Journal of IiME Volume 1 Issue 2 www.investinme.org Gene therapy for mitochondrial dysfunctions using optimized mRNA transport to the mitochondrial surface (continued) II. Rescue of respiratory chain defects in fibroblasts harboring mutations in ATP6 and ND4 genes (Bonnet et al., Rejuvenation Research : 10, 128-144 ; 2007) With the aim of determining whether allotopically expressed mtDNA-encoded genes could rescue mitochondrial dysfunction, we examined human cultured skin fibroblasts harboring either the NARP T8993G ATP6 mutation or the LHON G11778A ND4 mutation, allotopically expressing the recoded ATP6 or ND4 wild-type genes. Mitochondrial function was evaluated by the measurement of (i) cell ability to grow in galactose medium, which force them to rely on OXPHOS; (ii) in vitro ATP synthesis using respiratory chain substrates; (iii) enzymatic activity of respiratory chain complexes I and V 31. We were able to demonstrate that the allotopic expression of engineered ATP6 and ND4 genes in human fibroblasts harboring either of these genes mutated leads to a complete and long-lasting restoration of respiratory chain function 32 (Tables 1 and 2). Notably, we examined a second LHON patient harboring the G3460A substitution in the ND1 gene. Our optimized allotopic approach significantly rescued respiratory chain I deficiency in these cells. Therefore, our approach for ND1, ND4 and ATP6 genes ensures the efficient mitochondrial translocation of the corresponding precursors, probably via a co-translational pathway. The rescue of mitochondrial dysfunction indicated that the processed polypeptides were fully functional within their respective respiratory chain complexes and, therefore, able to compensate for the endogenous inactive proteins 32, and C.Bonnet, S. Augustin et al. (manuscript submitted, 2007) . (continued on page 26) Table 1: In vitro ATP synthesis rate µM ATP/min/106 cells Complex I substrats Complex II substratI P value ; n Complex I substrats Complex II substrats Control NARP NARP + nATP6 Control LHON LHON + nND4 2081.9 ± 138.1 805.6 ± 262.4 2045.52 ± 428.7 1962.1 ± 352.1 793.3 ± 493.7 1659.4 ± 245.5 1563.1 ± 214.0 400.7 ± 221.7 1659.6 ± 522.5 1266.3 ± 62.1 658.7 ± 185.2 1929.7 ± 480.2 0.004 ; n = 5 0.0013 ; n = 4 0.0003 ; n = 5 0.0022 ; n = 4 P values shown in the third column were obtained according to the Student’s t test for the pairs NARP/ NARP + nATP6 or LHON/ LHON + nND4 for data collected for either complex I or complex II substrates. "n" indicates the number of independent measurements performed. LHON fibroblasts showed a decreased ATP syhtesis rate when complexe II substrates were uses. This result suggests a general perturbation of the respiratory chain activity. Notably, this activity was fully restored by the allotopic expression of ND4. Invest in ME Charity Nr 1114035 Page 25/72

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