My project this summer is studying the effects of gene therapy for a mitochondrial trifunctional protein (TFP) deficiency. This autosomal recessive mutation effects the function of an octamer protein complex that is responsible for fatty acid oxidation as seen in the figure. Even though our mutation only effects one subunit, it alters the folding of the complex rending the entire complex inactive. This process not only breaks down fatty acids, but it also supplies the acetyl-CoA needed for the citric acid cycle which is produced by the step labeled 3 by the beta subunit. It effects the liver and heart heavily since both rely heavily on fatty acid oxidation as a source of energy. Since this process is unable to occur, those organs are not able to function optimally and have fat build up in their cells (the medical word for this is steatosis). Muscles, which sometimes depend on fatty acid oxidation, are also affected. This condition shortens one’s lifespan significantly, with cardiomyopathy being the typical cause of death.
Our lab’s goal is to correct this using gene therapy to replace the missing enzyme. We are currently trying to do this using an Adeno-associated viral vector (AAV). For anyone not familiar, this can be thought of as a trojan horse. We enclose the DNA coding for the protein missing within the virus and use the virus “machinery” to get inside the cell. The goal here is that the cells can now make the protein, hopefully curing or improving the condition. Some problems that can arise include the cells not taking up the virus, immune response, and the DNA being lost as the cells replicate.
We are testing this AAV therapy in a mouse model with a TFP deficiency. We administer the treatment in affected and control mice. We plan to perform a variety of tests to see if it is effective. This includes glucose tests, strength tests, tests to look at the amount of fat in the liver, Western blots to measure protein levels, and looking at the stained tissue under the microscope to name a few. My role this summer is to help with these tests and look at the data they produce. So far, I have done background reading on the condition, genotyped new litters using PCR and gel electrophoresis, ran some of these tests on our mice to assess the effectiveness of our therapy, and analyzed some of our results using graph software. In the next weeks I will learn how to run more of the tests we need to evaluate the treatment and perform some of them. I’m very excited to follow this project and see where it goes throughout this summer and beyond.
