Clinically-driven design of synthetic gene regulatory programs in human cells

Abstract

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.

Context-aware synthetic biology by controller design: Engineering the mammalian cell

“…Each of the processes can be further regulated by suitable inputs, which can change with time (Figure 3D). For example, the rate of transcription can be dynamically modulated by recombinases (Weinberg et al, 2017) and TFs (Donahue et al, 2020;Gaber et al, 2014;Israni et al, 2021;Kiani et al, 2014;Li et al, 2015;Nissim et al, 2014;Stanton et al, 2014); the rates of mRNA translation or degradation can be modulated by small molecules/aptamers (Yokobayashi, 2019), ribosome-binding proteins (RBPs) (DiAndreth et al, 2019;Wagner et al, 2018;Wroblewska et al, 2015), and miRNAs (Cottrell et al, 2017;Michaels et al, 2019); and protein degradation and activity levels can be modulated by proteases (Cella et al, 2018 (A) Zooming in on the genetic device constituting one node in a larger network. The basic genetic device represents one gene encoded on a strand of DNA D X , which is transcribed to generate an mRNA M X , which is translated to a protein X.…”

GAMES: A dynamic model development workflow for rigorous characterization of synthetic genetic systems

“…crTFs confer external control over the expression of a target gene in the presence of a chemical inducer. crTFs have been built using various mechanisms and genetic parts 5, 6, 40, 41 , and these systems can also confer titratable control over the expression of clinically relevant genes 41 . Typically, crTF protein-protein interactions are mediated by the association of cognate dimerization domains 5, 6, 40, 41 .…”

Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells

“…Activation of immune system responses upon cell transplantation in patients is also a major concern. Human-derived genetic circuits can reduce possible immunogenic responses upon cell implantation (Israni et al, 2021). Therefore, increased use of humanized genetic circuits in engineered cells can potentially minimize the risk of immune system responses.…”