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J. Liu, S.S. Tuli, D.C. Bloom, A.S. Lewin, G.S. Schultz; Ribozyme Gene Therapy of Herpes Simplex Infection in the Cornea . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3080.
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© ARVO (1962-2015); The Authors (2016-present)
Herpes Simplex Keratitis (HSK) is the leading cause of corneal blindness in developed nations. Despite active antiviral drugs, there is still no effective method for preventing recurrent HSK or treating stromal diseases. We tested the ability of ribozymes targeting the mRNA of essential HSV genes to inhibit viral infection in vitro and in vivo for gene therapy against HSK.
Hammerhead ribozymes were designed against mRNAs of four essential HSV genes and those with the most rapid cleavage kinetics were packaged in an adenoviral vector containing a CMV promoter and an IRES–GFP to localize ribozyme expression in rabbit skin cells (RSC). HSV–1 viral yields following Ad–ribozyme treatment were compared to control cells transduced with adenovirus vector without ribozyme. A ribozyme targeting the mRNA of an HSV–1 late gene (UL20 gene) was tested in vivo using a protected ribozyme containing 2’modifications of the ribose sugars of selected nucleotides. This modified ribozyme was delivered to the right eyes of 3 rabbits using iontophoresis, and an inactive ribozyme served as a control in left eyes, followed by inoculation of HSV–1 replicating virus that expressed ß–galactosidase. Three days post– infection, rabbits were sacrificed and corneas were harvested for X–gal staining to visualize the viral infection.
The UL20 ribozyme delivered by adenoviral vector reduced HSV–1 viral yield by 83% in the cell culture when cells were infected with HSV–1 at a moi of 10–3 for 24 hours in the presence of the ribozyme. In a time course study, the UL20 ribozyme reduced viral replication by 98% after 6 days of HSV–1 infection, indicating a cumulative effect of ribozyme cleavage. The chemically stabilized UL20 ribozyme reduced HSV–1 reduced dendrite lesion area by 57% compared with the rabbit eyes treated with an inactive ribozyme.
A hammerhead ribozyme targeting the mRNA of UL20 gene (a late essential gene HSV–1) significantly reduced the severity of an HSV–1 ocular infection in rabbits as well as significantly knocking down viral yield in cell culture. These results suggest a new gene therapy strategy targeting HSV–1 late genes that should lack the toxicity and resistance of antiviral drugs. Further studies evaluating the long term efficacy of different viral vectors to deliver therapeutic ribozymes during acute or recurrent HSV–1 infection are currently underway.
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