About This Project

Inherited and acquired mutations in the mitochondrial DNA can lead to several debilitating diseases, including blindness, neurodegeneration, and sarcopenia. Gene therapy can be a viable strategy to alleviate such diseases. We propose to identify variants of Cytochrome oxidase subunit 2 (COX2) and validate its utility in rescuing function in a model cell line derived from a multi-system disorder and COX2 deficient patient. Think of it as restoring the power plant that produces energy in a cell.

Ask the Scientists

What is the context of this research?

Initially evolving from ancient bacteria, mitochondria are tiny organelles within our cells that perform the activity of ATP production and other vital functions. Mitochondria are unique among animal organelles for their genome distinct from the nucleus and there are 13 genes essential to oxidative phosphorylation (OxPHOS) that still reside within the human mitochondrial genome. Mitochondrial DNA is highly prone to mutation due to a variety of factors and these mutations result in several pathologies. Our endeavor is in identifying gene therapy approaches to address these mutations. The COX2 gene is a core component of Complex IV in the OxPHOS relay. Mutations in this gene are associated with C IV deficiency affecting a critical step in the oxidation of cytochrome C using molecular oxygen.

What is the significance of this project?

Our lab has successfully produced proteins for all 13 genes found in mitochondria by allotopic expression, but only one (ATP8) has successfully restored function in a disease-model cell line. That natural evolution has already transferred >1000 genes from mitochondrial DNA to the nucleus. Our goal is to find variants of the COX2 gene that can help cells function properly by mimicking the natural evolutionary process. Results from such an experiment would not only be a significant step towards efficacious COX2 gene therapy but would also provide key insights into the genetic changes necessary for successful allotopic expression of all mitochondrial genes. This can also pave the way to treat myopathy, infertility, and coronary artery disease, among others due to COX2 mutations.

What are the goals of the project?

We propose to generate >1 million variants for the COX2 gene using error-prone PCR similar to a study performed in yeasts and test these variants in rescuing oxidative phosphorylation, a function that is crucial for ATP production in cells. The nutrients that we consume are metabolized to CO2 and high-energy electron donors that further combine with oxygen to convert ADP to ATP. We will test this in a human model cell line that is lacking the COX2 protein. This cell line is unable to grow in nutrient medium restrictive for oxidative phosphorylation. Upon placing the variants in such a medium, only cells that are capable of performing OxPHOS can survive. Such a screen would allow us to identify functional variants of the COX2 gene for further analysis.