360. Transmitochondrial Stem Cells: Toward Autologous Cybrid Stem Cell Therapy and Synergistic Transient Regenerative Gene Therapy for Mitochondrial (mt)DNA Diseases
Authors: Roger Bertolotti
Journal: Molecular Therapy (2006)

Mitochondrial (mt)DNA diseases are increasingly important both for inherited diseases and aging disorders (Wallace, 1999). They are currently approached by allotopic gene therapy that relies on nuclear transgene expression of mtDNA-encoded proteins engineered for mitochondrial location. Additional approaches involve endonucleases aimed at cleaving mutant mtDNA. Such a mtDNA gene therapy is restricted to a small fraction of mtDNA mutations and is hampered by random-integration hazards associated to current long-term nuclear gene therapy. We have devised a new approach based on autologous transmitochondrial stem cells (Bertolotti, 2005). It completes the long-term gene therapy arm of our stem cell gene therapy platform that comprises a synergistic transient regenerative gene therapy arm. Transmitochondrial embryonic stem (ES) cells are used for the genesis of mtDNA disease models (Sligh et al, 2000). They are cybrids resulting from the fusion of a somatic cytoplast (enucleated cell) with a totipotent ES cell that has been cured from its mtDNA content by rhodamine-6G (R6G). Under optimized conditions, R6G- treated cells are rescued by cytoplast fusion; such a repopulation of mtDNA-free cells by exogenous mitochondria results in homoplasmic cybrids with a pure mtDNA population. Consistent with somatic cell genetics (Ephrussi, 1972), neither the elimination of mtDNA nor the fusion with an enucleated somatic cell appear to interfere with the totipotent epigenotype of ES cells. Like ES cells, adult stem cells are thus expected to maintain their potentialities under appropriate growth conditions when fused to somatic cytoplasts, thereby opening mtDNA diseases to cybrid stem cell gene therapy. Cybrid stem cells should thus be obtained by fusing donor enucleated cells with R6G-treated patient stem cells, preferentially multi/pluripotent ones that can be extensivelly grown/expanded in vitro (eg MAPCs, hBMSCs). Such an extended in vitro expansion should be critical for the resulting cybrids in order to eliminate nuclear- encoded donor immunogens. True autologous stem cell cybrids would comprise wild-type mtDNA from the mother kindred since mtDNA is maternally inherited. A clinical platform could thus be envisioned for the production of autologous stem cell cybrids. With their differentiating/differentiated progeny, such stem cell cybrids could possibly convert their gain in energy metabolism efficiency into a selective in vivo growth advantage over resident mtDNA mutant cognates, thereby driving effective therapeutic repopulation dynamics into appropriate niches (long-term engraftment) and relevant tissues/organs (differentiated cell turn-over). Importantly enough, magnification of this repopulating/regenerative capability will be under the control of the transient regenerative gene therapy arm of our stem cell gene therapy platform. Cybrid stem cell gene therapy is currently discussed in terms of mtDNA diseases affecting targetable tissues such as the heart (eg homoplasmic hypertrophic cardiomyopathy) and, possibly, as part of the stem cell/gene therapy armamentarium aimed at myocardium regeneration.