June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Terminable cell-based GDNF delivery device for intravitreal applications
Author Affiliations & Notes
  • Amy CY Lo
    Department of Ophthalmology, The University of Hong Kong, Hong Kong, Hong Kong
    Research Center of Heart, Brain, Hormone, and Healthy Aging, The University of Hong Kong, Hong Kong, Hong Kong
  • Francisca S. Y. Wong
    Department of Ophthalmology, The University of Hong Kong, Hong Kong, Hong Kong
  • Kwok Ming Yau
    Department of Biochemistry, The University of Hong Kong, Hong Kong, Hong Kong
  • Barbara P Chan
    Department of Mechanical Engineering, The University of Hong Kong, Hong Kong, Hong Kong
  • Footnotes
    Commercial Relationships Amy Lo, None; Francisca Wong, None; Kwok Ming Yau, None; Barbara Chan, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 257. doi:
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      Amy CY Lo, Francisca S. Y. Wong, Kwok Ming Yau, Barbara P Chan; Terminable cell-based GDNF delivery device for intravitreal applications. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):257.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 Caspase-mediated 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 cell-encapsulating 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.

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