Gene Therapy of Herpes Simplex Keratitis Using Hammerhead Ribozymes

Abstract

Abstract: : Purpose: Herpes keratitis, generally caused by Herpes Simplex Virus type I (HSV–1), is the leading infectious cause of corneal blindness in industrialized countries. Although oral or topical therapies using antiviral medications and corticosteroids can help treat lesions, there is no effective therapy to prevent acute and recurrent infection. Our goal is to develop a gene therapy treatment for HSV keratitis using HSV–vectored ribozymes that target mRNAs of several genes that are essential for viral replication. These experiments are designed to test whether the ribozymes can block HSV–1 wt virus replication in tissue culture. Methods: Four essential genes (ICP4, ICP27, U_L20, and U_L30) were selected as targets for hammerhead ribozymes. In vitro kinetic analysis allowed us to choose the most active ribozyme for each mRNA. Rabbit skin cells were transfected with expression vectors in which synthesis of the ribozyme was driven by CMV enhancer and chicken ß–actin promoter. After brief selection using antibiotic G418 (48 hrs), cells were infected with wt HSV–1 virus (17syn+). At low multiplicity of infection (MOI of 0.001), after a 24–hour incubation, the ability of ribozymes to interfere with multiple rounds of viral replication was evaluated. At high MOI (1), one cycle of viral replication was permitted (12 to 18 hours) before determining the effect of ribozymes. Viral replication in different groups was compared using plaque assays. The reduction of target mRNA will be evaluated using reverse transcription PCR (RT–PCR). Results: The Kcat/K_M (a measure of enzymatic efficiency) of ICP4 and U_L30 ribozymes are 0.3 and 2.7 µM^–1min^–1, respectively, in 20mM Mg²⁺. The Kcat/K_M of U_L20 and ICP27 ribozymes are 15.9 µM^–1min^–1 and 11.7 µM^–1min^–1, respectively, in 5mM Mg²⁺. In tissue culture, the ICP4 ribozyme is able to reduce viral replication by 40% while the U_L20 and ICP27 ribozymes greatly reduced viral replication (>80%). Inhibition of replication by the U_L30 ribozyme is currently being evaluated. Conclusions: The efficacy of ribozymes in blocking viral replication in cell culture correlates with their relative kinetic activity in cell free reactions. Since the U_L20 and the ICP27 ribozymes efficiently block viral replication, genes encoding them are being cloned into a non–replicating HSV vector. The effects of virally delivered ribozymes will be tested in cell culture then in a rabbit model of HSV–1 recurrent ocular disease.

Keywords: gene transfer/gene therapy • herpes simplex virus • wound healing