Reprogramming endogenous mRNA by Crispr Associated Fragment Trans-splicing (CRAFT)
Mentors: David Fiflis, Aravid Asokan, Ph.D.
Departments of Biomedical Engineering, MGM, Surgery
Gene therapy is a strategy to correct monogenic disorders through the delivery of nucleic acids that encode a healthy copy of the mutant gene. Classically, a working copy of a defunct gene is introduced via viral vector; most commonly an adeno-associated viral capsid with a packaging capacity of 4.8kb. Hundreds of genes have coding regions larger than 4.8kb (Dystrophin ~11kb) and therefore cannot be delivered by this classical approach. Novel CRISPR Associated Fragment Trans-splicing (CRAFT) technology provides a new gene therapy strategy by efficient editing of RNA via trans-splicing with the help of Cas-13. By manipulating the splicing pathway and inducing trans-splicing, CRAFT is able to replace a full-length protein sequence without having to deliver the entire sequence. Currently, we are targeting Duchenne Muscular Dystrophy (DMD) and Myotonic Dystrophy (DM1) but aim to broaden CRAFT’s application. We are rationally engineering CRAFT to increase trans-splicing efficiency while also scaling up in-vitro testing by moving to patient-derived cardiomyocytes. Our many engineered versions of CRAFT are delivered into cells whose edited RNA can be extracted, reverse-transcribed into cDNA, PCR amplified, and sequenced. Our initial design screen shows strong candidates to move CRAFT forward by transducing it into patient-derived cardiomyocytes and eventually in-vivo.
This week’s “chalk talks” were very entertaining and it was exciting to see how my peers took ownership and pride in each of their respective projects. Last week, over the course of three days, each of the individuals in the BSURF program had to do an 8-minute presentation on the work they are currently doing in their labs and on their projects. At the whim of random pulling of names from a bucket, aided only by a whiteboard and an expo marker, we each took turns presenting in front of the whole program.
One of the chalk talks that caught my attention was the one done on the topic of nanoparticles by Joe Laforet. He and his mentor are currently working on using self-aggregating nanoparticles to use as a more efficient drug delivery system than the ones currently used. The contemporary design of nanoparticles is based on enveloping a drug of interest in a metallic shell at the molecular level. There are some major issues with this design though. It’s difficult to design and has a very low drug-carrying capacity (only ~5%). Additionally, the metallic envelope is toxic in high doses and affects solubility. A low solubility is bad because it is difficult for the body to dissolve and absorb the drug of interest.
Joe and his mentor are coming up with new designs for nanoparticles and have been using the tendency for some molecules to form nano-scale aggregates to their advantage. The drug of interest is paired with a molecule that serves as a natural vector that can target an organ or tissue of interest. It may sound simple but these nano-clusters of drug and excipient pairs have a drug loading capacity of 95% (remember the 5% of contemporary nanoparticles).
Joe works with machine learning algorithms to help generate simulations that he then analyzes to predict whether a drug and its excipient pair will form a nanoparticle. His mentor can then go ahead and test this nanoparticle in the lab. The simulations he’s created look great and are very satisfying to watch unfold. Additionally, this work has great potential in the medical field and seems very exciting. Nice job and best of luck Joe!
We have proof of concept that our RNA trans-splicing technology works in vitro. We’ve shown that we can efficiently edit pre-mRNA by transfecting and transducing Human Embryonic Kidney cells (HEK293) and are planning to move into patient-derived cardiomyocytes (human heart cells) which we have differentiated from induced pluripotent stem cells (iPSCs). These two in vitro models, HEK293 and cardiomyocytes, provide a testing platform through which we can spend time increasing the efficiency of our proposed pre-mRNA editing tool. The goal is to increase efficiency as much as we possibly can before we go on and test the technology in humanized mouse models (in vitro). The current experiments we are running are done in a controlled environment and simply cannot accurately model the numerous cell types and molecules that are present in an actual organ. So we know that once we begin in vivo testing, the efficiency of our tool will drop in magnitude because of the many unpredictable variables that come into play in mouse models.
