International audience; RNA interference (RNAi)-based gene therapy has great potential in cancer and infectious disease treatment to correct abnormal up-regulation of gene expression. We show a new original method uses synthetic microRNAs combined with a thermo-inducible promoter to reduce specific gene expression. The targeted gene is the luciferase firefly reporter gene overexpressed in a subcutaneous tumor which allows the RNAi monitoring by bioluminescence imaging (BLI). The inducible inhibition was first demonstrated in vitro using genetically modified cells lines and then in vivo using the corresponding xenograft model in mice. Achieving spatio-temporal control, we demonstrate the feasibility to induce, in vivo, a specific gene inhibition on demand. Future applications of this RNAi-based gene therapy, which can be restricted to pathological tissue, would offer wide-ranging potential for disease treatment. RNA interference (RNAi) is an evolutionarily-conserved mechanism which has become a revolutionary strategy to regulate gene expression. This biological process, mediated by small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs) or microRNAs (miRNAs), has rapidly emerged as a key experimental tool for gene function analysis and target validation in mammalian systems, both in vitro and in vivo. As a consequence, RNAi appears to be an attractive technology for gene therapy to down-regulate specific genes 1. However, RNAi induction mediated by siRNAs is limited by their pharmacokinetic properties, their poor cell penetration and their limited activity in vivo 2. Similarly, although not a prerequisite, experimental studies often use polymerase III-dependent promoters for applications that require shRNA synthesis. The development of shRNA embedded in a miRNA scaffold driven by RNA polymerase II makes tissue-specific or inducible RNAi possible by using a wide variety of promoters. Furthermore, biosynthesis of optimized shRNAs based on miRNA cell processing machinery provide efficient and safe therapeutic routes for RNAi induction in vitro and in vivo 3-5. MiRNAs are a class of non-coding RNA implicated in the regulation of around 30% of the cell transcriptome 6. MiRNAs can naturally direct gene silencing at a post-transcriptional level. Most gene therapies require restriction of the treatment to the targeted tissues to minimize unwanted side effects. Thus, RNAi-based therapies require spatial control of the induction. Heat-sensitive transgene expression systems have been proposed for use in gene therapy to enable spatial control of gene activity, also offering temporal control. HSP (heat shock protein)-related promoters are inducible and external heat treatment enables their activation. In vitro feasibility of the transcription of an artificial miRNA by HSP-related promoters has been reported 7 but only following transient cell transfection. However, the Hsp70B promoter has many advantages including its low basal activity and the heat-induced expression 8-11 which could create new approaches for Hsp-controlled RNAi systems in vivo. Moreover, the efficient control of gene expression in vivo using a HSP-related promoter has been reported using encoding genes such as HSV thymidine kinase, FAS ligand and cytokine genes 9,12-14 or reporter genes for in vivo monitoring 10,15,16. In this study, we propose a new in vivo method to combine the shRNA embedded in a miRNA scaffold inhib-itory effect with its thermo-induced expression driven by the Hsp70B promoter. This article demonstrates the feasibility and efficiency of this strategy in vitro followed by its implementation in vivo using the reporter gene firefly luciferase (LucF) as the target gene.