| Purpose:To assess intravitreal injection dose and safety of recombinant adeno-associated virus (AAV)-mediated gene delivery of human NADH dehydrogenase subunit4(ND4) in rabbit eyes.Methods:An open reading frame for human ND4or AAV-green fluorescent protein (GFP) were fused to the mitochondrial targeting sequence and packed into separate AAV capsids. Rabbits of three treatment groups were administered0.1mL AAV-ND4,0.1mL AAV-GFP, or0.1mL vehicle via intravitreal injection, respectively. The efficiency and safety of recombinant AAV-mediated gene delivery of human ND4in rabbit eyes was assessed with a slit lamp microscope and direct ophthalmoscope, measurements of intraocular pressure and flash visual evoked potential, and optical coherence tomography (OCT). The mRNA and protein expressions of human ND4in the retina of rabbits were determined with real-time PCR, immunofluorescence, and Western blot.Results:No complications occurred in any of the three treatment groups after the intravitreal injection. At one-month post-injection, no significant difference in the mean thickness of retinal nerve fiber layer (RNFL) was found among the three groups. the mean thickness of the RNFL detected by OCT was67.2±7.5μm in the AAV-ND4group,65.2±10.3μm in the AAV-GFP group, and68.2±8.8μm in the control group.Results of the visual evoked potential test showed that there was no difference in the latency of the visual P100wave among the three groups.:58.6±7.5ms in the AAV-ND4,55.3±6.2ms in the AAV-GFP, and57.6±4.8ms in the control. Real-time PCR, immunofluorescence, and Western blot analyses verified the expressions of ND4and GFP in the RNFL.Conclusions:Intravitreal injection of AAV-ND4expression vectors was effectively and safely performed in our study. The data on the dose and method of intravitreal injection from our study will provide a valuable reference for clinical intravitreal injection of AAV-ND4for the treatment of Leber’s hereditary optic neuropathy. |
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