Small-molecule-based regulation of RNA-delivered circuits in mammalian cells

Wagner, Tyler; Becraft, Jacob; Bodner, Katie; Teague, Brian; Zhang, Xin; Woo, Amanda Rui En; Porter, Ely; Alburquerque, Bremy; Dobosh, Brian; Andries, Oliwia; Sanders, Niek N.; Beal, Jacob; Densmore, Douglas; Kitada, Tasuku; Weiss, Ron

doi:10.1038/s41589-018-0146-9

Cited by 56 publications

(58 citation statements)

References 42 publications

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“…Because inducible translation systems enable setting the expression level and time at suitable ranges for therapies, they will be an important component for future gene therapies. Although a drug-regulatable translational repressor was recently reported 11 , to our knowledge, ours is the first report of a drug-regulatable translational activator for RNA circuits. Furthermore, because the domains of drug-regulatable CaVT are independent modules, the DmrA and DmrC used in this study can be exchanged with any other inducible hetero-dimerization domain 36,47 .…”

Section: Discussionmentioning

confidence: 78%

“…In the present study, we developed the novel translational activator, CaVT, which functions in RNA-based mammalian synthetic circuits. To date, several RBPs such as L7Ae, MS2CP, U1A, Lin28A, and TetR have been used to develop RNA circuits [9][10][11]37 . While the translational repression of mRNAs has been achieved by these RBPs, direct translational activation in mammalian cells is rather difficult.…”

Section: Discussionmentioning

confidence: 99%

“…While the translational repression of mRNAs has been achieved by these RBPs, direct translational activation in mammalian cells is rather difficult. Thus, the "repression of translational repressors" approach has been used for indirect translational activation [9][10][11] . However, this layered approach makes the design of circuits complicated.…”

Section: Discussionmentioning

confidence: 99%

“…Especially, while transcriptional activators are important components in DNA circuits [6][7][8] , the direct activation of gene expression is difficult in RNA circuits. Rather, in current RNA circuits, the activation of gene expressions has been achieved by the repression of translational repressors by combining multiple RNA-binding proteins (RBPs) 9,10,11 . However, in such indirect methods, the number of necessary components tend to increase, complicating the system.…”

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confidence: 99%

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Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Nakanishi

Saito

2020

Nat Commun

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Synthetic RNA-based gene circuits enable sophisticated gene regulation without the risk of insertional mutagenesis. While various RNA binding proteins have been used for translational repression in gene circuits, the direct translational activation of synthetic mRNAs has not been achieved. Here we develop Caliciviral VPg-based Translational activator (CaVT), which activates the translation of synthetic mRNAs without the canonical 5′-cap. The level of translation can be modulated by changing the locations, sequences, and modified nucleosides of CaVT-binding motifs in the target mRNAs, enabling the simultaneous translational activation and repression of different mRNAs with RNA-only delivery. We demonstrate the efficient regulation of apoptosis and genome editing by tuning translation levels with CaVT. In addition, we design programmable CaVT that responds to endogenous microRNAs or small molecules, achieving both cell-state-specific and conditional translational activation from synthetic mRNAs. CaVT will become an important tool in synthetic biology for both biological studies and future therapeutic applications.

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Section: Discussionmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Nakanishi

Saito

2020

Nat Commun

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“…Because they remain outside the nucleus and thereby avoid the potential risk for insertional mutagenesis, RNA viruses are attractive candidates for future synthetic circuit delivery vectors. In fact, while DNA vectors currently remain more prevalent in therapeutic applications, RNA viruses have received growing attention 40 , including from synthetic biologists seeking to improve their specificity [41][42][43] . Here we sought to address key challenges required to make RNA viruses into a more engineerable, and safer, alternative to DNA vectors.…”

Section: Discussionmentioning

confidence: 99%

Engineering multiple levels of specificity in an RNA viral vector

Gao

Chong

Ince

et al. 2020

Preprint

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Synthetic molecular circuits could provide powerful therapeutic capabilities, but delivering them to specific cell types and controlling them remains challenging. An ideal “smart” viral delivery system would enable controlled release of viral vectors from “sender” cells, conditional entry into target cells based on cell-surface proteins, conditional replication specifically in target cells based on their intracellular protein content, and an evolutionarily robust system that allows viral elimination with drugs. Here, combining diverse technologies and components, including pseudotyping, engineered bridge proteins, degrons, and proteases, we demonstrate each of these control modes in a model system based on the rabies virus. This work shows how viral and protein engineering can enable delivery systems with multiple levels of control to maximize therapeutic specificity.

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Lipid Nanoparticle Delivery of Chemically Modified NGF^R100W mRNA Alleviates Peripheral Neuropathy

Yang

Xia

et al. 2022

Adv Healthcare Materials

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Messenger RNA (mRNA) carries genetic instructions to the cell machinery for the transient production of antigens or therapeutic proteins and shows enormous potential in vaccine development, cancer immunotherapy, protein replacement therapy, and genome engineering. Here, the synthesis of chemically modified nerve growth factor mutant (NGF R100W ) mRNA through in vitro transcription is described. After the replacement of the original signal peptide sequence with the Ig Kappa leader sequence, codon-optimized NGF R100W mRNA yielded high secretion of mature NGF R100W , which promotes axon growth in PC12 cells. Using lipid nanoparticle (LNP)-delivery of N1-methylpseudouridine-modified mRNA in mice, NGF R100W -mRNA-LNPs result in the successful expression of NGF R100W protein, which significantly reduces nociceptive activity compared to that of NGF WT . This indicates that NGF R100W derived from exogenous mRNA elicited "painless" neuroprotective activity. Additionally, the therapeutic value of NGF R100W mRNA is established in a paclitaxel-induced peripheral neuropathy model by demonstrating the rapid recovery of intraepidermal nerve fibers. The results show that in vitro-transcribed mRNA has significant flexibility in sequence design and fast in vivo functional validation of target proteins. Furthermore, the results highlight the therapeutic potential of mRNA as a supplement to beneficial proteins for preventing or reversing some chronic medical conditions, such as peripheral neuropathy.

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Small-molecule-based regulation of RNA-delivered circuits in mammalian cells

Cited by 56 publications

References 42 publications

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Engineering multiple levels of specificity in an RNA viral vector

Lipid Nanoparticle Delivery of Chemically Modified NGF^R100W mRNA Alleviates Peripheral Neuropathy

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Small-molecule-based regulation of RNA-delivered circuits in mammalian cells

Cited by 56 publications

References 42 publications

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Engineering multiple levels of specificity in an RNA viral vector

Lipid Nanoparticle Delivery of Chemically Modified NGFR100W mRNA Alleviates Peripheral Neuropathy

Contact Info

Product

Resources

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Lipid Nanoparticle Delivery of Chemically Modified NGF^R100W mRNA Alleviates Peripheral Neuropathy