Mitochondria are the power plants of the cell, producing chemical energy stores used to power cellular activities. They come equipped with their own DNA, separate from that in the cell nucleus. Damage to that DNA that occurs as a side-effect of the processes necessary to generating chemical energy stores is thought to be one of the root causes of degenerative aging. The SENS Research Foundation funds research to work around this problem by putting copies of the most important mitochondrial genes into the cell nucleus, where they are better protected and will provide a backup source of the protein machinery needed for correct mitochondrial operation, thus eliminating this contribution to aging.
Other approaches are possible, however: replace the mitochondrial DNA or mitochondria entirely on a regular basis, for example, or as in the research noted here use a variant form of gene therapy to deliver individual replacement genes into the mitochondria:
We injected a modified self-complementary (sc) AAV vector [into] the mouse vitreous to deliver the human ND4 gene under the control of a mitochondrial heavy strand promoter (HSP) directly to the mitochondria of the mouse retina. Control viruses consisting of scAAV lacking the COX8 targeting sequence and containing human ND4, or scAAV containing green fluorescent protein (GFP), were also vitreally injected. Using next-generation sequencing of mitochondrial DNA extracted from the pooled mouse retinas of experimental and control eyes, we tested for the presence of the transferred human ND4, and any potential recombination of the transferred human ND4 gene with the endogenous host mitochondrial genome.
We found hundreds of human ND4 DNA reads in mitochondrial samples of MTS AAV-ND4-injected eyes, a few human ND4 reads with AAV-ND4 lacking the MTS, and none with AAV-GFP injection. Putative chimeric read pairs at the 5′ or 3′ ends of human ND4 showed only vector sequences without the flanking mouse sequences expected with homologous recombination of human ND4 with the murine ND4. Examination of mouse mitochondrial ND4 sequences for evidence of intra-molecular small-scale homologous recombination events yielded no significant stretches greater than three to four nucleotides attributable to human ND4. Furthermore, in no instance did human ND4 insert into other non-homologous sites of the 16 kb host mitochondrial DNA.
Our findings suggest that human ND4 remains episomal in host mitochondria and is not disruptive to any of the endogenous mitochondrial genes of the host genome. Therefore, mitochondrial gene transfer with an MTS-AAV is non-mutagenic and likely to be safe if used to treat Leber hereditary optic neuropathy patients with mutated ND4.