Gene Therapy for Corneal Cystinosis

Abstract

Purpose: : Corneal cystine crystal deposits are pathognomonic for cystinosis in human patients and are a virtually constant finding at the age of 1 year in cystinotic patients. The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin. The aim of this study is to create an AAV-Ctns vector and test its efficacy and safety in gene transfer studies

Methods: : Animal model of cystinosis was created by making a Ctns-/- mouse using a promoter trap approach. Ctns-/- mice and C57bl/6 mice were used in this study. A mixture of ketamine (150 mg/kg) and xylazine (10 mg/kg) was injected intraperitoneally for anesthesia. Proparacaine (0.5%) was used as an ocular topical anesthetic. AAV8-GFP and AAV2-GFP vectors were delivered by a single intrastromal corneal injection of 2.0 µl (vector titer= 2x10¹¹) into one eye of each mouse being studied. The contralateral eye received 2.0 µl PBS injections for control. Successful injection was gauged by observing that ≥70% of the cornea became whitened and edematous immediately following the injection. Antibiotic eye drops and lubricating gel were administered after the procedure. Tonometry, optical coherence tomography (OCT), slit-lamp examination, corneal histology and corneal cystine analysis were used as measures for efficacy and safety in this viral vector mediated gene transfer study.

Results: : Cystine crystals were detected in the corneas of 5-month-old Ctns-/- mice. The IOP in these knockout mice did not change significantly. Compared with AAV2, AAV8 vector had a higher efficiency transduction that resulted in high levels and uniform expression of reporter gene GFP, which started from day 3 and lasted at least for 8 months.

Conclusions: : Ctns-/- mice have some cystine accumulation in cornea and it can serve as an animal model for studying the corneal gene therapy. AAV8 can transduce the corneal stroma very efficiently, so it can be used in corneal gene therapy. An AAV8-CTNS vector was successfully created and injected into Ctns^-/- mice ranging from 1.5 to 4.5 months of age to test its efficacy in preventing, reducing, or eradicating crystals in corneas of Ctns^-/- mice.

Keywords: gene transfer/gene therapy • cornea: stroma and keratocytes • injection