Gene transfer to ovine corneal endothelium

Sonja Klebe, Pamela J. Sykes, Douglas J. Coster, David C. Bloom, Keryn A. Williams

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)

Abstract

Purpose: Modification of a donor cornea by gene therapy has potential to modulate irreversible rejection, the major cause of corneal graft failure. The sheep is a useful model for the human in this respect, as ovine endothelial cells are amitotic. The aim of the study was to investigate the ability of various non-viral and viral agents to transfer a reporter gene to ovine corneal endothelium. Methods: The non-viral agents Transfectin-10, Transfectin-20, Transfectin-50, SuperFect, Effectene and CLONfectin were used to deliver the reporter gene, Escherichia coli lacZ, to ovine corneal endothelium in vitro. A Herpes simplex virus-1 and an adenoviral vector each encoding E. coli lacZ were similarly tested. Infected corneas were organ-cultured for up to 7 days in vitro to allow transfection efficiency, duration of gene expression and toxicity attributable to each vector to be compared. Results: Scattered single or clusters of endothelial cells expressing the reporter gene were observed after transfection with CLONfectin, Transfectin-10, Transfectin-20 and Transfectin-50. SuperFect and Effectene were virtually ineffective. At best, the absolute number of infected cells per endothelial monolayer after 3 or 7 days of organ culture was estimated as < 0.01%. The Herpes simplex virus-1 vector also failed to transduce ovine corneal endothelium efficiently. In contrast, transfection rates of up to 70% of endothelial cells were observed with the adenoviral vector. Conclusion: Non-viral vectors and Herpes simplex virus-1 are unlikely to be suitable for gene therapy of corneal endothelium, because the efficiency of transfection is low compared with the rates achieved with adenoviral vectors.

Original languageEnglish
Pages (from-to)316-322
Number of pages7
JournalClinical and Experimental Ophthalmology
Volume29
Issue number5
DOIs
Publication statusPublished or Issued - 2001
Externally publishedYes

Keywords

  • Corneal endothelium
  • Gene therapy
  • Non-viral and viral vectors
  • Reporter gene

ASJC Scopus subject areas

  • Ophthalmology

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