Purchase this article with an account.
H. Yu, J. Guy; Modification of Adenoassociated Virus (aav) to Deliver Dna to Mitochondria. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3127.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Objective: To drive gene expression in mitochondria.Background: Diseases caused by mutated mtDNA are an untreatable group of disorders.Design/
We inserted ATPc and COX8 mitochondrial targeting sequences (MTS) into the VP2 capsid of the AAV viral envelope. We used the ATPc MTS (P1), in tandem (P1-P1), or fused in frame to GFP (P1 GFP), COX8 MTS alone or fused in frame to GFP (COX8 GFP). To determine the efficiency of these different capsid targeting sequences to drive red fluorescent protein (RFP) expression, we seeded RGC5 cells into 96 well plates. We used a double-stranded AAV vector where we had inserted RFP driven by the mitochondrial heavy strand (HSP) promoter. For each of the 5 different VP2 modifications we seeded 10 wells of cells then measured RFP fluorescence using a fluorospectrometer.
VP2 insertion of COX8GFP resulted in the highest levels of RFP fluorescence. Relative to P1 the COX8GFP fluorescence of RFP was 55% greater (p = 0.007). With a fluorescent cy3 tag to the self complementary sc-pTR-HSP-RFP we found cy3 fluorescence in a perinuclear and punctate pattern typical of mitochondrial localization. To prove mitochondrial import, we added MitoTracker Green, then examined for co-localization that varied from approximately 23%-53%. Western blots of mitochondrial isolates revealed COX8GFP. While the fluorescence of RFP could be detected by spectrophotometry it was not visualized by microscopy. Therefore we generated a mitochondrial Cherry (mCherry). Western blotting revealed mCherry in mitochondrial isolates. Relative to an average fluorescent intensity 5.67 for the D loop promoter the fluorescence intensity of mcherry was 10.55 for the heavy strand promoter. Thus, the heavy strand promoter was 86% more efficient in driving expression of mcherry in cells (P=0.00017). When driven by the HSP promoter mcherry was visualized in living cells co-expressing COX8GFP.Conclusions/Relevance: A mitochondrially targeted AAV vector may be useful for treatment of mitochondrial diseases.
This PDF is available to Subscribers Only