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Clinical science
Scientific reports
In vitro adenovirus mediated gene transfer to the human cornea
  1. C F Jessup,
  2. H M Brereton,
  3. D J Coster,
  4. K A Williams
  1. Department of Ophthalmology, Flinders University, Adelaide, Australia
  1. Correspondence to: Dr Keryn Williams Department of Ophthalmology, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia; keryn.williamsflinders.edu.au

Abstract

Background/aims: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas.

Methods: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72–96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants.

Results: Transduction of human corneas with high doses (5×107–3×108 pfu) of AdV caused eGFP expression in 12–100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2×109 pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm2 (compared to 2114 (716) nuclei/mm2 for all other groups). Transgenic protein production peaked at 2.4 (0.9) μg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) μg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm2.

Conclusion: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.

  • AdGFP, replication deficient E1, E3 deleted adenovirus serotype 5 encoding GFP
  • AdV, adenovirus based vector
  • eGFP, enhanced green fluorescent protein
  • FCS, fetal calf serum
  • MOI, multiplicity of infection
  • PBS, phosphate buffered saline
  • pfu, plaque forming units
  • scFv, single chain antibody fragment
  • humans
  • cornea
  • gene therapy
  • adenovirus
  • reporter gene
  • AdGFP, replication deficient E1, E3 deleted adenovirus serotype 5 encoding GFP
  • AdV, adenovirus based vector
  • eGFP, enhanced green fluorescent protein
  • FCS, fetal calf serum
  • MOI, multiplicity of infection
  • PBS, phosphate buffered saline
  • pfu, plaque forming units
  • scFv, single chain antibody fragment
  • humans
  • cornea
  • gene therapy
  • adenovirus
  • reporter gene

https://doi.org/10.1136/bjo.2004.061754

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    Footnotes

    • Funding: This study was supported by the National Health and Medical Research Council of Australia and the Ophthalmic Research Institute of Australia. CFJ is supported by the South Australian Joyner scholarship in Medicine. The funding bodies had no direct input into study design, collection, analysis or interpretation of data for this publication.

    • Competing interests: none declared.

    • Ethics approval: Necessary ethics committee approval was secured for the study from the Flinders Clinical Research Ethics Committee.

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