General Overview
Being a completely student-run team of first time students navigating the research and synthetic biology realm, we sought to thoroughly understand the effects and solidify the vision of our projects before we committed to implementing wet lab elements. Our goal was to not only understand the industry and pipelines for drug manufacturing, but also to fully digest the mechanisms associated with our protein of interest (G9a) in order to create an optimal therapeutic that took into account the numerous safety challenges.
We first defined a clear scope for our project. This included determining that most of this project’s extent would require us to understand the manufacturing of therapeutics on an industrial level rather than being community-based. We initially identified the problem within our community to be substance use disorder (SUD), but beyond that, we understood we would have to consult with those years ahead in experience to learn the full responsibility that came with manufacturing a product with the lives of people on the line.
However, our biggest obstacle was the limitations in both existing research applicable to the protein and the construction of our targeted therapeutic. Therefore, we clearly identified our goals which ultimately were divided into three parts, learning about: (1) the functionality of G9a and implementation into our protocol, (2) the application of our implemented RNAi technology and its implications on patients, and (3) the process of manufacturing a drug, including what it takes to get the drug out into just clinical studies. Our team’s philosophy lies heavily on quality compared to quantity, so we sought out each institution/company per our goals to consult with in these matters.
The functionality of G9a and implementations into our protocol
Professor Ethan M Anderson
Our protein G9a (also known interchangeably as EHMT2) was a very niche field of study which prompted us to look into multiple works published by professors in articles such as “The histone methyltransferase G9a mediates stress-regulated alcohol drinking[1]” and “Knockdown of the histone di-methyltransferase G9a in nucleus accumbens shell decreases cocaine self-administration, stress-induced reinstatement, and anxiety[2]”. A name which frequently occurred was Assistant Professor at Louisiana State University Ethan M. Anderson, who we found to be a specialist in the field of G9a study and a newly promoted PI. Therefore, we resolved to get in touch with him through email in late May through our expressed interest in wanting to learn more about his studies. His enthusiasm to help led to a few of our members meeting with him on July 1st, 2024.
In our meeting, we addressed parallels between his past experiments and our current project. We interviewed him about the conduct of his experiments, including questions in which we were able to gauge the viability of our project, modify our protocols to increase accuracy, and how we as high schoolers can confidently defend the credibility of our results.
Contextual Aspect
- We asked questions relating to how he experimented with G9a and the different techniques of inhibition he used. He explained how he used a G9a inhibitor, UNC0642, to carry out the experiment; however, the use of this drug as a therapeutic was not very practical due to the short-lived inhibition and the varied efficiency in binding to G9a enzymes and was not suggested for conducting our own project.
- As a result, we looked into different ways to potentially decrease the concentration of G9a and the concept of RNAi, specifically shRNA, came up. As we researched more into how we would buy and assemble the plasmid, we quickly realized that money and the precision required to build the plasmid with precise DNA exon-exon sequences were going to be far out of our capabilities. We ended up pivoting to siRNA technology which has a similar function; it is further elaborated in section (2) The application of our implemented RNAi technology and its implications on patients
Environmental Aspect
- All types of RNA may be more unstable than we predict, so it was crucial for us to have excess copies of shRNA (later changed to siRNA as we pivoted) on hand in the case of having to repeat the experiment again.
- Housekeeping genes are essential in a qPCR run in order to compare our results of the effect of siRNA on our gene (EHMT2) with the housekeeping gene (which Anderson recommended using GAPDH). This was to ensure our siRNA was causing only the EHMT2 to decrease in levels, not anything other protein.
- Therefore, aside from designing primers and probes for our qPCR detection of EHMT2, we also designed a separate set for the detection of GAPDH in our experiment, running our real-time PCR on a multiplex in order to mix both dyes (from EHMT2 and GAPDH) together into one reaction for better efficiency.
Research Appeal Aspect
We also asked for some advice regarding methods to present our research to an audience.
- Our biggest piece of advice received in this realm was to understand your audience. Maintaining that research etiquette depending on who our presentations are tailored to is crucial in the professional world.
- We kept this in mind as we approached different people in the industry. With large science companies like Thermofisher and academic institutions, we held a professional attitude when approaching them about the prospects of our project. However, to a younger audience and those with little exposure to the fields of study, we created engaging ways to simplify our information while promoting the same ideas.
- Keep explanations of data as simple as possible, and not only display but describe the results.
*consent was given to use this photo (above: Professor Ethan M Anderson)
The application of our implemented RNAi technology and its implications on patients
ONPATTRO, Thermofisher Scientific
To take it another step further into understanding the effect of our research in real-world applications, we reached out to ONPATTRO[3], the first company to utilize FDA approved RNA based therapy to treat a rare disease. The members of the team were able to listen in on one of their lectures, held by former Vice President of Alnylam Pharmaceuticals’s Central Nervous System Research Team, Dinah Sah, who lead researchers in the development of the world’s first siRNA therapeutic. After the lecture, we inquired further about their own discoveries as well as any input they could give to us on our project.
Some large ideas we drew from their product included:
- The concept based around short interfering RNA (siRNA), which was the standard RNA technology we were opting to use instead of short hairpin RNAs (shRNAs).
