Protease-based protein control and cellular reprogramming : approaches to advance gene and cell therapy

Our generation is witnessing staggering advancements in gene and cell therapies. These technologies hold great promise for the treatment of human diseases that are pharmacologically insurmountable. Yet, two major concerns have to be addressed: safety and efficacy. Here, I describe synthetic biology approaches to overcome these limitations and to realize the full potential of gene and cell therapy. Synthetic biology is an emerging discipline aimed at reprogramming living cells and organisms by integrating genetics, engineering principles, computational and quantitative analyses. More specifically, we have developed protease-based techniques that provide control over the duration, intensity, and context of the therapy. We have designed two systems based on the HCV NS3 protease that enable the followings: (1) pharmacological control of the therapeutic protein expression and (2) redirection of the tumorigenic signaling pathway to a designed therapeutic program. The first technique is termed "Small Molecule-Assisted Shutoff", or SMASh. It utilizes a self-cleaving degron that we engineered from NS3 protease. Clinically approved, non-toxic NS3 inhibitor can modulate the degron cleavage and thus determines the duration and amount of the protein production. Given its biocompatibility and simplicity, SMASh can readily be applied to medical research including but not limited to stem/immune cell engineering and therapeutic virus design. For example, SMASh has been adopted to control the replication of an oncolytic virus and to regulate the expression of the chimeric antigen receptors on T cells, providing a much-needed safety handle. At the same time, SMASh is also being widely used in the basic science projects as it enables conditional expression of a protein-of-interest in yeast and mammalian cell lines. The second technique I present here is "Rewiring of Aberrant Signaling to Effector Release" (RASER). RASER is novel in that cancer cells are targeted based on their most upstream driving mechanism, which is in most cases a hyperactivated signaling pathway. Instead of blocking the tumorigenic signaling with drugs, we rewired it to activate a therapeutic program. Again based on the NS3 protease, we developed a synthetic two-component system that redirects ErbB activity to the release of an membrane-sequestered effector cargo. The resulting ErbB-RASER system responds specifically and robustly to constitutively active ErbB and can be programmed to induce a variety of outputs such as apoptosis or CRISPR/Cas9 activation. Since the sensing and actuating modules of RASER are highly customizable, we envision that it can be used to treat various cancers. To summarize, this thesis introduces new protease-based technologies, their characterization, and potential applications. The techniques can be utilized not only to treat patients but also to answer biological questions. I believe that insights from my studies are applicable to other synthetic biology-based therapeutics, possibly leading to more creative and effective approaches for gene and cell therapy.

Keywords: Biology.
Advisor: Lin Michael Z. 
Dixon Scott
Ferrell James Ellsworth 
Kim Peter