Engineered riboregulators enable post-transcriptional control of gene expression

Isaacs, Farren J.; Dwyer, Daniel J.; Ding, Chunming; Pervouchine, Dmitri D.; Cantor, Charles R.; Collins, James J.

doi:10.1038/nbt986

Cited by 507 publications

(504 citation statements)

References 47 publications

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“…Alternative strategies with unregulated expression of growth-related genes pose the risk of uncontrolled lymphoproliferation and leukemic transformation (6). Thus, the ability to integrate growth-stimulatory gene expression with tightly controlled regulatory systems has the potential to greatly improve the safety and efficacy of adoptive T-cell therapy.Inspired by the diverse functional roles exhibited by regulatory RNAs in natural systems (12-14) and the relative ease by which RNA can be modeled and designed (15), researchers have begun developing synthetic RNA-based regulatory systems that integrate discrete gene-regulatory and sensing functions as genetic control strategies (16)(17)(18). However, the absence of successful adaptations of these earlier genetic devices to the regulation of functional responses in mammalian cells highlights remaining difficulties in translating designs that regulate reporter gene expression to functional control.…”

mentioning

confidence: 99%

Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

Chen

Jensen

Smolke

2010

Proc. Natl. Acad. Sci. U.S.A.

239

245

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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, ribozymebased 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 RNAbased 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. T he ability to control functional responses in mammalian cells with customizable and compact regulatory systems in vivo addresses a critical need in diverse clinical applications, particularly in cellular therapies (1). As an example, adoptive T-cell therapy seeks to harness the precision and efficacy of the immune system against diseases that escape the body's natural surveillance. The adoptive transfer of antigen-specific T cells can reconstitute immunity to viruses and mediate tumor regression (2-4). T cells engineered to express tumor-specific T-cell antigen receptors can achieve highly refined target recognition (5), thus minimizing toxic off-target effects associated with conventional chemotherapy. However, considerable research has shown that the persistence of transferred T cells in vivo is both central to therapeutic success and elusive to current technology (6, 7). The efficacy of adoptive immunotherapy in humans is often limited by the failure of transferred T cells to survive in the host (8, 9).Clonal expansion of T cells is a critical component of T-cell activation mediated by cytokines such as IL-2 and IL-15, which activate JAK-STAT signaling pathways and lead to the expression of genes involved in growth modulation (10) (Fig. 1A). Sustaining the survival and proliferation of Tcells following adoptive transfer is challenging because of the limited availability of homeostatic cytokines (IL-15/IL-7) and stimulatory antigen presenting cells. State-of-the-art strategies for improving the persistence of adoptively transferred lymphocytes require that patients be subjected to myeloablative total body irradiation/chemother...

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mentioning

confidence: 99%

Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

Chen

Jensen

Smolke

2010

Proc. Natl. Acad. Sci. U.S.A.

239

245

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“…A longer spacer means less output, as the bound ribosome must travel further to begin translation and therefore has a greater chance of failing to reach the start codon. Although ribosome binding site modification is a common tuning method in gene circuits (15)(16)(17), the strategy of Egbert and Klavins (1) stands out for its quantitative simplicity: to evaluate the strength, simply count the number of bases.…”

Section: Tuning Knobs For Genetic Circuitsmentioning

confidence: 99%

Making gene circuits sing

Prindle

Hasty

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Biomolecular implementations of finite automata 8,9 and logic gates 4,[10][11][12][13] have already been developed [14][15][16][17][18] . Here, we report an autonomous programmable molecular system based on the manipulation of DNA strands that is capable of performing simple logical deductions.…”

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confidence: 99%

Molecular implementation of simple logic programs

2009

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Autonomous programmable computing devices made of biomolecules could interact with a biological environment and be used in future biological and medical applications [1][2][3][4][5][6][7] . Biomolecular implementations of finite automata 8,9 and logic gates 4,10-13 have already been developed [14][15][16][17][18] . Here, we report an autonomous programmable molecular system based on the manipulation of DNA strands that is capable of performing simple logical deductions. Using molecular representations of facts such as Man(Socrates) and rules such as Mortal(X) Man(X) (Every Man is Mortal), the system can answer molecular queries such as Mortal(Socrates)? (Is Socrates Mortal?) and Mortal(X)? (Who is Mortal?). This biomolecular computing system compares favourably with previous approaches in terms of expressive power, performance and precision 2,4,8,9,11,12,19 . A compiler translates facts, rules and queries into their molecular representations and subsequently operates a robotic system that assembles the logical deductions and delivers the result. This prototype is the first simple programming language with a molecular-scale implementation.Our logic system consists of propositions, implications and queries, represented by short DNA molecules, some single-stranded and some double-stranded, as shown in Fig. 1a. A proposition p, such as 'Socrates is a Man', is represented by a double-stranded (ds) DNA molecule with a sticky end representing p on one end and a fluorophore (a fluorescent molecule) with a matching quencher (a molecule that absorbs excitation energy from a fluorophore) at its other end. A query p?, such as 'Is Socrates a Man?', is represented by a dsDNA molecule with a sticky end complementary to the representation of p and a recognition site (marked light red) for the restriction enzyme FokI. A molecular query p? can be positively answered by the molecular proposition p, because they have complementary sticky ends, as shown in Fig. 1b. The sticky end of the query molecule q? hybridizes with that of the proposition molecule q. FokI is attracted to its recognition site on the query (before hybridization or possibly following it) and cleaves the proposition molecule q. This results in the separation of its remaining sense and antisense strands, which in turn abolishes the quenching of the green fluorophore, allowing the fluorophore to emit green light in response to light excitation. This green emission can be interpreted by an outside observer as a positive response to the query.An implication p q (for example 'Socrates is Mortal if Socrates is a Man') is represented by a hairpin single-stranded (ss) DNA with three components: a sticky end representing the proposition p, a segment complementary to the representation of q and a segment that together with an auxiliary complementary strand forms a recognition site for FokI. The implication p q can be used to reduce the query p? to the query q?, meaning that to positively answer p? it suffices to positively answer q? This query reduction is justified by the d...

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Engineered riboregulators enable post-transcriptional control of gene expression

Cited by 507 publications

References 47 publications

Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

Making gene circuits sing

Molecular implementation of simple logic programs

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