Abstract 4812: Creation of a novel gene delivery system for high throughput candidate oncogene validation
Authors: Feddersen Charlotte R. 
Published: August 2015

Introduction

The purpose of this study is to develop an in vivo, high throughput candidate oncogene validation screen, focusing specifically on hepatocellular carcinoma (HCC) specific oncogenes.

Experimental Procedure

Liver cancer is globally the 6th most commonly diagnosed cancer and the 2nd most deadly cancer. There are few therapies available, especially for late stages of the disease, and most treatments only prolong survival by months. Although much research has been done to determine the mutations responsible for driving HCC, including large-scale analysis by TCGA, it is becoming obvious that HCC involves numerous cell pathways and that many driver mutations are found in a small percentage of tumors. As groups such as TCGA discover more candidate driver mutations, a high throughput method to validate those genes in vivo is needed. We have engineered a novel gene delivery system, called Ad-PB, that uses recombinant adenoviruses to deliver piggyBac transposons expressing individual candidate oncogenes. The piggyBac transposase (PBase) mediates transfer of the transposon cargo from the adenoviral to the host cell genome where it can be stably expressed.

Our current screen is designed to simultaneously test 31 candidate oncogenes previously discovered by characterizing recurrent copy number alterations in tumors from patients with HCC verses matched nontumor tissue. Expression of these 31 genes within the piggyBac transposon is Cre-inducible, and each candidate is tagged with a unique barcode to facilitate tracking of individual genes within adenoviral pools. The plasmids encoding the genes are grown up using a recombinant virus (adenovirus) which is then pooled, purified, and injected into Alb-Cre; Trp53R27OH/+ mice. Expression of PBase from a separate adeno-associated virus mediates gene transfer into hepatocytes. Cells that express bona fide oncogenes will then be predisposed to HCC formation. Tumors with matched nontumor tissue will be analyzed using the Illumina platform to sequence the unique barcodes associated with each gene. Statistical models using sequence reads from both tumor and nontumor tissue will allow us to determine which candidates are enriched in tumor tissue. Positive controls for the screen are known oncogenes, a β-cateninΔN90 and N-rasG12V that will be co-delivered using the Ad-PB system.

Results

Preliminary results using the Ad-PB system to deliver a mCherry transposon demonstrated that the Ad-PB system can cause stable transgene integration and expression for up to 20 weeks. In addition, a semi-quantitative method has been established to perform barcode scanning to assess the relative abundance of individual candidate genes within a pool of adenoviruses.

Conclusion

The Ad-PB system will allow simultaneous validation of candidate oncogenes and although we are verifying the system using HCC specific oncogenes, we expect that it will be adaptable for many gene delivery systems.

Citation Format: Charlotte R. Feddersen. Creation of a novel gene delivery system for high throughput candidate oncogene validation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4812. doi:10.1158/1538-7445.AM2015-4812