The avascular, slow evolving aAMD results mainly from inflammation and oxidative stress, causing cellular dysfunction that provokes accumulation of deposits and the decrease of RPE-synthesized neuroprotective factors, and finally, the loss of photoreceptors and retinal ganglion cells after RPE cells’ death.
RPE cells produce and secrete PEDF, which possesses neuroprotective and anti-angiogenic properties, in particular by inhibiting VEGF-mediated proliferation and migration of endothelial cells, and by protecting RPE cells and neurons from oxidative stress and ischemia. GM-CSF is produced by diverse types of cells including the RPE and is responsible for counteracting apoptosis, proliferation, differentiation and maturation of myeloid cells, and adaptive immune responses to inflammation and infection. PEDF alone is sufficient to effectively inhibit CNV in nAMD. Overexpressed together, PEDF and GM-CSF have the potential to prevent retinal degeneration in aAMD, by mitigating the effects of anti-oxidative stress, inhibiting inflammation, supporting cell survival, allowing the integration of cell transplants and protection of neural cells. To alleviate damages caused by both forms of AMD and /or to ease treatment, cell-based gene therapy might thus represent a powerful tool to overexpress these proteins exerting a protective effect on the retina.
We propose to transplant subretinally iris pigment epithelial (IPE) or RPE cells after genetic modification, to enable permanent overexpression of PEDF and/or GM-CSF. Transfection is successfully realized using the Sleeping Beauty (SB100x) transposon system, that offers an efficient and safe method for gene delivery. We have shown that IPE and RPE cells can be efficiently transfected with the PEDF and the GM-CSF genes expressed in vitro for longer than 18 months using SB100X and confirmed the cell protective, anti-angiogenic, ‑inflammatory and ‑oxidant effect of PEDF/GM-CSF in vitro and retina culture. We demonstrated that transplanted PEDF-transfected cells cause a significant reduction of neovascular lesions in vivo.
Safety of the vector system has been improved by using antibiotic-resistance-gene free miniplasmids. Additionally, we established the transfection using the transposase mRNA instead of DNA, which is degraded within approximately 2 minutes. Thus, a long-term expression of the transposase that poses the risk of re-mobilization and ‑integration of the transposon with an increased risk of adverse effects such as insertional mutagenesis is minimized.
According to the number of cells we can collect from an iris sample for transfection (about 10,000), our gene therapy is suited to treat early stages of AMD before cell loss.
For advanced stages, when RPE cells are lost, we aim to combine our gene therapy with stem cell approaches.
Ethically favorable, autologous iPS cells shall be reprogrammed from non-invasively collected renal tubular cells using the SB100x system and differentiated. The generated RPE cells will be enhanced to recover a healthy retinal environment to ensure the survival of themselves and retinal cells by aforementioned gene therapy approaches.