May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Targeted and Non-Targeted Small Interfering RNAs Equally Suppress Choroidal Neovascularization Through Activation of Toll-Like Receptor 3
Author Affiliations & Notes
  • M. E. Kleinman
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • K. Yamada
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • A. Takeda
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • M. Nozaki
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • R. Albuquerque
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • J. Smith
    Casey Eye Institute, Oregon Health and Sci Univ, Portland, Oregon
  • E. W. Taylor
    Chemistry, Univ of North Carolina, Chapel Hill, North Carolina
  • J. Ambati
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky
  • Footnotes
    Commercial Relationships  M.E. Kleinman, None; K. Yamada, None; A. Takeda, None; M. Nozaki, None; R. Albuquerque, None; J. Smith, None; E.W. Taylor, None; J. Ambati, None.
  • Footnotes
    Support  NEI/NIH, Research to Prevent Blindness, Burroughs Wellcome Fund, American Health Assistance Foundation
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3298. doi:https://doi.org/
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      M. E. Kleinman, K. Yamada, A. Takeda, M. Nozaki, R. Albuquerque, J. Smith, E. W. Taylor, J. Ambati; Targeted and Non-Targeted Small Interfering RNAs Equally Suppress Choroidal Neovascularization Through Activation of Toll-Like Receptor 3. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3298. doi: https://doi.org/.

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

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Abstract

Methods: : siRNA targeting non-mammalian genes, non-expressed genes, non-genomic sequences, both pro- and anti-angiogenic genes, or RNAi-incompetent siRNA were intravitreously injected into wild-type mice and mice deficient in TLR3 or its downstream mediators after laser photocoagulation. CNV volumes were assessed at 7 days. Spatial trafficking of fluorescent siRNA was monitored by immunofluorescence and flow cytometry (FACS). Interferon responses after siRNA treatment were analyzed by ELISA, and mRNA levels of anti-angiogenic factors were measured with real-time RT-PCR. Primary human choroidal endothelial cells (CECs) were interrogated for surface expression of TLR3 using FACS and confocal microscopy. Structural determinants of siRNA:TLR3 interaction were identified by docking simulations and molecular dynamics.

Results: : Non-targeted siRNA reduced CNV in wild-type mice as effectively as siRNA-Vegfa or siRNA-Vegfr1 without entering cells. CNV suppression by any siRNA required activation of surface TLR3, its adaptor TRIF, and induction of interferon-γ and interleukin-12. siRNA shorter than 21 nucleotides did not inhibit CNV; modelling studies suggested that this resulted from their inability to bridge effectively between dsRNA-binding residues of two TLR3 monomers. In vitro, surface TLR3 was found on all human CEC isolates tested.

Conclusions: : These results demonstrate that targeted siRNAs currently in clinical trials reduce CNV solely via TLR3 activation, that non-targeted siRNA are equally as effective in suppressing laser induced CNV, and that siRNA therapeutics may cause unanticipated vascular or immune effects due to the innate host response.

Keywords: choroid: neovascularization • immunomodulation/immunoregulation • drug toxicity/drug effects 
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