I felt this update was needed to give context to the things I do on a day-to-day or weekly basis. As I previously mentioned, our general aim is to increase the efficiency of our technology in vitro by running transfection and transduction experiments on HEK293 and differentiated cardiac cells. This means that I am in charge of keeping these cells alive and well so that we have plates of cells available to repeatedly run experiments on. The HEK293 cell lines are quite resilient and fairly easy to maintain, the stem cells are not. We cannot culture stem cells with antibiotics because it inhibits their proliferation and so we must be very careful to not contaminate them with bacteria and kill them. Whenever I deal with the stem cells I wear a lab coat, nitrile gloves, and protective sleeves. Also, 75% ethanol spray is a good friend of mine. You can never use too much ethanol spray to disinfect the items you work with within the cell culture hoods.
In addition to cell culture, I do transfections and transductions of the genetic material necessary for our technology to work. This means that I am responsible for introducing the technology into cells while also keeping them alive and well. A couple of days after introducing our tool into the cells, I am also responsible for extracting the RNA from each of the wells in the plates, synthesizing DNA from that RNA, running PCR amplification on the synthesized DNA, and then submitting that DNA for sequencing. This is roughly the entire pipeline, and the process of seeding cells all the way through submitting for sequencing can take 4 days in HEK293 cells and up to 19 days in our stem cells. The extra 15 days are included in the case of stem cells because that’s how long it can take to fully differentiate a place of stem cells into cardiomyocytes. On any given day, I could be working on any time point in this process.
In the future, my work will also include working on a directed evolution model to have the power of molecular evolution aid us in finding our prime construct.
(Here’s a pic of me with my stem cell drip)
Aravind Asokan, the principal investigator at Asokan Lab (Synthetic Virology & Gene Therapy), did not grow up knowing or even thinking that he’d become a scientific researcher. In fact, science never particularly interested him until much later in his university years. He grew up in India where the educational system tends to focus less on being well-rounded and more on striving to be at the top of your class and getting into a good college. So although he was taking science courses throughout his middle and high school years, the goal of attending a top university provided him with most of his direction.
He achieved this goal and after four and half years in a dual degree program in India, he acquired a master’s in biology and a bachelor’s in pharmaceutical sciences. This period in his life was very similar to his transition from high school to university, in that he had a broad sense that he wanted to be in BioTech and knew that he needed to go to graduate school in the United States. After his Masters at Ole Miss, his intellectual curiosity led him to an interest in the relatively new field of gene therapy. Now knowing his specific interests, he applied to and finished his Ph.D. program at UNC-Chapel Hill where he worked on synthesizing lipids for DNA delivery into cells. It was after his 3 years of postdoctoral fellowship that he was offered a role as a faculty member with his own lab and full control of his intellectual pursuits. This made all the difference.
Intellectual freedom is exciting but Asokan finds that bringing in the right people for the lab is equally as exciting. His lab members play a critically important role because his vision can only go so far and it’s the people coming into the lab that extend his vision and begin to build their own. This is why Asokan is inclined to bring individuals with diverse scientific backgrounds ranging from yeast cell biology to tuberculosis in zebrafish. During his years in school, Asokan admits to not really having a precise direction and allowing his excitement and desire to be involved in a field to take him wherever he may go. He looks for similar characteristics in his lab members and loves to take in those who deeply want to be involved and who will make the best of the opportunity. Bringing in people who are curious, driven, but often have no background in synthetic virology or gene therapy is what makes his lab successful. The diversity of perspectives allows challenges to be tackled from different angles, which is often what brings about breakthroughs.
It is also through his lab members that Asokan wishes to leave a legacy. When asked about scientific legacy he said he wasn’t concerned with his own and that he often doesn’t think about it. What he does think about is the impact that his graduate students will have on the world. He knows that these individuals will go and take on opportunities with an optimistic and curious drive. As he pointed to the names of several lab alumni which he had pinned on the wall behind his desk, he asked me to imagine not only the students currently working in the lab right now but also the ones who are to come and go in the future. The impact that these individuals will have when they leave the lab is his legacy. Regardless of what they decide to do after their degrees, whether they continue researching, go into BioTech consulting, or leave science altogether, it doesn’t matter to him because he knows they will make a beneficial impact.
