Journal of IiME Volume 1 Issue 2 www.investinme.org Gene therapy for mitochondrial dysfunctions using optimized mRNA transport to the mitochondrial surface (continued) Table 2 : Complexes I and V activity measurements in NARP and LHON fibroblasts after normalisation with the values obtained in control fibroblasts Complexe I Complexe V NARP 1 NARP + nATP6 1 LHON 0.6 ± 0,12 1.05 ± 0,23 0.47 ± 0.009 1 LHON + nND4 0.97 ± 0.24 1 < 0.0001 ; n = 9 P values shown in the fourth column were obtained according to the Student’s t test for the pairs NARP/ NARP + nATP6 or LHON/ LHON + nND4. "n" indicates the number of independent measurements performed. Complex I activity in NARP cells was identical to that measured in control fibroblasts (1). Complex V activiy in LHON fibrolbasts was not different to the one measured in control cells (1). Complex I and V activities were fully restored in LHON and NARP fibroblasts by the optimized allotopic expression of ND4 and ATP6 genes respectively. C. Research Project We are aware that the optimization of allotopic expression represents a real hope for patients suffering from diseases caused by mutations in mtDNA genes. However, all our efforts will remain unfruitful if the biosafety and the beneficial to mitochondrial function of our vectors are not proved in experimental models for mitochondrial diseases. Since, this proof is the mandatory step required before any attempt to the transfer to clinic, it becomes our highest priority. Unfortunately, only one animal model for mtDNA gene invalidation is available. These mice carry a mutation in the mitochondrial COXI gene leading to a decreased cytochome oxydase (COX) activity in several tissues 33. Even though, they do not have any visual impairment, we will try to rescue their muscle COX deficiency using our strategy. Additionally, we decided to use the optimized allotopic expression approach to create an animal model which will mimic LHON disease. First, we performed in vitro mutagenesis of the wild-type engineered human ND4 gene to obtain a nuclear version harboring the G11778A substitution. This mutation, responsible of 60% of LHON cases, converts a highly conserved arginine to histidine at codon 340 17. Each nuclear version of ND4 was combined with the two mRNA targeting sequences of the COX10 gene, which ensures the efficient delivery of the polypeptides inside the organelle 32 . We developed an in vivo electroporation (ELP) method to introduce either the wild-type or the mutated version of ND4 into retinal ganglion cells (RGCs) Invest in ME Charity Nr 1114035 of adult rats, as recently described 34. If we confirm that the animal model generated share an array of similarities with the clinical manifestations of LHON, we will assess the ability of our vector to protect RGCs. If we can demonstrate the proof-of-principle that our approach results in significant quantifiable improvements of RGC function in the experimental model of LHON that we generated we will open the door to gene therapy for retinal degenerations due to mutations in mtDNA. Expected consequences for knowledge in the field of medicine and public health Retinal dystrophies with mitochondrial etiology are inaccessible to curative or pallialtive therapy. Our knowledge on mRNA sorting to mitochondrial surface and its involvement in the organelle biogenesis makes this phenomenon a promising tool to fight these diseases. The transfer to clinic of our gene therapy protocol will undoubtedly represent a major step for the generation of a treatment aimed at improving life conditions of patients suffering for diseases such as LHON or DOA. We can envisage if these trails are successfull that clinical studies on other visual handicaps leading to blindness such as glaucoma 35 and devastating neurodegenerative disorders such as Charcot-Marie Tooth 36 could begin. (continued on page 27) Page 26/72 < 0.0001 ; n = 8 P value ; n

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