The applicant's laboratory has recently developed a novel SV40-based episomal expression vector for human gene therapy applications that is predicted to be safe while permitting high copy replication in human cells. This episomal plasmid utilizes a specifically designed SV40 large T antigen mutant to drive extrachromosomal replication in human cells while eliminating the undesired ability of large T antigen to bind and inactivate host p53 and RB tumor suppressor gene proteins. Because this vector replicates to thousands of copies by 2-3 days after gene transfer, the expression system is predicted to safely express target genes at higher levels per cell than any currently published gene therapy vector. To further modify this vector to include appropriate safety and replication-control elements, the applicant proposes to develop a novel """"""""colon cancer-specific"""""""" expression system which will amplify extrachromosomally and express target genes only in tumor cells.
Specific Aim 1 is to develop a noninfectious, safety-modified SV40 based episomal expression system to permit external control of tumor-specific vector amplification and gene expression. An SV40-based episomal expression system will be constructed which employs both tissue-specific and inducible promoters to limit expression of mutant T antigen, and hence vector amplification, to colon tumor cells.
Specific Aim 2 is to determine the effectiveness of the SV40-based episomal vector system encoding herpes simplex virus thymidine kinase to kill tumor cells in vitro. After exposure to GCV, the specificity and efficiency of tumor cell kill will be evaluated.
In Specific Aim 3 a preclinical colon cancer model will be employed to evaluate the ability of SV40-based episomes to eradicate established tumor xenografts in nude mice using liposome-mediated gene transfer. Mice will be followed for tumor regression and survival. In summary, this application supports key, initial """"""""proof-of-principle"""""""" experiments to evaluate """"""""tumor-specific"""""""" episomal vectors and gene transfer systems for gene therapy of colon cancer.
