Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

J Vis Exp. 2016 Apr 7;(110):e54058. doi: 10.3791/54058.

Abstract

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

Publication types

  • Research Support, N.I.H., Extramural
  • Video-Audio Media

MeSH terms

  • Adenoviridae / genetics*
  • Biomarkers / metabolism
  • Cathepsin F / genetics
  • Cell Count
  • Corneal Diseases / therapy*
  • Diabetic Neuropathies / therapy*
  • Epithelium, Corneal / cytology*
  • Epithelium, Corneal / metabolism
  • Genetic Therapy* / methods
  • Genetic Vectors
  • Humans
  • Limbus Corneae / cytology*
  • Limbus Corneae / metabolism
  • Matrix Metalloproteinase 10 / genetics
  • Organ Culture Techniques
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins c-met / genetics
  • Stem Cells / metabolism*
  • Wound Healing / physiology*

Substances

  • Biomarkers
  • MAS1 protein, human
  • Proto-Oncogene Mas
  • MET protein, human
  • Proto-Oncogene Proteins c-met
  • CTSF protein, human
  • Cathepsin F
  • Matrix Metalloproteinase 10