May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
In vivo Analysis of Cone Specific Transgene Expression Using Lentiviral Vectors
Author Affiliations & Notes
  • M. D. Fischer
    Institute for Ophthalmic Research, Ocular Neurodegeneration Research Group, Universität Tübingen, Germany
  • R. L. Bauer
    Institute for Ophthalmic Research, Ocular Neurodegeneration Research Group, Universität Tübingen, Germany
  • S. Michalakis
    Munich Center for Integrated Protein Science, Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Germany
  • G. B. Jaissle
    Centre for Ophthalmology, University Eye Hospital, Universität Tübingen, Germany
  • P. Szurman
    Centre for Ophthalmology, University Eye Hospital, Universität Tübingen, Germany
  • M. Biel
    Munich Center for Integrated Protein Science, Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Germany
  • M. W. Seeliger
    Institute for Ophthalmic Research, Ocular Neurodegeneration Research Group, Universität Tübingen, Germany
  • Footnotes
    Commercial Relationships  M.D. Fischer, None; R.L. Bauer, None; S. Michalakis, None; G.B. Jaissle, None; P. Szurman, None; M. Biel, None; M.W. Seeliger, None.
  • Footnotes
    Support  None.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5347. doi:https://doi.org/
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      M. D. Fischer, R. L. Bauer, S. Michalakis, G. B. Jaissle, P. Szurman, M. Biel, M. W. Seeliger; In vivo Analysis of Cone Specific Transgene Expression Using Lentiviral Vectors. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5347. doi: https://doi.org/.

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

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Abstract

Purpose: : Lentiviral vectors have become attractive delivery vehicles since they readily transfect non-dividing cells and provide stable and long-term gene expression in vivo. Clinical applications require a gene transfer vector capable of accurately targeting particular cell types in the retina. To develop such a vector, we compared the expression of the green fluorescent protein (GFP) gene after subretinal injections of lentiviral constructs with the transgene expression driven by unspecific and cone specific promoters.

Methods: : Lentiviral vectors were constructed expressing GFP under the transcriptional control of the murine cytomegalovirus (CMV), arrestin3 (arr3), middle-wavelength-sensitive (MWS) opsin and short-wavelength-sensitive (SWS) opsin promoters. Viral vectors were injected into the subretinal space of wild type (SV129) and knockout mice (CNGA3-/-). GFP expression was analyzed by confocal scanning laser ophthalmoscopy (cSLO) in vivo immediately after injection and at later time points. Cell specific transgene expression was confirmed by examination of GFP fluorescence in vertical retinal slices.

Results: : Use of the constitutive CMV promoter yielded unspecific GFP expression surrounding the injection site. First results revealed efficient and specific expression of GFP in cone photoreceptors when using constructs containing one of the cone-specific promoters. In vivo imaging showed confined retinal detachment immediately following subretinal injection. Fluorescence could be detected within one week post procedure.

Conclusions: : Confocal SLO imaging is a useful tool to document and follow up effects of subretinal injection, especially when using GFP as reporter gene. Cone specific gene expression can be achieved by using lentiviral vectors that contain regulatory elements of cone specific genes. This allows for precise delivery of transgenes to cone photoreceptors within the retinal ultrastructure. These observations have significance in devising gene therapy strategies for various cone and cone-rod dystrophies.

Keywords: gene transfer/gene therapy • photoreceptors • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) 
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