Ikeda Yasuhiro, Yonemitsu Yoshikazu, Miyazaki Masanori, Kohno Ri-Ichiro, Murakami Yusuke, Murata Toshinori, Tabata Toshiaki, Ueda Yasuji, Ono Fumiko, Suzuki Toshimichi, Ageyama Naohide, Terao Keiji, Hasegawa Mamoru, Sueishi Katsuo, Ishibashi Tatsuro
Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Hum Gene Ther. 2009 Jun;20(6):573-9. doi: 10.1089/hum.2009.009.
Abstract Gene therapy may hold promise as a therapeutic approach for the treatment of intractable ocular diseases, including retinitis pigmentosa (RP). Gene transfer vectors that are able to show long-lasting transgene expression in vivo are highly desirable to treat RP; however, there is a dearth of information regarding long-term transgene expression in the eyes of large animals. We previously reported that the simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vector showed efficient, stable, and safe retinal gene transfer, resulting in significant prevention of retinal degeneration by gene transfer of a neurotrophic factor, human pigment epithelium-derived factor (hPEDF), in rodents. Before applying this strategy in a clinical setting, we here assessed the long-lasting transgene expression of our third-generation SIVagm-based lentiviral vectors in the retinal tissue of nonhuman primates. Approximately 20-50 mul of SIV-EGFP (enhanced green fluorescent protein) or SIV-hPEDF was injected into the subretinal space via a glass capillary tube. To detect EGFP expression in the retina, we used a fluorescence fundus camera at various time points after gene transfer. Human PEDF expression was assessed by immunohistochemical analysis, Western blot assay, and enzyme-linked immunosorbent assay. The retinas demonstrated frequent EGFP expression that was preserved for at least 4 years without significant decline. The expression of hPEDF was stable, and occurred mainly in the retinal pigment epithelium. The secreted protein was detected in vitreous and aqueous humor. We thus propose that SIVagm-mediated stable gene transfer might be significantly useful for ocular gene transfer in a clinical setting.
摘要 基因治疗有望成为治疗包括色素性视网膜炎（RP）在内的难治性眼部疾病的一种治疗方法。能够在体内显示持久转基因表达的基因转移载体对于治疗RP非常理想；然而，关于大型动物眼中长期转基因表达的信息却很匮乏。我们之前报道过，基于非洲绿猴猿猴免疫缺陷病毒（SIVagm）的慢病毒载体显示出高效、稳定且安全的视网膜基因转移能力，通过在啮齿动物中转移神经营养因子人色素上皮衍生因子（hPEDF）基因，能显著预防视网膜变性。在将该策略应用于临床之前，我们在此评估了我们的第三代基于SIVagm的慢病毒载体在非人灵长类动物视网膜组织中的长期转基因表达情况。通过玻璃毛细管将约20 - 50微升的SIV - EGFP（增强型绿色荧光蛋白）或SIV - hPEDF注入视网膜下间隙。为了检测视网膜中的EGFP表达，我们在基因转移后的不同时间点使用荧光眼底相机。通过免疫组织化学分析、蛋白质印迹分析和酶联免疫吸附测定来评估人PEDF的表达。视网膜显示出频繁的EGFP表达，这种表达至少维持了4年且无明显下降。hPEDF的表达稳定，主要发生在视网膜色素上皮中。在玻璃体和房水中检测到了分泌蛋白。因此，我们认为SIVagm介导的稳定基因转移在临床环境中对于眼部基因转移可能具有显著的实用性。
