Purchase this article with an account.
J. Guy, X. Qi, L. Sun, A. Lewin, W. Hauswirth; Modification of AAV to Deliver DNA Directly to Mitochondria . Invest. Ophthalmol. Vis. Sci. 2006;47(13):734.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To determine if allotopic expression could be modified to direct import of AAV to deliver its DNA payload directly into mitochondria.
To direct the targeting of the AAV vector into the mitochondria we inserted GFP cDNA under the direction of a mitochondrial targeting sequence (MTS) into a modified AAV capsid. We modified this vector by adding the COX8 MTS that we had described previously for allotopic expression of GFP into mitochondria. Here it was linked to GFP and inserted into the VP2 capsid of AAV at unique EAGI sites. We then linked a normal human mitochondrial encoded ND4 subunit gene with an appended FLAG epitope to the mitochondrial promoter H strand promoter, inserting into the AAV backbone (pTR–UF) containing the inverted terminal repeats. This plasmid pTR–UF11mitoND4 enveloped by the mitochondrial targeted COX8–GFP AAV was delivered to human 293 cells and LHON cybrids homoplasmic for the G11778A mutation in mitochondrial DNA. Mitochondria were labeled by MitoTracker Red. To prove the entire AAV virion was directed to mitochondria we co–stained with a conformation antibody (A20) that recognizes only the fully assembled virus. ND4FLAG expression was detected in a one step incubation with anti–FLAG–Cy3 conjugated antibody. We also isolated mtDNA from the transfected LHON cybrids using PCR primers flanking the H strand promoter and the 3'mitoND4. With these primers the endogenous mitochondrial DNA would produce a gene product > 10 Kb, while our H strand ND4 should produce a 1.3 Kb product. We selected a PCR extension time of two minutes, too short for amplification of the endogenous mitochondrial DNA.
We found the translocation of AAV capsid particles expressing GFP to mitochondria, by co–localization with the mitochondrial specific dye MitoTracker. The A20 specific antibody co–localized with COX8–GFP suggested import of the fully assembled virion. ND4FLAG expression detected in the transfected cells was identical to our previously allotopic delivery of ND4FLAG from the cytoplasm. Agarose gel electrophoresis of mtDNA isolated from transfected LHON cybrids revealed the 1.3 Kb band, indicating that our modified AAV vector delivered the normal ND4 subunit gene to the mitochondria of homoplasmic LHON cybrid cells, thus creating a heteroplasmic allele expressing both wild–type and mutated mtDNA.
Modification of AAV to deliver a normal gene directly to mitochondria represents a major milestone in the quest to treat LHON. This technique may also be applied to deliver other mitochondrial genes and treat the multitude of diseases associated with mutated mitochondrial DNA.
This PDF is available to Subscribers Only