DiGI-T3
Many diseases result from dysfunctional proteins, but therapeutics directly targeting them are limited. Current treatments, mainly small molecules, cause side effects. Ohio State's iGEM team is exploring a solution using Type III Secretion Systems (T3SS) for continuous therapeutic protein delivery. Although native to some infectious bacteria, T3SS can be modified for safe use in Escherichia coli to deliver therapeutic proteins, particularly targeting gastrointestinal diseases. Our dry-lab successfully modeled optimization of T3 secretion sequences in Java, offering insights into T3SS function and sequence design for efficient secretion. We engaged in outreach events like COSI and WestFest, educating the public about our project and synthetic biology. Conversations with professionals informed our approach and emphasized our project's potential for GI cancer treatment. Challenges in lab access limited our wet-lab work, but we documented our plans, paving the way for future T3SS research. Our aim is advancing T3SS studies and spotlighting its therapeutic promise.
Proteins are the most functionally diverse and important molecules in the cell’s biophysical processes that are critical to maintain life. Whether it be carrying out an enzymatic function, facilitating transportation of nutrients, or transmitting signals, proteins are involved. Since proteins are so vital to the functioning and survival of cells and organisms, diseases can occur when these molecules accumulate structural defects, leading to unnatural activity.
Several diseases that affect millions have been associated with dysfunctional proteins. For example, amyloidosis is a disease characterized by a buildup of amyloid protein deposits around various organs, causing symptoms such as fatigue, swelling and numbness (Mohty M. et al. (2021)). Additionally, many cancers can be attributed to the misfolding of proteins responsible for controlling cell division, making these proteins a focal point of research interest.
Current treatments of similar diseases largely involve the administration of small-molecule drugs. While these therapeutics are sufficient in treatment, they often interfere with other biological processes, resulting in adverse side effects. The emerging application of therapeutic proteins aims to counter these issues.
Since the discovery of insulin in 1921, therapeutic proteins have been a central focus in modern medicinal research. By directly replacing or targeting abnormal proteins, these molecules have the capacity to treat several genetic diseases. Additionally, due to the specificity of their structure and function, they are less likely to negatively impact other reactions in the cell or provoke immune responses.
Ongoing research has been promising and many potential protein candidates for novel therapeutics have been identified, however the field is yet to achieve its full potential due to 3 main challenges:
This year, the 2023 Ohio State iGEM team has decided to overcome these barriers by introducing an efficient, inexpensive, and precise mechanism of therapeutic protein delivery via Type III Secretion Systems (T3SS).
T3SS is a protein complex expressed by various pathogenic strains of gram-negative bacteria, including E. coli, Salmonella and Shigella. Similar to a syringe, T3SS works by latching onto host mammalian cells and injecting effector proteins directly into the host cell’s cytoplasm. To be transported through the thin needle, effector proteins must be unfolded and stabilized during secretion, and then refolded immediately following secretion so that they can perform their intended functions (Deng W. et al. (2017)).
T3SS is naturally expressed in pathogenic bacteria, so injected effector proteins are typically virulence factors that interfere with the host cell’s immune responses and signaling pathways, establishing environments that are more suitable for their survival (LeBlanc M. et al. (2021)). Our team has negated this effect by referencing non-pathogenic strains of DH10B E. coli that have been engineered by experts to express the secretion system (Lynch P. et al.). In the context of our project, referencing these strains were crucial in demonstrating the therapeutic viability of our idea.
Our project also aims to digitally model the secretion optimization using software programs in Java. By developing this digital model, we hope to offer a powerful tool for optimizing the efficiency of protein delivery via T3SS, helping us overcome the stability, cost, and delivery challenges that have prevented the full application of therapeutic proteins in medical treatments. In conjunction with our team’s other work, our computational modeling seeks to revolutionize the field of therapeutic protein delivery.
Extensive discussions with experts helped us focus our initially ambitious idea to a more refined and feasible approach. These meetings guided our team to also focus on investigating nanobodies as a starting point, which have been shown to be capable of secretion. Nanobodies are small fragments of the functioning regions of antibodies derived from camelid species. They are composed of a single peptide, consisting of between 110-115 amino acids and lack complex structural elements (Yang E. et al. (2020)). These characteristics make nanobodies strong candidates for secretion. Due to their significantly small size, nanobodies can bind to a variety of target molecules with ease, allowing for several possible applications when coupled with secretion:
With our project, we also plan on providing healthcare providers with improved therapies for early stage colorectal cancer patients. Given that we use E. coli DH10B as the delivery vehicle for our therapeutic agents, tumor growths in the colon and rectum would be the most ideal targets as the GI system is naturally home to a diverse and complex microenvironment. In addition, the nanobodies that we inject into tumor cells could specifically target proteins that trigger tumor growth:
