7. The risk of integration: cell lines for determining the frequency and character of vector-mediated insertional gene activation
Authors: Andrea D. Converse, Nikunj V. Somia, R. Scott McIvor
Journal: Molecular Therapy (2004)

Many gene therapy applications require the use of an integrating vector in order to achieve stable, long-term expression. Retroviruses and lentiviruses have been tested extensively for this purpose, and non-viral approaches such as the use of transposons are also being explored. Although gene transfer using such vectors is quite effective, a potential risk associated with the use integrating vectors is that of insertional oncogenesis, which can result from vector integration into the chromosome causing the activation of a neighboring proto-oncogene or the inactivation of a tumor suppressor gene. The clinical relevance of this risk emerged recently during a gene therapy trial to treat X-linked severe combined immunodeficiency, in which two patients developed a T cell leukemia-like condition that is suspected to have been caused by retroviral insertional activation of the LMO2 proto-oncogene. In order to assess the risk of insertional gene activation, we set out to establish an in vitro system that could be used to investigate the frequency of such events mediated by different integrating vectors, and to study the character of insertional activation events brought about by different integrating vector types, such as the effect of distance and chromosomal position on insertional gene activation. Here we describe the establishment of cell lines which have been engineered to contain a newly introduced hypoxanthine phoshoribosyltransferase (HPRT) gene that can be activated by vector insertion nearby to the HPRT gene. We first isolated an HPRT-deficient HT1080 cell line by selecting a 6-thioguanine-resistant HT1080 clone and expanding it in culture. We designed a construct pfcFH which contains a minimal FOS promoter regulating a human HPRT gene with an upstream CMV enhancer flanked by loxP elements. The HT1080-HPRT- cell line was electroporated with pfcFH, positive transfectants were isolated in HAT medium, and single integrant clones were identified by Southern hybridization. These clones were then transfected with pCRE for expression of Cre recombinase, thereby excising the floxed CMV enhancer element, inactivating expression of the HPRT gene, and allowing for selection of HPRT- clones in 6-thioguanine. These clones (designated HT-fH) contain an inactive, minimal FOS-regulated HPRT gene integrated into different chromosomal positions, and can be used to quantitate the frequency of insertional gene activation by exposure of the cells to a test integrating vector and scoring for activation of the FOS-HPRT gene by subculturing the transduced cells in HAT medium. In addition, by mapping the distances between vector integration sites and the FOS-HPRT gene in different HAT-resistant clones, the constellation of integration distances causing insertional gene activation can be determined. These cell lines are currently being used to test the relative frequency of insertional gene activation events mediated by different retroviral and lentiviral vector designs, such as LTR-regulated vs. self-inactivating vector designs. These cell lines should be useful in characterizing the relative safety of many different integrating vector designs with respect to the risk of insertional gene activation.