The Wong lab is a DESIGN driven lab where we apply synthetic biology to engineer desired properties in mammalian cells, with application in understanding cellular design principles and to develop cellular therapy. Our research program can be broadly categorized into three areas: (1) Advanced chimeric antigen receptor (CAR) design, (2) Drug-inducible gene and signaling switches, and (3) Biocomputer engineering. Our works have appeared in some of the most prominent journals such as Cell and Nature Biotechnology.
- Advanced chimeric antigen receptor design
CAR T cells have shown phenomenal successes in clinical trials against B cell malignancies, leading to two FDA-approved therapies. Many CARs are being developed to target various antigens for different cancers. For instance, my lab developed the first CAR against Axl, a receptor tyrosine kinase commonly overexpressed in breast, lung, colon, and pancreatic cancers (Cho et al, Scientific Reports, 2018). Despite encouraging results in the clinics against blood-based cancer, safety and efficacy continue to be major hurdles that hinder CAR T cell therapy development. Therefore, to address these challenges, there is an urgent need to endow CARs with advanced functionalities, such as the ability to finely tune T cell activation, enhance tumor specificity, and independently control different signaling pathways and cell types.
To expand the capability of CAR T cells, we have created a split, universal, and programmable (SUPRA) CAR system composed of a universal receptor expressed on T cells and a tumor-targeting single-chain variable fragment (scFv) adaptor molecule. The SUPRA CAR system is the most feature-rich CAR system, simultaneously encompassing multiple critical “upgrades” including the ability to switch targets, finely tune T cell activation strength, and logically respond to multiple antigens (Cho JH, et al., Cell, 2018 ). These features are useful to combat relapse, mitigate over-activation, and enhance specificity. Furthermore, we have extended the orthogonal SUPRA CAR system to regulate different T cell subsets independently, demonstrating a dually-inducible CAR system. Together, our design of and results with SUPRA CARs illustrate that multiple advanced logic and control features can be implemented into a single, integrated system.
- Drug-inducible gene and signaling switches
While SUPRA enables inducible control of T cell activation and combinatorial antigen logic sensing, the use of a protein to activate CAR T cells is not the easiest or most cost-effective strategy. Furthermore, not all cancers require combinatorial antigen logic detection for successful therapy. As such, a system that enables drug-controllable expression of CARs using FDA-approved small molecule would also be highly desirable. Using a tamoxifen-inducible Flp recombinase, we developed ON, OFF, and Expression Level switches to control CAR expression in human T cells. Furthermore, since recombinases permanently modify the DNA, we showed that these switches naturally have memory characteristics and can permanently alter CAR expression with transient exposure to tamoxifen. We also showed that we can tune the percentage of cells that changed state based on the dose or duration of drug addition. Our switches are the first gene switches with memory for CAR expression control (Chakravarti D. et al., ACS Synthetic Biology, 2019).
In addition to a gene switch, which could have slow kinetics due to the need for protein synthesis, we have also developed a dual-gated small molecule inducible switch based on control over the activity of a critical tyrosine kinase in the TCR signaling pathway, ZAP-70 (Wong and Wong, ACS Synthetic Biology, 2018). To the best of our knowledge, this is the first dual gated kinase switch in human T cells. Our results indicate that the ZAP70 switch introduces tight temporal control over T cell activity, with activation in less than 2 minutes and inhibition within 1 minute. The ON-OFF switch can also be used to modulate the strength of T cell activation by varying concentrations of the activator and inhibitor. This switch could provide rapid and flexible control over T cell activity for adoptive T cell therapy.
- Engineering biocomputers in mammalian cells
Synthetic genetic circuits have many important applications, such as improving the specificity of tissue-specific gene expression and enhancing the safety profile of cell-based immunotherapy. For instance, we have leveraged miRNA to specifically express genes in inhibitory neurons in live mice (Sayeg et al., ACS Synthetic Biology, 2015). However, designing successful genetic circuits has proven to be very challenging, and one key problem in genetic circuit design is that they often do not behave as intended. Many genetic circuits are assembled from cascades of simpler genetic elements, connected via biological components, but these connections have proven to be highly unpredictable and are difficult to implement in single cells. Therefore, if genetic circuits can be designed with minimal layers, the probability of creating circuits that exhibit the intended behavior should greatly increase. This design approach represents a new paradigm from the conventional layered approach.
We have recently developed a DNA recombinase-based genetic circuit platform called Boolean Logic and Arithmetic through DNA Excision (BLADE) that can address some of the key challenges in engineering circuits (Weinberg et al., Nature Biotechnology, 2017). BLADE is the first generalizable design framework for engineering genetic circuits in mammalian cells that does not require extensive optimization from the user. We used BLADE to build 113 circuits in human embryonic kidney and Jurkat T cells, devising 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. Without the BLADE platform, most logic functions described here would have taken years to implement successfully in a biological host. Therefore, this work represents a significant achievement in synthetic biology based on the scale and sophistication of our circuits and the new paradigm in genetic circuit design.
As shown by our work and others, site-specific recombinases such as Cre and Flp are very powerful tools for regulating gene expression and have been applied towards many applications in engineering sophisticated genetic circuits and animal model development. Chemical and light-inducible recombinases are particularly instrumental in providing spatial and temporal control of gene expression. However, due to the challenge of developing inducible recombinases with acceptable properties (low basal activity, high dynamic range), there are very limited inducible recombinases available, and most of them were developed for Cre only. Therefore, while the current collection of inducible recombinases has been valuable in many studies, more high-performance inducible recombinases in addition to Cre, along with a systematic characterization of their activity, are urgently needed.
We have developed the largest collection of inducible recombinases (>20), which has demonstrated significantly enhanced performance (in revision). Our collection is based on the systematic identification of suitable split sites within a recombinase and combines them with several chemical and light-inducible dimerization (CID or LID) systems. Furthermore, we have engineered inducible split Cre systems with much better performance than existing inducible Cre systems. To demonstrate novel capability with our split recombinases, we created a tripartite-inducible Flp and a 4-Input AND gate. We have performed extensive quantitative characterization of the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs regarding signal-to-noise ratio (SNR). To facilitate sharing of this set of reagents, we have initiated the deposition of our library to Addgene. This library thus significantly expands capabilities for precise and multiplexed mammalian gene expression control.