Establishment of a Reliable Reporter System

One of our most significant achievements was the creation of a reliable mScarlet-based DUX4 reporter system that allowed us to visualize and quantify DUX4 activity in real time in muscle cells. Adapted from an existing luciferase reporter, our system provided critical data on the pathogenic activity of DUX4 in a cellular environment and enabled us to measure the efficacy of our DUX4-DBD inhibitory construct. The development of this system was pivotal, as it served as the primary tool for evaluating the success of our therapeutic approach.

Successful Expression and Validation of DUX4-DBD Inhibition

We validated our competitive inhibition strategy by engineering and overexpressing a truncated version of the DUX4 protein, termed DUX4-DBD (DNA-binding domain). By co-expressing DUX4 and DUX4-DBD alongside our reporter constructs, we assessed the therapeutic potential of DBD as an inhibitor of DUX4 pathogenicity. Through fluorescence and luciferase assays, as well as flow cytometry, we demonstrated that DUX4-DBD successfully competed with full-length DUX4 for target sites in DNA, effectively reducing the activation of downstream genes. This was a key milestone in showing that DUX4-DBD could function as a therapy for FSHD, as we consistently observed reduced levels of DUX4 reporter expression in the presence of our modified construct.

Enhancement of Inhibition Using KRAB Domain Fusion

To enhance the repressive capabilities of DUX4-DBD, we incorporated the KRAB transcriptional repressor domain, a powerful gene silencing tool. Essentially, we engineered a “reverse DUX4,” which silences rather than activates downstream genes. This fusion construct further suppressed the expression of DUX4 target genes beyond what we observed with DUX4-DBD alone. This enhancement was validated through a series of transfection experiments in HEK293T and C2C12 muscle cells, where the KRAB domain addition resulted in more robust inhibition of DUX4’s pathogenic activity.

Development of a Reporter Cell Line and Inducible Full-Length DUX4 Cell Line

To investigate the activity of DUX4 and the effectiveness of our inhibitory constructs, we successfully established a reporter cell line by transient transfection utilizing the mScarlet fluorescent protein. This cell line was engineered to express mScarlet under the control of DUX4 target gene promoters, allowing us to visualize DUX4 activation in real time. In parallel to the reporter system, we developed an inducible cell line for the expression of full-length DUX4, which served as a critical model for studying the pathological effects of DUX4 activation in muscle cells.

Transcription Factor Binding Model

We developed a Transcription Factor Binding Model that successfully established a foundational understanding of the competitive inhibition dynamics between DUX4-FL and DUX4-DBD at the single-cell level. By employing a biophysical competition equation, the model allowed us to simulate and quantify the binding interactions, revealing how variations in DUX4-DBD concentrations influence DUX4-FL expression. The model provided insights into the required concentration of DUX4-DBD to achieve a target inhibition level, which is crucial for preventing transcriptional activation of DUX4-target genes that lead to muscle degeneration. By integrating data from our wet lab experiments, such as transcriptional outputs in HEK cells, we were able to refine the model and validate our predictions, making the approach a benchmark for future studies. The transcription factor binding model serves as an excellent reference for other research teams facing similar challenges in competitive inhibition and gene regulation, demonstrating the efficacy of computational modeling in complementing experimental data.

Ordinary Differential Equation (ODE) Model

Building on our previous findings, we developed an ODE model that extended the analysis to consider the temporal dynamics between DUX4-FL and DUX4-DBD concentrations across cell populations. This allowed us to simulate the progression of competitive interactions over time, enhancing our understanding of DUX4-driven transcription. By incorporating parameters such as protein stability and half-life derived from experimental data, we improved the model's accuracy and relevance to physiological conditions. The ODE model's output helps predict how variations in DUX4-DBD concentrations can be adjusted in response to patient-specific DUX4-FL levels, making it a powerful tool for designing personalized therapeutic strategies.

Markov Model

We explored the Markov model as an alternative to the ODE model, providing a different perspective on the stochastic dynamics of DUX4-FL expression. Although still in progress, preliminary insights from this model may highlight unique pathways of expression and inhibition that warrant further investigation. By potentially integrating additional biological variables, this model could enhance our understanding of DUX4 dynamics in the context of disease variability.

