1056. Failure of an Insulator Element To Prevent Retroviral Insertional Activation of Oncogenes and Lymphomagenesis

Abstract

Gene therapy vectors that insert into the human genome are proving successful in treating human inherited disorders. However, studies to date, such as trials of murine gammaretrovirus (MLV) vectors in humans, have encountered the complication of insertional activation of host oncogenes by the retroviral vectors leading to tumorigenesis. One potential strategy to prevent this complication and improve the safety of gene therapy vectors is the use of insulator elements to prevent transcriptional enhancers in insertional vectors from activating the promoters of adjacent oncogenes. We have tested the ability of the chicken beta-globin HS4 insulator (cHS4) element to prevent insertional activation of oncogenes and T-cell lymphomagenesis by an MLV. MLVs have been extensively used for genome-based, high throughput molecular identification of oncogenes (http://rtcgd.ncifcrf.gov/). Enhancer activation of oncogenes, usually from distances>10 kbp, is by far the most frequent mechanism of tumorigenesis by these retroviruses. We inserted the cHS4 insulator into the LTR of a T-cell lymphomagenic, replication-competent MLV. The cHS4 element was stable within the viral genome for multiple rounds of viral replication in cultured cells and mice. Infection of mice induced a modest, though statistically significant, slowing of the appearance of lymphomas in infected mice. We then applied inverse PCR and genomic analysis to determine the insertion sites of MLVs in the genomes of the tumor cells. This analysis showed that inserted proviruses were able to activate flanking oncogenes (Myc, Rras2, Rasgrp1, Ccnd3, Kras, Runx1, Fos, Gfi1, Pim1) by an enhancer mechanism even when the cHS4 element was positioned between the viral enhancer and the promoter of the mouse oncogene. Moreover, the percentage of tumors with proviruses positioned in that orientation was the same with either the insulated or the wild type virus. Thus the cHS4 element did not prevent insertional activation of numerous oncogenes. These results mean that the use of insulators to block oncogene activation by insertional gene therapy vectors is not a simple issue. Instead, careful experimentation will be required to devise an effective approach.