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Joseph Paul, Timothy Colgan, Vicki Chrysostomou, Peter van Wijngaarden, Jonathan G Crowston; Intra-optic nerve delivery of AAV-mediated gene therapy in mice. Invest. Ophthalmol. Vis. Sci. 2018;59(9):315.
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© ARVO (1962-2015); The Authors (2016-present)
To validate an optic nerve injection technique for delivery of adeno-associated virus (AAV) gene therapies in mice. Once validated, we used this approach investigate the effect a vector targeting MCT1 knockdown in the optic nerve on visual function.
3-month-old male C57Bl/6J mice were used for all experiments, under 70:7 mg/kg ketamine:xylazine anaesthesia. In n=8 mice, a 1mm incision was made in the bulbar conjunctiva and the extraocular muscles resected to expose the optic nerve. An incision was made in the dural sheath with a 29G needle, and 1µL AAV-CMV-eGFP was injected via glass micropipette (100nl/min). One optic nerve per animal was treated, contralateral nerves served as controls. Animals were culled for tissue harvest two weeks after injection. Optic nerves were resin embedded, sectioned (1µm) cross-sectionally or longitudinally and stained with methylene blue for structural analysis.In a second experiment, n=12 mice had cranial electrodes implanted and baseline VEPs and ERGs recorded. Using the procedure above, 1µL AAV-MBP-Slc16A1 (1e10vg, treatment) or AAV-MBP-LacZ (1e10vg, control) was injected into one optic nerve per animal. Two weeks later VEP and ERGs were recorded and tissue harvested. Optic nerves were processed for immunofluorescence, and retinas flatmounted for ganglion cell counts. Data expressed as group mean ± SD. T-tests were used for statistical comparison.
In treated nerves, eGFP expression was limited to an approximately 500µm section of tissue around the injection site, and seen in 47±8% of oligodendrocytes in that area. G-ratios were unchanged between treated (0.59±0.08) and control (0.59±0.08) nerves (p=0.87) and degenerative axons were seen.Two weeks after injection, no differences were seen in pSTR (treated 85.0±17.7% of baseline, control 88.8±19.8%, p=0.4). There were significant differences in VEP N1 and P1N1 latencies in treated eyes (N1 119.36±26.5% of baseline p=0.04, P1N1 124.8±26.1% p=0.02), P1 latency did not change (105.0±34.4%, p=0.2). No significant changes were seen in control eyes (P1 106±41.1% N1 100.8±10.1 p=0.04, P1N1 113.6±63.1% p=0.02). No change was seen in ganglion cell counts (treated 3541±822 cells/mm2, control 3981±535 cells/mm2, p=0.13).
Intra-optic nerve injection is a safe and effective procedure for delivery of AAV2-mediated gene therapies. Treatment with a vector targeting MCT1 knockdown caused delays in VEP latency measurements.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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