Claims
- 1. A process for selecting a promoter for inclusion in a plasmid used to transfect a target cell, the process comprising the steps of:
identifying a transcription product in high abundance in a target cell; identifying a promoter associated with the transcription product; inserting the promoter into a gene expression plasmid construct, the plasmid construct having a therapeutic gene to be expressed; transfecting the target cell with the gene expression plasmid construct; and verifying gene expression of the therapeutic gene in the target cell.
- 2. The process of claim 1, wherein the transcription product is a specific mRNA.
- 3. The process of claim 1, wherein the transcription product is a protein.
- 4. The process of claim 1, wherein a Serial Analysis of Gene Expression is used to identify the transcription product.
- 5. The process of claim 2, wherein a SAGE database is used to identify the mRNA.
- 6. The process of claim 1, wherein a cDNA hybridization is used to identify the transcription product.
- 7. The process of claim 1, further comprising the step of identifying an enhancer associated with the promoter.
- 8. A process for selecting a promoter for inclusion in a plasmid to be used in gene therapy, the process comprising the steps of:
determining a gene expression level for a plurality of transcription products in a diseased tissue; selecting a transcription product in high abundance in the diseased tissue and a target cell associated with the diseased tissue; identifying a promoter associated with the transcription product; inserting the promoter into a gene expression plasmid construct, the plasmid construct having a therapeutic gene to be expressed; transfecting the target cell with the gene expression plasmid construct; and verifying gene expression of the therapeutic gene in the target cell.
- 9. The process of claim 8, further comprising the steps of transfecting the diseased tissue and verifying gene expression of the therapeutic gene in the diseased tissue.
- 10. The process of claim 8, wherein a microarray analysis is used to identify the gene expression level for the transcription products in the diseased tissue.
- 11. The process of claim 8, wherein a SAGE database is used to select the transcription product.
- 12. The process of claim 8, further comprising the steps of identifying an enhancer associated with the promoter and inserting the enhancer into the gene expression plasmid construct.
- 13. The process of claim 8, wherein the diseased tissue is a breast cancer.
- 14. A process for designing a plasmid for transfecting a target tissue, the process comprising the steps of:
selecting a gene expression plasmid having an origin of replication, a multiple cloning site, a therapeutic gene, a polyadenylation signal sequence, and an antibiotic resistant gene; identifying a transcription product in high abundance in a cell line associated with a target tissue; identifying a promoter associated with the transcription product; inserting the promoter into the gene expression plasmid in various locations close to the therapeutic gene to form a plurality of plasmid constructs; transfecting the target cell line with each plasmid construct; measuring gene expression of the therapeutic gene in the target cell line transfected with each plasmid construct; selecting the plasmid constructs that provide efficient gene expression in the transfected target cell line; and verifying gene expression of the therapeutic gene from the selected plasmid constructs in the target tissue.
- 15. The process of claim 14, further comprising the steps of identifying an enhancer associated with the promoter and inserting the enhancer into the gene expression plasmid.
- 16. The process of claim 14, wherein the antibiotic resistant gene is a Kanamycin resistant gene.
- 17. The process of claim 14, wherein the therapeutic gene is a p53 gene.
- 18. The process of claim 14, wherein the target tissue is breast tissue.
- 19. The process of claim 14, wherein the gene expression plasmid further comprises a constitutive promoter.
- 20. The process of claim 19, wherein the constitutive promoter is a viral promoter.
- 21. The process of claim 20, wherein the viral promoter is the cytomegalovirus promoter.
- 22. The process of claim 14, wherein a SAGE database is used to identify the transcription product.
- 23. An expression plasmid comprising:
an origin of replication gene; a polyadenylation site; an antibiotic resistant gene; a multiple cloning site; a therapeutic gene; and a promoter selected according to the process of claim 1.
- 24. The expression plasmid of claim 23, wherein the antibiotic resistant gene is a Kanamycin resistant gene.
- 25. The expression plasmid of claim 23, wherein the therapeutic gene is a p53 gene.
- 26. The expression plasmid of claim 23, wherein the promoter is a glycerealdehyde 3-phosphate dehydrogenase promoter.
- 27. The expression plasmid of claim 23, further comprising an enhancer.
- 28. The expression plasmid of claim 27, wherein the enhancer is a hypoxia enhancer.
- 29. The expression plasmid of claim 23, further comprising more than one enhancer.
- 30. The expression plasmid of claim 27, wherein the expression plasmid contains more than one copy of the enhancer.
- 31. The expression plasmid of claim 23, further comprising a viral constitutive promoter.
- 32. The expression plasmid of claim 23, further comprising a CMV promoter-enhancer.
- 33. The expression plasmid of claim 23, wherein the expression plasmid contains more than one promoter selected according to the process of claim 1.
- 34. An expression plasmid for gene therapy in breast cancer, the plasmid comprising:
an origin of replication gene; a polyadenylation site; an antibiotic resistant gene; a multiple cloning site; a therapeutic gene; a GADPH promoter; and a hypoxia enhancer.
- 35. The plasmid of claim 34, further comprising a keratin-8 promoter-enhancer.
- 36. The plasmid of claim 34 having multiple copies of the hypoxia enhancer.
- 37. The plasmid of claim 34, wherein the GAPDH promoter and the hypoxia enhancer are situated 5′ of the therapeutic gene.
- 38. The plasmid of claim 37, wherein the GAPDH promoter and the hypoxia enhancer are adjacent the therapeutic gene.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application, pursuant to 35 U.S.C. 111(b), claims the benefit of the filing date of provisional application Serial No. 60/370,900 filed Apr. 8, 2002, entitled “Enhanced Transcription for Robust Cancer Treatment” and of provisional application Serial No. 60/430,780 filed Dec. 4, 2002 entitled “Promoter Selection Process for Enhanced Transcription.”
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Research leading to this invention was federally supported, in part, by the Breast Cancer SPORE, National Institutes of Health, through Grant P50CA58183, and the U.S. Government has certain rights thereunder in this invention.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60370900 |
Apr 2002 |
US |
|
60430780 |
Dec 2002 |
US |