Viral Gene Delivery Methods for the Cornea

Abstract

Abstract: : Purpose: Both inherited and viral diseases affecting the cornea might be treated through gene therapy. Adeno–associated virus (AAV) has the advantages of long term delivery of genes to both dividing and non dividing cells. The goal of this study includes (1) to compare gene transfer efficiency of different AAV vectors in the rabbit cornea, and (2) to locate transgene expression in different cell layers of corneal tissue. Methods: An AAV2 vector expressing GFP driven by chicken– ß–actin promoter and CMV enhancer was packaged in 5 different capsid types (1, 2, 5, 7, 8). Both eyes of NZ white rabbits were infected with the same serotype. Equal numbers of AAV particles (10¹¹ vector genomes) were diluted in buffer and applied to the cornea for 10 minutes following excimer ablation of the corneal epithelium to 25 microns. Seven days were allowed for gene expression before corneas were removed. The corneas were fixed in 4% formaldehyde before cryosectioning at 10um thickness. One sample was analyzed at every third section. Immunostaining using biotinylated antibody against GFP and an alkaline phosphatase detection as well as a peroxidase detection system were employed. Bright field and fluorescence micrographs were taken for each section using a morphometric microscope, and the level of GFP immunostaining was measured using MCID software. Results: By 7 days all the serotypes of AAV led to expression of the transgene in all layers of the corneal tissue. Epithelial cells were heavily stained compared with stroma and endothelial cells. AAV type 1 was 20 –30% more efficient in gene delivery in comparison with other serotypes, followed by AAV type 8 which exhibited the highest level of penetration to deep layers of the cornea. Conclusions: Among all the serotypes that have been tested AAV type 1 and 8 can provide long lasting and highly efficient transduction. These experiments point the way for delivery of potentially therapeutic genes to the cornea.

Keywords: cornea: basic science • gene transfer/gene therapy • wound healing