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Hong Yu, Huijun Yuan, Gabriel S Gaidosh, John Guy; A Single Mito-targeted AAV Containing Multiple Mitochondrial Genes: Expression and Rescue Studies In Mice and LHON Cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3308.
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© ARVO (1962-2015); The Authors (2016-present)
To treat LHON caused by mutated ND4, ND1 and ND6 with a single mitochondrially targeted AAV.
The wild-type hND4 gene with a FLAG tag, followed by mitochondrial encoded Cherry, under control of the mitochondrial promoter (sc-HSP-ND4mCherry), or ND4 followed by ND1 and ND6 (sc-HSP-ND416), were packaged with mito-targeted AAV2 (COX8). A nuclear-encoded Cherry, fused in frame at the N terminus with the 23AA COX8 presequence, under control of the CMV promoter (sc-CMV-cox8Cherry) was packaged with wild type AAV2. The rAAV (mtAAV-HSPND4mCherry) was injected intravitreally into right eyes of normal mice. Contralateral left eyes received allotopic cherry (AAV-CMVcox8Cherry). The presence of mCherry was evaluated by serial confocal laser scanning ophthalmoscopy (LSO) and ND4FLAG by immuohistochemistry. Rescue of G11778A LHON cybrids with mtAAV-HSPND416was compared to the single gene construct mtAAV- HSPND4 at MOIs of 0, 500, 1000, 5000 and 10000.
LHON Cybrids. Cells infected with mtAAV-HSPND416 survived better in restrictive glucose free media containing galactose compared to uninfected LHON cybrids and those infected with mtAAV- HSPND4. The differences were significant at the MOI of 1000 (P=0.0004) and 5000 (P=0.02). Mice. Two days after injection, mice were examined using LSO; allotopic Cherry was expressed OS and red fluorescent particles were also found in the retinal ganglion cell (RGC) layer and optic nerve head OD. The number of red fluorescent particles increased as the mouse aged. Post-mortem cell counting of RGCs, showed the fraction of mito-targeted Cherry expressing cells, increased from 82.5% at 13 days to 91.1% at 1 month post injection. Similarly, the fraction of allotopically expressed Cherry in RGCs, increased from 80.3% at 13 days to 90.9% at 1 month post injection. Retinal flat mounts and longitudinal sections reacted with anti-FLAG, Porin and Thy1.2 antibodies, demonstrated expression of ND4FLAG and mCherry in the RGCs. The ND4FLAG and mCherry colocalized with RGC marker Thy1.2 and mitochondrial marker Porin. At higher magnification, the mCherry was evident surrounding DAPI labeled nuclei in the RGC layer.
As 95% of LHON cases are due to mutations in ND1, ND4 and ND6, a single mtAAV expressing multiple mitochondrial encoded genes provides a new way to develop effective gene therapy for the blindness of almost all cases of LHON.
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