Our laboratory has developed techniques for directly targeting intracellular oncogene products. Preliminary studies showed that a K- ras antisense (AS-K-ras) expression vector can specifically eliminate expression of K-ras p21.
The specific aims outlined below are designed to study transduction of both AS-K-ras and the tumor suppressor wildtype p53 cDNA (wtp53) with viral vectors. The viral vectors LNSX, N2A, and adeno-associated virus (AAV) will be used to prepare the following constructs: LNSX (S-K-ras, AS-K-ras, p53), N2A- (S-K-ras, AS-K-ras, p53), AAV (S-K-ras, AS-K-ras). The GP+envAM-12 amphotropic packaging cell line will be used for production of infectious retroviral particles. These viral constructs will be used to transduce the following human cancer cell lines: H460a (wtp53 and mut K-ras), H358 (del p53 and wt K-ras), H322a (mut p53 and wtp53). The following parameters will be assessed for each transduced cell line: 1) Growth rate of cells by cell counts and [3H]Thd uptake; 2) Level of expression by mRNA; 3) Differential expression of other related genes such as H-ras and N-ras; 4) Level of protein expression by Western blot; 5) tumorigenicity of cells in nu/nu mice. The efficiency of transduction will be determined by assessment of colony formation in selective media. Changes in efficiency mediated by changes in viral titer and by multiple cycles of infection will be determined. The biodistribution of constructs in non-tumor bearing immunocompetent and nu/nu mice will be assessed. We will study the distribution both short (hrs) and long-term (wks and months). The distribution and expression of the constructs in different tissues will be studied after systemic and regional administration. The long-term effects on the whole animal will be determined. The effect of systemic and regional administration of viral constructs on subcutaneous and endobronchial tumor growth will be determined. Systemic administration of protamine will be evaluated as an adjunct for increasing efficiency of transduction in vivo.
Showing the most recent 10 out of 77 publications
