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 3

CAR expression and function in peripheral T lymphocytes is fully restored by 2-MRE copy regulation.

Options: View larger image (or click on image) Download as PowerPoint
CAR expression and function in peripheral T lymphocytes is fully restore...
(A) Lentiviral vectors used in these experiments express 19z1 CAR driven by hPGK promoter under regulation by either no (19z10), 2 (19z12), or 4 (19z14) copies of miR-181a–specific MRE. (B) Mean fluorescence intensity (MFI) expression of 19z1 in DN, DP, CD4SP, and CD8SP thymocytes as well as CD4+ and CD8+ splenocytes normalized to expression in CD4–CD8– splenocytes for each mouse. Data are averaged from 4 mice harboring each of vectors 19z10, 19z12, and 19z14 analyzed 3–4 months after BM transplantation. Error bars denote SEM. (C) Expression of 19z1 in peripheral blood 19z1+CD3+ T cells in a total of 23 mouse chimeras harboring vectors 19z10, 19z12, and 19z14 analyzed 4 weeks after BM transplantation. Expression levels in CD3– cells indicate comparable levels of gene transfer. Error bars denote SD. Repeated analysis at week 10 after BM transplantation (not shown) corroborated the data presented here. (D) T cells expressing a miR-181–regulated 19z1 receptor specifically lysed a hCD19+ tumor cell line. 51Cr-release assays of T cells isolated from spleens of mouse chimeras harboring vectors 19z10, 19z12, and 19z14 targeting EL4-hCD19+ tumor cells or unmodified EL4 (hCD19–) tumor cells, as well as T cells from a mouse reconstituted with untransduced BM cells. E/T ratio, effector-to-target ratio of CD8+19z1+ T cells to tumor cells.