microRNA-Mediated Gene Regulation Effectively Restricts In Vivo Transgene Expression in Hematopoietic Stem Cell Progeny.
Authors: Papapetrou Eirini P. , Kovalovsky Damian , Beloeil Laurent , Sant’Angelo Derek , Sadelain Michel 
Published: April 2020

Stem cell engineering and targeted in vivo gene delivery increasingly require tight control of transgene expression. Lineage- and differentiation stage-specific gene regulation is classically afforded by pol-II-dependent transcript regulation. A super-imposed layer of post-transcriptional control would be valuable to correct undesirable expression patterns or fine-tune developmentally regulated or inducible gene expression. microRNAs (miRNAs) have recently emerged as potent repressors of gene expression at the post-transcriptional level. In this study, we investigate the potential of using miRNA regulation to provide lineage-restricted expression, exploiting miRNAs with distinctive expression patterns in hematopoietic tissue. miR-223 is preferentially expressed in granulocytes and monocytes (80-and 110-fold, respectively). miR-181a is highly expressed in B-lymphocytes and, particularly, in thymocytes, but down-regulated (∼1000-fold) in post-thymic T cells. We, therefore, constructed lentiviral vectors encoding either green fluorescent protein (GFP) or an antigen-specific receptor, placed under the transcriptional control of the ubiquitous EF1a promoter and tagged with miRNA-recognition elements (MREs) complementary to the mature miR-223 or miR-181a. In a panel of murine and human cell lines expressing varying levels of the two miRNAs we find that GFP knockdown is dependent on the presence of the miRNA and directly proportional to the number of MRE repeats. Four copies of the repeat permit better down-regulation than 2 copies (albeit depending on the level of endogenous miRNA). Two different MREs can be combined in tandem, resulting in additive down-regulation. In vivo, in mouse bone marrow chimeras harboring the miR-223-responsive vector, GFP expression is specifically repressed in myeloid cells (&gt;85% compared to the control vector lacking miRNA target sequences). Reciprocally, chimeras harboring the miR-181a-regulated vector express GFP in myeloid and erythroid lineages, but transgene expression is profoundly repressed in thymocytes and B-cell progenitors. A vector harboring a composite MRE confers GFP expression almost exclusively confined to the erythroid lineage. These results demonstrate for the first time that transgene expression can be selectively regulated at the post-transcriptional level within the hematopoietic tissue. In mouse chimeras expressing a CD19-specific antigen receptor (which we and others are currently using in clinical trials in B cell malignancies), we analyze receptor expression in the 4 double negative subsets (DN1, DN2, DN3 and DN4), the double-positive subset (DP), and the CD4+ and CD8+ single positive (SP) subsets. We detail and quantify receptor knock-down at each developmental stage, and show that miR-181a-mediated regulation prevents receptor expression at critical stages of positive and negative thymic selection. Antigen receptor expression is dramatically repressed in DN and DP cells, while rising in CD4+ and CD8+ SP thymocytes. Importantly, expression is fully restored in post-thymic T cells, and maintained in activated T cells. miRNA-mediated post-transcriptional regulation is thus proving to be a powerful means to direct lineage- and differentiation stage-specific transgene expression in genetically modified stem cells.