May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Optimization of Lentiviral Gene Delivery to the Retinal Pigment Epithelium in vivo
Author Affiliations & Notes
  • S.E. Haire
    Ophthalmology, University of FLorida, Gainesville, FL, United States
  • I.A. Elder
    Ophthalmology, University of FLorida, Gainesville, FL, United States
  • D.R. Saban
    Ophthalmology, University of FLorida, Gainesville, FL, United States
  • J.J. Janssen
    Ophthalmology, University of Nijmegen, Nijmegen, Netherlands
  • A.M. Timmers
    Ophthalmology, University of Nijmegen, Nijmegen, Netherlands
  • Footnotes
    Commercial Relationships  S.E. Haire, None; I.A. Elder, None; D.R. Saban, None; J.J. Janssen, None; A.M.M. Timmers, None.
  • Footnotes
    Support  AFAR, RPB
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 452. doi:
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      S.E. Haire, I.A. Elder, D.R. Saban, J.J. Janssen, A.M. Timmers; Optimization of Lentiviral Gene Delivery to the Retinal Pigment Epithelium in vivo . Invest. Ophthalmol. Vis. Sci. 2003;44(13):452.

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

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Abstract

Abstract: : Purpose: Since RPE cells are at a crucial junction in diseases such as AMD, it is feasible that gene delivery to RPE can be applied to affect the pathogenesis of the disease. Lentivirus was chosen as our delivery vehicle due to its high insert capacity, non-immunogenic nature, strong preference for RPE and its ability to elicit rapid and continuous gene expression. In this in vivo study, we test promoters for expression of reporter genes in RPE. Methods: Promoter constructs include a 0.8 kb upstream fragment of the 11-cis retinol dehydrogenase gene (11c-RDH) and a 1.5 kb upstream fragment of the elongation factor 1α gene (EF1α). These promoters flanked by the reporter gene GFP were cloned in self-inactivating FLAP lentiviral transducing vector (pTYF). Lentiviral vectors were packaged according a protocol developed by Coleman and Huentelman (pers. commun). This method typically yields titers of 109 to 1010 infectious particles/ml of high purity. Approximately 106 infectious particles of each virus were subretinally injected into the right eye of the rats. Non-injected, left eyes served as the control. Full field scotopic ERG analysis was done 7 or 14 days post injection. Dark-adapted rats were exposed to white light flashes (-4.4 to 0.4 log cd.s.m-2). The ratios of A-wave and B-wave amplitudes of injected to non-injected eyes were determined for electrophysiological evaluation of viral injection and reporter gene expression. Subsequently, rats were sacrificed and eyecups were sectioned on a cryostat. Sections of 14µm were analyzed for GFP expression. Results: In agreement with our previously reported results, histological analysis showed GFP expression exclusively in RPE cells. Eyes injected with pTYF.EF1α.GFP showed more robust GFP expression, detectable 7 days post injection. GFP expression with pTYF.11c-RDH.GFP was detected 14 days post injection. No histological abnormalities were detected in the injected eyes. ERG data revealed that neither lentiviral vector detrimentally affected retinal electrophysiology. There were no significant differences in A and B wave amplitudes between injected and non-injected eyes. Conclusions: We have produced lentivirus highly specific for gene delivery to the RPE cells. Previous studies have shown long-term reporter gene expression (at least 20 months). Given the success of these vectors in transducing RPE cells and the absence of any detrimental electrophysiological changes, we will apply this lentiviral gene delivery system to test candidate genes to create animal models for diseases, such as AMD.

Keywords: gene transfer/gene therapy • retinal pigment epithelium • age-related macular degeneration 
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