule are in an inverted orientation with respect to one another.

14. The isolated cell of claim 13, wherein said signal sequences in said second nucleic acid molecule flank an inverted positive regulatory element of said target gene or an inverted coding region of said target gene, so that expression of said recombinase results in inversion of said inverted positive regulatory element or inversion of said inverted coding region, and activation of expression of said target gene.

15. The isolated cell of claim 13, wherein said signal sequences in said second nucleic acid molecule flank an inverted negative regulatory element of said target gene or a coding region of said target gone, so that expression of said recombinase results in inversion of said inverted negative regulatory element or inversion of said coding region, inactivation of expression of said target gene.

19. A isolated cell comprising two nucleic acid molecules, wherein the first nucleic acid molecule, comprising a recombinase gene operably linked to an expression control sequence and signal sequences recognized by a recombinase and the second nucleic acid molecule, comprising a target gene and signal sequences recognized by a recombinase, are present in separate vectors.