Gold Nanoparticles: A Potent Vector for Non-Viral Corneal Endothelial Gene Therapy

Abstract

Purpose: : Healthy corneal endothelium is essential for corneal clarity. Treatment of defective corneal endothelial function often requires corneal transplant. Gene therapy is an attractive approach to treat/cure corneal endothelial diseases. We investigated the gene transfer efficacy of adeno-associated virus (AAV) and gold nanoparticles stabilized in polyethyleneimine (GNP-PEI) vectors for delivering therapeutic genes into human corneal endothelial cells.

Methods: : HPV16-E6/E7 transformed human corneal endothelial (HCN) cultures were incubated with AAV6, AAV8, AAV9 or GNP-PEI vector expressing reporter gene under control of Rous sarcoma virus or CMV+chicken beta actin promoter for 6 hours. The AAV titer had 109genomic copies/µl and GNP-PEI transfection solution had 5µg DNA with PEI nitrogen to DNA phosphate ratio180. The amount of transgene delivery was quantified with cytochemistry, immunostaining and/or real-time polymerase chain reactions. Commercial kits were used to prepare RNA and cDNA. Toxicity was tested using Trypan blue assay and morphological examinations.

Results: : Significant gene delivery into human corneal endothelial cells was detected with tested AAV and GNP-PEI vectors. Quantification of experimental data revealed 0.6-4.4% transgene delivery by AAV vectors into HCN. AAV6 vector showed highest transduction (4.4%; p <0.05) among tested AAV vectors. Contrary to AAV vectors GNP-PEI vector showed remarkably high transgene delivery into HCN (19-45%; p <0.001). Quantitative real-time PCR analysis showed GNP-PEI vector delivered up to 13 times more reporter gene in HCN. The AAV and GNP-PEI vectors showed 2-10% and 29-41% decrease in cellular viability, respectively. The AAV and GNP-PEI vectors did not induce significant phenotype change.

Conclusions: : Tested AAV6 and GNP-PEI vectors are efficient for delivering therapeutic genes into human corneal endothelial cells. Substantial loss of cellular viability by the GNP-PEI vector is most likely due to the long vector exposure (6 hours) to HCN. Studies are underway to define dose and toxicity of selected vectors for corneal endothelial gene therapy.

Keywords: cornea: endothelium • gene transfer/gene therapy • cornea: basic science