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Ling Luo, Hironori Uehara, Subrata K Das, Bonnie Archer, Jacquelyn M. Simonis, Nirbhai Singh, Ying Liu, Thomas Olsen, Judd Cahoon, Balamurali K. Ambati; Short Hairpin RNA Delivered By Adeno-associated Virus Vectors (aav.shRNA) Induces Retinal Degeneration Via Extracellular And Intracellular TLR3. Invest. Ophthalmol. Vis. Sci. 2011;52(14):10.
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To determine whether short hairpin RNA delivered by adeno-associated virus vectors (aav.shRNA) induces retinal neurotoxicity.
An adeno-associated viral vector encoding shRNA conjugated and green fluorescent protein (aav.siRNA.GFP, 0.3X109GC/ul) was performed as treatment and the same concentration of adeno-associated viral vectors expressing GFP (aav.GFP) without shRNA or PBS were performed as controls. 10 days after intravitreal or subretina injection(1ul) in wild type or TLR3-/- mice, fundi were examined in vivo by autofluorescence and optical coherence tomography (OCT) using the Heidelberg Spectralis. IFN-gamma and IL-12 levels were evaluated by ELISA. Retina was also observed in vitro by transmission electron microscope and stained by Tunnel staining. In situ hybridizationwas performed to show the shRNA localization.The primary human RPE cells were transfected by the same set of aav.shRNA.GFP (1x109GC/8cm2)and then the cytotoxcity was evaluated. The rescued experiment was performed by TLR3 antibodies and chloroguine. hRPE barrier function was tested by trans Epithelium Resistance(TER).
Retinal degeneration and ruptured RPE layer RPE were easily found at 10days after aav.shRNA.GFP treatment in wild type mice but not in controls and in TLR3-/- mice. The levels of IFN-gamma and IL-12 treated with aav.shRNA.GFP increased significantly (n=8, P< 0.05) than the controls but no differences among each group in TLR3-/-. Tunnel staining showed the photoreceptors apoptosis and retinal degeneration. TEM disclosed the increased vacuoles and pigments lost in RPE and abnormal conjunction between the PRE and disorganized segments. These abnormalities were only found in aav.siRNA.GFP treatment group but not in aav.GFP or PBS control in wild type mice. In situ hybridization showed shRNA localized in the outer nuclear layers primarly after intravitreal injection. The density of RPE cells dramatically decreased after aav.shRNA.GFP transfection 5 days than aav.GFP (n=6, p=3.2E-17) and PBS (N=6, P<3.4E-17). TLR3 antibodies & Chloroquine increased (1 hour ) both extracellularly and intracellularly the density of RPE cells (n=6, p<0.0005) . RPE barrier function dramatically and consistently decreased during one month after aav.shRNA.GPF transfection.
aav.shRNA induces significant retinal neurotoxicity via activation of extracellular and intracellular TLR3.
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