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)