May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Novel Physical Methods for Nonviral DNA Transfection of RPE
Author Affiliations & Notes
  • T.W. Chalberg
    Genetics,
    Stanford University, Stanford, CA
  • A. Vankov
    Ophthalmology,
    Hansen Experimental Physics Laboratory,
    Stanford University, Stanford, CA
  • P. Huie
    Ophthalmology,
    Hansen Experimental Physics Laboratory,
    Stanford University, Stanford, CA
  • F.E. Molnar
    Ophthalmology,
    Stanford University, Stanford, CA
  • M.P. Calos
    Genetics,
    Stanford University, Stanford, CA
  • D.V. Palanker
    Ophthalmology,
    Hansen Experimental Physics Laboratory,
    Stanford University, Stanford, CA
  • Footnotes
    Commercial Relationships  T.W. Chalberg, None; A. Vankov, Stanford University P; P. Huie, None; F.E. Molnar, None; M.P. Calos, Stanford University P; Poetic Genetics LLC C; D.V. Palanker, Stanford University P.
  • Footnotes
    Support  NIH DK58187, HL68112 to MPC and EY012888 to DVP; Whitaker Foundation RG–03–0042 to DVP.
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 4690. doi:
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      T.W. Chalberg, A. Vankov, P. Huie, F.E. Molnar, M.P. Calos, D.V. Palanker; Novel Physical Methods for Nonviral DNA Transfection of RPE . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4690.

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

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Abstract

Abstract: : Purpose:We recently demonstrated nonviral gene transfer to rat RPE by using electroporation and long–term transgene expression mediated by the phiC31 site–specific integrase [1]. In these experiments, however, the procedure caused collateral damage and could not be directly translated to clinical applications. We now demonstrate safe and effective DNA delivery to the mammalian retina in a large animal model. Methods:Chorioallantoic membranes (CAM) and RPE sheets from chicken embryos were used as model tissues for DNA delivery in vitro. We used electroporation, ultrasound, and mechanical stress to deliver DNA into cells and monitored gene expression with bioluminescence imaging. For in vivo experiments with rabbits, 100 µl of plasmid DNA was injected into the subretinal space. The RPE was electroporated trans–sclerally with an array of microelectrodes placed outside the eye. A combination of mechanical stress and high local electric field was generated on an array of microelectrodes using Pulsed Electron Avalanche Knife (PEAK) [2]. Results:Electroporation alone produced expression ∼100–fold above background levels in CAM. Addition of ultrasound or mechanical stress additionally enhanced gene delivery 10–100 fold. However, using protocols optimized on the model systems in vitro, electroporation alone was not sufficient for good DNA delivery to RPE, and ultrasound caused damage to the rabbit retina and RPE. Using 300 V pulses of PEAK a rapid vaporization and ionization was achieved on a microelectrode array producing stress waves synchronized with the electric field. This approach resulted in efficient DNA delivery over a wide area without visible damage to retina or RPE in vivo. Signal was localized to the site of the retinal bleb where the electrode array was applied. Conclusions:Subretinal injection followed by a combination of electric pulse and mechanical stress in situ provides efficient, localized, surgically compatible, non–viral DNA delivery to rabbit retina in vivo. Future experiments will focus on continued characterization of the safety and efficacy of the delivery method and evaluation of long–term transgene expression in the presence of phiC31 integrase. References: 1. Chalberg TW, Genise HL, Vollrath D, and Calos MP. "PhiC31 integrase confers site–specific integration and long–term transgene expression in rat retina." Submitted, 2004. 2. Palanker DV et al. "Effects of the pulsed electron avalanche knife on retinal tissue." Arch Ophthalmol, 2002; 120: 636–40.

Keywords: gene transfer/gene therapy • retinal pigment epithelium • vitreoretinal surgery 
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