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Rajeshwari D. Koilkonda, Tsung-Han Chou, Marco Ruggeri, Vittorio Porciatti, William W. Hauswirth, Vince Chiodo, Sanford L. Boye, John Guy; Early Functional and Long-term Rescue of the LHON Mouse With Wild-type Human ND4 Gene Delivery. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1416.
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© ARVO (1962-2015); The Authors (2016-present)
To demonstrate the feasibility of gene therapy with a wild-type (wt) human ND4 subunit of complex I to rescue visual loss and optic neuropathy.
To induce optic neuropathy single-stranded (ss) AAV containing the mutant human R340H ND4 responsible for most cases of LHON caused by G11778A mutated mitochondrial DNA was injected into the vitreous of both mouse eyes. Self-complementary rescue vectors containing the wt human ND4 with an appended FLAG epitope were packaged in single phenylalanine (F) for tyrosine (Y) substitutions in the VP3 AAV capsid (position 444) or triple F-Y substitutions (positions 444,500,730). For rescue either construct was injected into the right eyes. Contralateral eyes received scAAV-GFP. To gauge expression another series of mice received only the wt ND4 into the right eyes and GFP into the left eyes. ND4FLAG or GFP expressing cells relative to Thy1.2 labeled RGCs were quantitated 1, 3 and 7 days post injection (PI). Pattern electroretinogram (PERG) assessed rescue of visual function. Thickness of the optic nerve head and retina was evaluated by OCT. 1yr PI, rescue was gauged by ATP synthesis, TUNEL positive RGCs and optic nerve (ON) fiber counts.
Expression- Confocal microscopy revealed punctate and perinuclear expression of wt ND4FLAG. Relative to Thy1.2+ RGCs, quantification of scAAV-ND4FLAG+ RGCs was 21%, 50% and 85% by 1, 3 and 7 days respectively. Rescue- PERG at 1 and 3m PI showed a 28% and 48% reduction of amplitude in the rescued eyes compared to baseline. In contralateral eyes the reduction was 44% (p<0.05) and 54%. In addition, the control eyes showed latency delay of 17% and 20% (p<0.05) compared to rescued eye 1 and 3m PI. OCT images showed optic disc (OD) swelling in 60% of the controls at 1m. WT-ND4 reduced the OD swelling to 28%. A year after injections ON sections revealed significant thinning in control eyes (AVG±SD) 317±23µm, relative to wt ND4 rescued eyes 347±20µm (p<0.05). WT-ND4 rescued eyes had 30% less ON fiber loss (p<0.05) and less TUNEL + RGCs/mm2 (AVG±SE) 39±9 relative to 262±36 for control eyes (p<0.05). ATP synthesis in rescued ONs of 190 nM ATP/min/mg was similar to 192 nM ATP/min/mg for normal uninjected ONs and it was greater than 89 nM ATP/min/mg for unrescued control left eyes.
Human ND4 delivered by self-complementary Y-F AAVs improved oxidative phosphorylation, suppressed visual loss and optic neuropathy, thus providing proof of concept of this therapy for LHON patients.
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