803. Endogenous microRNA Regulation Suppresses Transgene Expression in Hematopoietic Lineages and Enables Stable Gene Transfer
Authors: Brian D. Brown, Mary Anna Venneri, Anna Zingale, Lucia Sergi Sergi, Luigi Naldini
Journal: Molecular Therapy (2006)

One of the major barriers to stable gene transfer is the development of anti-transgene immunity. In studies of gene therapy for inherited diseases, such as the hemophilias, successful outcome has been precluded by the development of immune responses against the vector and transgene product. Several factors contribute to the induction of an immune response following gene transfer. Chief amongst these factors is the expression of the transgene product within antigen presenting cells (APCs). Tissue-specific promoters (TSPs) can be used to prevent expression within APCs. Unfortunately, even using TSPs, neutralizing antibodies against the transgene product and immune-mediated vector clearance can often be observed. This may be due to off-target transcriptional activity of the promoter within APCs. Restricting transgene expression to a particular cell type may also decrease the potential efficacy of gene transfer by limiting the pool of cell types expressing the transgene. Recently, a complex network of gene regulation was uncovered, which is mediated by small non-coding RNAs known as microRNAs (miRNA). In an effort to develop an improved system for systemic gene transfer, we sought to exploit the endogenous miRNA machinery, which de-targets rather than targets gene expression and functions at the post-transcriptional level, for regulating transgene expression. A lentiviral vector (LV) encoding GFP was modified to contain a sequence with perfect complementarity to mir-142-3p, a hematopoietic-specific miRNA. Using this design, we reasoned that transgene expression would be prevented in hematopoietic lineages, while high levels of expression would be permitted in non- hematopoietic cells. Indeed, we demonstrated that by adding the mir-142-3p target sequence, GFP expression could be completely prevented in both intra- and extra-vascular hematopoietic lineages, while expression was maintained in non-hematopoietic cells (Brown et al. Nature Medicine in press). This expression profile, which could not be attained until now, enabled stable gene transfer (>120 days) and prevented immune-mediate vector clearance in immunocompetent mice, thus overcoming a major hurdle to successful gene therapy. In sharp contrast, there was rejection of transgene positive cells when mice were treated with an LV encoding antigen under control of the hepatocyte-specific albumin promoter. The improved restriction of transgene expression from the miRNA- regulated LV compared to the albumin promoter likely occurred because post-transcriptional regulation can overcome off-target expression due to positional effects of insertion and/or imperfect reconstitution of the TSP. In addition to preventing an immune response, miRNA de-targeting, unlike TSPs, enabled transgene expression in a larger population of cells, including fibroblasts and endothelium. We are now expanding the use of this system for the treatment of hemophilia B. miRNA-regulated LVs encoding human Factor IX have been developed, and studies are now underway to evaluate the effectiveness of this strategy for long-term correction of hemophilia B mice.