Steven Dowdy and RNA Therapeutics

By Kaila Gabriel


The public interest and knowledge in genetic engineering is increasing rapidly, with many taking time to consider the ethics and morality of therapeutics involved in affecting the genome. The public seems to either fear or embrace the idea of changing our DNA through technologies like CRISPR, however not many have been exposed to the rapidly advancing world of RNA therapeutics, which has more imminent prospects of curing countless diseases. I was given the opportunity to discuss this technology with Prof. Steven Dowdy, a researcher at the University of California in San Diego, who has been focusing his lab on solving the major challenges with delivering RNA therapies into the cell for almost 20 years. The conversation I had with Dowdy opened my eyes to the quickly advancing field, and the realities of the future. Currently sitting on 5 scientific advisory boards at innovative biotechnology companies, Dowdy is part of a scientific journey at the forefront of a huge breakthrough in RNA therapeutics.





The Journey into Becoming a Scientist


At the age of 8, Dowdy knew that science was his calling, and he fueled his growing passion by studying biology in his undergraduate degree. Given the opportunity in his third and fourth year to work in a lab, he discovered his passion for research and made the decision to continue his academic journey into graduate school, where he thought he would become an immunologist. In graduate school however, he quickly realized that immunology was not his calling, and he became interested in the cloning of tumour suppressor genes. After completing his PhD working on cloning a tumour suppressor gene, Dowdy went to MIT for his Post Doctoral Degree, where he learned lots about biochemistry and molecular biology. His many years in school all indirectly connected and prepared him for the ultimate goal for his lab, to deliver RNA therapeutics into the cell.



The Original Goal


Dowdy originally set the goal of his lab and post doctoral degree to disprove a widely accepted theory regarding a major player in cell cycle control, the Cyclin D/CDK4 complex. The accepted theory that Cyclin D/CDK4 inactivated the tumour suppressor protein Rb using phosphorylation did not have strong evidence behind it, and Dowdy hypothesized that Cyclin D/CDK4 activated Rb with phosphorylation. For 20 years, Dowdy and his lab stood by their hypothesis, despite disapproval from the scientific community, and were finally able to generate a rigorous data set that disproved the original theory, and directly supported their hypothesis. They found that Cyclin D/CDK4 mono-phosphorylated Rb at one of the fourteen possible phosphorylation sites, with activation of 14 different isoforms.



Shifting Focus


In order to test the hypothesis surrounding the role of Cyclin D/CDK4, Dowdy and his team needed to inactivate CDK 4 within synchronized cells (cells all in the same stage of the cell cycle) to observe the subsequent consequences. Cyclin D /CDK4 was inactivated by the introduction of the p16 protein. The only time efficient way to introduce p16 into the cell was by fusing it with a viral peptide. The viral peptide allowed for the p16 protein to undergo endocytosis and endosomal escape, thus being available for control of the cell cycle.


The main challenge in RNA therapeutics is the delivery of siRNA into the cell, which can only be achieved through endosomal escape since RNA is too large to enter the cell through the cell membrane. The endosome is not meant to allow its contents inside the cytoplasm because they are destined for the lysosome, the digestive organelle of the cell.

Although we now understand how to manipulate siRNA for targeted RNA therapeutics, no researcher has been successful in allowing for efficient endosomal escape of siRNA.

Dowdy’s prior research paved the way for solving the process of endosomal escape of an siRNA, because he was already successful in inducing it for a protein.


Current research and the possible applications


In recent advances of their research, Dowdy and his team have created a synthetic molecule that will have the capability to mimic viral endosomal escape in order to deliver an siRNA into the cell. Once delivered, the siRNA will perform RNA Interference and silence any gene of interest. This technology has the potential to cure or treat any disease currently identified as undruggable. The other exciting aspect of this technology is that it is able to pharmaco-evolve with disease progression and mutation, meaning it is able to keep pace with any changes in the DNA of the disease. In most cancer treatments, for example, a drug only works for a certain period of time before the cancer is able to mutate and the receptors no longer recognise the ligands, therefore stripping the drug of any therapeutic properties. With siRNA that is equipped with the ability of endosomal escape however, the tumour can be biopsied and a drug can be administered before the cancer has time to significantly mutate, rendering the drug extremely effective. The applications of RNA therapeutics RNAi also includes treatment of genetic disorders, viral infections, bacterial diseases, producing transgenic animals, and countless others.


