ObjectiveTo investigate the feasibility of immoribund skin fibroblast cell line derived from Leber′s hereditary optic neuropathy (LHON) patients as a cell model. MethodsA basic research. Two LHON patients and 2 healthy volunteers were recruited from Department of Ophthalmology of Genetic Clinic of Henan Provincial Eye Hospital. The skin tissue of participants was obtained, and the 4 immortalized skin fibroblasts were constructed by SV40 virus infection, including 2 LHON patient cells (LHON-1 and LHON-2 cells) and 2 healthy volunteers cells (NC-1 and NC-2 cells). Mitochondrial morphology in cells was observed by electron microscope. The levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide-oxidation state (NAD+), nicotinamide adenine dinucleotide-reduction state (NADH) and adenosine triphosphate (ATP) in fibroblasts were detected. Cellular oxygen consumption was measured by seahorse mitochondrial pressure assay. Cell viability was detected using cell counting kit-8 (CCK8). One-way ANOVA was performed to compare the levels of ROS, NAD+, NADH and ATP in LHON and NC cells, as well as basal oxygen consumption, maximal oxygen consumption, ATP-coupled oxygen consumption, and cell viability. ResultsCompared with NC-1 and NC-2, the number of mitochondrial crest in LHON fibroblasts was significantly reduced, indicating abnormal mitochondrial morphology. Biochemical analysis showed that ROS levels in LHON cells increased, but NAD+/NADH and ATP levels decreased, and the oxygen consumption was significantly inhibited, indicating the presence of mitochondrial damage and respiratory dysfunction. The results of CCK-8 detection showed that the survival ability of LHON-1 and LHON-2 cells was worse under stress conditions. ConclusionImmortalized skin fibroblast cell lines from LHON patients presented mitochondrial dysfunction.
Objective To observe the ocular and systemic safety and tissue distribution of adeno-associated virus vector 8-PDE6B (AAV8-PDE6B) after subretinal injection in C57BL/6J mice. Methods An animal experimental study. Thirty healthy C57BL/6J mice, 15 females and 15 males, aged 8 weeks, weighing 15-25 g, without specific pathogens, were randomly divided into solvent group, low-dose group, medium-dose group and high-dose group, with 6, 8, 8 and 8 mice respectively. The right eye was used as the experimental eye. Mice in the solvent group were injected with 1 μl of solvent under the retina; mice in the low-dose group, medium-dose group and high-dose group were injected with 1 μl of AAV8-PDE6B vector solution with viral titers of 1.70×1012, 8.50×1012 and 4.25×1013 vg/ml respectively under the retina. On day 28 after injection, retinal rod cGMP-specific 3',5'-cyclic phosphodiesterase 6β (PDE6B) expression was detected by immunofluorescence staining. Ophthalmic examination, fluorescein angiography (FFA), optical coherence tomography (OCT), hematoxylin-eosin (H&E) staining, and electroretinography (ERG) were performed to evaluate ocular morphology and retinal function. Body weight monitoring, hematological, liver function tests and biochemical analyses, and H&E staining of major tissues were used to evaluate systemic safety. Dot-blot and quantitative real-time polymerase chain reaction (qPCR) were used to detect serum AAV8 neutralizing antibodies and vector distribution, respectively. One-way analysis of variance was used for comparisons among multiple groups. ResultsCompared with the vehicle group, PDE6B expression in the photoreceptor outer segments was significantly increased in the low-, medium-, and high-dose groups (F=44.027, P＜0.001). On day 28 after injection, no obvious abnormalities were found on ophthalmic examination in any dose group. FFA showed no retinal vascular leakage, and OCT and H&E staining showed intact retinal structure. There were no significant differences in retinal thickness (including total thickness of inner nuclear layer, outer plexiform layer and outer nuclear layer), or ERG amplitudes of dark-adapted a-wave, dark-adapted b-wave, and light-adapted b-wave between the dose groups and the vehicle group (P＞0.05). The mice in all groups remained in good general condition, and no death occurred. There were no significant differences in body weight or major hematological and biochemical parameters among groups (F=1.475, 0.532, 0.285, 1.331, 0.814, 2.199, 2.342, 2.384, 2.650, 2.339, 1.305, 0.555, 1.259; P＞0.05). H&E staining showed no obvious abnormalities in major tissues. No AAV8 neutralizing antibody was detected in serum samples from any group. Except for one spleen sample in the high-dose group with vector gene copy number (11.3 copies/μg DNA) slightly above the lower limit of quantification (10.0 copies/μg DNA), vector gene copy numbers in other major extraocular tissues were below the lower limit of quantification. ConclusionSubretinal injection of low-, medium-, and high-dose AAV8-PDE6B in C57BL/6J mice shows good safety and tolerability, low extraocular tissue distribution, and no obvious humoral immune response.