Publications

Fulltext BibTeX Abstract
Ho, P. and Chen, Y.Y.
Current Opinion in Chemical Biology. 2017. DOI: 10.1016/j.cbpa.2017.06.003

Fulltext Options

View on External Site http://www.sciencedirect.com/science/article/pii/S1367593117300091
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.

BibTeX

Fulltext BibTeX Abstract
Ho, P., Ede, C., and Chen, Y.Y.
ACS Synthetic Biology. 2017. DOI: 10.1021/acssynbio.6b00392

Fulltext Options

View on External Site http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00392
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.

BibTeX

Fulltext BibTeX Abstract
Chang, Z.L. and Chen, Y.Y.
Trends in Molecular Medicine. 2017;23(5):430-450.

Fulltext Options

View on External Site http://www.cell.com/trends/molecular-medicine/abstract/S1471-4914(17)30040-0?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1471491417300400%3Fshowall%3Dtrue
Chimeric antigen receptors (CARs) are versatile synthetic receptors that provide T cells with engineered specificity. Clinical success in treating B-cell malignancies has demonstrated the therapeutic potential of CAR-T cells against cancer, and efforts are underway to expand the use of engineered T cells to the treatment of diverse medical conditions, including infections and autoimmune diseases. Here, we review current understanding of the molecular properties of CARs, how this knowledge informs the rational design and characterization of novel receptors, the successes and shortcomings of CAR-T cells in the clinic, and emerging solutions for the continued improvement of CAR-T cell therapy.

BibTeX

Fulltext BibTeX Abstract
Zah, E., Lin, M.Y., Silva-Benedict, A., Jensen, M.C., Chen, Y.Y.
Cancer Immunology Research. 2016;4(7):639-641.

Fulltext Options

View on External Site http://cancerimmunolres.aacrjournals.org/content/4/6/498.long
In the June 2016 issue of Cancer Immunology Research, we reported the design and optimization of bispecific, OR-gate chimeric antigen receptors (CARs) that can trigger robust T-cell activation in response to target cells that present either CD19 or CD20 (1), thus preventing malignant B cells from escaping T-cell therapy through loss of CD19 expression. In our original study, although OR-gate CAR T cells could significantly delay tumor expansion and prolonged survival, tumors were not eradicated. Tumor persistence was not due to the loss of both CD19 and CD20 antigens, nor due to the inability of the adoptively transferred T cells to survive or expand in vivo. We concluded that the failure to eradicate tumors was due to suboptimal tumor or T-cell dosing and/or the aggressiveness of the Raji tumor model. As the Raji tumor model had been used in other studies at dosages comparable with those employed in our own experiments, complete tumor clearance should be possible in this xenograft model using adoptively transferred CAR T cells. Here, we report follow-up studies showing that the viability levels of CAR T cells, both immediately after thawing and after 1-2 days of in vitro culture, are an important indicator of the CAR T cells' antitumor capability in vivo. Healthy T cells with OR-gate CARs could efficiently eradicate established lymphoma xenografts even if the lymphoma cells had spontaneously lost CD19 expression, whereas single-input CD19 CAR T cells succumbed to the selective expansion of CD19- mutants.

BibTeX

Fulltext BibTeX Abstract
Zah, E., Lin, M.Y., Silva-Benedict, A., Jensen, M.C., Chen, Y.Y.
Cancer Immunology Research. 2016;4(6):498-508.

Fulltext Options

View on External Site http://cancerimmunolres.aacrjournals.org/content/4/6/498.long
The adoptive transfer of T cells expressing anti-CD19 chimeric antigen receptors (CARs) has shown remarkable curative potential against advanced B-cell malignancies, but multiple trials have also reported patient relapses due to the emergence of CD19-negative leukemic cells. Here, we report the design and optimization of single-chain, bi-specific CARs that trigger robust cytotoxicity against target cells expressing either CD19 or CD20, two clinically validated targets for B-cell malignancies. We determined the structural parameters required for efficient dual-antigen recognition, and we demonstrate that optimized bi-specific CARs can control both wild-type B-cell lymphoma and CD19- mutants with equal efficiency in vivo. To our knowledge, this is the first bi-specific CAR capable of preventing antigen escape by performing true OR-gate signal computation on a clinically relevant pair of tumor-associated antigens. The CD19-OR-CD20 CAR is fully compatible with existing T-cell manufacturing procedures and implementable by current clinical protocols. These results present an effective solution to the challenge of antigen escape in CD19 CAR T-cell therapy, and they highlight the utility of structure-based rational design in the development of receptors with higher-level complexity.

