May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Efficient and Sustained Long-term Transgene Expresion in Cultured Human and Rat Iris Pigment Epithelium Via Lentiviral Transduction
Y. Zhang; T.J. McFarland; B. Appukuttan; S. Chen; D. Burke; J.T. Stout
Author Affiliations & Notes
  • Y. Zhang
    Retina, Casey Eye Inst, Portland, OR, United States
  • T.J. McFarland
    Retina, Casey Eye Inst, Portland, OR, United States
  • B. Appukuttan
    Retina, Casey Eye Inst, Portland, OR, United States
  • S. Chen
    Ophthalmology, Casey Eye Inst, Portland, OR, United States
  • D. Burke
    Ophthalmology, Casey Eye Inst, Portland, OR, United States
  • J.T. Stout
    Ophthalmology, Casey Eye Inst, Portland, OR, United States
  • Footnotes
    Commercial Relationships  Y. Zhang, None; T.J. McFarland, None; B. Appukuttan, None; S. Chen, None; D. Burke, None; J.T. Stout, None.
  • Footnotes
    Support  Clayton Foundation for Research and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 441. doi:
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      Y. Zhang, T.J. McFarland, B. Appukuttan, S. Chen, D. Burke, J.T. Stout; Efficient and Sustained Long-term Transgene Expresion in Cultured Human and Rat Iris Pigment Epithelium Via Lentiviral Transduction . Invest. Ophthalmol. Vis. Sci. 2003;44(13):441.

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Abstract

Abstract: : Purpose: Autologous transplantation of iris pigment epithelium (IPE) cells into the subretinal space can replace lost or dysfunctional retinal pigment epithelium, however IPE cells cannot restore RPE's function completely. Our hypothesis is that IPE cells, transduced with a lentiviral vector containing anti-angiogenic genes, transplanted into the subretinal space can help to treat age-related macular degeneration or other macular diseases. The goal of this study was to observe and test transgene expression in cultured human and rat IPE cells transduced with lentiviral vectors containing the anti-angiogenic genes soluble neuropilin-1 (sNRP1) and soluble kinase insert domain-containing receptor (sKDR). Methods: Human and rat IPE cells were isolated by enzyme-assisted microdissection, and cultured in supplemented Ham's F12 Nutrient Mixture (F12). An eGFP cDNA, sNRP1-FLAG or sKDR-FLAG fusion cDNA was cloned into the plasmid pHR' under the control of the CMV promoter. Replication-defective lentiviral vectors were prepared by three plasmid co-transfection into 293T cells. Human and rat IPE cell cultures were transduced by these lentiviral vectors. Expression of eGFP was analyzed by fluorescence microscopy; RT-PCR utilizing primers specific to the fusion-gene was used to confirm expression within transduced IPE cells. Results: EGFP expression in IPE cells was observed via fluorescent light microscopy, initially at 24 hours after lentiviral transduction. Sustained expression of eGFP was evident for at least 3 months. RT-PCR of transduced IPE cells indicated the presence of sNRP1-FLAG and sKDR-FLAG mRNAs. Conclusions: In vitro, human and rat IPE cells can be efficiently transduced with recombinant lentiviral vectors containing eGFP, sNRP1-FLAG or sKDR-FLAG gene. Genetically modified IPE cells may prove useful in the therapeutic treatment of macular disease.

Keywords: gene transfer/gene therapy • iris • transplantation 
© 2003, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2025 Association for Research in Vision and Ophthalmology.
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