Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors

Abstract

The possible activation of cellular proto-oncogenes as a result of clonal transformation is a potential limitation in a therapeutic approach involving random integration of gene vectors. Given that enhancer promiscuity represents an important mechanism of insertional transformation, we assessed the enhancer activities of various cellular and retroviral promoters in transient transfection assays, and also in a novel experimental system designed to measure the activation of a minigene cassette contained in stably integrating retroviral vectors. Retroviral enhancer-promoters showed a significantly greater potential to activate neighboring promoters than did cellular promoters derived from human genes, elongation factor-1alpha (EF1alpha) and phosphoglycerate kinase (PGK). Self-inactivating (SIN) vector design reduced but did not abolish enhancer interactions. Using a recently established cell culture assay that detects insertional transformation by serial replating of primary hematopoietic cells, we found that SIN vectors containing the EF1alpha promoter greatly decrease the risk of insertional transformation. Despite integration of multiple copies per cell, activation of the crucial proto-oncogene Evi1 was not detectable when using SIN-EF1alpha vectors. On the basis of several quantitative indicators, the decrease in transforming activity was highly significant (more than tenfold, P < 0.01) when compared with similarly designed vectors containing a retroviral enhancer-promoter with or without a well-characterized genetic insulator core element. In this manner, the insertional biosafety of therapeutic gene vectors can be greatly enhanced and proactively evaluated in sensitive cell-based assays.

Gene Therapy of Mpl Deficiency: Challenging Balance Between Leukemia and Pancytopenia

“…This is in agreement with the low transforming activity of cellullar internal promoters in the in vitro immortalization assay as well as the Cdkn2a −/− mouse tumor model. 24,27 However, insertional mutagenesis was not the major cause of side effects induced by Mpl vectors and the PGK promoter was not sufficient to prevent the phenotoxic effects.…”

Lentiviral vector system for coordinated constitutive and drug controlled tetracycline-regulated gene co-expression

“…6A,B) and thus did not show major differences from previously described LV SIN vectors with constitutive (PGK; spleen focus forming virus (SFFV)) [11,36,42] or tetracyclineregulated [23,10] promoters. Importantly, in contrast to replication-deficient gammaretroviral-based vectors studies in vitro [37,38], our newly developed T11/mPGK LV SIN vector did not show integrations near potent proto-oncogenes (i.e. Mecom locus) during similar observation periods.…”

“…contrast to replication-deficient gammaretroviral (RV)-based vector studies in vitro [37,38], T11/mPGK LV SIN vectors did not reveal integrations upstream of potent proto-oncogenes (i.e. Evi1 or Mecom locus) during similar observation periods (Supplementary Table S1).…”

Partially corrected X-linked severe combined immunodeficiency: long-term problems and treatment options

“…Retroviral enhancer-promoters also have a significantly greater potential to activate neighboring promoters than cellular promoters derived from human genes such as elongation factor-1 (EF1) and phosphoglycerate kinase (PGK) [31], as implicated by the lack of activation of the crucial proto-oncogene Evi1 activation despite integration of multiple copies per cell, and were not detectable when using SIN-EF1 vectors. The decrease in transforming activity was significantly different regardless of the presence of a well-characterized genetic insulator core element [106].…”