Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Abstract

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 103 primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD.

Going non-viral: theSleeping Beautytransposon system breaks on through to the clinical side

“…Importantly, cells transfected with a PEDF-transposon and the SB100X transposase have been found to express recombinant PEDF long-term for the duration of 16 months that the cells have been in culture ( Figure 4(C)) (Johnen et al, 2012). Genome-wide insertion site analysis in human RPE cells established a close-to-random insertion profile of SB and a frequency of inserting into GSHs similar to that found in human T cells (Gogol-Doring et al, 2016;Monjezi et al, 2016;Thumann et al, 2017). Importantly, no insertions were observed in genes critical to RPE cell function, including the angiogenic factor VEGF-A, the visual cycle protein CRALBP, the lysosomal enzyme cathepsin D, the tight-junction protein ZO-1 and the cytokeratin KRT8.…”

“…Since the isolation of IPE or RPE cells from a patient's biopsy yields a limited number of cells, and since safety is a major concern of any therapy, and of gene therapy in particular, we have developed a protocol for the efficient delivery of the PEDF gene in as few as 5.000-10.000 freshly isolated IPE and RPE cells from human donor eyes by vectorizing the PEDF-encoding SB transposon and the SB100X transposase in pFAR miniplasmids (Marie et al, 2010;Thumann et al, 2017). Using this protocol, a standard operating procedure has been established that i) consistently shows highly efficient transfer of the PEDF gene in RPE and IPE cells obtained from donor eyes, ii) enables expression of recombinant PEDF at high levels of recombinant protein in cultured PEDF-transfected cells, and iii) allows for sustained transgene expression (for over one year that the cells have been in culture) in genetically engineered cells.…”

Gene Therapy with the Sleeping Beauty Transposon System

“…A variant of the MC technology is represented by 'free of antibiotic resistance markers' (pFAR) miniplasmids [67] that, similar to MCs, lack antibiotic-resistance genes, thereby significantly enhancing the safety profile of nonviral gene delivery in clinical settings. Importantly, the pFAR and SB technologies were successfully combined [68], and are planned for use in a Phase I clinical trial to treat agerelated macular degeneration [69].…”

Transposons: Moving Forward from Preclinical Studies to Clinical Trials

“…In particular, a clinical trial is imminent based on the use of SB100modified pigment epithelial cells expressing antiangiogenic factors to treat age-related macular degeneration (AMD). 174,180 …”