Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

Abstract

RNA molecules perform diverse regulatory functions in natural biological systems, and numerous synthetic RNA-based control devices that integrate sensing and gene-regulatory functions have been demonstrated, predominantly in bacteria and yeast. Despite potential advantages of RNA-based genetic control strategies in clinical applications, there has been limited success in extending engineered RNA devices to mammalian gene-expression control and no example of their application to functional response regulation in mammalian systems. Here we describe a synthetic RNA-based regulatory system and its application in advancing cellular therapies by linking rationally designed, drug-responsive, ribozyme-based regulatory devices to growth cytokine targets to control mouse and primary human T-cell proliferation. We further demonstrate the ability of our synthetic controllers to effectively modulate T-cell growth rate in response to drug input in vivo. Our RNA-based regulatory system exhibits unique properties critical for translation to therapeutic applications, including adaptability to diverse ligand inputs and regulatory targets, tunable regulatory stringency, and rapid response to input availability. By providing tight gene-expression control with customizable ligand inputs, RNA-based regulatory systems can greatly improve cellular therapies and advance broad applications in health and medicine.

Fig. 1.

An engineered T-cell proliferation regulatory system utilizing a synthetic RNA device to achieve drug-mediated modulation of cell signaling and proliferation. (A) The common γ-chain T-cell proliferation pathway and integration of a synthetic controller targeted to the upstream signaling events. (B) An engineered T-cell proliferation regulatory system on the basis of programmable drug-mediated regulation of cytokine expression from a synthetic ribozyme switch. Ribozyme color scheme is as described in ref. .

Fig. 2.

Modularly constructed ribozyme switches exhibit tunable, drug-mediated regulation of gene expression and cell growth in mammalian T cells. (A) A modular insertion strategy allows the implementation of multiple copies of ribozyme switches to tune regulatory stringency. Spacer sequences (Orange and Green) provide structural insulation and maintain the functional independence of each switch. (B) Ribozyme switches provide tunable, small-molecule-mediated regulatory systems. Cell viability levels are reported for constructs encoding theo-responsive switches (L2bulge1, 8, 9) in one (1×), two (2×), three (3×), and four (4×) copies through transient transfections in CTLL-2 cells grown in 0 and 1 mM theo. No IL-2 control, construct not encoding a proliferative cytokine; sTRSV ribozyme, construct encoding a nonswitch hammerhead ribozyme. The gray bar indicates background viability level of cells in the absence of cytokine. (C) Ribozyme switches provide titratable regulatory systems. Cell viability levels are reported for the L2bulge9 regulatory systems at various theo concentrations. (D and E) Tailoring of the input responsiveness of the RNA-based regulatory system through direct replacement of the sensor component generates titratable, tet-responsive switches (L2bulge18tc). (F and G) Tailoring of the output of the RNA-based regulatory system through direct replacement of the target gene (cd19-tk-t2a-il15) achieves enhanced survival response compared to IL-2-based systems. All viability and fluorescence values were normalized to those of controls expressing the appropriate transgene regulated by an inactive ribozyme and cultured at corresponding theo concentrations. Reported values are mean ± SD from at least two replicate samples.

Fig. 3.

T cells stably expressing ribozyme switch systems exhibit drug-mediated regulation of growth over extended time periods in vitro. (A) Clonal cell lines stably expressing the ribozyme switch system exhibit drug-mediated growth. Cell growth was monitored by counting viable cells, and data for a representative clone [1264-48, expressing cd19-tk-t2a-il15-L2bulge9(3×)] are shown. (B) The ribozyme switch system regulates gene expression in response to changes in input concentrations. Duplicate sets of the clonal cell line 1264-48 were cultured in the presence (Red and Orange) and absence (Light Blue and Dark Blue) of theo over 18 days. Expression levels were monitored by staining with PE-CD19 antibody, and values were normalized to those from the inactive ribozyme control. The highest expression level was set to 100%. The red dashed line indicates background staining level of a cell line without a CD19 construct.

Fig. 4.

T cells stably expressing the ribozyme switch systems exhibit drug-mediated regulation of growth over extended time periods in vivo. (A) Both cytokine expression and functional ribozyme switches are required for effective regulation of T-cell proliferation. Images are shown for day 14 postinjection of the negative control (no IL-15 Control, CffLuc), positive control (inactive Rz control, stable cell line expressing inactive ribozyme), and stable cell line expressing the ribozyme switch system [L2bulge9(3×), clone 1264-48] in the presence and absence of 500 μM theo. (B) Total luciferase signal flux over 14 days after injection of T cells is reported from the negative control (CffLuc) and clone 1264-48 [L2bulge9(3×)].

Fig. 5.

The ribozyme switch system effectively regulates gene expression and cell fate in primary human T_CM cells. (A) CD19 expression levels are elevated in the presence of theo. The populations of cells that are (B) live and CD19+ or (C) apoptotic and CD19+ indicate an increase in live cells and decrease in apoptotic cells in response to theo. Values for the L2bulge9(3×) sample are normalized to those of the inactive ribozyme control cultured at the same theo concentration. Reported values are mean ± SD from triplicate samples. The highest level is set to 100%.