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Microvesicle delivery of a lysosomal transport protein to ex vivo rabbit cornea
Jess G Thoene1, Monte A DelMonte2, Jodi Mullet1
1Department of Pediatrics, Division of Pediatric Genetics, Metabolism and Genomic Medicine, University of Michigan, Ann Arbor, MI 48109, USA.
Abstract:
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.
Insights
Microvesicles carrying functional cystinosin offer a promising new therapy for cystinosis. These engineered microvesicles may reduce painful corneal cystine crystals, improving treatment frequency for patients.
Area of Science:
- Biochemistry
- Cell Biology
- Ophthalmology
Background:
- Transmembrane proteins are challenging to use therapeutically due to denaturation outside lipid membranes.
- Mutations in the CTNS gene cause cystinosis, a disorder leading to lysosomal cystine accumulation and painful corneal crystals.
- Current treatment for cystinosis involves daily cysteamine eye drops.
Purpose of the Study:
- To investigate the therapeutic potential of microvesicles containing functional cystinosin for treating cystinotic corneal keratopathy.
- To evaluate the efficacy of these microvesicles in ex vivo ocular models.
Main Methods:
- Functional cystinosin was produced in microvesicles by infecting Spodoptera frugiperda (Sf9) cells with a baculovirus vector.
- Microvesicles were characterized for yield and size.
- The uptake and localization of cystinosin-containing microvesicles were assessed in ex vivo rabbit ocular globes.
Main Results:
- Infection of Sf9 cells yielded a high concentration of microvesicles (10^11/ml) with a modal diameter of 90 nm.
- Addition of these microvesicles to cystinotic fibroblasts depleted cystine for up to two weeks.
- In ex vivo rabbit corneas, microvesicles containing cystinosin-green fluorescent protein (GFP) showed punctate perinuclear green fluorescence in corneal keratocytes.
Conclusions:
- Microvesicles containing functional cystinosin demonstrate potential for treating cystinotic corneal keratopathy.
- This approach may allow for less frequent administration (twice/month) compared to current daily eye drops.
- Engineered microvesicles represent a novel strategy for delivering therapeutic transmembrane proteins like cystinosin.
