(2gn) Improving Biological Molecule Delivery through Understanding the Endomembrane System

Biological molecules are increasing in clinical use to treat and prevent disease with the FDA approving 2 mRNA vaccines for COVID-19, 4 small interfering (siRNA) therapeutics and over 100 antibody therapies since 2015. Small molecule drugs work by altering catalytic pockets of disease-causing proteins and are easy to produce and for patients to use, but around 85% of disease-causing proteins are thought to be difficult to drug or undruggable. Biological molecules have the benefit of being able to alter protein function separately from catalytic activity such as blocking cellular entry, blocking protein-protein interactions, decreasing mRNA levels, increasing degradation, etc. The key limitations to biological molecules to date are expensive synthesis and storage, administration to patients, and delivering the large, usually charged, molecules to therapeutic sight in the body and the therapeutic compartment of cells. Solving these challenges will unlock treatments for countless diseases.

At Michigan State University under the advice of Dr. S. Patrick Walton and Dr. Christina Chan I worked on questions revolving around RNA delivery and biotherapeutic protein production. In one project, I investigated how endoplasmic reticulum (ER) stress alters siRNA mediated gene silencing. ER stress occurs when a cellular condition causes dysregulation in proteostasis in the ER leading to a build up of unfolded and misfolded proteins. Obesity, diabetes, smoking, infection, and cancer are becoming increasingly common comorbidities in the general population, thus, understanding the impact of ER stress on RNA delivery will inform future RNA based therapeutics. In response to ER stress, cells initiate the Unfolded Protein Response (UPR) to restore proteostasis by decreasing protein expression, increasing chaperone protein expression, or increasing protein degradation; if proteostasis is not restored, apoptosis is initiated. Using human cell line culture, Confocal-Laser Scanning Microscopy, and Flow cytometry, I demonstrated that siRNA accumulates more in cells experiencing ER stress but is sequestered in autophagosomes -- bi-layer membrane bound vesicles that degrade bulk cellular material -- and achieves less therapeutic effect. These results will inform the design of future RNA-based therapeutics targeting diseases known to increase ER-associated autophagy such as diabetes and other obesity-linked diseases. In another project, I investigated the role of ER stress in therapeutic protein production. Tasking cells with producing recombinant proteins throws off ER proteostasis, initiating the UPR. The ER is the synthesis organelle for secreted proteins and membrane components and with the Golgi apparatus makes secretory vesicles. The UPR affects this system by increasing ER and secretory machinery size while decreasing overall protein expression and increasing protein degradation. Using a secreted and non-secreted luciferase protein I was able to optimize conditions and cell line genetics to debottleneck secretion in ER stressed cells. These results will assist the cost reduction efforts of recombinant protein therapeutic producers. Finally, I investigated the effect of circularization of mRNA on cellular entry and escape. mRNA has a short half-life in vivo due to degradation by exonucleases leading to increased numbers of doses needing to be made and administered to achieve therapeutic effect. This increases costs of manufacture and storage and the likelihood that a patient will miss needed doses. One method other researchers and our lab have used to increase mRNA half-life is reacting the 3’ and 5’ ends of the mRNA together to form a circular mRNA (circRNA) that binds less effectively to exonucleases, taking longer to degrade in vivo. Because circRNA has no ends and a different secondary structure, the electrostatic interactions with lipoplexes and cationic lipids for delivery along with electrostatic interactions with the cell membrane change. Using small molecule inhibitors of endocytosis, circRNA and linear RNA encoding for luciferase, and Confocal-Laser Scanning Microscopy, I demonstrated the circRNA has different endocytosis patterns and endosomal escape efficacies that limit the bioavailability of circRNA in the cytoplasm to achieve therapeutic effect. These results indicate that increased half-life does not mean increased therapeutic effect and that RNA delivery vehicles need to be designed for the specific RNA structure.

