DNA polymerase gene expression system utilizing an RNA polymerase co-delivered with the gene expression vector system

• US 5,591,601 A
• Filed: 05/14/1993
• Issued: 01/07/1997
• Est. Priority Date: 05/14/1993
• Status: Expired due to Term

• Current Assignee: Ohio University Edison Animal Biotechnology Institute
• Original Assignee: Ohio University Edison Animal Biotechnology Institute
• Inventors: Wagner, Thomas E., Chen, Xiaozhuo, Li, Yunsheng
• Primary Examiner(s): LeGuyader, John L.
• Application Number:
  US08/062,657
• Time in Patent Office: 1,334 Days
• Field of Search: 435/69.1, 435/183, 435/69.4, 435/91.31, 435/91.21, 435/172.3, 435/320.1, 435/240.1, 435/252.3, 435/254.11, 435/257.2, 435/240.2, 536/23.1, 536/24.1, 935/34, 935/36, 935/38, 935/39, 514/44
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)

Abstract

The present invention relates to a gene expression system in eukaryotic cells. In particular, it relates to a self-initiating and self-sustaining gene expression system utilizing an RNA polymerase prebound to a DNA construct before the introduction of the complex into cells. This system is capable of functioning in the cell cytoplasm without integration into the genome of host cells, and does not require host cell factors for initiation and maintenance of gene expression. Therefore, the invention has a wide range of applications in in vitro and in vivo gene expression, including gene therapy in resting cells.

Claims

1. A gene expression system in mammalian cells comprising a recombinant DNA molecule which comprises a coding sequence for an RNA polymerase having transcriptional activity that does not require mammalian host cell factors or a functional derivative thereof, operably linked to its cognate promoter;

and an exogenous RNA polymerase which transcribes said coding sequence from said cognate promoter, and which polymerase is co-delivered with said DNA molecule into mammalian cells.

2. The expression system of claim 1 is which the RNA polymerase coding sequence is derived from a bacteriophage selected from the group consisting of T7, T3, SP6 and K11.

35. The expression system of claim 1, 3 or 11 in which the DNA molecular further comprises a capping independent sequence that allows the translation of uncapped RNA in mammalian cells.

36. The expression system of claim 35 in which the capping independent sequence is derived from encephalomyocarditis virus.

3. A gene expression system in mammalian cells comprising a recombinant DNA molecule which comprises a coding sequence for a gene of interest or a functional derivative thereof, operably linked to a cognate promoter for an RNA polymerase having transcriptional activity that does not require mammalian host cell factors;

and an exogenous RNA polymerase which transcribes said coding sequence from said cognate promoter, and which polymerase is co-delivered with said DNA molecule into mammalian cells.

4. The expression system of claim 3 in which the RNA polymerase is derived from a bacteriophage selected from the group consisting of T7, T3, SP6 and K11.

5. The expression system of claim 4 in which the gene of interest encodes a protein.

6. The expression system of claim 5 in which the gene of interest encodes human growth hormone.

7. The expression system of claim 5 in which the gene of interest encodes chloramphenicol acetyltransferase.

8. The expression system of claim 5 in which the gene of interest encodes luciferase.

9. The expression system of claim 4 in which the gene of interest encodes an anti-sense RNA.

10. The expression system of claim 4 in which the gene of interest encodes a ribozyme molecule.

11. A gene expression system in mammalian cells comprising a recombinant DNA molecule which comprises a coding sequence for an RNA polymerase having transcriptional activity that does not require mammalian host cell factors or a functional derivative thereof, operably linked to its cognate promoter, and a coding sequence for a gene of interest or a functional derivative thereof, operably linked to a second copy of the cognate promoter;

and an exogenous RNA polymerase which transcribes said coding sequences from said cognate promoter, and which polymerase is co-delivered with said DNA molecule into mammalian cells.

12. The expression system of claim 11 in which the RNA polymerase coding sequence is derived from a bacteriophage selected from the group consisting of T7, T3, SP6 and K11.

13. The expression system of claim 12 in which the gene of interest encodes a protein.

14. The expression system of claim 13 in which the gene of interest encodes human growth hormone.

15. The expression system of claim 13 in which the gene of interest encodes chloramphenicol acetyltransferase.

16. The expression system of claim 13 in which the gene of interest encodes luciferase.

17. The expression system of claim 12 in which the gene of interest encodes an anti-sense RNA.

18. The expression system of claim 12 in which the gene of interest encodes a ribozyme molecule.

19. A method for expressing an exogenous gene in mammalian cells comprising:

(a) co-delivering into mammalian host cells;

(i) a recombinant DNA molecule which comprises a coding sequence for an RNA polymerase having transcriptional activity that does not require mammalian host cell factors or a functional derivative thereof, operably linked to its cognate promoter;

(ii) a second recombinant DNA molecule which comprises a coding sequence for a gene of interest or a functional derivative thereof, operably linked to a second copy of the cognate promoter of (i); and

(iii) an exogenous RNA polymerase which transcribes said coding sequences from said cognate promoters; and

(b) inducing expression of the gene of interest in the cells.

20. The method of claim 19 in which the RNA polymerase coding sequence is derived from a bacteriophage selected from the group consisting of T7, T3, SP6 and K11.

21. The method of claim 20 in which the gene of interest encodes a protein.

22. The method of claim 21 in which the gene of interest encodes human growth hormone.

23. The method of claim 21 in which the gene of interest encodes human chloramphenicol acetyltransferase.

24. The method of claim 21 in which the gene of interest encodes luciferase.

25. The method of claim 20 in which the gene of interest encodes an anti-sense RNA.

26. The method of claim 20 in which the gene of interest encodes a ribozyme molecule.

37. The method of claim 19 or 27 in which the DNA molecular further comprises a capping independent sequence that allows the translation of uncapped RNA in mammalian cells.

38. The method of claim 37 in which the capping independent sequence is derived from encephalomyocarditis virus.

27. A method for expressing an exogenous gene in mammalian cells comprising:

(a) co-delivering into mammalian host cells;

(i) a recombinant DNA molecule which comprises a coding sequence for an RNA polymerase having transcriptional activity that does not require mammalian host cell factors or a functional derivative thereof, operably linked to its cognate promoter, and a coding sequence for a gene of interest or a functional derivative thereof, operably linked to a second copy of the cognate promoter; and

(ii) an exogenous RNA polymerase which transcribes said coding sequences from said cognate promoters; and

(b) inducing expression of the gene of interest in the cells.

28. The method of claim 27 in which the RNA polymerase coding sequence is derived from bacteriophage T7.

29. The method of claim 28 in which the gene of interest encodes a protein.

30. The method of claim 29 in which the gene of interest encodes human growth hormone.

31. The method of claim 29 in which the gene of interest encodes chloramphenicol acetyltransferase.

32. The method of claim 29 in which the gene of interest encodes luciferase.

33. The method of claim 28 in which the gene of interest encodes an anti-sense RNA.

34. The method of claim 28 in which the gene of interest encodes a ribozyme molecule.