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X. Qi, A. Lewin, W. Hauswirth, J. Guy; Long-Term Outcomes for the Mouse Eye with AAV Mediated Gene Delivery of the Human ND4 Complex I Subunit. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1443.
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To evaluate the long-term effects of AAV mediated allotopic gene expression of a normal human ND4 (wt-ND4) of complex I subunit in the mouse visual system.
wt-ND4 was made compatible with the universal genetic code and a mitochondrial targeting sequence (ATPc) was appended to the N terminus and an epitope (FLAG) added to the C terminus. This construct was packaged into AAV2 capsids. AAV containing wt-ND4, mutant (R340H) ND4 (mut-ND4), and GFP were intravitreously injected into the mice eyes. One year later, gene expression and potential toxicities were assessed by fluorescence, light and electron microscopy.
One year after the gene inoculation fluorescence microscopy revealed 15.0% FLAG positive and 5.3% GFP-positive retinal ganglion cells (RGC) in flat mounted retina of AAV-wt-ND4 injected eyes, relative to the mean number of RGCs. The level of AAV-mut-ND4 gene expression was similar that the FLAG positive RGCs was 16.0% and GFP-positive RGCs was 5.4%. AAV- wt-ND4 inoculated retina had mean RGC of 3100+142 cells/mm2, a 3.5 % less than the normal untreated eyes with mean of 3314+138 cells/mm2, p>0.05. In contrast, AAV-mut-ND4 caused 40% loss of RGC with mean of 1956+143 cells/mm2, p<0.01. Morophometric analysis revealed that AAV-GFP inoculation caused 7% bigger in area of optic nerve head (ONH) relative to normal eyes, p>0.05. AAV-wt-ND4 gene inoculation showed the ONH swelling by 14%, p>0.05. AAV-mut-ND4 inoculation caused optic head swelling by 30 %, p<0.05. In the retrobulbar optic nerve cellular infiltration was increased almost two-fold (p < 0.01) in mut-ND4 treated eyes as compared to normal eyes. Delivery of the AAV- wtND4 or GFP did not change the cellular infiltration. To detect any demyelination in the retrobulbar optic nerve, the area of axonal myelin fiber was measured. As compared to normal eyes, AAV- wtND4 or GFP decreased the area of axonal myelin fiber by 3-5%, both p>0.05. The mut-ND4 inoculation caused axonal myelin fiber thinner by 36% (p<0.01), meaning the loss of myelin. Counting the number of axons, in contrast to the normal untreated eyes, AAV-GFP or wt-ND4 decreased the axons by 5% or 9%, respectively, both p>0.05. The severe loss of axons was evident after mut-ND4 gene inoculation, caused 41% loss of axons (p<0.01).
A nuclear-encoded, importable version of wt-ND4 delivered by AAV2 can be detected one year after gene transfer. Expression of normal human ND4 in murine did not induce significant loss of RGCs and the axonal demyelination suggesting it may safe for treatment of LHON.
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