Model-guided design of mammalian genetic programs

Muldoon, Joseph J.; Kandula, Viswajit; Hong, Mihe; Donahue, Patrick S.; Boucher, Jonathan; Bagheri, Neda; Leonard, Joshua N.

doi:10.1126/sciadv.abe9375

Cited by 26 publications

(31 citation statements)

References 73 publications

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“…Mathematical modeling validated with experimental measurements can provide a rapid characterization of parts and a reliable prediction of circuit performance under a broad range of conditions that are challenging and costly to achieve by experiments. The success of using mathematical modeling to quantify and guide the design of synthetic gene circuits has been reported in various systems from bacteria to mammalian cells [29][30][31][32][33]. Given previous successes with model-guided design with the evaluation of the gene pulse generator and IFFL circuits [25,[34][35][36], here, we develop ordinary differential equation (ODE)-based mathematical models to first explore the capability of an RNA-only circuit functioning as an IFFL circuit for pulse generation and then, to guide the design and experimental realization of an RNA-protein hybrid IFFL circuit with predictable dynamics.…”

Section: Plasmid Construction and E Coli Strains Usedmentioning

confidence: 99%

Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits

et al. 2021

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RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toehold switch (THS), and three-way junction (3WJ) repressor, have been utilized to construct RNA-based synthetic gene circuits in living cells. In this work, we utilized these regulators to construct type 1 incoherent feed-forward loop (IFFL) circuits in vivo and explored their dynamic behaviors. A combination of a STAR and 3WJ repressor was used to construct an RNA-only IFFL circuit. However, due to the fast kinetics of RNA–RNA interactions, there was no significant timescale difference between the direct activation and the indirect inhibition, that no pulse was observed in the experiments. These findings were confirmed with mechanistic modeling and simulation results for a wider range of conditions. To increase delay in the inhibition pathway, we introduced a protein synthesis process to the circuit and designed an RNA–protein hybrid IFFL circuit using THS and TetR protein. Simulation results indicated that pulse generation could be achieved with this RNA–protein hybrid model, and this was further verified with experimental realization in E. coli. Our findings demonstrate that while RNA-based regulators excel in speed as compared to protein-based regulators, the fast reaction kinetics of RNA-based regulators could also undermine the functionality of a circuit (e.g., lack of significant timescale difference). The agreement between experiments and simulations suggests that the mechanistic modeling can help debug issues and validate the hypothesis in designing a new circuit. Moreover, the applicability of the kinetic parameters extracted from the RNA-only circuit to the RNA–protein hybrid circuit also indicates the modularity of RNA-based regulators when used in a different context. We anticipate the findings of this work to guide the future design of gene circuits that rely heavily on the dynamics of RNA-based regulators, in terms of both modeling and experimental realization.

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Section: Plasmid Construction and E Coli Strains Usedmentioning

confidence: 99%

Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits

et al. 2021

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“…The most straightforward is to insert suicide genes, leading to cell death after the administration of defined molecules [ 69 ]. Alternatively, more refined ways to improve safety, control and precision based on the Boolean logic have emerged [ 70 , 71 ]. For instance, the detection of a pathological state can be improved by the dual recognition of two inducing signals, both required to trigger effector outputs: this is an AND-gate ( Figure 3 c) [ 63 , 65 ].…”

Section: Synthetic Circuit To Control Cellular Responsesmentioning

confidence: 99%

Exploiting B Cell Transfer for Cancer Therapy: Engineered B Cells to Eradicate Tumors

Page

Hubert

Fusil

et al. 2021

IJMS

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Nowadays, cancers still represent a significant health burden, accounting for around 10 million deaths per year, due to ageing populations and inefficient treatments for some refractory cancers. Immunotherapy strategies that modulate the patient’s immune system have emerged as good treatment options. Among them, the adoptive transfer of B cells selected ex vivo showed promising results, with a reduction in tumor growth in several cancer mouse models, often associated with antitumoral immune responses. Aside from the benefits of their intrinsic properties, including antigen presentation, antibody secretion, homing and long-term persistence, B cells can be modified prior to reinfusion to increase their therapeutic role. For instance, B cells have been modified mainly to boost their immuno-stimulatory activation potential by forcing the expression of costimulatory ligands using defined culture conditions or gene insertion. Moreover, tumor-specific antigen presentation by infused B cells has been increased by ex vivo antigen loading (peptides, RNA, DNA, virus) or by the sorting/ engineering of B cells with a B cell receptor specific to tumor antigens. Editing of the BCR also rewires B cell specificity toward tumor antigens, and may trigger, upon antigen recognition, the secretion of antitumor antibodies by differentiated plasma cells that can then be recognized by other immune components or cells involved in tumor clearance by antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity for example. With the expansion of gene editing methodologies, new strategies to reprogram immune cells with whole synthetic circuits are being explored: modified B cells can sense disease-specific biomarkers and, in response, trigger the expression of therapeutic molecules, such as molecules that counteract the tumoral immunosuppressive microenvironment. Such strategies remain in their infancy for implementation in B cells, but are likely to expand in the coming years.

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“…His group has developed mathematical models about why COMET works that have revealed quantitative design rules for building and implementing transcriptional control. Leonard showed that these rules enable predictive design of mammalian genic programs without tuning or refinement 14 …”

Section: Keynote Address: New Tools For Engineering Mammalian Cellsmentioning

confidence: 99%

Synthetic biology: at the crossroads of genetic engineering and human therapeutics—a Keystone Symposia report

Cable¹,

Leonard

et al. 2021

Annals of the New York Academy of Sciences

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Synthetic biology has the potential to transform cell‐ and gene‐based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease‐related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium “Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics,” which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell‐based therapies.

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Model-guided design of mammalian genetic programs

Cited by 26 publications

References 73 publications

Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits

Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits

Exploiting B Cell Transfer for Cancer Therapy: Engineered B Cells to Eradicate Tumors

Synthetic biology: at the crossroads of genetic engineering and human therapeutics—a Keystone Symposia report

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