Autogene nucleic acids encoding a secretable RNA polymerase

Claims

What is claimed is:

1. A secretable RNA polymerase expression cassette comprising: (a) a eukaryotic promoter and a first RNA polymerase promoter operably linked to a nucleic acid encoding a secretable RNA polymerase (sRNAP) comprising a RNA polymerase and a secretion domain; and (b) a second RNA polymerase promoter operably linked to a nucleic acid encoding a product of interest, wherein the sRNAP enters the cytoplasm of a cell and carries out template dependent synthesis of RNA.

2. The expression cassette of claim 1, wherein said RNA polymerase is a non-host RNA polymerase.

3. The expression cassette of claim 1, wherein said RNA polymerase is a member selected from the group consisting of: a phagemid RNA polymerase, a prokaryotic RNA polymerase, an archaebacterial RNA polymerase, a plant RNA polymerase, a fungal RNA polymerase, a eukaryotic RNA polymerase, a viral RNA polymerase, mitochondrial RNA polymerase, and a chloroplast RNA polymerase.

4. The expression cassette of claim 3, wherein said phagemid RNA polymerase is a member selected from the group consisting of: a SP6 RNA Polymerase, a T7 RNA Polymerase, a K11 RNA Polymerase, and a T3 RNA Polymerase.

5. The expression cassette of claim 1, wherein said product of interest is a member selected from the group consisting of: a protein, a nucleic acid, an antisense nucleic acid, ribozymes, tRNA, snRNA, and an antigen.

6. The expression cassette of claim 1, wherein said product of interest encodes a protein selected from the group consisting of: a herpes simplex virus thymidine kinase (HSV-TK), a cytosine deaminase, a xanthine-guaninephosphoribosyl transferase, a purine nucleoside phosphorylase, a carboxylesterase, a deoxycytidine kinase, a nitroreductase, a thyrnidine phosphorylase, and a cytochrome P450 2B1.

7. The expression cassette of claim 1, wherein said product of interest encodes a protein selected from the group consisting of: p53, a retinoblastoma susceptibility gene product (RB1), a calmodulin-dependent serine threonine (DAP kinase), p16, a protein from an alternative reading frame of p16 (ARF), an adenomatous polyposis coli gene product (APC), neurofibromin, a phosphatase and tensin homologue deleted from chromosome 10 (PTEN), a Wilms= tumor gene product (WT1), a neurofibromatosis type 1 gene product (NF1), and a von Hippel-Lindau tumor suppressor gene product (VHL).

8. The expression cassette of claim 1, wherein said product of interest encodes a protein selected from the group consisting of: angiostatin, endostatin, and vascular endothelial growth factor (VEGF)-R2.

9. The expression cassette of claim 1, wherein said product of interest encodes apoptin.

10. The expression cassette of claim 1, wherein said product of interest encodes a protein selected from the group consisting of: IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IFN-.alpha., IFN-.beta., IFN-.gamma., TNF-.alpha., GM-CSF, G-CSF, and Fms-like tyrosine kinase 3 (Flt3)-Ligand.

11. The expression cassette of claim 1, wherein said product of interest is a protein selected from the group consisting of: a restriction endonuclease, a single chain antibody, a peptide hormone, erythropoietin (EPO), and single-chain insulin.

12. The expression cassette of claim 1, wherein said eukaryotic promoter is a member selected from a group consisting of: a cytomegalovirus promoter, a von Willebrand factor (vWf) promoter, a Clara cell secretory protein (CCSP/UG) promoter, an osteoblast-specific osteocalcin promoter, an albumin promoter, a muscle creatine kinase (MCK) promoter, a mucin-1 (Muc-1) promoter, a carcinoembryonic antigen (CEA) promoter, a prostate specific antigen (PSA) promoter, an epidermal growth factor receptor (HER-2) promoter, a Myc promoter, a L-plastin promoter, an alpha-fetoprotein (AFP) promoter, a hypoxia-responsive element (HIRE) promoter, an early growth response (egr-1) promoter, a multidrug resistance (mdr-1) promoter, a heat shock protein 70 (hsp70) promoter, a tetracycline induced promoter, a simian virus 40 (SV40) promoter, an alcohol dehydrogenase (ADH1) promoter, a GAL4 promoter, a LexA promoter, a rous sarcoma virus (RSV) promoter, a human imniunodeficiency virus (HIV) promoter, and a simian foamy virus (SFV) promoter.

13. A lipid-nucleic acid composition comprising: a nucleic acid-lipid particle comprising a lipid portion and a nucleic acid portion, wherein said nucleic acid portion comprises a secretable RNA polymerase expression cassette comprising: (a) a eukaryotic promoter and a first RNA polymerase promoter operably linked to a nucleic acid encoding a secretable RNA polymerase comprising a RNA polymerase and a secretion domain; and (b) a second RNA polymerase promoter operably linked to a nucleic acid encoding a product of interest.

14. The nucleic acid of claim 13, wherein a RNA polymerase promoter is operably linked to said nucleic acid encoding a secretable RNA polymerase.

15. The lipid-nucleic acid composition of claim 13, wherein said nucleic acid-lipid particle is a serum-stable nucleic acid-lipid particle comprising a nucleic acid fuily encapsulated within said lipid portion.

16. The lipid-nucleic acid composition of claim 13, wherein said lipid portion comprises a protonatable lipid having a pKa in a range of about 4 to about 11.

17. The lipid-nucleic acid composition of claim 13, wherein said lipid portion comprises a cationic lipid.

18. The lipid-nucleic acid composition of claim 17, wherein said cationic lipid is a member selected from the group consisting of DODAC, DODAP, DODMA, DOTAP, DOTMA, DC-Chol, DMRIE, and DSDAC.

19. The expression cassette of claim 1, wherein said secretion domain is an importation competent signal peptide.

20. The expression cassette of claim 1, wherein said secretion domain is a protein transduction domain.

21. The expression cassette of claim 20, wherein said protein transduction domain is a TAT polypeptide.

22. The expression cassette of claim 21, wherein said TAT polypeptide comprises the amino acid sequence X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-A- rg-X.sub.10, wherein X.sub.1 is Tyr or Ala; X.sub.2 is Gly or Ala; X.sub.3, X.sub.6, X.sub.7, X.sub.9 and X.sub.10 are Arg or Ala; and X.sub.8 is Gln, Arg or Ala.

23. The expression cassette of claim 22, wherein said TAT polypeptide comprises SEQ ID NO:1.

24. The lipid-nucleic acid composition of claim 13, wherein said secretion domain is an importation competent signal peptide.

25. The lipid-nucleic acid composition of claim 13, wherein said secretion domain is a protein transduction domain.

26. The lipid-nucleic acid composition of claim 25, wherein said protein transduction domain is a TAT polypeptide.

27. The lipid-nucleic acid composition of claim 26, wherein said TAT polypeptide comprises the amino acid sequence X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-A- rg-X.sub.10, wherein X.sub.1 is Tyr or Ala; X.sub.2 is Gly or Ala; X.sub.3, X.sub.6, X.sub.7, X.sub.9 and X.sub.10 are Arg or Ala; and X.sub.8 is Gln, Arg or Ala.

28. The lipid-nucleic acid composition of claim 27, wherein said TAT polypeptide comprises SEQ ID NO:1.

29. The expression cassette of claim 1, wherein the RNA polymerase is a T7 RNA polymerase.

30. The lipid-nucleic acid composition of claim 13, wherein the RNA polymerase is a T7 RNA polymerase.