The Chemical and Biological Engineering Department is pleased to present student seminars by Gauri Bora and Amparo Cosio of the Leonard lab as part of our ChBE Seminar Series.

Gauri Bora will present a seminar titled "Engineering synthetic epigenetic regulation to enable state-switching genetic programs in mammalian cells."

Engineered cell therapies are a promising frontier, with early successes in cancer treatment demonstrating the transformative potential of this approach. Most cell therapies today are programmed to express outputs at a steady-state; however, dynamic control of cell gene expression, such as state-switching, could enable implementation of novel therapeutic functionalities, such as autonomous stem cell differentiation or pulsating therapeutic delivery. In nature, eukaryotic systems have evolved epigenetic control to induce, maintain, and switch states, but these behaviors are challenging to implement in a synthetic, orthogonal context when building novel genetic programs. A reason this challenge arises is the lack of understanding as to how specific biological mechanisms interact; it remains unknown how the use of synthetic epigenetic- or chromatin-modifying domains can be composed into genetic programs to modulate the behavior of a cell. While previous work has generated quantitative insights for mapping biological understanding into these mechanisms, fundamental design principles for implementing epigenetic regulation remain unexplored. This leaves biologists with few tools regarding how to construct genetic programs to encode cells with dynamic behaviors.

To address this need, I characterized and employed synthetic transcription factors (synTFs) that incorporate a library of repressing chromatin regulatory domains (CRD) fused to a panel of DNA binding domains that can be implemented with precise local composition using cognate promoters. I explored key design criteria for using these domains in genetic programs, including: (1) effect on gene expression and memory when recruiting both activating and repressing synTFs to a promoter; (2) synTF dominance due to temporal and spatial organizations; (3) variation in promoter binding site composition to modulate cooperative interactions. Though previous literature suggests synTFs with CRDs will silence most activated genes, I found varied levels of repression across different promoter types. Moreover, when recruited with an activating synTF, the spatial orientation and stoichiometric ratios of the synTFs greatly impact the level of conferred silencing, revealing previously unknown rules of dominance of competing biological mechanisms. Lastly, using our unique testbed that allows for intricate promoter architecture, we discovered combinations of CRDs that can be leveraged to make versatile gene expression profiles with distinct memory and potency. This type of deep characterization will enable us to build more complex programs that require multiple handles to tune dynamic gene expression. Overall, these exhaustive design principles will guide the next phase of the project, in which we construct a small panel of state-switching programs with therapeutic utility. Achieving these important and sophisticated functionalities will be a crucial step towards building safer and more effective cell-based therapies.

Amparo Cosio will present a seminar titled "Ultra-Compact Synthetic Transcription Factor Platform for Small-Molecule Regulated Control of AAV Transgene Expression."

Gene therapies often employ ubiquitous or tissue-specific promoters to drive constitutive transgene expression. However, the ability to fine-tune transgene expression in response to patient-specific needs is crucial for optimizing therapeutic efficacy. Inducible gene expression systems offer this control, yet the packaging constraints of widely used AAV vectors precludes the use of existing systems for such vectors. Developing compact, small-molecule-inducible systems would address these challenges and enhance the versatility of gene therapies across a range of clinical applications. In this study, we describe the development of new ultra-compact, potent, and tunable, small molecule-induced gene regulation systems optimized for use in AAV vectors. We adapted minimal zinc finger transcription factors to make them controllable by FDA-approved drugs, which we termed minimal inducible synthetic transcription factors (mini-synTFs). We demonstrated the ability to employ multiple small-molecule controllable modalities to conditionally regulate transgene expression from single AAV vectors. This approach identified promising compositions employing multiple small molecule-controllable systems. These technologies will enable precise control over transgene expression in gene therapy applications.

Bagels and coffee will be provided at 9:30am, and the seminar will start at 9:40am. Please plan to arrive on time to grab a bagel and mingle!

*Please note that there will be no Zoom option for seminars this year.