Challenges in GTMP process validation for a non-viral gene therapy

Abstract

Purpose : Under the aegis of the TargetAMD project, a gene therapeutic treatment for neovascular age-related macular degeneration (nAMD), a major cause of blindness in elderly, is being developed by transfecting autologous iris pigment epithelial (IPE) cells with the pigment epithelium-derived factor (PEDF) gene using the Sleeping-Beauty transposon system, to avoid the challenges associated with viral vectors. The transfected cells will be immediately transplanted subretinally to the same patient to prevent choroidal neovascularization (CNV). GMP protocols for the non-viral approach have been developed to manufacture the transfected cells (the Gene Therapy Medicinal Product, GTMP). Here we report on the process validation for the GTMP manufacture, a requirement for a planned clinical trial.

Methods : 50,000 freshly isolated human IPE cells (hIPE) were electroporated to develop the necessary protocols for the GTMP production using clinical-grade Cliniporator^TM, buffer and plasmids. Feasibility and safety were established in rabbits and mice, and efficacy in a CNV rat model. By transplanting 5,000-20,000 transfected IPE cells sub-retinally or -conjunctivally, GTMP-biodistribution, vector integration, PEDF expression and toxicity were determined by performing clinical, genomic, proteomic and immunohistological analyses. Production simulations enabled the development of SOPs and controls.

Results : Using GMP-grade buffer and plasmids, 50,000 PEDF-transfected hIPE cells showed an 11.3-fold increase in gene and a 7.6-fold increase in PEDF protein expression. Transplanted in rats, rat PEDF-transfected cells cause a reduction in CNV area of >50%. Methodological feasibility could be video documented in rabbits transplanted subretinally with freshly isolated PEDF-transfected rabbit IPE cells with no indication of toxicity or presence of the transplanted cells in any organ other than the eye determined by cllinical, immunohistological, and RT-qPCR analyses of 43 organs. The integration profile of the PEDF gene (25% in safe-harbors, 50% in non-replicating regions, circa 1 gene copy/genome) indicates minimal risk to the patient. The process, protocols and SOPs have been established from data of over 60 simulations.

Conclusions : The TargetAMD consortium delivered the pre-clinical proof-of-concept for a non-viral gene therapy to treat nAMD that is ready to be tested in patients in a phase Ib/IIa clinical trial.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.