AAV8 mediated gene therapy for corneal cystinosis

Abstract

Purpose: Cystinosis is a lysosomal storage disorder that results from mutations in the gene CTNS, which encodes cystinosin, the lysosomal membrane transporter for cystine. Corneal accumulations of intracellular cystine crystals lead to debilitating ocular complications. The aim of this study is to deliver an adeno-associated virus (AAV8)-Ctns vector into the cornea of Ctns-/- mice to study the efficacy of AAV8-mediated CTNS gene transfer.

Methods: 1.5 to 4.5-month old Ctns-/- mice from the Cherqui Laboratory at The Scripps Research Institute and 2-month old wildtype C57BL6 mice were used. AAV8 vectors cloned with either green fluorescent protein (GFP) or CTNS were delivered by single intrastromal corneal injections of 1.5 µl (vector titer= 2x1011) with a Hamilton micro-syringe. Successful injection was gauged by observing that ≥70% of the cornea became whitened and edematous immediately following the injection. The non-injected contralateral eyes were used as controls. At 11 to 15 months post-injection, mice were sacrificed and eyes collected. CTNS mRNA expression was measured using real-time polymerase chain reaction (q-PCR). Optical coherence tomography and corneal histology were used to evaluate changes in corneal crystal content.

Results: AAV8 vector had a high transduction efficiency that resulted in high levels and uniform expression of reporter gene GFP in the cornea, starting from day 3 and lasted up to 11 months at the point of eye collection. At 12 to 15 months post-injection, there was a significant difference in mRNA expression of CTNS as measured by qPCR in AAV8-Ctns eyes (n=4) when compared to that in the untreated eyes (n=4) (2225±1102.02 fold increase vs. 138.5±241.33 fold increase respectively, P-value = 0.0051).

Conclusions: AAV8 is a reliable vector with high transduction efficiency for gene transfer in Ctns-/- mice, producing extended expression of cystinosin for 12 to 15 months and likely longer after a single injection. Current study is aiming at characterizing corneal histology and biochemistry in treated and untreated mice.

Keywords: 538 gene transfer/gene therapy • 484 cornea: stroma and keratocytes • 480 cornea: basic science