Adenovirus - mediated gene transfer to human cornea


Larkin D., ORAL H. B. , George A.

Investigative Ophthalmology and Visual Science, vol.37, no.3, 1996 (SCI-Expanded) identifier

  • Publication Type: Article / Article
  • Volume: 37 Issue: 3
  • Publication Date: 1996
  • Journal Name: Investigative Ophthalmology and Visual Science
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Bursa Uludag University Affiliated: Yes

Abstract

Purpose: To examine the feasibility of gene transfer to human cornea by adenovirus vectors, and the effect of viral promoters on transfer efficiency and gene expression. Methods: 10mm diameter eye bank corneal discs were divided into 6 specimens. These were incubated for 3h in solutions of recombinant replication-deficient type 5 adenovirus containing a marker gene encoding E.coli β-galactosidase (β-gal): (i) AdCB2, driven by a CMV promoter, at 1.5×108 pfu/specimen, or (ii) AdRL, driven by a Rous sarcoma virus (RSV) promoter, at 4.0×108 pfu/specimen. Control specimens were incubated in virus-free medium. Following ex vivo storage in culture medium at 37°C, gene expression was examined by histology and a colorimetric assay of β-gal production normalised for total corneal protein. Results: Under influence of both promoters, gene expression was restricted to the endotheuum (75-100% of cells positive at 3d) and migrating epithelium at the stromal edge. Following storage, β-gal assay indicated CMV-driven expression to be maximal at 7 days, falling to undetectable levels at 28d. RSV-driven expression was quantitatively lower at all timepoints other than at 3d (when maximal), falling to undetectable levels at 21d. No β-gal expression was found in control specimens. Conclusions: Transgene expression driven by both CMV and RSV promoters was similar and attenuated by 21-28d. However, these results indicate the feasibility of short-term recombinant virus-mediated transfer of novel genes, in an 'eye bank' setting.