Patrick Ho’s research while affiliated with University of California System and other places

In response to the changing landscape of medical challenges, pioneers in molecular biology and genetic engineering launched a new paradigm of pharmaceutical development. Unlike chemical pharmaceuticals and biologics, cellular therapeutics have the potential to establish prolonged proliferation in the patient and provide continual surveillance against disease relapse without repeated drug administration. This chapter discusses some of the challenges facing cell‐based therapeutics ‐ particularly cell‐based immunotherapies ‐ and highlights solutions that have been developed through the application of synthetic biology. Genetically engineered stem cells have been programmed to deliver cytotoxic molecules, angiostatic factors, and immunostimulatory cytokines to tumor cells, demonstrating the versatility and programmability of living cells as therapeutic agents. The ability to efficiently design, construct, and optimize synthetic biological systems that modify and/or interface with living cells is expanding new possibilities in the development of cellular therapeutics and offering enticing views of next generation strategies for disease treatment.

The recent expansion of molecular tool kits has propelled synthetic biology toward the design of increasingly sophisticated mammalian systems. Specifically, advances in genome editing, protein engineering, and circuitry design have enabled the programming of cells for diverse applications, including regenerative medicine and cancer immunotherapy. The ease with which molecular and cellular interactions can be harnessed promises to yield novel approaches to elucidate genetic interactions, program cellular functions, and design therapeutic interventions. Here, we review recent advancements in the development of enabling technologies and the practical applications of mammalian synthetic biology.

Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen‒responsive therapeutics that can complement surface-targeted therapies.