Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States.
Department of Ophthalmology, Gazi University Medical Faculty, Ankara, Turkey.
Invest Ophthalmol Vis Sci. 2018 Jan 1;59(1):298-310. doi: 10.1167/iovs.17-22074.
To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS).
TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 μM MG132 and transduced with FIV.GFP (green fluorescent protein)- or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 μM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA.
In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 μM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus.
Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.
确定蛋白酶体抑制剂 MG132 是否能提高 FIV 载体在人眼小梁网（TM）-1 细胞和猴器官培养眼前节（MOCAS）中的转导效率。
TM-1 细胞用 0.5%二甲基亚砜（DMSO；载体对照）或 5 至 50 μM MG132 预处理 1 小时，然后以 20 的多重转导（MOT）转导 FIV.GFP（绿色荧光蛋白）或 FIV.mCherry 表达载体。24 小时后，用 GFP 抗体固定和染色细胞，手动或通过荧光激活细胞分选（FACS）计数阳性细胞。仅转导 FIV.GFP 颗粒的细胞用作对照。还通过荧光显微镜评估了 20 μM MG132 处理对高剂量（2×107 和 0.8×107 转导单位[TU]，分别）和低剂量（2×107 和 0.8×107 转导单位[TU]，分别）FIV.GFP 转导的影响，有无 MG132。通过 qPCR 对细胞和组织中的载体基因组当量进行定量。
在 MG132 处理组中，在所有测试浓度下，转导细胞的百分比均呈显著的剂量依赖性增加。MG132 处理的 TM-1 细胞中的载体基因组当量也增加了。用 20 μM MG132 和高剂量载体处理的 MOCAS 中也观察到 TM 中 FIV.GFP 表达增加。MOCAS 组织中的载体基因组当量也显著增加。低剂量病毒未观察到转导增加。
蛋白酶体抑制增加了 FIV 颗粒在 TM-1 细胞和 MOCAS 中的转导效率，可能是通过慢病毒载体将治疗基因递送至 TM 的有用辅助手段。
