Stable Transformation of CHO Cells and Human NARP Cybrids Confers Oligomycin Resistance (oli^r) Following Transfer of a Mitochondrial DNA–Encoded oli^rATPase6 Gene to the Nuclear Genome: A Model System for mtDNA Gene Therapy

Abstract

Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

Delivery of drugs and macromolecules to mitochondria☆

“…This chimeric protein eventually replaced the defective ATPase6 component in the complex. Later Manfredi et al and Zullo et al separately showed that ATPase6 subunit can be expressed in cytosol and be brought to mitochondria [78,79]. More work will have to be done to determine how general were these findings.…”

Cell and animal models of mtDNA biology: progress and prospects

“…Possibly because of their high hydrophobicity, to date only four mitochondrial genes have been corrected in this manner, in plants (94) and yeast (99) and in cell culture (43,85,140). This approach is limited by the general difficulties of nuclear gene therapy, as well as problems with aggregation of some of the allotopic proteins (85).…”

Forces maintaining organellar genomes: is any as strong as genetic code disparity or hydrophobicity?

“…Such reservations have been voiced by very senior figures in the field, (59,60) possibly contributing to the lack of manpower that has hitherto been applied to developing allotopic expression in mammals. The obstacles to functional allotopic expression are anyway highly challenging-modulating these proteins' hydrophobicity without impairing function will require much experimentation-but evolution does not, after all, appear to be telling us that recent progress in this area (61)(62)(63)(64) is a false dawn. The biomedical potential of successful, comprehensive allotopic expression is considerable: it would provide a cure for many presently incurable diseases caused by mtDNA mutations.…”