Journal of IiME Volume 1 Issue 2 www.investinme.org Gene therapy for mitochondrial dysfunctions using optimized mRNA transport to the mitochondrial surface (continued) common mitochondrial disorder with an estimated prevalence of 1 in 25,000 in north-east England. The pathology is characterized by selective death of RGCs leading to central vision loss and optic nerve atrophy, prevalently in young males. The age of onset of visual loss ranges from 8 to 60, typically occurs between ages of 15 and 35 years. The course of visual loss is generally acute or subacute, both eyes are involved sequentially. The average time interval between affected eyes is approximately two months, the duration of progression of visual loss in each eye averaged approximately four months 17. LHON is a devastating disorder with the majority of patients showing no functional improvement and remaining within the legal requirement for blind registration. The three most common pathogenic mutations found in about 95% of LHON’s patients are located in ND1 (G3460A), ND4 (G11778A) or ND6 (T14484C) genes. They encoded subunits of the respiratory chain complex I and the mutations have the double effect of lowering ATP synthesis and increasing oxidative stress chronically 17. Although, extensive studies were conducted since more than 15 years, the pathogenesis of LHON is poorly understood. One recent hypothesis suggests that the pathophysiology of optic neuropathies does not just involve the disorder of ATP production by mitochondria but that the non-maintenance of the sharp mitochondrial gradient at the optic nerve head constitutes the first step in a vicious event cycle that further compromises neuronal respiration and that would eventually lead to profound energy depletion, the increased production of toxic free radicals and neuronal cell death through apoptosis 18, 19. LHON, as the other mitochondrial diseases, is resistant to treatments with quinone analogs, vitamines or oxygen radical scavengers, which were harmless but very inefficient in most of the cases 20. Therefore, the allotopic expression (expression of mitochondrial genes transferred to the nucleus) of some of mtDNA genes has been tried in cybrid cells as a possible therapeutic option to cure mitochondrial diseases. However, several attempts failed to obtain a complete and long-lasting rescue of the mitochondrial defect in cells harboring mutations of mtDNA genes 21, 22, 23. Probably, the highly hydrophobic nature of proteins encoded by the mitochondrial genome represents a physical impediment to mitochondrial import. Therefore, up until today important limitations are found to the allotopic expression as a therapeutic approach for mtDNA-related diseases 24. In previous studies, we demonstrated that in the yeast Saccharomyces cerevisiae, 47% of mRNAs encoding mitochondrialproteins are transported to the organelle surface 25. This phenomenon represents a key step to ensure the proper import and functionality of the corresponding polypeptides inside the organelle 26 and is conserved in human cells 27. The delivery of mRNAs to the organelle surface depends on two sequences: the region coding for the mitochondrial targeting sequence (MTS) and the 3’ untranslated region (3’UTR) 28. Invest in ME Charity Nr 1114035 Thus, we decided to optimize the allotopic expression for mtDNA genes by ensuring the delivery of corresponding mRNAs to the organelle surface. This optimization will prepare the development of an effective treatment for mitochondrial disorders due to mtDNA mutations. The research project of our team is conducted since 2004 along the following complementary axes: Optimize the allotopic expression of mtDNA genes. Rescue of respiratory chain defects in cells harboring different mutations in mtDNA encoded genes. B. Previous activities of our team: 2004-2007 I. Optimization of the allotopic expression of mtDNA genes (Kaltimbacher et al., RNA : 12, 1408-1417 ; 2006) Recently, we have shown that a protein which is normally encoded by mtDNA was efficiently translocated into the mitochondria of HeLa cells by the use of signals that force its mRNA, transcribed in the nucleus, to localize to the organelle surface. We constructed a nuclear version of the mtDNA-encoded ATP6 gene flanked by cis-acting elements of either COX10 or SOD2 mRNAs, which localizes to the mitochondrial surface in HeLa cells 27, 29. The rationale behind this was that mRNA targeting to the mitochondrial surface will lead to a tight coupling between both translation and translocation processes, which should be required for highly hydrophobic proteins, such as ATP6. Noteworthy, when both the MTS and the 3’UTR of SOD2 or COX10 a highly efficient mitochondrial translocation of the ATP6 was observed (Fig.1). Notably, ATP6 protein was insensitive to proteolysis in the presence of detergent, suggesting that it probably was assembled in the complex V of the respiratory chain 30. (continued on page 24) ME Facts Mitochondrial dysfunction provides a physiological basis for the debilitating and overwhelming fatigue suffered by ME/CFS patients whilst the changes in the NTE (neuropathy target esterase) gene provide an intriguing link with OP poisoning and nerve agent exposure found in GWS. - Group for Scientific Research into ME 2006 (http://www.erythos.com/gibsonenquiry/Repor t.html) Page 23/72

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