In addition to research conducted toward my PhD, I have other relevant experiences that inform my research interests. I worked as a post-bachelor research assistant in the lab of Dr. Ravi Saraf at the University of Nebraska where I developed protocols to synthesize monodisperse 10-100 nm citrate capped gold nanoparticles that could be self-assembled with DNA, RNA, or proteins into nanoparticle changes that act as electrochemical detectors of complementary biological molecules. While working on gold nanoparticle devices I learned scanning electron microscopy, transmission electron microscopy, atomic force microscopy, and UV-Vis spectroscopy. In 2018 I was awarded NIH T32 fellowship with the Integrative Pharmacological Sciences Training Program (IPSTP). The IPSTP allowed me to take classes and training programs that are not usually available to chemical engineers. During an intensive summer bootcamp I learned the FDA approval process for drugs and biological compounds, how targets are selected, standard preclinical in vitro assays for drug development, Good Laboratory Practices, mouse and rat drug and behavioral testing, and bioinformatics. Combining knowledge sets from chemical engineering, cellular biology, electrochemistry, and pharmacology will lay the foundation for my future career.

Research I would like to pursue focuses on improving the biodistribution and bioavailability of RNA-based therapeutics. The liver is the blood filtering organ of the body and target of all four FDA approved siRNA therapeutics. Developing methods to screen for liver accumulation compared to other organs will improve the success rate of RNA-based therapeutics clinical trials. One way to do this would be to use liver organoids and organoids of target material in mono- or co-culture. The short-term goals of this project will be to determine how RNA/lipid complex variations alter accumulation in cell types within liver organoids and develop protocols to co-culture liver organoids with kidney, spleen, heart, and tumor organoids. The long-term goals of this project will be to test co-treatment of small molecule drugs and RNA therapeutics to achieve better biodistribution. Once arrived at target cells, RNA must initiate an endocytosis response and escape the membrane bound endocytic vesicles to enter the cytoplasm to achieve therapeutic effect. Recycling to the cell membrane and degradation of endocytic vesicles may limit RNA escape into the cytoplasm. RNA escapes membrane bound vesicles by making the membrane more “leaky” through proposed mechanisms: delivery vehicle membrane fusion, osmotic pressure swelling, and protein transport. In order to design a delivery vehicle for RNA delivery, a better understanding of the molecular conditions i.e. lipid composition and protein composition during endosome maturation is required. The short-term goals of this project are to isolate and identify membrane bound cellular components, determine the proteins and lipids in each component, and test how commercially available transfection reagents interact with the isolates. The long-term goals of this project are to use computational modeling to predict delivery vehicle/endosome interactions, identify differences in endocytosis machinery in different cell populations, and develop novel RNA delivery vehicles that mimic endosomes.

Teaching Interests

As an engineer, scientist, and educator, my career goals are to provide students and mentees with problem solving skills, critical thinking challenges, and a sense of community that lead me to develop my passion for learning. While at Michigan State University I have had the opportunity to be a teaching assistant for Biochemical Engineering Laboratory, Process Design and Optimization, and Engineering Design. I have developed a philosophy that learning is done best when students are given the tools to succeed and then challenged. In the Biochemical Engineering Laboratory class, we used a semi-flipped classroom model where the professor would lecture on Tuesdays and the students would present on current events or subjects relating to the laboratory portion. This model allowed us to present the basics of bacteria culture and then have a student teach the classroom about how bacteria are used to make hand sanitizer, adding real world application. Collaboration and technology are an integral part of being a chemical engineer and the classroom. I would like to teach classes that instill the basics of engineering problem solving through drawing a diagram while listing assumptions, knowns, unknowns, and equations such as Introduction to Chemical Engineering, Mass and Energy Balances, and Transport Phenomena. I would also like to teach interdisciplinary classes like cell engineering, drug delivery, and biochemical process engineering. I have mentored 5 undergraduate and high school students in laboratory research. With lab mentees, I try to make sure they get everything they want out of the experience. One wanted to know what grad school was like and I allowed them to shadow me whenever they were free. Another had a project in mind and I made sure they had all the material they needed and helped with protocols. My main goal when mentoring in the lab is to show good safety practices, professionalism, and hypothesis driven research.