3.1. Allotopic expression-mediated

3.1. Allotopic expression-mediated gene therapyEndosymbiotic gene transfer from the mitochondria to the nucleus is a ubiquitous ongoingevolutionary process that occurs at a substantial frequency (Timmis et al., 2004). Thisphenomenon is emulated for allotopic expression-mediated gene therapy for defective mtDNAgenes for the integration of the corresponding corrective mitochondrial genes into the nucleus(Guy et al., 2002; Manfredi et al., 2002; Shokolenko et al., 2010; Cwerman-Thibault et al.,2015). Transport of the subsequently expressed protein into the mitochondria is facilitated bya mitochondria-targeting sequence (MTS; which helps in transporting a protein of interest intothe mitochondria) (Shokolenko et al., 2010). nDNA-encoded proteins that are destined to belocalized in the mitochondria contain an MTS at their N-terminal ends, which is recognized bymitochondrial protein import channels, thereby allowing protein import (Chin et al., 2018).Moreover, specific 3’- untranslated regions (3′-UTRs) are used to locate mRNAs on the mitochondrial surface and help in co-translational import of these nDNA-encodedmitochondrial proteins (Kaltimbacher et al., 2006; Bonnet et al., 2007a). However, it has beenrecently reported that it is the choice of MTS that determines the efficiency of delivery ofallotopically expressed proteins to the mitochondria, rather than the selected 3’-UTR (Chin etal., 2018). This method was first tested for its efficacy in mitochondrial ATP8 gene-deficientmodels of Saccharomyces cerevisiae, where the incorporation of the WT mtDNA gene in thenucleus resulted in successful restoration of function (Nagley et al., 1988). This strategy ofintegrating mtDNA genes in the nDNA has been applied to correct defects arising from severalmtDNA mutations (Fig. 2A).As a key strategy for treating LHON, allotopic expression-mediated gene therapy has beenaccomplished in vitro in cybrid cells (derived from homoplasmic osteosarcoma) harboringm.11778G>A mutation in the ND4 gene (Guy et al., 2002). Through this method, the WT ND4gene was integrated into the nucleus by recombinant AAV (rAAV) vector. The plasmid wasconstructed by fusing the ND4 gene with an MTS (from the P1 isoform of human ATP synthasesubunit c), and a FLAG epitope tag. This sequence was then integrated with green fluorescenceprotein (GFP) via an internal ribosomal entry site (IRES) sequence, a chicken β-actin promoterand a cytomegalovirus enhancer. The final construct was cloned into pTR-UF12 plasmid vectorand packaged to generate rAAV particles. The cybrid cells were then stably transfected with3×107 rAAV particles. The expression of the fused protein was driven by the chicken β-actinpromoter and the enhancer. The transfected cells subsequently showed GFP and FLAGexpression in the cytoplasm and mitochondria respectively. The FLAG localization to themitochondria implied successful transport of the ND4 gene, which was further confirmed bythe observed three-fold increase in ATP synthesis in the cybrid cells (Guy et al