Abstract
Purpose:
Despite advances in antiviral therapies, past history of HSV-1 keratitis is associated with poor prognosis of subsequent penetrating keratoplasty. With the aim of installing in the transplant a permanent protection against herpetic infection, we checked the potential of meganuclease gene therapy in corneal grafts
Methods:
Either cultured rabbit and human corneas or rabbit eyes were treated with a meganuclease targeting HSV-1 genome and expressed from a recombinant adeno-associated virus. Treatment was done by incubation or intracamerular injection. Administration of a non-coding vector was used as control. Two weeks after, the organs were infected with the HSV-1 F(1) strain recombinant for a LacZ expression cassette, through incubation in the medium or intracamerular injection. Corneas were examined during late phase of second lytic cycle of HSV-1 in the endothelium, with various techniques including macroscopic observation of X-Gal staining. Meganuclease effect on endothelial infection was finally checked in rabbit eyes transplanted with meganuclease-treated corneas following keratoplasty.
Results:
For organ cultures, treatment reduced the X-Gal staining surface or the number of plaques, that is the number of infected endothelial cells after inoculation, by 64% in rabbit and by 75% in human. In rabbit, meganuclease restricted by 39% the number of infected cells in each plaque, those contaminated at the end of the first lytic cycle and then for high MOI. Extension of herpetic infection was also limited by a decrease in particle synthesis, as a 78% decrease of X-Gal staining intensity is registered for rabbit. In the Rabbit in vivo model of endothelitis, plaque number was reduced by 56% and the quantity of plaques associated with keratic precipitates was lowered accordingly. There was a decrease by half in the extension of focal oedemas. Meganuclease effects upon erosion and herpetic particle synthesis concur finally in reducing by 70% the number of particles in aqueous humour. For transplanted eyes, treatment limited plaque number by 77%. Quantities of PK-associated plaques and particles in the anterior chamber were reduced by 80%.
Conclusions:
Altogether these results demonstrate the potential of meganuclease as antiviral agent and suggest that human transplants treated with a vector inducing sufficient amount of meganuclease could be protected against endothelitis.