The second of our two students this past summer was Michaela Copp. We were able to invite Michaela to join us this summer because of the generous support of Michael Antonov, who wanted to support a high-impact short-term infrastructure project.
The objective of my research project is to establish a safe harbor landing site – a position in the genome capable of integrating new genetic material without posing a risk to the host cell– in the nucleus for the expression of engineered mitochondrial genes. Mitochondria generate the cellular energy consumed by mammalian cells through the process of oxidative phosphorylation. Like the nucleus, mitochondria possess their own DNA, termed mtDNA, which encode for 13 proteins critical to cellular respiration. Unfortunately, mitochondria do not have an efficient system for repairing damaged DNA, leading to mutation rates 10 times greater than that detected in nuclear DNA. Scientists believe evolutionary forces have driven mitochondrial genes from the mitochondria into the nucleus, where they are protected from the highly-reactive oxygen molecules produced by oxidative phosphorylation. The SRF Mitochondrial team hopes to mimic this evolutionary process by providing cells with a modified “backup” copy of the remaining mitochondrial genes at a safe harbor within the nucleus. The procedure of expressing genes in the nucleus originating from the mitochondria is called allotopic expression.
Prior to this project, allotopic expression studies on mitochondrial genes had been performed via traditional transfection / virus induction procedures which integrate the new DNA randomly into the host genome. The goal of this study is to express the mitochondrial genes from an identified safe-harbor site in the nucleus, the hROSA26 locus, in order to minimally disrupt the host genome and ensure the gene functions predictably. This will be accomplished by targeting the hROSA26 locus using the CRISPR system, creating a double-stranded break, and introducing a new strand of DNA containing a selection marker flanked by the attp sites of two different integrases (in this case Bxb1 and PhiC31) on either ends. These attp sites will function as "landing sites" to introduce different allotopic constructs. Integrases catalyze the site-specific integration of foreign DNA into a chromosome, and the dual integrase system will allow us to insert our mitochondrial genes with a directionality and specificity of 100%. The benefit of using this allotopic expression approach is that the integrase system has no size limitations and provides for the integration of the mitochondrial genes at a precise location in the genome. Ultimately, this project will allow the SRF Mitochondrial team to establish a safe harbor landing site with the capability of integrating the entire mitochondrial genome into the nucleus.
We are following up on this work to determine whether we can use this technology to fundamentally improve mitochondrial gene therapy.