- After hearing the advice given to us by ONPATTRO, we decided to move forward with using siRNA instead of shRNA as it was a lot more feasible to be developed into a therapeutic drug compared to shRNA which carries the implications involved with gene editing.
- The incorporation of lipid nanoparticles as an effective delivery system.
- Therefore, we resolved to apply this into our experiment by providing a concept model that can be built upon in future usage. This model incorporates the potential procedures which we would have executed upon if we had worked with constructed LNPS from scratch, following similarly to the ones made by ONPATTRO (For more information, go to the “modeling” page).
- For our current protocol, we still wanted to resemble lipid nanoparticles as close as we could in an in vitro study for the transfection process. Therefore, we communicated with Thermofisher Scientific[4], where we sourced the best alternate that performed with the same theory as lipid nanoparticles did in HEK293-T cells, which ended up being Lipofectamine™ RNAiMAX Transfection Reagent[5].
- SORT molecules for targeted efficiency.
- Onpattro wanted to target a hereditary disease known as transthyretin-mediated amyloidosis caused by faulty TTR proteins that is produced in the liver and builds up in surrounding organs. This causes damage specifically to the heart, as faulty TTR proteins build up, the left ventricle becomes more and more weak.
- LNP are naturally absorbed in the liver so using LNPs to mediate to different organs could be challenge, but by using specific SORT (selective organ targeting) molecules we are target different organs outside of the liver.
When we inquired about the potential use of LNPs to mediate the siRNA therapeutic, Dr. Sah advised us against it as we were only dealing with in vitro experiments. Instead, she suggested that we shift our focus to developing an siRNA in the most optimal concentration and time frame as that information would be vital in the research of a siRNA mediated therapeutic for G9a. She described how incorporating LNPs as the therapeutic medium should only come after thorough in vitro studies regarding optimal concentration, cell tissue type, siRNA exposure time in cell, and the variant of EHMT2 we wanted to silence.
The process of manufacturing a drug & the pipeline into discovery and delivery
ONPATTRO, XENCOR
The development of a commercial drug is a long and often unrewarding process that we knew would take extensive research and rigorous cross examination. We took inspiration from the extensive pipeline involved in the process of manufacturing drugs, and took the liberty of drawing insights from real-world examples such as ONPATTRO and Xencor. These companies helped us understand the complex procedure behind creating a successful therapeutic. Through our motive of understanding the industry, we have detailed some key details of the development stage and applied it to our approaches.
- ONPATTRO: Stages of Drug Development
- In our project, we mirrored this research-heavy approach by delving into a great extent of scientific literature and cross comparing sources over the web. Owing to the insight gained from ONPATTRO, we understood that the consequences of rushing through the discovery phase included a yield of faulty conclusions. We recognized the responsibility that came with spontaneously rushing into in vivo studies too early, but also the importance of applicational experiments. As a result, we performed extensive in vitro experiments to validate our protein of interest. By comparing multiple studies found by different members of the team and maintaining open communication, we ensured that the gene we targeted was well-validated across a variety of scientific sources.
- The development phase in our project meant refining our gene-targeting strategy and strengthening our concept model which could be carried out
by designing a delivery system tailored to the biological conditions in which the accessibility of our treatment method would be considered.
I. Developing a commercial drug consists of two main stages: discovery and development.
During the discovery phase which can take 1.5 to 8 years, researchers focus on identifying a gene of interest and a target ID responsible for the disease. Validation through cross-referencing of sources is performed to ensure that the target gene is appropriate for therapeutic intervention and to confirm the gene’s involvement in the disease. Within the research phase, before any safety or patient tests are conducted, there must be sufficient invitro and invivo studies to showcase a proof of concept for a new drug. This involves selectively screening various types of the disease protein and identification of the most common variant of the gene that causes the most expression of the faulty or excess protein.
II. The second step of developing a commercial drug is development, which encompasses lead optimization, preclinical testing, and adjusting the drug to function at its optimal level.
In this stage, researchers aim to design a therapeutic candidate optimized for potency, selectivity, and stability. It is a crucial step to ensure the therapy works effectively in the right conditions. Once a committee of researchers agree on an idea and model, the creation of the drug and tests for its ideal concentration, optimal functioning conditions, time frame, and logistics that would define the drug are implemented.
References
[1] Anderson, Ethan M., Marcelo F. Lopez, Abigail Kastner, Patrick J. Mulholland, Howard C. Becker, and Christopher W. Cowan. 2022. “The Histone Methyltransferase G9a Mediates Stress-Regulated Alcohol Drinking.” Addiction Biology 27 (1): e13060. https://doi.org/10.1111/adb.13060.
[2] Anderson, Ethan M., Haosheng Sun, Daniel Guzman, Makoto Taniguchi, Christopher W. Cowan, Ian Maze, Eric J. Nestler, and David W. Self. 2019. “Knockdown of the Histone Di-Methyltransferase G9a in Nucleus Accumbens Shell Decreases Cocaine Self-Administration, Stress-Induced Reinstatement, and Anxiety.” Neuropsychopharmacology: Official Publication of the American College of Neuropsychopharmacology 44 (8): 1370–76. https://doi.org/10.1038/s41386-018-0305-4.
[3] https://www.onpattro.com.
[4] https://www.thermofisher.com/us/en/home.html
[5] https://www.thermofisher.com/order/catalog/product/13778075