Development of a Ribozyme Gene Therapy Against Herpes Simplex Virus Type I (HSV-1) Infection

Abstract

Purpose:: Herpes keratitis is the most common cause of corneal blindness in the U.S. We proposed a ribozyme gene therapy approach to eliminate HSV infection by inhibiting the expression of HSV essential genes.

Methods:: A hammerhead ribozyme (Rz) was designed targeting mRNA of U_L20, an essential HSV-1 gene. The U_L20 ribozyme was packaged in an adenovirus vector, and an adenovirus vector expressing GFP was used as control. The Ad-U_L20 Rz was used to block viral replication of several HSV-1 strains, including wild-type HSV-1 (17syn+ and KOS) and several drug resistant strains (PAAr5, tkLTRZ1, and ACGr4) in tissue culture. The Ad-U_L20 Rz was tested in vivo in a mouse HSV-encephalitis model. Three groups including PBS, Ad-GFP, and Ad-U_L20 ribozyme treatments (10 Swiss mice each) were included. Ad-U_L20 Rz, PBS control or Ad-GFP were injected sub-epidermally and applied topically in rear footpads. HSV-1 infection was conducted right after at a dose of 10⁴ pfu (20-fold >LD₅₀). In a separate experiment, animals from either treatment (GFP or ribozyme) were sacrificed and feet, dorsal root ganglion (DRG), and spinal cord were harvested. Viral DNA levels in each tissue were measured using real-time PCR.

Results:: U_L20 Rz significantly inhibited viral replication of all the HSV-1 strains tested including three drug resistant strains, up to 99%. Ad-U_L20 ribozyme treatment consistently led to a 90% protection for mice from lethal HSV-1 infection, while survival rate in PBS group was 45% and survival in the Ad-GFP group was 40%. Levels of viral DNA in feet, DRG, and spinal cord from animals treated with U_L20 ribozyme were less than viral DNA levels in tissues from Ad-GFP treated animals.

Conclusions:: This study suggests that ribozymes targeting essential herpes genes of the late kinetic class may be a new therapeutic strategy for inhibiting HSV infection. It is possible that knocking down other essential late genes of HSV-1 may provide similar effect. A combination of ribozymes targeting different essential HSV-1 genes might provide synergistic effect and prevent the development of HSV mutants that escape therapy with a single ribozyme. Delivery approach for ribozymes into cornea to prevent HSK is currently under investigation.

Keywords: gene transfer/gene therapy • herpes simplex virus • keratitis