BibTeX

Fulltext BibTeX Abstract
Ede, C., Chen, X., Lin, M.Y., Chen, Y.Y.
ACS Synthetic Biology. 2016;5(5):395-404.

Fulltext Options

View on External Site http://pubs.acs.org/doi/abs/10.1021/acssynbio.5b00266
Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

BibTeX

Fulltext BibTeX Abstract
Chen, Y.Y.
Trends in Immunology. 2015;36(11):667-669.

Fulltext Options

View on External Site http://www.sciencedirect.com/science/article/pii/S1471490615002185
Recent advances in T-cell therapy for cancer, viral infections, and autoimmune diseases highlight the broad therapeutic potential of T-cell engineering. However, site-specific genetic manipulation in primary human T cells remains challenging. Two recent studies describe efficient genome editing in T cells using CRISPR and TALEN approaches.

BibTeX

Fulltext BibTeX Abstract
Chang, Z., Silver, P.A., Chen, Y.Y.
Journal of Translational Medicine. 2015;13(1):161.

Fulltext Options

View on External Site http://www.translational-medicine.com/content/13/1/161/
Background: T cells expressing chimeric antigen receptors (CARs) have shown exciting promise in cancer therapy, particularly in the treatment of B-cell malignancies. However, optimization of CAR-T cell production remains a trial-and-error exercise due to a lack of phenotypic benchmarks that are clearly predictive of anti-tumor functionality. A close examination of the dynamic changes experienced by CAR-T cells upon stimulation can improve understanding of CAR–T-cell biology and identify potential points for optimization in the production of highly functional T cells. Methods: Primary human T cells expressing a second-generation, anti-CD19 CAR were systematically examined for changes in phenotypic and functional responses to antigen exposure over time. Multi-color flow cytometry was performed to quantify dynamic changes in CAR-T cell viability, proliferation, as well as expression of various activation and exhaustion markers in response to varied antigen stimulation conditions. Results: Stimulated CAR-T cells consistently bifurcate into two distinct subpopulations, only one of which (CARhi/CD25+) exhibit anti-tumor functions. The use of central memory T cells as the starting population and the resilience—but not antigen density—of antigen-presenting cells used to expand CAR-T cells were identified as critical parameters that augment the production of functionally superior T cells. We further demonstrate that the CARhi/CD25+ subpopulation upregulates PD-1 but is resistant to PD-L1-induced dysfunction. Conclusions: CAR-T cells expanded ex vivo for adoptive T-cell therapy undergo dynamic phenotypic changes during the expansion process and result in two distinct populations with dramatically different functional capacities. Significant and sustained CD25 and CAR expression upregulation is predictive of robust anti-tumor functionality in antigen-stimulated T cells, despite their correlation with persistent PD-1 upregulation. The functionally superior subpopulation can be selectively augmented by careful calibration of antigen stimulation and the enrichment of central memory T-cell type.

BibTeX

Fulltext BibTeX Abstract
Wei, K.Y., Chen, Y.Y., Smolke, C.D.
Biotechnology and Bioengineering. 2013;110(4):1201-10.

