title: Gene transfer of integration defective anti-HSV-1 meganuclease to human corneas ex vivo.
abstract: Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.
authors.affiliation: The Veneto Eye Bank Foundation (FBOV), Venice, Italy.
authors.forename: H M
authors.initials: HM
authors.lastname: Elbadawy
authors.affiliation: Cellectis Therapeutics SAS, Paris, France.
authors.forename: M
authors.initials: M
authors.lastname: Gailledrat
authors.affiliation: Cellectis Therapeutics SAS, Paris, France.
authors.forename: C
authors.initials: C
authors.lastname: Desseaux
authors.affiliation: The Veneto Eye Bank Foundation (FBOV), Venice, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Salvalaio
authors.affiliation: 1] The Veneto Eye Bank Foundation (FBOV), Venice, Italy [2] Department of Molecular Medicine, University of Padova, Padova, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Di Iorio
authors.affiliation: The Veneto Eye Bank Foundation (FBOV), Venice, Italy.
authors.forename: B
authors.initials: B
authors.lastname: Ferrari
authors.affiliation: The Veneto Eye Bank Foundation (FBOV), Venice, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Bertolin
authors.affiliation: The Veneto Eye Bank Foundation (FBOV), Venice, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Barbaro
authors.affiliation: The Veneto Eye Bank Foundation (FBOV), Venice, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Parekh
authors.affiliation: Vectalys-Vectalys SAS, Canal Biotech II, Toulouse, France.
authors.forename: R
authors.initials: R
authors.lastname: Gayon
authors.affiliation: Department of Molecular Medicine, University of Padova, Padova, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Munegato
authors.affiliation: Department of Molecular Medicine, University of Padova, Padova, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Franchin
authors.affiliation: Department of Molecular Medicine, University of Padova, Padova, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Calistri
authors.affiliation: Department of Molecular Medicine, University of Padova, Padova, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Palù
authors.affiliation: Department of Molecular Medicine, University of Padova, Padova, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Parolin
authors.affiliation: The Veneto Eye Bank Foundation (FBOV), Venice, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Ponzin
authors.affiliation: The Veneto Eye Bank Foundation (FBOV), Venice, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Ferrari
mesh_terms: D003315:Cornea / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000235:genetics* / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D003850:Deoxyribonuclease I