Rescue of mtDNA mutations by allotopic expression of engineered mitochondrial genes - Matthew O'Connor
Mutations in mitochondrial genes occur as we age, and the human body does not have a good system to repair them. At SENS Research Foundation we are in the early stages of creating an exciting and innovative system to repair mitochondrial mutations. In this project, engineered mitochondrial genes are being used to restore function to cells that have defective mitochondrial genes.
Recent research suggests that the use of specific targeting sequences attached to engineered mitochondrial genes expressed in the nucleus allows for functional rescue of mutations in mitochondrial DNA. These targeting sequences localize corrected mRNA to the surface of the mitochondria and improve the efficiency of protein import into the mitochondria. Several challenges remain that inhibit the use of allotopic expression as a viable strategy, however, including efficient and stable long-term protein expression necessary for permanent rescue of deleterious mutations.
Our strategy is as follows: (1) recode the mitochondrial genes for nuclear codon usage, (2) target the mRNA to the surface of the mitochondria using 5' and 3' end targeting signals, (3) achieve efficient import using a canonical N-terminal mitochondrial targeting sequence (MTS), and (4) demonstrate functional rescue of mitochondrial mutations.
We have analyzed localization of the targeted RNA sequences to the mitochondria by qRT-PCR from isolated mitochondria. To evaluate the ability of these engineered mitochondrial genes to rescue mtDNA mutations, these constructs are being stably expressed in WT and mutant patient-derived cell lines. The successful rescue of mtDNA mutations by targeting exogenous mitochondrial gene products into the mitochondria has tremendous potential for the treatment of the maladies caused by mitochondrial mutations.