Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras
Eirini P. Papapetrou, … , Derek Sant’Angelo, Michel Sadelain
Eirini P. Papapetrou, … , Derek Sant’Angelo, Michel Sadelain
Published December 1, 2008
Citation Information: J Clin Invest. 2009;119(1):157-168. https://doi.org/10.1172/JCI37216.
View: Text | PDF
Research Article Genetics

Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3′ untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a–specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell–based therapies, including cancer immunotherapy.

Authors

Eirini P. Papapetrou, Damian Kovalovsky, Laurent Beloeil, Derek Sant’Angelo, Michel Sadelain

×

Figure 2

miR-181a MRE restricts tumor antigen–specific receptor expression in developing thymocytes.

Options: View larger image (or click on image) Download as PowerPoint
miR-181a MRE restricts tumor antigen–specific receptor expression in dev...
(A) Lentiviral vectors bidirectionally expressing EGFP (as the neutral reporter) and the 19z1 CAR under posttranscriptional regulation by miR-181a. (B) Gating example of flow cytometry analysis of thymocyte subpopulations: DN (CD4–CD8–), DN1 (CD4–CD8–CD44+CD25–), DN2 (CD4–CD8–CD44+CD25+), DN3 (CD4–CD8–CD44–CD25+), DN4 (CD4–CD8–CD44–CD25–), DP (CD4+CD8+), CD4SP (CD4+CD8–), and CD8SP (CD4–CD8+). (C) Expression of miR-181 family miRNAs in sorted murine thymocyte subsets relative to expression in Ter119+ BM cells. ISP, intermediate SP. Error bars denote SD. (D) Representative FACS profile of thymocytes from 2 mice harboring the miR-181a–regulated vector G19z14×181a, 1 mouse harboring the control vector G19z10, and 1 nontransplanted mouse. Shown are cells gated on subpopulations exemplified in B. Numbers within plots denote percentage of cells in the respective quadrants. (E) Average 19z1 expression, quantified by FACS analysis, at different stages of thymocyte development relative to expression in DN1 stage in mouse chimeras reconstituted with BM cells transduced with vectors G19z10 and G19z14×181a. Data are mean ± SEM from 20 mice.