Introduction
COVID-19 has been one of the most lethal pandemics in human history and does not need anymore of an introduction to understand its impact on daily life across the globe. The pandemic has created an emergency situation that created great precedent to develop a vaccine as fast as possible. Messenger RNA (mRNA) vaccines have revolutionized vaccines as a whole for their ease of modification and simplicity.
However, very little time was put into exploring novel regulatory mechanisms that the vast field of RNA folding can provide for in vivo delivery of the coding sequence (what we affectionately refer as “cargo”) into mammalian cells. We are honored to have the guidance of Dr. Albeck’s and Dr. Fraser's work with real time translation and RNA chemistry were invaluable to accomplish Project Cargo’s goal to utilize a specific RNA structure known as an Iron Response Element (IRE) that binds to an Iron Response Binding Proteins (IRBP). There are two primary IRBP proteins in the body: IRBP1 and IRBP2. When these proteins bind to the IRE, it blocks translation of the mRNA transcript. IRBP is iron sensitive and has an allosteric site for iron-sulfur clusters. When iron binds to the IRBP, it induces a conformational change that results in IRBP unbinding from the IRE. This opens up the 40s ribosomal subunit to be recruited and initiate translation of the cargo sequence.
Intention
The intention is to introduce a modified IRE sequence to the 5’ UTR of an mRNA vaccine in order to reduce the magnitude of initial translation rates by “slow dripping” the mRNA transcripts to ribosomes in mammalian cells. The amount of coding sequence protein, in the case of COVID-19 being coronavirus spike protein, being introduced to the immune system less suddenly. The innate immune response typically responds more aggressively to larger stimuli and we believe our IRE system can mitigate this to reduce the severity of standard mRNA vaccine side effects of headaches, soreness, and fevers. This still provides the body the same amount of time to build adaptive immunity. Our modified IRE mRNA vaccines can function by either binding to endogenous IRBP proteins in cells or pre-packaged with a bound IRBP protein in the vaccine itself.
There is a catch however, one simply can not insert a relatively large sequence such as an IRE into a 5’ UTR without any complications. Inserting an IRE sequence or any secondary RNA structure at random can result in conflicts with already present RNA structures that may reduce the folding of the structure itself and impact biological function. It can also lead to an increased affinity to downstream portions of the RNA that cause a phenomenon known as “global kissing” where the RNA strand folds onto itself. To ensure the efficient insertion, Project Cargo designed a computational pipeline under the guidance of Dr. Aviran that will iterate a desired RNA secondary structure through a given sequence to identify the best location to place an IRE. This is done by folding the RNA sequence automatically through the aid of the Vienna RNA folding package and the work of Dr. Radecki (read more about how this exactly works here!)
Project Cargo sought to address the issue of vaccine hesitancy in Black and/or disabled communities. These communities are more likely to get the vaccine at later dates due to inequity issues, concerns of side effects, and historical events that create extra stigma to use standard US healthcare systems. We wanted to lay down the groundwork for future research efforts into better understanding these communities to improve vaccine equity and distribution in the US which will lead to more effective pandemic responses. We worked closely with Mr. Glenn Ellis to connect us to people who could share their pandemic and vaccine experiences from Alabama and Pennsylvania. One of our topmost priorities was ensuring ethical data collection from participants and making sure to fully inform participants of any essential background information, maintain identity confidentiality, and offer to share our findings with participants if they were interested in how their data would be used.
This sure was a lot for a summer still recovering from COVID-19 and Team UC Davis still feel that there is still so much more that can be done with Project Cargo. As such we have documented everything we can so that future iGEM teams and research inquiries can continue the work. We have packaged our folding pipeline into a neat GitHub bundle that includes the code, operation guide, and other essentials so that people of all familiaties with coding and RNA structure can fold their own RNA sequences. We have documented our wet lab engineering notebook, so students can have a prime example of the verification steps that are essential to plasmid assembly. As a supplementary piece to that, we have created a comprehensive CHO DG44 cell culturing guide. Team UC Davis has been working with mammalian cell systems for many years now and we would love to share our experiences. Mammalian cell culturing can seem daunting and difficult at first, and as such our guide includes many images and troubleshooting tips to get your first plate of CHO DG44 cells growing. Finally we have documented our interview methods and human outreach theory to help other students who want to collect data from the community do so ethically and extract usable data.
Concluding Remark
Please go visit our pages for more details about each topic. It has been a great pleasure working this summer and we hope we have created the tools and interest to continue the work of optimizing mRNA vaccines for public health efforts. Happy RNA folding everyone!