| dc.description.abstract | Synthetic biology is an interdisciplinary field of science that involves the redesign and fabrication of biological components to synthesize a modified and advanced biological system to solve problems in medicine, agriculture, or bio-products. Expanding the repertoire of synthetic biology tools will enable us to remotely control gene expression and cellular behaviors in living organisms, which is useful for interrogating gene regulatory networks and controlling biological processes, as well as for engineering therapeutic immune cells for precision medicine. We describe herein the development of a set of optogenetic and chemogenetic devices, including a synthetic calcium (Ca2+)-responsive transcriptional reprogramming device (CaRROT), a light-switchable chimeric antigen receptor (LiCAR), and designer receptors exclusively activated by designer drugs (DREADDs).
CaRROT is a chemical/light-inducible transcriptional reprogramming device composed of an optogenetically engineered, genetically-encoded Ca2+ channel actuator and a calcium-responsive dCas9 fusion construct (NFAT1−460-dCas9-VP64). In the presence of blue light or chemically-induced Ca2+ depletion, CaRROT undergoes light-inducible nuclear translocation and turns on gene expression at endogenous genomic loci in the presence of small guide RNAs (sgRNAs). This reprogramming tool was created to avoid the off-target effects involved in the current platform of gene transcription reprogrammers made of CRISPR/Cas9 (dCas9) fused with repressive or activating effectors only.
In addition, our study includes a novel light-switchable chimeric antigen receptor (CAR) that can be remotely controlled through near infrared (NIR) light-converting upconvension nanoplates for nano-optogenetic immunotherapy. LiCAR was designed by splitting the extracellular antigen-binding modules or the intracellular signal transduction modules from a conventional CAR. Each component was fused with one part of a pair of optical dimerizers, which undergo reversible light-dependent heterodimerization. Upon light stimulation, functional CARs will be reassembled to elicit T cell effector activity. LiCAR-expressing T-cells can selectively produce anti-tumor immune responses in the dual presence of tumor antigen and light in a spatiotemporally controlled manner, thus mitigating the side-effects and safety concerns associated with conventional CAR T-cells. Finally, the incorporation of DREADDs to T cells can either boost or suppress the strength and viability of engineered cells, thereby providing a chemogenetic tool for tunable activation or suppression of therapeutic immune cells. Together, we have created a toolbox that permits the precise control of the dose, location, and duration of immune responses to benefit future personalized immunomodulatory therapies. | |
