Preventing endothelial cell death

Transferring a gene producing the anti-apoptotic protein p35 to corneal tissues could prolong donor cornea storage by preventing endothelial cell death and improve allograft survival by preventing epithelial cell death and related immune response, Thomas A Fuchsluger MD, PhD, of the Schepens Eye Research Institute at Harvard University, and Düsseldorf University Eye Hospital, Germany, told the second EuCornea Congress.

Human corneas in 4 degree C hypothermic storage that were treated with the p35 gene using a lentiviral vector lost epithelial cells more slowly than untreated corneas, retaining enough cells to be viable for more than 11 weeks compared with seven weeks for untreated tissues. Another gene, producing the anti-apoptotic protein bcl-xL, also slowed cell loss in storage, but was not as effective as p35.

Moreover, the endothelial cells expressing anti-apoptotic genes retained physiological cell morphology. Maintaining denser corneal tissues with functional morphology through anti-apoptotic gene therapy could increase the number of high-quality grafts in storage and reduce graft failure after transplantation, Dr Fuchsluger said.

Similarly, p35 gene-treated corneal epithelial sheets transplanted in a mouse model resisted induced apoptosis and associated opacity better and displayed enhanced epithelial barrier integrity compared with untreated allografts, leading to higher graft survival rates at 51 days in the treated group, Dr Fuchsluger reported. Over-expression of the p35 protein was observed in the treated group for at least two weeks. In addition to preserving cells, reduced apoptosis also appeared to reduce immune response toward the allograft. This research demonstrates the crucial role apoptosis plays in graft survival and suggests that preventing apoptosis by introducing p35 genes may improve survival rates.

A better vector

However, vectors remain problematic, with HIV-derived lentiviruses potentially inducing pathology. Dr Fuchsluger tested several adeno-associated virus, or AAV, variants in a murine model to determine suitability. Three of the AAV serotypes showed much higher proportions of endothelial cells expressing GFP than untreated corneas, suggesting that less-pathogenic AAV may be an alternative to HIV-based lentiviral vectors.

Calcium phosphate nanoparticles may be a viable non-viral vector, Dr Fuchsluger said. He noted that they are highly biocompatible, biodegrade easily, and low-cost preparations are stable in storage for up to two weeks. After transfection, the particles dissolve into calcium and phosphate, and the small increase in Ca2+ levels has been shown to not affect cell viability.

Dr Fuchsluger has demonstrated that calcium nanoparticles can be used to efficiently transfect endothelial cells of human cornea. He used a preparation of nanoparticles coated with polyethylenimine surrounding nucleic acids to produce EGFP to treat the corneas. After 12 hours, 55 per cent of cells in the treated group expressed EGFP compared with none in a negative control and about 15 per cent in a positive control. Polyethylenimine to the calcium phosphate nanoparticles increased the transfection efficiency, he noted.

'Calcium phosphate nanoparticles are suitable tools for transfection of corneal endothelial cell,' Dr Fuchsluger said. Since they produce little apoptosis, they may be an alternative to viral vectors.

References

1. A new tool for the transfection of corneal endothelial cells: Calcium phosphate nanoparticles. Hu J, Kovtun A, Tomaszewski A, Singer BB, Seitz B, Epple M, Steuhl KP, Ergün S, Fuchsluger TA. Acta Biomater. 2011 Sep 17. [Epub ahead of print]

2. [Viral vectors for gene delivery to corneal endothelial cells]. Fuchsluger TA, Jurkunas U, Kazlauskas A, Dana R. Klin Monbl Augenheilkd. 2011 Jun;228(6):498-503. Epub 2011 Jun 7. German.

3. Anti-apoptotic gene therapy prolongs survival of corneal endothelial cells during storage. Fuchsluger TA, Jurkunas U, Kazlauskas A, Dana R. Gene Ther. 2011 Aug;18(8):778-87. doi: 10.1038/gt.2011.20. Epub 2011 Mar 17.

4. Corneal endothelial cells are protected from apoptosis by gene therapy. Fuchsluger TA, Jurkunas U, Kazlauskas A, Dana R. Hum Gene Ther. 2011 May;22(5):549-58. Epub 2011 Mar 17.

Contact

Thomas Fuchsluger - thomas.fuchsluger@med.uni-duesseldorf.de