TARGETING CONNEXIN 43 BY GENE THERAPY

TITLE: TARGETING CONNEXIN 43 BY GENE THERAPY FOR SCAR REDUCTION AND REGENERATIVE HEALING OF HUMAN CORNEA
SPONSOR:

FP7 Marie Curie COFUND Programme

CO-SPONSOR:
TIMEFRAME:

May 2013 – May 2015

THEME: Molecular Therapies
STATUS: ONGOING
GRANT AMOUNT:

130.000 €

FBOV ROLE: Host Institution
PARTNERS INVOLVED: University of Perugia
P.I. : Hossein Elbadawy, Ph.D

 

SUMMARY: The cornea serves as a barrier and contributes to the maintenance of corneal transparency necessary for good vision. In most cases, injuries resulting in corneal defects are recovered promptly. Conventional treatment with Mitomycin C or 5-Fluorouracil can reduce the inflammation but the side effects are potentially sight threatening. Procedures such as anterior lamellar keratolplasty, commonly performed by the Veneto eye bank, selectively replace the injured front part of the cornea. This project aims at using gene therapy to target connexin 43 (Cx43) gene expression for reducing inflammation and accelerating wound healing after transplantation of corneas to improve corneal graft survival rates. Additionally, scar formation exerts negative effects on the vision and severe scars may require allograft transplantation. Reduction of scar formation via improved wound healing is an ideal solution for these patients.

 

OBJECTIVES/SCOPE : This ultimate goal is to employ gene therapy approaches to accelerate corneal healing and improve allograft survival rates.

This will be achieved by:

  1. Optimising an ex vivo model of wound healing
  2. Analysing molecular mechanisms operating in ocular ulcers
  3. Defining and analysing biomarkers of human epithelial immune response
  4. Targeting specific proteins using gene therapy is cutting-edge techniques 

SUMMARY OF METHODS: To optimise the conditions for corneal injury and recovery in vitro, several models were tested. We developed a new three-dimensional organ culture system to mimic the corneal natural environment. The system is composed of a sterile artificial chamber made of a heat resistant material (Teflon). The artificial chamber has three inlets to organise the artificial tears dripping. The irrigation of each cornea is controlled by a peristaltic pump. Injury of the ocular surface and subsequent healing are investigated by means on immunofluorescence staining and confocal microscopy. Changes in the histology of corneal sections are also illustrated in using specific staining including Masson’s Trichrome and hematoxylin and eosin.  Corneal opacity, swelling, and epithelial integrity are indicators of the healing rate. Additionally, epithelial proliferation and cell borders antibodies against proteins, including Ki67, P63 and ZO-1, which we found to be expressed in distinct patterns during the healing process Analysis of gene loss/gain of function is studied using gene knockdown/overexpression, respectively. Illustration of the inflammatory response map for the healing process is performed by monitoring markers of inflammation, such as F4/80, a specific marker of macrophages and myeloperoxidase (MPO), a specific marker of neutrophils. Reduction in neovascularisation is evaluated as indicated by the expression of the vascular endothelial growth factor (VEGF). Cytotoxicity and cell apoptosis are measured using caspase detection probes, colorimetric and fluorometric assays.

 

EXPECTED RESULTS: Finding, testing and optimising the effectiveness of novel agents to significantly reduce corneal ulcers and scar formation, followed by designing a suitable dosage form, which can be used for future clinical trials.

 

PUBLICATIONS:  

 

ABSTRACTS/POSTERS TO CONFERENCES:

 

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