Recently, comprehensive characterization of over 300 ovarian serous adenocarcinomas identified more than five thousand genes that are recurrently affected by somatic point mutation, copy number alteration or epigenetic modification. Likely this means that individual tumors harbor mutations affecting hundreds of genes. Unfortunately, only a small number of well-characterized genes (e.g. TP53, MYC) are mutated at a high rate in ovarian tumors. The majority of somatic mutations in an individual human ovarian tumor affect genes that are rarely mutated and thus shared by only a small percentage of tumor samples. With this degree of genetic heterogeneity, distinguishing rare driver mutations from the more abundant passenger events is a daunting task. Clearly additional experimental evidence is needed to validate the large number of genes that are recurrently mutated in human tumors. The long-term goal is to develop a system to achieve a gene transfer efficiency that would facilitate in vivo validation experiments capable of testing a large number o candidate genes in a single experiment. The objective of this application is to develop an Ad-PB hybrid vector system to combine the gene delivery efficiency of recombinant adenoviruses with the ability of the piggyBac transposon system to provide stable integration and long-term expression of transgenes. The central hypothesis is that such a hybrid gene transfer system would accelerate the pace at which candidate cancer genes can be validated using in vivo models of cancer. The rationale for the proposed work is that piggyBac transposon system has been shown to efficiently deliver up to four transposons simultaneously into cells. Moreover, we have recently shown that the piggyBac transposase is capable of mobilizing transposons directly from an adenoviral genome - something that has never been achieved with a cut-and-paste transposon previously.
Two aims are proposed to develop the Ad-PB system.
Aim1 will focus on improving the efficiency of gene delivery and determining the rate of transposon co-delivery using adenoviral co-transduction in cultured cells.
Aim 2 will perform experiments in vivo to determine the rate of stable gene delivery into ovarian surface epithelium in the mouse. Finally, we will perform experiments to demonstrate the ability of the Ad-PB system to validate candidate cancer genes identified by high throughput genetic analysis of human ovarian tumors. This application is innovative since the development of the Ad-PB system would provide a unique technology that could be used to accelerate the pace at which candidate cancer genes are validated in vivo. Such an approach is preferred to cell cultured-based assays that fail to model the complex tumor microenvironment found in human tumors. The application is significant as there is currently great need for approaches to rapidly evaluate dozens to hundreds of candidate genes being identified by ongoing efforts to characterize the human cancer genome.
Recently work by the Cancer Genome Atlas has documented a tremendous amount of genetic diversity among human serous ovarian carcinomas. With somatic mutations affecting over five thousand genes in over 300 tumor samples, the genetic complexity of these tumors confounds ongoing efforts to identify the causal events that lead to tumor initiation and progression. We propose a hybrid vector system using recombinant adenoviruses to delivery piggyBac transposons into cells of the ovarian surface epithelium. This gene delivery system will be used to accelerate in vivo experiments to validate candidate genes identified by high throughput analysis of human ovarian tumors.