Dunaief J L, Kwun R C, Bhardwaj N, Lopez R, Gouras P, Goff S P
Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Hum Gene Ther. 1995 Sep;6(9):1225-9. doi: 10.1089/hum.1995.6.9-1225.
In this preliminary report, we describe a technique for gene transfer into the retina using a retrovirus vector. We transferred the bacterial LacZ gene and the neomycin-resistance gene into pigmented wild-type rat retinal pigment epithelial (RPE) cells in culture. The RPE culture was exposed to retrovirus, and infected cells were selected with a neomycin analog (G418). The LacZ gene product was detected by X-Gal histochemistry in 95-100% of drug-resistant cells. These genetically labeled cells were transplanted into the subretinal space of two 15- to 25-day-old albino RCS rats, which have an inherited retinal degeneration syndrome. The retinas were fixed and stained with X-Gal at 3 and 6 weeks after transplantation. At both time points, pigmented, LacZ-containing cells were seen in the subretinal space. Further, there were several rows of photoreceptor nuclei in the transplant area of the approximately 2-month-old rats, while in the control contralateral eye the photoreceptor nuclei were virtually absent, as for untreated animals, suggesting that the transplanted LacZ-marked, wild-type RPE cells may have helped preserve photoreceptors. The technique for gene transfer into RPEs followed by transplantation thus provides a means for gene therapy in organisms with a genetic defect in RPE cells.
在本初步报告中，我们描述了一种使用逆转录病毒载体将基因导入视网膜的技术。我们将细菌LacZ基因和新霉素抗性基因导入培养的野生型有色大鼠视网膜色素上皮（RPE）细胞。将RPE培养物暴露于逆转录病毒，并使用新霉素类似物（G418）筛选感染细胞。通过X-Gal组织化学在95%-100%的耐药细胞中检测到LacZ基因产物。将这些经基因标记的细胞移植到两只15至25日龄患有遗传性视网膜变性综合征的白化RCS大鼠的视网膜下间隙。在移植后3周和6周，将视网膜固定并用X-Gal染色。在两个时间点，均在视网膜下间隙中观察到有色的、含有LacZ的细胞。此外，在大约2月龄大鼠的移植区域有几排光感受器细胞核，而在对侧对照眼中，光感受器细胞核几乎不存在，如同未处理的动物一样，这表明移植的LacZ标记的野生型RPE细胞可能有助于保护光感受器。因此，先将基因导入RPE细胞然后进行移植的技术为患有RPE细胞遗传缺陷的生物体提供了一种基因治疗手段。
