Ray J, Wolfe J H, Aguirre G D, Haskins M E
James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, USA.
Invest Ophthalmol Vis Sci. 1998 Aug;39(9):1658-66.
To determine the versatility of retroviral vector-mediated rat beta-glucuronidase cDNA expression in the normal retinal pigment epithelium (RPE) of eyes of various species and in RPE of eyes with three types of mucopolysaccharidosis (MPS types I, VI, and VII) and to evaluate the effect of multiple transductions and long-term stable expression in the RPE.
A retroviral construct containing a rat beta-glucuronidase cDNA (NTK-BGEO) was used to infect RPE cells at subconfluence. The transduced cells were selected in G418, an antibiotic toxic to normal mammalian cells. Beta-glucuronidase activity was measured in transduced cells and media, using a fluorogenic substrate. Glycosaminoglycan profiles were examined by metabolically labeling RPE with Na2(35)SO4.
Transduced RPE cells, regardless of species or disease status, expressed rat beta-glucuronidase. The expressed enzyme restored normal levels of glycosaminoglycans in the RPE cells of homozygous MPS VII-affected dogs by metabolizing stored glycosaminoglycans. The expressed enzyme failed to metabolize stored glycosaminoglycans of MPS I and MPS VI, indicating that overexpression could not bypass the exoglycosidase restriction. Multiple transductions increased beta-glucuronidase activity several times in the cell layer and in the media. The expression was stable in vitro for at least 12 weeks.
A retroviral vector can mediate transfer of beta-glucuronidase in various species of normal and MPS-affected RPE. The expression is stable in vitro. The metabolism of stored glycosaminoglycans in MPS needs replacement of only the deficient enzyme to reverse the storage.
确定逆转录病毒载体介导的大鼠β-葡萄糖醛酸酶cDNA在不同物种眼睛的正常视网膜色素上皮（RPE）以及患有三种黏多糖贮积症（I型、VI型和VII型黏多糖贮积症）的眼睛的RPE中的通用性，并评估多次转导和在RPE中长期稳定表达的效果。
使用含有大鼠β-葡萄糖醛酸酶cDNA的逆转录病毒构建体（NTK-BGEO）感染亚汇合状态的RPE细胞。在对正常哺乳动物细胞有毒性的抗生素G418中筛选转导的细胞。使用荧光底物测量转导细胞和培养基中的β-葡萄糖醛酸酶活性。通过用Na2(35)SO4对RPE进行代谢标记来检查糖胺聚糖谱。
无论物种或疾病状态如何，转导的RPE细胞均表达大鼠β-葡萄糖醛酸酶。表达的酶通过代谢储存的糖胺聚糖，使纯合VII型黏多糖贮积症患病犬的RPE细胞中的糖胺聚糖水平恢复正常。表达的酶未能代谢I型和VI型黏多糖贮积症中储存的糖胺聚糖，表明过表达不能绕过外切糖苷酶的限制。多次转导使细胞层和培养基中的β-葡萄糖醛酸酶活性增加了几倍。该表达在体外至少12周内稳定。
逆转录病毒载体可介导β-葡萄糖醛酸酶在各种正常和黏多糖贮积症影响的不同物种的RPE中转移。该表达在体外稳定。黏多糖贮积症中储存的糖胺聚糖的代谢仅需替换缺陷酶即可逆转储存。