Genetics intrigues me because of its ability to explain the mysteries of biology. It helps us understand the biological programming behind all life forms, including ourselves. In the past 100 years we have discovered DNA, developed ways to read it, and now we are working on methods to write and edit this code. It is the growing understanding of the universal language of life that provides us the incredible power to shape the future of humanity. This is a scientific revolution that, with the right amount of careful consideration, will change the human condition for the best. Most obviously it will transform healthcare.
A person’s healthcare treatment today is based on what works for the average human in a population of 7 billion. But with the decreasing cost of genome sequencing and a better understanding of the genome itself, a person’s treatment is becoming increasingly based on their biology. Healthcare is therefore beginning to shift from generalized to precise.
One of these novel precise treatments is gene therapy and the researchers at Asokan Lab work to find novel ways of improving it. Gene therapy is a technique that targets the cause of the disease by finding genetic solutions for genetic disorders. The classic model of gene therapy is to use a viral vector to deliver a working copy of a defunct gene. The introduction or change of genetic material into the cells of a patient is all about changing how a protein or group of proteins is produced by the cell. For example, one of the genetic disorders targeted by the project I’m currently working on is Duchenne muscular dystrophy (DMD). Patients with DMD have severely reduced muscle strength as a result of alteration to a protein called dystrophin that helps keep muscle cells intact. The goal of the project I’m working on is to increase levels of functional dystrophin expression in DMD patients through RNA editing.
The central dogma of biology is that the pattern of information flow in our cells goes like this: From DNA to new DNA (DNA replication), from DNA to new RNA (transcription), from RNA to new proteins (translation). RNA gene therapy targets disease-causing mutations at the translation step. In our project, we aim to edit RNA via trans-splicing by manipulating the splicing pathway by which pre-mRNA turns into mRNA. In doing so, we can replace a mutant exon in the DMD mRNA transcript, which is known to abolish dystrophin production, with a functional version of that exon. When this correction is made, therapeutic levels of dystrophin restoration can occur. DMD is only one of the multiple genetic disorders we will be targeting with this RNA editing mechanism. I’m very excited to be working on a project with this level of therapeutic novelty and medical relevance.
It’s a simple answer but, my expectation from this research experience is to learn A LOT. I’m feeling fortunate for the opportunity to get involved in genetic engineering; something that I am truly passionate about and that I think is absolutely fascinating. Over a year ago now I applied to the BSURF program and although a lot has changed since the end of my freshman year, my scientific interests have stayed consistent. The top three labs in my preference list were the same and in the same order as they were last year so it’s safe to say that I’d been waiting a long time for Summer 2021 and I’m excited it’s finally here.
As I previously expressed, I want to learn as much as I can this Summer. More specifically, I want to learn how to do proper science, both in theory and in practice. Learning the theory of the techniques used in a molecular biology lab will be my job behind the scenes (aka a lot of reading outside the lab). But alongside knowledge and a deeper understanding of the theory, I would like to build a strong foundation of practical laboratory skills. In these next 7 weeks, I hope to learn the laboratory techniques necessary to build my competence and independence in a genetic engineering lab. Designing a relevant experiment and being able to understand it and its difficulties from both a theoretical and technical standpoint is my ultimate goal.
In the following weeks, I’d also like to learn more about myself. I’ve known I wanted to pursue a career in science since I was little. I was a particularly curious child and asked a lot of questions. “Where do volcanoes come from?”, “What’s the biggest number?”, “Why is the sky blue?”. Although simple and often poorly stated, I remember my parents and teachers encouraging my daily flurry of curious questions. In elementary school, many children dressed as superheroes or cowboys for Halloween; I put on a white lab coat and glasses. I loved that in science class, when a question was asked that didn’t yet have an answer, the knowledge gap was admitted and the topic was labeled as one that required further investigation so that maybe one day we might find an answer. I love science and the way it allows us to engage with what we do and do not know in a way that fosters a deeper understanding and a desire to pursue new knowledge, but I still don’t know what it really means to be a scientist. Only experience in the lab can teach you that. Without a doubt, my interests naturally attract me to research and this Summer I will find out what it really means to follow my childhood dreams. Soon enough I’ll be answering questions that previously had no answer.