Sleeping Beauty (SB100X) clinical study for Macular Degeneration
Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo
Sandra Johnen, 1 Yassin Djalali-Talab, 1 Olga Kazanskaya, 1 Theresa Möller, 1 Nina Harmening, 2 Martina Kropp, 2 Zsuzsanna Izsvák, 3 Peter Walter, 1 and Gabriele Thumann 1 , 2
Abstract
Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth.
To avoid the side effects associated with virally mediated gene delivery, we have used the hyperactive Sleeping Beauty (SB100X) transposon system to transfect pigment epithelial cells with the PEDF gene [41]. SB100X has a number of advantages over virally mediated gene delivery: it integrates transgenes exclusively into TA dinucleotides with specific predilection for dinucleotides that have distinct structural features, integrates transgenes with high efficiency, results in stable integration, integrates large inserts, and does not primarily integrate into transcriptional sequences [42–45].
This study was designed to investigate whether the SB100X transposon system can efficiently transfect primary pigment epithelial cells with the human PEDF gene and whether the secreted rPEDF is functional as an inhibitor of VEGF-mediated endothelial cell function and as a neuroprotective agent in vitro and whether PEDF-transfected pigment epithelial cells have the potential to inhibit neovascularization in vivo in a model of CNV.
Discussion
The retinal pigment epithelium maintains the avascularity of the retina and a healthy choroidal vasculature by expressing and maintaining a balance between angiogenic and antiangiogenic factors, in particular VEGF and PEDF. The evidence that in neovascular AMD the equilibrium is shifted in favor of the angiogenic VEGF [51, 52] led to the development of inhibitors of VEGF, for example, the humanized monoclonal antibody bevacizumab (Avastin), its Fab fragment ranibizumab (Lucentis), and aflibercept (Eylea), which engendered a new era for the treatment of neovascular AMD and other ocular neovascular diseases [53–55]. We theorized that the side effects, resulting from frequent intravitreal injections of anti-VEGF antibodies to prevent CNV, could be alleviated by subretinal transplantation of genetically modified pigment epithelial cells that continuously secrete antiangiogenic PEDF. Autologous pigment epithelial cells transplanted to the subretinal space in neovascular AMD patients [28, 29] were well tolerated over a long period of time. However, no significant beneficial effects have been shown on vision. We postulate that vision improvement requires the inhibition of neovascularization by higher levels of PEDF. To insure that the transplanted cells secrete continuously a sufficient quantity of recombinant PEDF, we have developed an efficient nonviral transfection protocol, based on the Sleeping Beauty (SB100X) transposon system, and have shown that recombinant PEDF secretion in culture by PEDF-transfected cells was stable and constant for more than 1 year [41].
PEDF is a multifunctional protein that exhibits varied cellular actions, indicating that there are distinctive receptors that elicit diverse events. In fact, the two functional epitopes identified, a 34-mer peptide (residues 24–57) and a 44-mer peptide (residues 58–101) [50], appear to bind to two different receptors: the 44-mer peptide binds to a 80?kDa protein found in retinoblastoma Y-79 cells, neuronal cells, and retinal cells [56, 57] and induces neuroblastoma cell differentiation but does not prevent vascular leakage, whereas the 34-mer peptide binds to a 60?kDa receptor on endothelial cells [58] and prevents vascular leakage, suppresses new but not established blood vessels, and has no effect on retinoblastoma cells. Since the ultimate goal of transplanting PEDF-transfected pigment epithelial cells subretinally is for the cells to secrete active PEDF to inhibit CNV, it is critical that the recombinant PEDF secreted by the transfected cells is fully functional. In this study, we analyzed the functionality of purified recombinant human PEDF secreted by ARPE-19 and rat RPE cells transfected with the PEDF gene using the SB100X transposon system. Affinity-purified recombinant PEDF showed neurotrophic activity, as evidenced by the differentiation of human neuroblastoma cells in culture and protection of photoreceptor cells from degeneration in organ cultures of neural retinas. In vitro, the antiangiogenic properties of recombinant PEDF were confirmed by the inhibition of VEGF-stimulated sprouting and chemotaxis: rPEDF, secreted by RPE cells transfected using the SB100X transposon, is a potent inhibitor of HUVEC sprouting, the inhibition is concentration dependent, and the inhibitory activity is approximately 2000 times more potent than the activity of bevacizumab.
Conclusions
The results demonstrate that SB100X-transfected pigment epithelial cells secrete constant levels of rPEDF. Homologous PEDF-transfected rat RPE cells transplanted to the subretinal space prevent development of severe CNV lesions and limit the area of neovascularization in a rat model of CNV. Since SB100X-mediated transfection integrates the transgene and since RPE cells survive in the subretinal space, autologous SB100X-mediated PEDF-transfected cells transplanted to the subretinal space of neovascular AMD patients should secrete recombinant PEDF and inhibit CNV for the life of the patients.
