Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells

Love, Kasey S.; Johnstone, Christopher P.; Peterman, Emma L.; Gaglione, Stephanie; Galloway, Kate E.

doi:10.1101/2024.06.25.600629

Cited by 2 publications

(1 citation statement)

References 66 publications

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“…58,77 While the DIAL framework provides many insights on engineering synthetic promoter systems, there remain limitations and opportunities to expand the capabilities. 50,59,60,[85][86][87] In its current form, DIAL allows us to increase but not decrease the level of the setpoint, limiting application to systems where a reduction in the setpoint is needed. Although permanent DNA-level editing of DIAL offers stability and long-term, hereditable memory, setpoint changes are irreversible.…”

Section: Dial Is Portable To Primary Cells and Ipscs And Regulates Di...mentioning

confidence: 99%

Programmable promoter editing for precise control of transgene expression

Kabaria,

Bae,

Ehmann

et al. 2024

Preprint

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Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes requires tools for precisely titrating expression. To this end, we developed a framework called DIAL for building editable promoters that allows for fine-scale, heritable changes in transgene expression. Using DIAL, we increase expression by recombinase-mediated excision of spacers between the binding sites of a synthetic zinc-finger transcription factor and the core promoter. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of unimodal setpoints from a single promoter construct. Through small-molecule control of transcription factors and recombinases, DIAL supports temporally defined, user-guided control of transgene expression. Integration of DIAL promoters into lentivirus allows for efficient delivery to primary cells. As promoter editing generates stable states, DIAL setpoints are heritable, facilitating mapping of transgene levels to phenotypes. The highly modular and extensible DIAL framework opens up new opportunities for screening and tailoring transgene expression to regulate gene and cell-based therapies.HighlightsPromoter editing generates a range of unimodal setpoints from DIAL, a synthetic promoter systemLength of the excisable spacer and identity of the zinc-finger activator tune setpointsNested, excisable spacers expand the number of unimodal setpointsDIAL generates stable setpoints that are robust to varying transactivator levelsDIAL transmits transient inputs into heritable statesThe TET-DIAL system enables small molecule activation of defined setpointsDIAL controls expression in primary cells and iPSCs; regulates physiologically-relevant transgenesOne Sentence SummaryDIAL offers an extensible framework for designing synthetic promoters that generate heritable setpoints of gene expression and performs across a range of cell types and delivery systems.OverviewDIAL offers an extensible framework for designing synthetic promoters that generate heritable setpoints of gene expression and perform across a range of cell types and delivery systems.

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Section: Dial Is Portable To Primary Cells and Ipscs And Regulates Di...mentioning

confidence: 99%

Programmable promoter editing for precise control of transgene expression

Kabaria,

Bae,

Ehmann

et al. 2024

Preprint

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Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems

Katz,

An,

Lee

et al. 2024

Preprint

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SummaryGene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery. We demonstrate dosage control for activation and inhibition, post-delivery tuning, and RNA-based delivery, for a genome-integrated marker. We then target theRAI1gene, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes, Smith-Magenis-Syndrome (SMS) and Potocki-Lupski-Syndrome, respectively. We demonstrate dosage control for RAI1 activation in HEK293s, Neuro-2As, and mouse cortical neurons via AAVs and lentiviruses. Finally, we activate the intactRAI1copy in SMS patient-derived cells to an estimated two-copy healthy range, avoiding the harmful three-copy regime. Our circuit paves the way for viable therapy in dosage-sensitive disorders, creating precise and tunable gene regulation systems for basic and translational research.

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Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells

Cited by 2 publications

References 66 publications

Programmable promoter editing for precise control of transgene expression

Programmable promoter editing for precise control of transgene expression

Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems

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