Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer Brian D Brown 1 , Mary Anna Venneri 1 , Anna Zingale 1 , Lucia Sergi Sergi 1 & Luigi Naldini 1,2 MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidence indicates that miRNAs have distinct expression profiles and play crucial roles in numerous cellular processes, although the extent of miRNA regulation is not well known. By challenging mice with lentiviral vectors encoding target sequences of endogenous miRNAs, we show the efficiency of miRNAs in sharply segregating gene expression among different tissues. Transgene expression from vectors incorporating target sequences for mir-142-3p was effectively suppressed in intravascular and extravascular hematopoietic lineages, whereas expression was maintained in nonhemato- poietic cells. This expression profile, which could not be attained until now, enabled stable gene transfer in immuno- competent mice, thus overcoming a major hurdle to successful gene therapy. Our results provide novel in situ evidence of miRNA regulation and demonstrate a new paradigm in vector design with applications for genetic engineering and therapeutic gene transfer. Recently, a complex network of gene regulation was uncovered, which is mediated by small 21–23-nucleotide noncoding RNAs known as miRNAs 1 . miRNAs act as a guide for the RNA-induced silencing com- plex (RISC) to repress translation of target cellular transcripts 2 . Mole- cular analysis has shown that miRNAs have distinct expression profiles in different tissues 3,4 , indicating, together with functional studies 5 , important roles for miRNAs in establishing cell identity 6 . As the targets of most miRNAs have not been identified, however, the extent and level of regulation mediated by individual miRNAs is not well known. Recent studies have used reporter systems to follow miRNA expression in situ 7,8 . Although these studies show tracking of miRNA expression, the degree and robustness of miRNA-mediated suppression was not specifically examined. Here, we set out to address these issues, and develop a miRNA-regulated gene expression system that is responsive to lineage commitment and suitable for in vivo gene transfer. Currently, one of the major barriers to stable gene transfer is the development of transgene-specific immunity 9 . In studies of gene therapy for inherited diseases, such as the hemophilias, a successful outcome has been precluded by the development of immune responses against the vector and transgene product 10 . Several factors contribute to the induction of an immune response following gene transfer 11 . Chief amongst these factors is the direct expression of the transgene product within professional antigen-presenting cells (APCs) of the immune system 12 . Previously, we and others have used tissue-specific promoters to target transgene expression to hepatocytes and prevent expression within APCs 13–15 . This strategy reduces the incidence and extent of the transgene-specific immune response. Unfortunately, even when tissue-specific promoters are used, neutralizing antibodies against the transgene product and immune-mediated vector clearance can still be observed 14,16 . This may owe to uptake and cross-presentation of the transgene product by nontransduced APCs and/or off-target transgene expression within APCs. The latter can occur because of nonspecific activity of the reconstituted transgene promoter, or, in the case of integrating vectors, because of promoter-enhancer trapping at insertions near active regions of transcription 17 . Restricting transgene expression to a particular cell type may also decrease the potential efficacy of gene transfer by limiting the pool of cells expressing the transgene. miRNA regulation, which de-targets rather than targets gene expression and functions at the post-transcriptional level, may provide a unique means for overcoming the limitations of current gene delivery systems. We reasoned that, by preventing transgene expres- sion in hematopoietic lineages while permitting high levels of expres- sion in nonhematopoietic cells, miRNA regulation could enable stable gene transfer in the absence of an immune response. RESULTS miRNA regulation prevents expression in hematopoietic cells We constructed a miRNA-regulated lentiviral vector by inserting four tandem copies of a 23-bp sequence (mirT) with perfect complemen- tarity to either mir-30a, mir-142-5p or mir-142-3p into the 3¢-untranslated region (3¢-UTR) of a green fluorescent protein (GFP) expression cassette driven by the ubiquitously expressed phos- phoglycerate kinase (PGK) promoter (Fig. 1a). This design, using multiple copies of a perfectly complementary target, is intended to Received 17 October 2005; accepted 9 January 2006; published online 23 April 2006; corrected 28 April 2006 (details online); doi:10.1038/nm1398 1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), 2 Vita Salute San Raffaele University, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy. Correspondence should be addressed to L.N. (naldini.luigi@hsr.it). NATURE MEDICINE VOLUME 12 [ NUMBER 5 [ MAY 2006 585 TECHNICAL REPORTS © 2006 Nature Publishing Group http://www.nature.com/naturemedicine