In vivo Viral Infusion Method for Gene Delivery to Trabecular Meshwork Cells in Rat Eyes

Abstract

 

Purpose:

 

To evaluate a method for gene delivery to trabecular meshwork (TM) cells that minimizes corneal endothelium uptake using an in vivo infusion method. Additionally, non-selective promoter cytomegalovirus (CMV) was compared with the promoter α2u, which is preferentially expressed in TM cells, to determine whether the ability of the infusion method to target the TM was independent of the selectivity of the promoters.

 

Methods:

 

A 1-mL syringe filled with CMV-LOX-GFP (1.3*10⁷ ifu/uL) and either α2u-CRE (1*10⁶ ifu/uL) or CMV-CRE (1*10⁵ ifu/uL) was placed in a syringe pump. A tube connected the syringe to a microneedle. The microneedle was placed in the anterior chamber of anesthetized rats. Adenoviruses were then infused into the anterior chamber at a rate of 0.75 uL/min for 40 minutes. Four days after infusion, the rats were sacrificed and the eyes were enucleated to determine relative luminosity based on GFP expression in the TM and corneal tissue using a fluorescent microscope and ImageJ software. One-tailed, paired t-tests were used to determine whether relative luminosity was increased in the TM compared to the cornea and two-tailed, unpaired t-tests were used to compare the relative luminosity of promoters α2u and CMV.

 

Results:

 

The relative luminosity was significantly increased in the TM when compared with the cornea for both adenovirus mixtures (table). There were no significant differences in relative luminosity between promoters α2u and CMV for the TM (p= 0.4, n=3) and the cornea (p=0.25, n=3).

 

Conclusions:

 

This in vivo viral infusion method effectively delivered high transgene expression to TM cells with minimal uptake by corneal endothelial cells in rats, regardless of the selectivity of the promoters used in this study. Slow infusion of recombinant adenoviruses provides a useful method for delivering genes to the TM, while minimizing the expression in the corneal endothelium. This method provides a stepping stone to deliver genes to the TM that may alter the outflow resistance of the eye.  

 

Keywords: gene transfer/gene therapy • trabecular meshwork • cornea: endothelium