Gene therapy of mitochondrial DNA mutations: A brief, biased history of allotopic expression in mammalian cells

Successful treatment of mitochondrial DNA (mtDNA) mutations might be possib le by construction of mtDNA-encoded protein genes so that they can be inser ted into the nuclear genome and the protein expressed in the mitochondria ( allotopic expression). This technique would require individual assembly of all 13 mtDNA-encoded protein genes with an aminoterminal leader peptide tha t directs the cytoplasmic translated protein to the mitochondrial membrane. The 13 allotopic genes could be inserted into the nuclear genome of a pati ent's stem cell that had been "cured" of its nascent mtDNA via ethidium bro mide treatment (tho-zero cell). The rho-zero cell would be a uridine auxotr oph, and recovery from uridine auxotrophy would indicate successful transfo rmation. The patient's own cells could then be returned to the patient's bo dy. With a selective advantage of recovered oxidative phosphorylation, the transformed cells could replace cells with mtDNA mutations. Results of expe riments by us on allotopically expressed CHO ATPase6 and of experiments by other workers suggest that there might be competition with endogenous mtDNA -encoded proteins if the particular protein gene is not removed from the en dogenous mitochondrial genomes. Thus, it is likely that an 13 mtDNA-encoded protein genes will need to be allotopically expressed, with concomitant re moval of all mtDNA genomes, in order for this form of mtDNA gene therapy to be successful.