Synthetic RNA-based post-transcriptional expression control methods and genetic circuits

Pardi, Malvin L.; Wu, Juanqi; Kawasaki, Shunsuke; Saito, Hirohide

doi:10.1016/j.addr.2022.114196

Cited by 12 publications

(6 citation statements)

References 128 publications

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“… 9 , 25 However, this strategy is only compatible with synthetic mRNAs that contain specific chemical modifications and might therefore be limited to applications such as short-lived RNA drugs or various in vitro cell purification procedures. 54 So far, this approach remains inapplicable for either genetically encoded expression over longer time periods or integration into complex multi-layered gene circuits to achieve programmable activity in vivo. In contrast, STIF-dependent genetic sensors not only enable generalizable detection of intracellular target signals with high spatial resolution (Fig.…”

Section: Discussionmentioning

confidence: 99%

Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells

Shao,

Li,

Qiu

et al. 2024

Cell Res

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Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A) signal of target genes and replacing it with a synthetic control region harboring RNA-binding protein (RBP)-specific aptamers, cap-dependent translation is rendered exclusively dependent on synthetic translation initiation factors (STIFs) containing different RBPs engineered to conditionally associate with different eIF4F-binding proteins (eIFBPs). This modular design framework facilitates the engineering of various gene switches and intracellular sensors responding to many user-defined trigger signals of interest, demonstrating tightly controlled, rapid and reversible regulation of transgene expression in mammalian cells as well as compatibility with various clinically applicable delivery routes of in vivo gene therapy. Therapeutic efficacy was demonstrated in two animal models. To exemplify disease treatments that require on-demand drug secretion, we show that a custom-designed gene switch triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice. For diseases that require instantaneous sense-and-response treatment programs, we create highly specific sensors for various subcellularly (mis)localized protein markers (such as cancer-related fusion proteins) and show that translation-based protein sensors can be used either alone or in combination with other cell-state classification strategies to create therapeutic biocomputers driving self-sufficient elimination of tumor cells in mice. This design strategy demonstrates unprecedented flexibility for translational regulation and could form the basis for a novel class of programmable gene therapies in vivo.

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Section: Discussionmentioning

confidence: 99%

Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells

Shao,

Li,

Qiu

et al. 2024

Cell Res

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“…In the case of the miR-206-responsive circRNA switch, 2-copy insertion into both the 5′ and 3′ UTRs (2 × 2 insertion, 4 copies total) showed the best fold-change ( Supplementary Figure S13 , left). These observations suggest that sequence dependency in circRNA systems is more pronounced than in linRNA systems, and that target miRNA-specific optimization steps will be required to maximize performance for circRNA switches ( 59 ). Recently, Chen et al reported the sequence elements that enhance circRNA performance ( 41 ).…”

Section: Discussionmentioning

confidence: 99%

Synthetic circular RNA switches and circuits that control protein expression in mammalian cells

Kameda

Ohno

Saito

2023

Nucleic Acids Research

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Synthetic messenger RNA (mRNA) has been focused on as an emerging application for mRNA-based therapies and vaccinations. Recently, synthetic circular RNAs (circRNAs) have shown promise as a new class of synthetic mRNA that enables superior stability and persistent gene expression in cells. However, translational control of circRNA remained challenging. Here, we develop ‘circRNA switches’ capable of controlling protein expression from circRNA by sensing intracellular RNA or proteins. We designed microRNA (miRNA) and protein-responsive circRNA switches by inserting miRNA-binding or protein-binding sequences into untranslated regions (UTRs), or Coxsackievirus B3 Internal Ribosome Entry Site (CVB3 IRES), respectively. Engineered circRNAs efficiently expressed reporter proteins without inducing severe cell cytotoxicity and immunogenicity, and responded to target miRNAs or proteins, controlling translation levels from circRNA in a cell type-specific manner. Moreover, we constructed circRNA-based gene circuits that selectively activated translation by detecting endogenous miRNA, by connecting miRNA and protein-responsive circRNAs. The designed circRNA circuits performed better than the linear mRNA-based circuits in terms of persistent expression levels. Synthetic circRNA devices provide new insights into RNA engineering and have a potential for RNA synthetic biology and therapies.

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“…Wroblewska et al [75] used RBPs to function as both the input and the output of RNA regulatory devices in post-transcriptional circuits, thus making it possible to design circuits that would have control over cellular behavior without genetic modifications. Numerous reports have further reviewed these biological parts and their functions within a given circuit [76][77][78][79][80][81][82][83].…”

Section: Design Of Genetic Circuitsmentioning

confidence: 99%

A Genetic Circuit Design for Targeted Viral RNA Degradation

Bello,

Popoola,

Okpuzor

et al. 2023

Bioengineering

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Advances in synthetic biology have led to the design of biological parts that can be assembled in different ways to perform specific functions. For example, genetic circuits can be designed to execute specific therapeutic functions, including gene therapy or targeted detection and the destruction of invading viruses. Viral infections are difficult to manage through drug treatment. Due to their high mutation rates and their ability to hijack the host’s ribosomes to make viral proteins, very few therapeutic options are available. One approach to addressing this problem is to disrupt the process of converting viral RNA into proteins, thereby disrupting the mechanism for assembling new viral particles that could infect other cells. This can be done by ensuring precise control over the abundance of viral RNA (vRNA) inside host cells by designing biological circuits to target vRNA for degradation. RNA-binding proteins (RBPs) have become important biological devices in regulating RNA processing. Incorporating naturally upregulated RBPs into a gene circuit could be advantageous because such a circuit could mimic the natural pathway for RNA degradation. This review highlights the process of viral RNA degradation and different approaches to designing genetic circuits. We also provide a customizable template for designing genetic circuits that utilize RBPs as transcription activators for viral RNA degradation, with the overall goal of taking advantage of the natural functions of RBPs in host cells to activate targeted viral RNA degradation.

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Synthetic RNA-based post-transcriptional expression control methods and genetic circuits

Cited by 12 publications

References 128 publications

Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells

Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells

Synthetic circular RNA switches and circuits that control protein expression in mammalian cells

A Genetic Circuit Design for Targeted Viral RNA Degradation

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