Fulltext Options

View on External Site http://onlinelibrary.wiley.com/doi/10.1002/bit.24792/full
Programming genetic circuits in mammalian cells requires flexible, tunable, and user-tailored gene-control systems. However, most existing control systems are either mechanistically specific for microbial organisms or must be laboriously re-engineered to function in mammalian cells. Here, we demonstrate a ribozyme-based device platform that can be directly transported from yeast to mammalian cells in a "plug-and-play" manner. Ribozyme switches previously prototyped in yeast are shown to regulate gene expression in a predictable, ligand-responsive manner in human HEK 293, HeLa, and U2OS cell lines without any change to device sequence nor further optimization. The ribozyme-based devices, which exhibit activation ratios comparable to the best RNA-based regulatory devices demonstrated in mammalian cells to-date, retain their prescribed functions (ON or OFF switch), tunability of regulatory stringency, and responsiveness to different small-molecule inputs in mammalian hosts. Furthermore, we observe strong correlations of device performance between yeast and all mammalian cell lines tested (R(2)  = 0.63-0.97). Our unique device architecture can therefore act as a rapid prototyping platform (RPP) based on a yeast chassis, providing a well-developed and genetically tractable system that supports rapid and high-throughput screens for generating gene-controllers with a broad range of functions in mammalian cells. This platform will accelerate development of mammalian gene-controllers for diverse applications, including cell-based therapeutics and cell-fate reprogramming.

BibTeX

Fulltext BibTeX Abstract
Chen, Y.Y.*, Galloway, K.E.*, Smolke, C.D.
Genome Biology. 2012;13(2):240. *These authors contributed equally.

Fulltext Options

View on External Site http://genomebiology.com/content/13/2/240
Advances in synthetic biology are contributing to diverse research areas, from basic biology to biomanufacturing and disease therapy. We discuss the theoretical foundation, applications, and potential of this emerging field.

BibTeX

Fulltext BibTeX Abstract
Chen, Y.Y. & Smolke, C.D.
Science Translational Medicine. 2011;3(106):106ps42.

Fulltext Options

View on External Site http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285276/
Synthetic biology aims to make biological engineering more scalable and predictable, lowering the cost and facilitating the translation of synthetic biological systems to practical applications. Increasingly sophisticated, rationally designed synthetic systems that are capable of complex functions pave the way to translational applications, including disease diagnostics and targeted therapeutics. Here, we provide an overview of recent developments in synthetic biology in the context of translational research and discuss challenges at the interface between synthetic biology and clinical medicine.

BibTeX

Fulltext BibTeX Abstract
Beisel, C.L., Chen, Y.Y., Culler, S.J., Hoff, K.G., & Smolke, C.D.
Nucleic Acids Research. 2011;39(7):2981-94.

Fulltext Options

View on External Site http://nar.oxfordjournals.org/content/39/7/2981.long
MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure-function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer-small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine.

BibTeX

Fulltext BibTeX Abstract
Chen, Y.Y., Jensen, M.C., & Smolke, C.D.
Proceedings of the National Academy of Sciences. 2010;107(19):8531-6.

Fulltext Options

View on External Site http://www.pnas.org/content/107/19/8531.long
RNA molecules perform diverse regulatory functions in natural biological systems, and numerous synthetic RNA-based control devices that integrate sensing and gene-regulatory functions have been demonstrated, predominantly in bacteria and yeast. Despite potential advantages of RNA-based genetic control strategies in clinical applications, there has been limited success in extending engineered RNA devices to mammalian gene-expression control and no example of their application to functional response regulation in mammalian systems. Here we describe a synthetic RNA-based regulatory system and its application in advancing cellular therapies by linking rationally designed, drug-responsive, ribozyme-based regulatory devices to growth cytokine targets to control mouse and primary human T-cell proliferation. We further demonstrate the ability of our synthetic controllers to effectively modulate T-cell growth rate in response to drug input in vivo. Our RNA-based regulatory system exhibits unique properties critical for translation to therapeutic applications, including adaptability to diverse ligand inputs and regulatory targets, tunable regulatory stringency, and rapid response to input availability. By providing tight gene-expression control with customizable ligand inputs, RNA-based regulatory systems can greatly improve cellular therapies and advance broad applications in health and medicine.

BibTeX

Fulltext BibTeX Abstract
Jewett, M.C., Miller, M.L., Chen, Y.Y., & Swartz, J.R.
Journal of Bacteriology. 2009;191(3):1083-91.

Fulltext Options

View on External Site http://jb.asm.org/content/191/3/1083.long
One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energy-intensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] (K(m)(ATP), 27 +/- 4 microM; K(m)(GTP), 14 +/- 2 microM). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level K(m)(ATP) is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.

BibTeX