MRI Preprocessing Pipeline

As we explored non-invasive approaches to assess DUX4-FL expression in vivo, the development of an MRI preprocessing pipeline emerged as a promising avenue for advancing our understanding of DUX4 dynamics in living tissues. Magnetic resonance imaging (MRI) is a powerful imaging modality that provides detailed anatomical information and, when paired with advanced processing techniques, can reveal functional insights into tissue health and pathology. By integrating imaging data with our computational models, we aim to create a holistic framework for evaluating DUX4-FL expression and its implications in the context of facioscapulohumeral muscular dystrophy (FSHD).

Stanford iGEM Bioengineering Research Program (SIBRP)

The Stanford iGEM Bioengineering Research Program (SIBRP) provided global access to bioengineering education through interactive webinars, a speaker series, and mentorship, reaching over 300 participants from 36 countries. We emphasized inclusivity and democratizing access to bioengineering education to historically marginalized populations, with 74% of participants identifying as women or non-binary and 51.3% as first-generation students. Through lectures and collaborative workshops, students learned foundational principles of synthetic biology and developed their own research proposals. A diverse lineup of expert speakers also offered insights into cutting-edge fields like machine learning in evolutionary design and engineering T cells to fight cancer. Through providing direct 1-on-1 mentorship and guiding students through their research proposal, we fostered a sense of community and ensured a personalized and supportive learning experience.

Phil’s Laberia Laboratory Simulation Game

Phil’s Laberia is an interactive educational game developed by the Stanford iGEM Team 2023 to make complex bioengineering concepts more accessible. By simulating gene editing and tissue engineering, the game provided an immersive and hands-on learning experience, making difficult topics easier to understand through real-world problem-solving challenges.

This year, the Stanford iGEM Team beta-tested Phil's Laberia with over 600 high school students and launched a pilot translation program, allowing students worldwide to volunteer and translate the game into 16 languages, including Mongolian and Kyrgyz! Our efforts expanded the game's accessibility, fostering a more inclusive and diverse educational experience for students from different linguistic and cultural backgrounds. Participants praised the game’s engaging design, which included interactive dialogue and detailed animations, enhancing both the fun and educational aspects. By offering a novel approach to learning synthetic biology, Phil's Laberia played a key role in demystifying bioengineering for a broad, global audience, particularly for students new to the field.

Society of Women Engineers Workshop

Partnering with the Santa Clara Valley Chapter of the Society of Women Engineers (SWE), Stanford iGEM hosted a two-day synthetic biology workshop through SWE’s GetSET program. This workshop targeted STEM-interested female-identifying high school students, introducing them to synthetic biology concepts and hands-on lab skills through activities like Bio-Art, where students created bacterial art on LB plates. Day two included a comprehensive tour of Stanford's engineering and science facilities, exposing students to cutting-edge research and educational opportunities in higher education. Through the SWE workshop, we provided an empowering environment that fostered curiosity and confidence in pursuing careers in science and engineering.

Stanford BioHacks

We hosted Stanford BioHacks, a bioengineering and biomedical computation hackathon designed to encourage collaboration and innovation in solving real-world challenges related to muscular dystrophy. Participants worked across disciplines to develop creative solutions, providing an engaging platform for both seasoned and novice bioengineers to contribute ideas. BioHacks promoted a spirit of teamwork, learning, and exploration of bioengineering applications, inspiring participants to explore new technologies and methodologies in tackling genetic disorders like FSHD.

FSHD Chatbot

To further raise awareness about Facioscapulohumeral Muscular Dystrophy (FSHD), Stanford iGEM developed an FSHD Chatbot that provided accessible and personalized information about the disease. This chatbot was designed to empower patients, caregivers, and the broader community by offering clear, concise explanations of FSHD and its impact, alongside updates on ongoing therapeutic research. By making this information readily available, the chatbot aimed to demystify FSHD and promote a better understanding of the condition and the importance of therapeutic interventions.

Internship Program

Stanford iGEM’s Internship Program offered high school students a hands-on opportunity to engage with bioengineering research, providing valuable practical experience in the field. Our interns, Chris and Edithe, worked closely with the Stanford iGEM Team and mentors, gaining insights into the research process and contributing to ongoing initiatives. This internship experience not only allowed the interns to develop technical skills, but also fostered their ability to actively contribute to a moving dynamic project and contribute meaningfully to the field of bioengineering, preparing them for future academic and professional endeavors.