Terminable cell-based GDNF delivery device for intravitreal applications

Abstract

PURPOSE: Encapsulated-cell therapy can potentially ameliorate retinal degeneration through localized and sustained delivery of freshly synthesized neuroactive factors in the posterior eye. We have previously designed an injectable collagen-alginate drug delivery platform that has achieved prolonged glial cell-derived neurotrophic factor (GDNF) delivery and photoreceptor rescue in rats with retinal degeneration. Here, we further introduced a Tet-On genetically controlled apoptotic termination control for safer applications. METHODS: Stable clones of modified fibroblasts with GDNF and Tet-On inducible Caspase-8 expressions were established by Lipofectamine™2000. Clones with the highest Dox-inducibility and the lowest background transgene expressions were identified with Dual Luciferase Reporter Assay and selected for further studies. Dox-induced cell death over 72 hours at 0-2ug/ml was accessed by MTS cell viability assay, immunoblotting of activated Caspase 8 and Caspase-Glo® 3/7 Assay. Cell death upon Dox exposure on cell-encapsulating collagen type I-alginate gel was assayed by MTS. Biocompatibility and stability of the gel system was evaluated on 7 and 14 days post intravitreal implantation into Royal College of Surgeons (RCS) rats on postnatal day 28 by phase contrast microscopy. RESULTS: GDNF-expressing HEK293 cells were stably transfected with pcDNA™6/TR. Among the stable clones collected, those with strong tet-repressor expression were isolated through Western Blot analysis. Clones with above 10 folds of Dox-induced luciferase activity were further identified, followed by stable transfection of recombinant Caspase 8 plasmid. Complete cell death via the Caspasemediated apoptotic pathway, with elevated expressions of activated Caspase 8 and 3/7, was achievable within 72 hours of 0-2ug/ml Dox induction. Higher Dox dosages resulted in faster termination of the device. Intravitreal injection of the device in RCS rats for 7 and 14 days showed the biocompatibility, encapsulated cell viability and mechanical stability of the device after prolonged implantation. CONCLUSIONS: We have established a Dox-terminable cellencapsulating intravitreal drug delivery device that showed good biocompatibility and stability after prolonged implantation in rats. Our work demonstrated the potential of this device as a sustainable drug release platform for any therapeutic protein of interest, targeting different ocular diseases.