A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

Win, Maung Nyan; Smolke, Christina D.

doi:10.1073/pnas.0703961104

Cited by 363 publications

(384 citation statements)

References 32 publications

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“…We developed a RNA-based regulatory system for mammalian T-cell proliferation on a platform for assembling RNA devices from modular sensor (aptamer) and gene-regulatory (hammerhead ribozyme) components. The activity of this ribozyme switch platform had been shown in the microorganism Saccharomyces cerevisiae (19,20); however, the activity of these genetic regulatory elements in mammalian cells had not been previously examined.…”

Section: Resultsmentioning

confidence: 99%

“…The cytokine and reporter proteins were linked through a self-cleaving T2A peptide (SI Text) to ensure that the ribozyme switch activity was equally effective on the linked target genes but that the proteins fold and function as independent molecules. Three theophylline (theo)-responsive ribozyme switches (L2bulge1, 8, and 9; SI Text), which had been tuned through sequence modifications to exhibit different regulatory response properties (19), were inserted into the 3′ UTR of the egfp-t2a-il2 fusion gene. Plasmids incorporating this regulatory system were transiently transfected into the CTLL-2 mouse T-cell line, which, like primary human T cells, is dependent on common γ-chain signaling for survival and proliferation (21).…”

Section: Resultsmentioning

confidence: 99%

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

Chen

Jensen

Smolke

2010

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

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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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

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.

Self Cite

242

245

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“…22 ), RNA cleavage (see refs. [23][24] ), ribosome shunting (see ref. 25 ) or trans-regulation of transcription.…”

Section: Tandem Arrangement Allows Fast and Easy Enhancement Of Activmentioning

confidence: 99%

Design criteria for synthetic riboswitches acting on transcription

et al. 2015

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Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

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“…Protein designers have used domain insertion to create allosteric switches with mechanisms ranging from subtle conformational changes to mutually exclusive folding (7)(8)(9). A conceptually similar approach has been used successfully to engineer highly-modular RNA switches (10). The other type of indel, end-to-end fusion, imposes fewer constraints on the relative orientation of the domains, especially if the linker region is unstructured.…”

mentioning

confidence: 99%

Light-activated DNA binding in a designed allosteric protein

Strickland

Moffat

Sosnick

2008

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

283

268

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An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an ␣-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical ''allosteric lever arm'' is a general scheme for coupling the function of two proteins.allostery ͉ LOV domain ͉ protein design ͉ trp repressor ͉ alpha helix

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A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

Cited by 363 publications

References 32 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

Design criteria for synthetic riboswitches acting on transcription

Light-activated DNA binding in a designed allosteric protein

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