An enduring goal of synthetic biology is to engineer cells to perform increasingly sophisticated therapeutic functions. Inspired by natural gene expression programs that elegantly govern diverse cellular behaviors, numerous technological advances have employed synthetic transcriptional programs to coordinate therapeutic cellular activities. These programs are mediated by heterologous or engineered transcription factors that orthogonally regulate the expression of therapeutic agents. However, key features of existing transcriptional components and programs render them unsuitable for therapeutic applications, including high potential for immunogenic or off-target effects and challenging delivery of large genetic payloads. There remains a need for well-designed elements that overcome these fundamental barriers to translation and ultimately enable customizable, tunable, and effective therapeutic responses.
Here, we describe an advanced platform for synthetic transcriptional control poised for diverse therapeutic applications. We first engineered a class of programmable transcriptional regulators based upon zinc finger DNA-binding domains, which are highly advantageous due to their compact size and derivation from native mammalian transcriptional systems. We constructed a library of humanized synthetic transcription factors with binding motifs that were unique and putatively orthogonal to the human genome. Our evaluation of cellular transcriptome response when these synthetic components were expressed in cell lines revealed highly specific on-target and low off-target regulation.
Furthermore, we developed two highly useful classes of regulatable gene expression programs, by connecting our synthetic transcription factors to domains responsive to favorable exogenous or endogenous signals. We first generated synthetic transcription programs responsive to safe and FDA-approved small molecules, and demonstrated how these programs could tunably and dynamically regulate the expression of therapeutic agents in relevant in vitro and in vivo contexts. Moreover, we constructed synthetic transcription programs responsive to desirable endogenous signals using customizable synthetic Notch receptors. Taken together, we envision that our advanced platform for synthetic transcriptional regulation will facilitate the design of sophisticated therapeutic programs for a wide range of gene and cell therapy applications. / 2022-01-18T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/41884 |
| Date | 19 January 2021 |
| Creators | Israni, Divya V. |
| Contributors | Khalil, Ahmad S. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0017 seconds