690. Permanent Epigenetic Silencing of Human Genes With Artificial Transcriptional Repressors
Authors: Angelo Amabile, Alessandro Migliara, Tiziano Di Tomaso, Mauro Biffi, Luigi Naldini, Angelo Lombardo
Journal: Molecular Therapy (2015)

There are several diseases whereby the goal of gene therapy is to silence rather than replace a gene function. Paradigmatic examples are diseases caused by a dominant negative mutation or those in which silencing of a host gene confers resistance to a pathogen or compensates the function of the missing gene. Yet, gene silencing can be used to enhance efficacy of cell therapy and for biotechnological applications. Until now, two technologies have been used to silence gene expression, namely RNA interference with short harping RNAs (shRNA) and gene disruption with Artificial Nucleases (ANs). Although some promising pre-clinical and clinical data have been already obtained, the low efficiency of knock-down with shRNA and of biallelic disruption with ANs may limit efficacy of these treatments, especially when residual gene activity can exert a biological function. To overcome this issue, we have developed a novel modality of gene silencing that exploits endogenous epigenetic mechanisms to convey robust and heritable states of repression at the desired target gene. We have generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of a chromatinmodifying enzyme involved in silencing of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in human cell lines and primary cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains that, when transiently co-assembled on the promoter of the reporter cassette, fully abrogated transgene expression in up to 90% of treated cells. Importantly, silencing was maintained for more than 250 days in cultured cell lines, was resistant to in vitro differentiation or metabolic activation of primary cells, and was confined to the reporter cassette. Silencing was associated with high levels of de novo DNA methylation at the targeted locus and was dependent on this epigenetic mark for its propagation. Finally, transient transfection of 3 ATRs targeted to the promoter region of the Beta-2-microglobulin (B2M) gene resulted in the loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of treated cells. This phenotype was associated with a switch in the epigenetic and transcriptional state of the constitutively active B2M gene, which became highly decorated with DNA methylation and deprived of RNA PolII and of its transcript. Of note, silencing was resistant to IFN-γ treatment, a potent B2M inducer. Overall, these data provide the first demonstration of efficient and stable silencing of an endogenous gene upon transient delivery of ATRs. This result was made possible by repurposing the machinery involved in silencing of ERVs, which instructs self-sustaining repressive epigenetic states on the gene of interest. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for the treatment of several diseases. (LN & AL co-authorship)