Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential2017-10-18 14:51:41
Journal of Virology
Volume 71, January 2018, Pages 126-134
Michael A. Goodman, Paritha I. Arumugam, Devin M. Pillis, Anastacia Loberg, Md Nasimuzzaman, Danielle Lynn, Johannes C.M. van der Loo, Phillip J. Dexheimer, Mehdi Keddache, Thomas R. Bauer Jr., Dennis D. Hickstein, David W. Russell and Punam Malik
Strong viral enhancers in ?-retrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating use of cellular promoters in ‘enhancer-less’ self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for non-genic integrations than ?-retroviruses/lentiviruses and preferential integration near transcriptional start sites, like ?-retroviruses. We found that strong viral enhancer/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous ?-retrovirus/lentivirus vectors carrying the same enhancer/promoters; an effect not explained solely by foamy virus' modest insertional site preference for non-genic regions, compared to ?-retrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrated a sequence specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus LTR that has high affinity binding for the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus, and significantly reduced when this insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancer/promoters are required.
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