Abstract
BackgroundThe rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host's immune system.Methodology/Principal FindingsIn the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol.Conclusions/SignificanceWe demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.
Highlights
Defects in the mitochondrial NADH-quinone oxidoreductase have been shown to lead to many human diseases [1,2]
We showed that injection of recombinant adeno-associated virus carrying the NDI1 gene into the brain and skeletal muscles of rats and mice resulted in functional expression of the transgene and that the expressed Ndi1 had protective effects against Parkinsonian symptoms in the rotenone-treated rats [5] and MPTP-treated mice [6]
We did a single injection of recombinant adeno-associated virus (rAAV) carrying the NDI1 gene or the GFP gene into the rat skeletal muscle and examined the sections using hematoxylin and eosin (H&E) staining 1 week or 1 month post-injection
Summary
Defects in the mitochondrial NADH-quinone oxidoreductase (complex I) have been shown to lead to many human diseases [1,2]. We have developed a gene therapy strategy that utilizes the NDI1 gene encoding the yeast rotenone-insensitive internal NADH-quinone oxidoreductase (Ndi1) [2,3,4]. The principle of this approach is that the yeast Ndi enzyme can replace functionality of defective complex I in the respiratory chain of mammalian mitochondria. Ndi acted as a member of the respiratory chain in the rodent mitochondria and was able to compensate for the malfunctioning complex I. The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. Long term expression of Ndi observed in rodents with no apparent damage to the target tissue was indicative of no action by the host’s immune system
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