The Ultimate Goal


Prof. Dowdy’s lab is currently working towards developing a universal endosomal escape domain (UEED). This domain will allow any RNA therapies to be directly imported into the cell with a newfound ability to escape the endosome by using the synthetic molecule his lab is working to create.

The plan, Dowdy said, is to make this UEED available to any company using RNA therapeutics struggling with the same issue of endosomal escape, so that the potential life saving treatments can reach as many people as possible.

Currently sitting on 5 scientific advisory boards, Dowdy is doing his best to collaborate internationally in efforts to solve the problem of endosomal escape.


Typical Work Day


Dowdy described his typical day as arriving at the lab at 9am to oversee and brainstorm with his coworkers. Dowdy believes he achieves work-life balance by surfing and sailing on the weekends. He described his job equivalent to being an artist because he is able to do what he loves and “talk science all day”. The self declared lab rat enjoys the challenging environment where no problem is solved without countless days, months, or even years of hard and dedicated work. No day at the lab is the exact same, and Dowdy said he loves the work he does with his team as they collaborate to solve one giant puzzle at a time.


Advice for Students


As a professor, Dowdy feels he has a responsibility to inspire the new and upcoming generation of scientists. His main advice for students currently looking to pursue a career in biotechnology or research was to stay passionate and interested because, as he put it, “it is a great time to be in science”. In the hiring process Dowdy looks for an intelligent, creative, and persistent student that has a passion to learn and has the courage to persevere through adversity.


Professor Dowdy and his team at the University of California have already and continue to accomplish extraordinarily innovative things, and I am personally very excited to see what they do next. With his research and the research of others like him, I believe that we are all about to bear witness to one of the greatest breakthroughs in medicine. I had an amazing time talking with Dr. Dowdy, and I have truly not met another person as infectiously passionate and excited about their work.


Background Info


For those interested, here is a brief description on some of the science behind Dowdy’s lab. For further information visit Dowdy’s lab page at: http://dowdylab.ucsd.edu/


The Cell Cycle

The progression of the cell cycle is regulated by many proteins, with the most important being Cyclins and Cyclin Dependent Kinases (CDKs). A specific cyclin will appear in the cell cycle at certain points, and will be degraded or deactivated at other points. CDKs on the other hand are always present and require a specific cyclin to bind to be activated. Once activated, CDKs will perform a kinase reaction, a reaction that adds a phosphate group to another protein, which is the basis for activation and deactivation of proteins in the cell cycle. The cyclin present determines which CDK will be active, and that determines which protein will be activated or deactivated depending on its response to phosphorylation. For example, Cyclin D/CDK4 has been shown, by Dowdy’s lab, to activate the Retinoblastoma protein (Rb), a protein responsible for a major checkpoint in the cell cycle, with mono phosphorylation, or adding one phosphate group.


The p16 protein is also a protein of interest because it has a deactivating effect on CDK 4 by preventing the binding of Cyclin D. This deactivation of CDK 4 prevents the progression of the cell cycle, therefore making p16 a tumour suppressor gene.


RNAi

RNA interference (RNAi) is a process our cells use to silence any gene of interest using Small Interfering RNA molecules (siRNA) that becomes loaded in Ago2 protein that will bind to and cleave selectively targeted mRNAs in the cytoplasm. In 2001, researchers discovered that RNAi could be used in mammals to target oncogenes (cancer causing genes). In recent years, researchers have been able to mimic RNAi using a modified siRNA molecule in order to one day treat cancers, viruses, and diseases.


Endosomes

When a cell performs uptake of material from its environment it is called endocytosis. Most molecules, however, cannot freely diffuse through the plasma membrane of the cell, so the cell developed a system of cell membrane invagination in order to bring the outside in. The result of endocytosis is a membrane bound “bubble” called an endosome/vesicle, which contains the content from outside of the cell. The endosome is then trafficked to the digestive organelle of the cell, the lysosome, and the contents will be degraded and “recycled”. Some viruses are able to enter the cell using the endosome and escape before they are digested by the lysosome, a process in which researchers such as Prof. Dowdy are trying to mimic in order to deliver RNAi therapeutics into the cell.