Herpes simplex virus mediated gene transfer to primate ocular tissues
Herpes simplex virus mediated gene transfer to primate ocular tissues
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DOI：
10.1006/exer.1999.0711
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发表时间：
1999-10-01
影响因子：
3.4
通讯作者：
Kaufman, PL
中科院分区：
文献类型：
--
作者：
Liu, XY;Brandt, CR;Kaufman, PL
我们评估了使用表达大肠杆菌LacZ基因的复制能力强的单纯疱疹病毒(HSV)1型核糖核苷酸还原酶突变体(HrR3)将基因导入猴眼的可行性。用hrR3感染体外培养的人眼小梁细胞(HTM)和人睫状肌细胞(HCM)，用组织化学方法检测β-半乳糖苷酶活性，以确定体外HSV介导基因转移的效率。对6只食蟹猴的前房(2×10(7)pfu)和(或)玻璃体(5×107pfu)注射病毒，观察表达LacZ的细胞的分布。体外培养的HTM和HCM细胞均表现出多样性依赖的β-半乳糖苷酶活性。在体内，房内和/或玻璃体内注射导致转基因在TM细胞和无色素睫状上皮细胞(NPE)中表达，但在CM细胞中不表达。视网膜色素上皮(RPE)细胞和散发性视网膜神经节细胞(RGC)中也检测到转基因表达。我们观察到注射病毒的眼的前房、TM和CM有明显的炎症反应，同时伴有轻微的玻璃体炎和视网膜炎。这项研究证明了使用hrR3作为载体在人眼细胞和活猴子的眼组织中成功地进行了基因转移。进一步研究炎症反应的病因、可能的细胞毒性和转基因表达的有限持续时间是必要的，以便使这项技术在临床上应用。(C)1999年学术出版社。
We evaluated the feasibility of delivering a gene into monkey eyes using a replication-competent herpes simplex virus (HSV) type 1 ribonucleotide reductase mutant (hrR3) expressing the Escherichia coli lacZ gene. To determine the efficiency of in vitro HSV-mediated gene transfer, cultured human trabecular meshwork (HTM) and human ciliary muscle (HCM) cells were infected with hrR3 and beta-galactosidase activity was measured histochemically. Six cynomolgus monkey eyes received viral injections into the anterior chamber (2 x 10(7) pfu) and/or the vitreous (5 x 107 pfu), and the distribution of cells expressing lacZ was evaluated. In vitro, both cultured HTM and HCM cells displayed multiplicity-dependent beta-galactosidase activity. In vivo, intracameral and/or intravitreal injection resulted in transgene expression in TM cells and in non-pigmented ciliary epithelial cells (NPE), but not in CM cells. Transgene expression was also detected in retinal pigmented epithelial (RPE) cells and sporadic retinal ganglion cells (RGC) in eyes receiving virus intracamerally and intravitreally respectively. We observed significant inflammation in the anterior chamber, TM and CM in virus-injected eyes, along with mild vitritis and retinitis. This study demonstrates successful gene transfer using hrR3 as a vector in human ocular cells and in ocular tissues in living monkeys. Further investigation of the etiology of the inflammatory response, possible cytotoxicity, and limited duration of transgene expression is necessary in order to make this technique clinically applicable. (C) 1999 Academic Press.