May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Lentiviral-Mediated Delivery of Short Hairpin RNA Targeting VEGF in Human RPE
Author Affiliations & Notes
  • G. Lo Giudice
    Department of Ophthalmology, Conegliano Hospital, Conegliano, Italy
  • G. Lombardi
    Department of Histology, Microbiology and Medical Biotechnologies,
    University of Padova, Padova, Italy
  • E. Vendramin
    Department of Histology, Microbiology and Medical Biotechnologies,
    University of Padova, Padova, Italy
  • A. Calistri
    Department of Histology, Microbiology and Medical Biotechnologies,
    University of Padova, Padova, Italy
  • S. Piermarocchi
    Department of Ophthalmology,
    University of Padova, Padova, Italy
  • C. Parolin
    Department of Biology,
    University of Padova, Padova, Italy
  • G. Prosdocimo
    Department of Ophthalmology, Conegliano Hospital, Conegliano, Italy
  • Footnotes
    Commercial Relationships G. Lo Giudice, None; G. