Thoene Jess G, DelMonte Monte A, Mullet Jodi
Department of Pediatrics, Division of Pediatric Genetics, Metabolism and Genomic Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Ophthalmology and Visual Sciences, Division of Pediatric Ophthalmology, Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA.
Mol Genet Metab Rep. 2020 Apr 7;23:100587. doi: 10.1016/j.ymgmr.2020.100587. eCollection 2020 Jun.
Therapeutic use of transmembrane proteins is limited because of irreversible denaturation when away from their native lipid membrane. Mutations in lysosomal membrane transport proteins cause many lethal disorders including cystinosis which results from mutations in CTNS, which codes for the lysosomal cystine transport protein, cystinosin. Cystinosin-deficient fibroblasts, including keratocytes (corneal fibroblasts) accumulate lysosomal cystine. Cystinosis patients develop highly painful corneal cystine crystals, resulting in severe visually debilitating photophobia. The only available therapy is daily treatment with cysteamine eye drops. We have previously shown that microvesicles containing functional cystinosin are spontaneously produced by infecting cells (Sf9) with baculovirus containing human wt CTNS. Infecting Sf9 cells for 3 days at a MOI of 1 yields 10microvesicles /ml with a modal diameter of 90 nm. Addition of these vesicles to cultures of cystinotic fibroblasts produces cystine depletion over the course of 96 h, which persists for 2 weeks. In this paper we show that addition of such microvesicles containing cystinosinGFP to ex vivo rabbit ocular globes yields punctate perinuclear green fluorescence in the corneal keratocytes. These results support potential therapeutic use of these cystinosin containing microvesicles in treating cystinotic corneal keratopathy with the advantage of administering twice/month instead of daily topical administration.
跨膜蛋白的治疗用途有限，因为其离开天然脂质膜时会发生不可逆变性。溶酶体膜转运蛋白的突变会导致许多致命疾病，包括胱氨酸病，该病由编码溶酶体胱氨酸转运蛋白胱氨酸转运体的CTNS基因突变引起。缺乏胱氨酸转运体的成纤维细胞，包括角膜细胞（角膜成纤维细胞）会积累溶酶体胱氨酸。胱氨酸病患者会出现高度疼痛的角膜胱氨酸晶体，导致严重的畏光，使视力严重受损。唯一可用的治疗方法是每天用半胱胺眼药水治疗。我们之前已经表明，通过用含有人类野生型CTNS的杆状病毒感染细胞（Sf9）可自发产生含有功能性胱氨酸转运体的微泡。以1的感染复数感染Sf9细胞3天可产生每毫升10个微泡，其模态直径为90纳米。将这些微泡添加到胱氨酸病成纤维细胞培养物中，在96小时内可使胱氨酸消耗，这种消耗可持续2周。在本文中，我们表明，将这种含有胱氨酸转运体绿色荧光蛋白的微泡添加到离体兔眼球中，角膜角膜细胞中会出现点状核周绿色荧光。这些结果支持这些含有胱氨酸转运体的微泡在治疗胱氨酸病性角膜病变方面的潜在治疗用途，其优点是每月给药两次，而不是每天局部给药。
