Browsing by Author "Brenner, Daniel A."

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    Compact engineered human mechanosensitive transactivation modules enable potent and versatile synthetic transcriptional control
    (Springer Nature, 2023) Mahata, Barun; Cabrera, Alan; Brenner, Daniel A.; Guerra-Resendez, Rosa Selenia; Li, Jing; Goell, Jacob; Wang, Kaiyuan; Guo, Yannie; Escobar, Mario; Parthasarathy, Abinand Krishna; Szadowski, Hailey; Bedford, Guy; Reed, Daniel R.; Kim, Sunghwan; Hilton, Isaac B.
    Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR–Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here, we defined and optimized minimal TADs built from human mechanosensitive transcription factors. We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS and eN3x9) and to build the CRISPR–dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific and robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II and V CRISPR systems, transcription activator-like effectors and zinc finger proteins. Further, as proofs of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into induced pluripotent stem cells and demonstrated that mechanosensitive transcription factor TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules and delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.
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    Persistent tailoring of MSC activation through genetic priming
    (Elsevier, 2024) Beauregard, Michael A.; Bedford, Guy C.; Brenner, Daniel A.; Sanchez Solis, Leonardo D.; Nishiguchi, Tomoki; Abhimanyu; Longlax, Santiago Carrero; Mahata, Barun; Veiseh, Omid; Wenzel, Pamela L.; DiNardo, Andrew R.; Hilton, Isaac B.; Diehl, Michael R.; Chemistry; Bioengineering
    Mesenchymal stem/stromal cells (MSCs) are an attractive platform for cell therapy due to their safety profile and unique ability to secrete broad arrays of immunomodulatory and regenerative molecules. Yet, MSCs are well known to require preconditioning or priming to boost their therapeutic efficacy. Current priming methods offer limited control over MSC activation, yield transient effects, and often induce the expression of pro-inflammatory effectors that can potentiate immunogenicity. Here, we describe a genetic priming method that can both selectively and sustainably boost MSC potency via the controlled expression of the inflammatory-stimulus-responsive transcription factor interferon response factor 1 (IRF1). MSCs engineered to hyper-express IRF1 recapitulate many core responses that are accessed by biochemical priming using the proinflammatory cytokine interferon-γ (IFN-γ). This includes the upregulation of anti-inflammatory effector molecules and the potentiation of MSC capacities to suppress T cell activation. However, we show that IRF1-mediated genetic priming is much more persistent than biochemical priming and can circumvent IFN-γ-dependent expression of immunogenic MHC class II molecules. Together, the ability to sustainably activate and selectively tailor MSC priming responses creates the possibility of programming MSC activation more comprehensively for therapeutic applications.