April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Immunhistochemical Characterization of AAV Transduced Retinas Following Subretinal Injection in Rats
Author Affiliations & Notes
  • K. Stieger
    Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany
  • B. Giers
    Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany
  • A. Mendes-Madeira
    INSERM U649, Laboratory for Gene Therapy, University of Nantes, Nantes, France
  • B. Lorenz
    Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany
  • F. Rolling
    INSERM U649, Laboratory for Gene Therapy, University of Nantes, Nantes, France
  • S. Haverkamp
    Department of Neuroanatomy, MPI for Brain Research, Frankfurt/M, Germany
  • Footnotes
    Commercial Relationships  K. Stieger, None; B. Giers, None; A. Mendes-Madeira, None; B. Lorenz, None; F. Rolling, None; S. Haverkamp, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 3100. doi:
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      K. Stieger, B. Giers, A. Mendes-Madeira, B. Lorenz, F. Rolling, S. Haverkamp; Immunhistochemical Characterization of AAV Transduced Retinas Following Subretinal Injection in Rats. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3100.

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

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Abstract

Purpose: : Ocular gene therapy studies have been developed for a variety of different diseases. In particular, gene therapy trials for RPE65 mutations have started recently and more than a dozen patients have been enrolled so far. In contrast to murine and canine studies, where vision and objective electrophysiological measurements such as ERG improved dramatically, the therapeutic benefit in human patients remained somewhat limited to improved light sensitivity and better performance of an obstacle parcours in single cases. The reason for this discrepancy remains unclear. Synaptic circuits in the outer (OPL) and inner plexiform layer (IPL) are dynamic and receptive to environmental alterations, as it has been demonstrated in nonhuman primates that changed from dichromatic to trichromatic vision following gene transfer of the missing chromophore. In this study, the synaptic architecture in the OPL is characterized in rat retinas following AAV mediated gene transfer.

Methods: : Two rats were injected subretinally with AAV2/5.CMV.gfp in both eyes. Two rats that did not receive any injection were used as control animals. Eyecups were fixed in 4% PFA and the neuroretina was prepared as flat mount to subsequently localize the GFP expressing area. Trimmed blocs were cryoprotected and cut vertically on a cryostat. Different primary antibodies were used for immunohistochemistry, including CtBP2, CaBP, PKCα and GFP. In addition, inflammatory and pro-apoptotic marker were tested and the Tunel Assay for the detection of apoptotic nuclei was performed.

Results: : The transduced area in the retina of rats can easily be detected and localized by anti-GFP immunolabeling. Pre- and postsynaptic structures are not altered and absolute numbers of synaptic ribbons do not differ significantly between injected and control retinas.

Conclusions: : The neuronal circuits in the OPL of healthy rat retinas undergoing AAV mediated gene transfer are not altered by the presence of viral particles or the expression of GFP as transgene. This observation likely requires further investigations in the dog model for RPE65 deficiency in order to determine the impact of RPE65 transgene expression on diseased retinas in animals and men.

Keywords: gene transfer/gene therapy • retina: distal (photoreceptors, horizontal cells, bipolar cells) • synapse 
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