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Conference Paper: Terminable cell-based GDNF delivery device for intravitreal applications

TitleTerminable cell-based GDNF delivery device for intravitreal applications
Authors
Issue Date2015
Citation
The 2015 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Denver, CO., 3-7 May 2015. How to Cite?
AbstractPURPOSE: 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.
DescriptionSession - 110 Gene therapy, drug delivery
Persistent Identifierhttp://hdl.handle.net/10722/218605

 

DC FieldValueLanguage
dc.contributor.authorLo, ACY-
dc.contributor.authorWong, SY-
dc.contributor.authorYao, KM-
dc.contributor.authorChan, BP-
dc.date.accessioned2015-09-18T06:48:01Z-
dc.date.available2015-09-18T06:48:01Z-
dc.date.issued2015-
dc.identifier.citationThe 2015 Annual Meeting of the Association for Research in Vision and Ophthalmology (ARVO), Denver, CO., 3-7 May 2015.-
dc.identifier.urihttp://hdl.handle.net/10722/218605-
dc.descriptionSession - 110 Gene therapy, drug delivery-
dc.description.abstractPURPOSE: 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.languageeng-
dc.relation.ispartofAnnual Meeting of the Association for Research in Vision and Ophthalmology, ARVO 2015-
dc.titleTerminable cell-based GDNF delivery device for intravitreal applications-
dc.typeConference_Paper-
dc.identifier.emailLo, ACY: amylo@hku.hk-
dc.identifier.emailYao, KM: kmyao@hku.hk-
dc.identifier.emailChan, BP: bpchan@hku.hk-
dc.identifier.authorityLo, ACY=rp00425-
dc.identifier.authorityYao, KM=rp00344-
dc.identifier.authorityChan, BP=rp00087-
dc.identifier.hkuros251440-

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