Programming gene and engineered-cell therapies with synthetic biology

Abstract

Gene and engineered-cell therapies promise to treat diseases by genetically modifying cells to carry out therapeutic tasks. Although the field has had some success in treating monogenic disorders and hematological malignancies, current approaches are limited to overexpression of one or a few transgenes, constraining the diseases that can be treated with this approach and leading to potential concerns over safety and efficacy. Synthetic gene networks can regulate the dosage, timing, and localization of gene expression and therapeutic activity in response to small molecules and disease biomarkers. Such "programmable" gene and engineered-cell therapies will provide new interventions for incurable or difficult-to-treat diseases.

Fig. 1. Building blocks for therapeutic programs.

A) Logic gates with inputs A and/or B) and outputs X) can be used to represent molecular processes and reactions. B) Conventional gene and cell therapies require just one exogenous molecular input and lack precise control over the output. Such modules function as buffer gates i.e., control devices whose output levels correspond to their input levels), because the RNA output will be produced in any cell that the DNA input is delivered to and the therapeutic protein will be translated correspondingly. C) Engineerable modules can regulate the production. conversion, or loss of specific DNA blue). RNA red), or protein yellow) species by using more than one molecular input. TE transcription factor: RBP RNA-binding protein.

Fig. 2.. Small molecule regulation enables control over strength of therapeutic activity and facilitates new applications.

A) Traditional CARs are activated when the T cell encounters a target antigen. ON-switch CARs respond to antigens only when a small molecule, such as rapalog, is administered. B) The pancreatic progenitor-to Hike cell differentiation circuit is controlled by VA. Increasing levels of VA establish three different gene-expression profiles for the transcription factors PDX1, NGN3, and MAFA to drive differentiation. The final concentration of PDX1 is a summation of translation from two mRNA sources akin to a wired-OR operation in electronic logic circuits. Dashed arrows indicate multiple steps. The same drawing conventions are used as in Fig. 1.

Fig. 3.. Genetically encoded therapeutic programs Incorporate cell specific biomarkers for localized activity.

A) In an AND-gate CAR T cell. the activation of a synNotch receptor by a first antigen induces the expression of a CAR. which in turn is activated by a second antigen to ultimately activate the T cell. B) RNA-encoded miRNA-classifier circuit selectively kills cancer cells characterized by high levels of miR-21 and low levels of miR-141. miR-142 3p). and miR-146a. The same drawing conventions are used as in Fig. 1.

Fig. 4.. Gene circuits that use feedback regulation to sense systemic biomarkers and secrete systemically acting effector molecules enable homeostasis.

A) A closed-loop circuit to treat obesity responds to fatty acids and produces pramlintide to slow gastric emptying, reduce glucagon, and modulate satiety. B) A cytokine converter circuit to treat psoriasis responds to inflammatory singnals TNF- and IL-22 and produces anti-psoriatic and anti-inflammatory cytokines, IL-4 and IL-10, respectively. Dashed arrows indicate multiple steps. The same drawing conventions are used as in Fig.1.