Protecting corneal endothelial cells

Emerging therapies may improve graft survival, reduce transplant need

Howard Larkin

Posted: Tuesday, September 1, 2020

Thomas A Fuchsluger MD, PhD

Gene and cell technologies now in development would protect corneal endothelial cells (EC) from dying, potentially extending the life of donated corneas in eye banks and improving endothelial graft survival, Thomas A Fuchsluger MD, PhD, told the 37th Congress of the ESCRS in Paris, France.
The innovative technologies might even reduce the need for transplantation by protecting patients’ own endothelium in vivo, said Dr Fuchsluger, who is chairman and professor of ophthalmology at Rostock University Hospital, Germany.
Several viral and non-viral vectors have proven effective in delivering genes and proteins that slow cell death of EC in donated corneas, said Dr Fuchsluger. These include lentiviruses, optimised adeno-associated viruses and calcium phosphate nanoparticles. This could help reduce the approximately 30% loss of corneas due to EC deterioration in eye banks, as well as the 25% loss after transplantation.
Similarly, magnetic nanoparticles have successfully penetrated and treated EC in explanted corneas, protecting them from early failure, Dr Fuchsluger noted. If this technique is successful when injected into the anterior chambers of living patients, it could avoid the need for transplants.
“This is an unmet need in ophthalmology,” he said.

Blocking cell death
EC apoptosis is triggered internally through mitochondrial action in response to stresses including oxidation and DNA damage, and externally through surface receptors receiving death ligands including Fas and TNFR1. The protein Bcl-xL has been found to block both apoptosis pathways, Dr Fuchsluger said. Transfecting genes that express Bcl-xL into ECs is effective in reducing cell death and maintaining EC physiological morphology and function.
In a 2011 study, Dr Fuchsluger and colleagues used a lentivirus to transfect genes for Bcl-xL into donor EC. A baculovirus that causes EC to express the broad-spectrum anti-apoptotic protein p35 was also tested. Both extended EC cell life and morphology well beyond untreated cells out to 11 weeks under eye bank storage conditions (Fuchsluger TA et al. Gene Ther. 2011 Aug;18(8):778-87. Fuchsluger TA et al. Human Gene Ther. 2011; 22:549-558).
Experiments with HIV transfection showed a preference for EC over stromal cells and may be a possible vector. Trials with various adeno-associated viruses (AAV) found AAV-2 and AAV-9 most efficient for gene transduction, making them a potential alternative vector to the much larger lentivirus. (Fuchsluger TA et al. Klin Monbl Augenheilkd. 2011(June);228(6):498-503.)
Efficiency of AAV transfection was further increased using a self-complementary approach that doubled the transfected DNA strand, Dr Fuchsluger said. The approximately two-week expression achieved with this method might make it useful for protecting EC immediately after transplantation, which is when the greatest loss occurs, Dr Fuchsluger said. (Gruenert et al. PLOS ONE March 29, 2016.)

Non-viral vectors
Among transfection solutions more elegant than viral vectors are calcium phosphate nanoparticles, Dr Fuchsluger said.
“They are cheap to produce, they can be stored on the shelf and you can produce them so they are custom designed for mammalian cells.”
Techniques include coating the calcium phosphate particle surrounding the DNA to be inserted with etoposide, which increases cellular uptake, delaying apoptosis in more cells, Dr Fuchsluger added.
“You can get up to about one in every six cells, which is a lot for a non-viral vector,” he noted (Hu J et al. Acta Biomateriala 2012;8:1156-1163).
Dr Fuchsluger is currently working with nanoparticles to transfect siRNA designed to downregulate two pro-apoptotic proteins. He reported a 25% reduction in Bak expression and 20% cut in Bax in experimental corneal tissue, which should translate into less EC self-destruction.

In vivo treatment
Magnetic nanoparticles could be used to protect EC in patients with failing endothelium, possibly avoiding the need for transplantation, Dr Fuchsluger said. The particles surround a magnetic core with DNA to be transfected and lipoplexes to aid adherence and penetration. The technique has decreased EC apoptosis up to 56% (Czugala et al. Nanomedicine. 2016 Jul;11(14):1787-800). In future practice, the particles would be injected into the anterior chamber and drawn into contact with the corneal endothelium using a magnetised contact lens.
Talks are under way with German regulators to launch phase I clinical trials of these technologies, Dr Fuchsluger reported.

Thomas A Fuchsluger :