April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Blocking RPE Transduction Following Sub-Retinal Injection of AAV Enhances Photoreceptor Targeted Gene Transfer
Author Affiliations & Notes
  • T. C. Cronin
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania
  • D. Chung
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania
  • E. Nandrot
    Institut de la Vision, Paris, France
  • A. Lyubarsky
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania
  • J. Bennett
    Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania
  • Footnotes
    Commercial Relationships  T.C. Cronin, Centre for Technology Transfer, University of Pennsylvania, P; D. Chung, None; E. Nandrot, None; A. Lyubarsky, None; J. Bennett, None.
  • Footnotes
    Support  Hope for vision, FFB
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 6430. doi:
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      T. C. Cronin, D. Chung, E. Nandrot, A. Lyubarsky, J. Bennett; Blocking RPE Transduction Following Sub-Retinal Injection of AAV Enhances Photoreceptor Targeted Gene Transfer. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6430.

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

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Purpose: : Sub-retinal injection of the common AAV2 pseudotypes frequently results in a bias to Retinal Pigment Epithelial transduction over neural retina. This has been of benefit to date in human clinical trials using AAV, where the disease target is in the RPE. However, many mutations predisposing to retinal disease are not of RPE origin, as such the sub-retinal injection route causes an effective ‘loss’ of virus to the RPE when strategies for AAV-mediated therapy are tested. We wished to test whether we could maximise the viral titre specific for the neural retina by blocking viral transduction of the RPE.

Methods: : The alphaVBeta5 integrin receptor is highly expressed on the apical surface of the RPE, and is essential to phagocytosis. It is also a prominent coreceptor for AAV5 transduction of cells. We initially tested AAV transduction in beta5-/- mice. Following on from this we examined transduction where RGD peptides or MFG-EGF8, a ligand essential for diurnal phagocytosis, was included as viral adjuvant to block RPE access. In vitro and in vivo results prompted further testing in rd1 and rd10 models of retinal degeneration.

Results: : Following sub-retinal injection of AAV2/5-EGFP, fluorescence was found to be stronger and more widespread in the neural retina of beta5-/- mice compared to WT with greatly reduced fluorescence in the RPE. The addition of a saturating RGD-based peptide for the integrin receptor facilitated a blocking of the RPE transduction which did not however, prevent retinal transduction. Western blot analysis confirms the blocking capacity of the peptide in vitro, where human RPE cells are transduced with virus in the presence of peptide. The therapeutic benefit of the approach was assessed in models of retinal degeneration by betaPDE6 assay and by ERG.

Conclusions: : FACS-analysis revealed almost complete ablation in the level of EGFP expression in the dissociated RPE and a corresponding 2-fold increase in transduction of the retina. The saturating peptides thereby enhanced the therapeutic benefit of delivering AAV2/5-(hum)betaPDE to mouse models of retinal degeneration.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • photoreceptors 

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