Inducible Gene Switches with Memory in Human T Cells for Cellular Immunotherapy.

Cell-based therapies that employ engineered T cells-including those modified to express chimeric antigen receptors (CARs)-to target cancer cells have demonstrated promising responses in clinical trials. However, engineered T cell responses must be regulated to prevent severe side effects such as cytokine storms and off-target responses. Here we present a class of recombinase-based gene circuits that will enable inducible, one-time state switching in adoptive T cell therapy using an FDA-approved drug, creating a generalizable platform that can be used to control when and how strongly a gene is expressed. These circuits exhibit memory such that induced T cells will maintain any changes made even when the drug inducer is removed. This memory feature avoids prolonged drug inducer exposure, thus reducing the complexity and potential side effect associated with the drug inducer. We have utilized these circuits to control the expression of an anti-Her2-CAR, demonstrating the ability of these circuits to regulate CAR expression and T cell activity. We envision this platform can be extended to regulate other genes involved in T cell behavior for applications in various adoptive T cell therapies.

High-performance chemical- and light-inducible recombinases in mammalian cells and mice.

Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells.

Engineered genetic circuits for mammalian cells often require extensive fine-tuning to perform as intended. We present a robust, general, scalable system, called 'Boolean logic and arithmetic through DNA excision' (BLADE), to engineer genetic circuits with multiple inputs and outputs in mammalian cells with minimal optimization. The reliability of BLADE arises from its reliance on recombinases under the control of a single promoter, which integrates circuit signals on a single transcriptional layer. We used BLADE to build 113 circuits in human embryonic kidney and Jurkat T cells and devised a quantitative, vector-proximity metric to evaluate their performance. Of 113 circuits analyzed, 109 functioned (96.5%) as intended without optimization. The circuits, which are available through Addgene, include a 3-input, two-output full adder; a 6-input, one-output Boolean logic look-up table; circuits with small-molecule-inducible control; and circuits that incorporate CRISPR-Cas9 to regulate endogenous genes. BLADE enables execution of sophisticated cellular computation in mammalian cells, with applications in cell and tissue engineering.