Leveraging microRNAs for cellular therapy.

Owing to Karl Landsteiner's discovery of blood groups, blood transfusions became safe cellular therapies in the early 1900s. Since then, cellular therapy made great advances from transfusions with unmodified cells to today's commercially available chimeric antigen receptor (CAR) T cells requiring complex manufacturing. Modern cellular therapy products can be improved using basic knowledge of cell biology and molecular genetics. Emerging genome engineering tools are becoming ever more versatile and precise and thus catalyze rapid progress towards programmable therapeutic cells that compute input and respond with defined output. Despite a large body of literature describing important functions of non-coding RNAs including microRNAs (miRNAs), the vast majority of cell engineering efforts focuses on proteins. However, miRNAs form an important layer of posttranscriptional regulation of gene expression. Here, we highlight examples of how miRNAs can successfully be incorporated into engineered cellular therapies.

Forced expression of the non-coding RNA miR-17∼92 restores activation and function in CD28-deficient CD4+ T cells.

CD28 provides the prototypical costimulatory signal required for productive T-cell activation. Known molecular consequences of CD28 costimulation are mostly based on studies of protein signaling molecules. The microRNA cluster miR-17∼92 is induced by T cell receptor stimulation and further enhanced by combined CD28 costimulation. We demonstrate that transgenic miR-17∼92 cell-intrinsically largely overcomes defects caused by CD28 deficiency. Combining genetics, transcriptomics, bioinformatics, and biochemical miRNA:mRNA interaction maps we empirically validate miR-17∼92 target genes that include several negative regulators of T cell activation. CD28-deficient T cells exhibit derepressed miR-17∼92 target genes during activation. CRISPR/Cas9-mediated ablation of the miR-17∼92 targets Pten and Nrbp1 in naive CD28-/- CD4+ T cells differentially increases proliferation and expression of the activation markers CD25 and CD44, respectively. Thus, we propose that miR-17∼92 constitutes a central mediator for T cell activation, integrating signals by the TCR and CD28 costimulation by dampening multiple brakes that prevent T cell activation.

Plasmid- or Ribonucleoprotein-Mediated CRISPR/Cas Gene Editing in Primary Murine T Cells.

The CRISPR/Cas technology allows for genome editing in primary T cells. We herein describe the activation of primary murine CD4+ or CD8+ T cells, followed by electroporation with plasmid or ribonucleoproteins (RNP) for gene modification. Gene edited T cells can subsequently be transferred to host mice for in vivo studies or cultured in vitro for further characterization. This protocol enables sophisticated genetic analysis of T cells using commonly available virus-free reagents.