PT  - JOURNAL ARTICLE
AU  - Israni, Divya V.
AU  - Li, Hui-Shan
AU  - Gagnon, Keith A.
AU  - Sander, Jeffry D.
AU  - Roybal, Kole T.
AU  - Keith Joung, J.
AU  - Wong, Wilson W.
AU  - Khalil, Ahmad S.
TI  - Clinically-driven design of synthetic gene regulatory programs in human cells
AID  - 10.1101/2021.02.22.432371
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.02.22.432371
4099  - http://biorxiv.org/content/early/2021/06/07/2021.02.22.432371.short
4100  - http://biorxiv.org/content/early/2021/06/07/2021.02.22.432371.full
AB  - Synthetic biology seeks to enable the rational design of regulatory molecules and circuits to reprogram cellular behavior. The application of this approach to human cells could lead to powerful gene and cell-based therapies that provide transformative ways to combat complex diseases. To date, however, synthetic genetic circuits are challenging to implement in clinically-relevant cell types and their components often present translational incompatibilities, greatly limiting the feasibility, efficacy and safety of this approach. Here, using a clinically-driven design process, we developed a toolkit of programmable synthetic transcription regulators that feature a compact human protein-based design, enable precise genome-orthogonal regulation, and can be modulated by FDA-approved small molecules. We demonstrate the toolkit by engineering therapeutic human immune cells with genetic programs that enable titratable production of immunotherapeutics, drug-regulated control of tumor killing in vivo and in 3D spheroid models, and the first multi-channel synthetic switch for independent control of immunotherapeutic genes. Our work establishes a powerful platform for engineering custom gene expression programs in mammalian cells with the potential to accelerate clinical translation of synthetic systems.Competing Interest StatementD.V.I., J.D.S., J.K.J, and A.S.K. are inventors on a patent related to the synZiFTR technology; D.V.I., H.S.L., K.T.R., W.W.W, and A.S.K. have filed patent applications related to inducible synZiFTRs. J.K.J. is a co-inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies. K.T.R. is a co-founder of Arsenal Biosciences, was a founding scientist/consultant and stockholder in Cell Design Labs, now a Gilead Company, and holds stock in Gilead. J.K.J. has financial interests in Beam Therapeutics, Chroma Medicine (f/k/a YKY, Inc.), Editas Medicine, Excelsior Genomics, Pairwise Plants, Poseida Therapeutics, SeQure Dx, Inc., Transposagen Biopharmaceuticals, and Verve Therapeutics (f/k/a Endcadia). J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. W.W.W. is a scientific co-founder of and holds equity in Senti Biosciences. A.S.K. is a scientific advisor for and holds equity in Senti Biosciences and Chroma Medicine, and is a co-founder of Fynch Biosciences and K2 Biotechnologies.