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Conference Paper: Terminable cell-based GDNF delivery device for intravitreal applications
Title | Terminable cell-based GDNF delivery device for intravitreal applications |
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Authors | |
Issue Date | 2015 |
Citation | The 2015 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Denver, CO., 3-7 May 2015. How to Cite? |
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. |
Description | Session - 110 Gene therapy, drug delivery |
Persistent Identifier | http://hdl.handle.net/10722/218605 |
DC Field | Value | Language |
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dc.contributor.author | Lo, ACY | - |
dc.contributor.author | Wong, SY | - |
dc.contributor.author | Yao, KM | - |
dc.contributor.author | Chan, BP | - |
dc.date.accessioned | 2015-09-18T06:48:01Z | - |
dc.date.available | 2015-09-18T06:48:01Z | - |
dc.date.issued | 2015 | - |
dc.identifier.citation | The 2015 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Denver, CO., 3-7 May 2015. | - |
dc.identifier.uri | http://hdl.handle.net/10722/218605 | - |
dc.description | Session - 110 Gene therapy, drug delivery | - |
dc.description.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. | - |
dc.language | eng | - |
dc.relation.ispartof | Annual Meeting of the Association for Research in Vision & Ophthalmology, ARVO 2015 | - |
dc.title | Terminable cell-based GDNF delivery device for intravitreal applications | - |
dc.type | Conference_Paper | - |
dc.identifier.email | Lo, ACY: amylo@hku.hk | - |
dc.identifier.email | Yao, KM: kmyao@hku.hk | - |
dc.identifier.email | Chan, BP: bpchan@hku.hk | - |
dc.identifier.authority | Lo, ACY=rp00425 | - |
dc.identifier.authority | Yao, KM=rp00344 | - |
dc.identifier.authority | Chan, BP=rp00087 | - |
dc.identifier.hkuros | 251440 | - |