Abstract
Mitochondrial gene therapy and diagnosis have the potential to provide substantial medical benefits. However, the utility of this approach has not yet been realized because the technology available for mitochondrial gene delivery continues to be a bottleneck. We previously reported on mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. HLV injection, a useful method for nuclear transgene expression, involves the rapid injection of a large volume of naked plasmid DNA (pDNA). Moreover, the use of a condensed form of pDNA enhances the nuclear transgene expression by the HLV injection. The purpose of this study was to compare naked pDNA and condensed pDNA for mitochondrial association in skeletal muscle, when used in conjunction with HLV injection. PCR analysis showed that the use of condensed pDNA rather than naked pDNA resulted in a more effective mitochondrial association with pDNA, suggesting that the physicochemical state of pDNA plays a key role. Moreover, no mitochondrial toxicities in skeletal muscle following the HLV injection of condensed pDNA were confirmed, as evidenced by cytochrome c oxidase activity and mitochondrial membrane potential. These findings have the potential to contribute to the development for in vivo mitochondrial gene delivery system.
Highlights
Mutations and defects in the mitochondrial genome form the basis of a variety of human diseases, many of which involve mitochondrial dysfunctions [1,2,3,4]
We focused on the condensation of plasmid DNA (pDNA) to enhance mitochondrial association, since it has been reported that, when condensed pDNA is used in hydrodynamic limb vein (HLV) injection, it is even more effective than naked pDNA in achieving nuclear transgene expression in skeletal muscle [10,11,12]
We showed that mitochondrial delivery of naked pDNA by HLV injection achieved the localization of exogenous pDNA in inside a mitochondrion, based on the DNase I digestion experiment [5]
Summary
Mutations and defects in the mitochondrial genome form the basis of a variety of human diseases, many of which involve mitochondrial dysfunctions [1,2,3,4]. Injection for achieving mitochondrial gene delivery targeted to mammalian skeletal muscle tissue [5]. Skeletal muscle represents an attractive target tissue for mitochondrial gene therapy, because mitochondrial genomic dysfunctions in skeletal muscle are largely associated with various mitochondrial diseases [2,4]. The HLV injection procedure, a useful method for nuclear transgene expression in skeletal muscle, involves the rapid injection of a large volume of naked plasmid DNA (pDNA) into the distal vein of a limb [6,7,8,9]. Using PCR analysis, we demonstrated the HLV injection technique could be used to deliver naked pDNA into myofibrillar mitochondria and that it had no influence on mitochondrial function [5]
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