Cytotoxic T Lymphocytes (CTLs) express highly specific receptor molecules that recognize disease markers and serve as the body's natural guard against infections. However, many diseases, notably cancer cells, have developed elaborate tactics to avoid detection by CTLs and evade immune responses. To address this challenge, T cells have been engineered to express synthetic receptors with specificity toward unnatural substrates, such as tumor-restricted antigens, thus redirecting the immune system against novel targets. While it has been demonstrated that engineered T cells can effectively target diseased cells, sustaining and regulating the growth of ex vivo expanded T cells in the tumor microenvironment remain major challenges. Our orthotopic model systems have demonstrated that without paracrine T cell growth factors, such as IL-2, CTL survival is of limited duration and insufficient for tumor control. Conversely, CTL genetic modification to constitutively express gamma-c cytokine transgenes such as IL-2 and IL-15 poses the risk of uncontrolled expansion. Therefore, a stringent regulatory system that is capable of fine-tuned control over growth factor production and the resultant T cell proliferation pattern is critical to the success of cellular immunotherapeutics.
We have developed a novel method to regulate T cell proliferation and survival by controlling cytokine transgene expression through small molecule-responsive RNA regulatory systems. The growth of IL-2 dependent CTLL-2 mouse T cells is controlled by the transgenic expression of cytokines such as IL-2 and IL-15, whose production levels are modulated by RNA-based regulatory elements implemented in the 3' untranslated region (UTR) of the cytokine transgenes. RNA regulatory elements are composed of three modular domains: (i) sensors consisting of RNA aptamers with high specificity for small-molecule ligands such as theophylline and tetracycline, (ii) actuators consisting of functional, noncoding RNA species such as microRNAs and self-cleaving ribozymes, and (iii) information transmitters consisting of rationally designed RNA sequences that transmit ligand-binding events from the sensor domain to changes in the activity of the actuator domain. By coupling small molecule-responsive RNA regulatory elements with target transcripts such as the egfp-t2a-il2 fusion gene, we construct systems in which transgene expression is stringently controlled and turned ON in the presence of the regulating small-molecule ligand. In a transient transfection system, we demonstrate ligand-dependent growth regulation through monitoring both fluorescence and cell culture viability. Stable integration of the regulatory systems into clonal T cell populations confirms their long-term ligand-responsive regulatory activities. Furthermore, we demonstrate the ability to fine-tune the response properties of these regulatory systems through sequence modifications targeted to the transmitter domains based on thermodynamic design strategies as well as combinatorial implementation of different classes of RNA-based regulatory elements. Progress is underway to examine small molecule control of the proliferation of engineered T cells harboring these regulatory systems in animal models, which will yield further insight into the in vivo functionality and possible improvements for RNA-based regulatory designs.
Coupled with technologies to generate new RNA sensors responsive to clinically relevant small molecule effectors, the platform reported here enables the design and construction of RNA-based regulatory systems tailored for applications that demand stringent control of cellular behavior and flexibility in the tuning of regulatory activities. This research highlights the exciting potential of new RNA-based regulatory systems in generating tightly-regulated, dynamic protein expression systems adaptable to pharmaceutical drugs with a high therapeutic index that are suitable for advanced clinical applications such as cellular therapeutics employing genetically modified T cells.