Microvesicle delivery of a lysosomal transport protein to ex vivo rabbit cornea.

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 Spodoptera frugiperda cells (Sf9) with baculovirus containing human wt CTNS. Infecting Sf9 cells for 3 days at a MOI of 1 yields 1011microvesicles /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.