Mason Eye Institute, School of Medicine, University of Missouri-Columbia, 1 Hospital Dr. Columbia, MO 65212, USA.
Brain Res Bull. 2010 Feb 15;81(2-3):273-8. doi: 10.1016/j.brainresbull.2009.07.005. Epub 2009 Jul 16.
In the present study, cellular tropism and relative transduction efficiency of AAV2/6, AAV2/8 and AAV2/9 vectors have been tested for the cornea using primary cultures of human corneal fibroblasts. The AAV6, AAV8 and AAV9 serotypes having AAV2 ITR plasmid encoding for alkaline phosphatase (AP) gene were generated by transfecting HEK293 cell line with pHelper, pARAP4 and pRep/Cap plasmids. Primary cultures of human corneal fibroblasts were exposed to AAV infectious particles at two different doses (1 x 10(5) and 2 x 10(5) MOI). Cytochemistry and enzyme assays were used to measure delivered transgene expression in samples collected at 4 and 30 h after AAV infection by counting AP-stained cells or quantifying AP enzyme activity. Cellular toxicity of AAVs was evaluated with TUNEL and trypan blue assays. All three AAV serotypes transduced human corneal fibroblasts. The order of transduction efficiency was AAV2/6>>>AAV2/9>AAV2/8. The transduction efficiency of AAV2/6 was 30-50-fold higher (p < 0.001) for the human corneal fibroblasts compared to the AAV2/8 or AAV2/9 at two tested doses. The level of transgene expression at 4h was considerably low compared to 30 h suggesting that the transgene delivery did not reach its peak at 4h. Cultures exposed to any of the three AAV serotypes showed more than 97% cellular viability and less than 5 TUNEL positive cells suggesting that tested AAV serotypes do not induce significant cell death and are safe for corneal gene therapy.
在本研究中，使用原代培养的人角膜成纤维细胞测试了 AAV2/6、AAV2/8 和 AAV2/9 载体对角膜的细胞嗜性和相对转导效率。AAV6、AAV8 和 AAV9 血清型使用含有碱性磷酸酶 (AP) 基因的 AAV2 ITR 质粒的 pHelper、pARAP4 和 pRep/Cap 质粒转染 HEK293 细胞系生成。将原代培养的人角膜成纤维细胞暴露于两种不同剂量（1 x 10(5) 和 2 x 10(5) MOI）的 AAV 感染颗粒下。在 AAV 感染后 4 和 30 小时收集样本，通过计数 AP 染色细胞或定量 AP 酶活性，使用细胞化学和酶测定来测量转导基因的表达。通过 TUNEL 和台盼蓝测定法评估 AAV 的细胞毒性。三种 AAV 血清型均转导了人角膜成纤维细胞。转导效率的顺序为 AAV2/6>>>AAV2/9>AAV2/8。与 AAV2/8 或 AAV2/9 相比，在两种测试剂量下，AAV2/6 对人角膜成纤维细胞的转导效率高 30-50 倍（p < 0.001）。与 30 小时相比，4 小时时的转基因表达水平相当低，表明在 4 小时时转导基因的传递未达到峰值。暴露于三种 AAV 血清型之一的培养物显示超过 97%的细胞活力和少于 5 个 TUNEL 阳性细胞，表明测试的 AAV 血清型不会引起明显的细胞死亡，并且对角膜基因治疗是安全的。
