Talk abstracts

Talk on Tuesday 09:00-09:15am submitted by Patrick Westmoreland

Role of Truncated Recombinant AAV Genomes in Tumor Formation

Patrick Westmoreland (MCDB Graduate Program The Ohio State University), Harkness Kuck (The Institute for Genomic Medicine Nationwide Childrens Hospital), Vijay Nadella (The Institute for Genomic Medicine Nationwide Childrens Hospital), Douglas M. McCarty (Department of Pediatrics The Ohio State University, Center for Gene Therapy Nationwide Childrens Hospital), Peter White (Department of Pediatrics The Ohio State University,The Institute for Genomic Medicine Nationwide Childrens Hospital)

Abstract:
Gene therapy utilizing recombinant adeno-associated virus (rAAV) has been shown to be a safe and effective treatment in mouse models of human disease. Albeit less likely to promote genotoxicity than obligate integrating vectors such as retroviruses, studies have shown an increase in hepatocellular carcinoma (HCC) in mice treated with rAAV. The focus of this study is to determine what features of rAAV vectors could contribute to tumor promotion by recovering vector genome junctions from HCC in a mouse model. Male C3H/HeJ mice, in which liver tumors grow rapidly, were used to achieve the sensitivity needed to detect rAAV-associated oncogenic events. Genomic DNA from tumors of mice treated with a GFP vector driven by a cytomegalovirus (CMV) promoter was extracted, fragmented, and enriched for vector sequence using biotinylated RNA probes. The enriched DNA was sequenced on the HiSeq platform and analyzed by NCH Institute for Genomic Medicine staff for vector genome junctions, defined as any split sequence read that aligned to both the mouse and vector genomes. 5 of 8 tumors tested had integration sites within the second intron of the gene encoding the E3 ubiquitin ligase, ITCH, a suspected oncogene. All five integrations contained only the CMV promoter element and the SV40 intron splice donor site, lacking the splice-acceptor and GFP coding region, suggesting that the vector was either truncated during or prior to integration. Analysis of episomal vector sequences from normal adjacent liver tissue showed that 14% of vector genomes were less than full-length, and 2% of the vector sequence contained only the CMV promoter and the SV40 intron splice donor site, similar to the integrated structures. The juxtaposition of the truncated vector intron within the second intron of ITCH, upstream of the ATG start codon, is predicted to give rise to a dysregulated fusion transcript, leading to ITCH overexpression and contributing to tumor formation. Given the size of ITCH intron 2 (25 kb), the dose of vector (2.5E10/kg), and an estimated integration frequency of 0.1-0.5%,each treated mouse is predicted to have approximately 10 vector integrations within that region. However, only truncated vector genomes were associated with tumors, suggesting that they pose a specific risk for genotoxicity.

Keywords: AAV, Gene Therapy, Genotoxicity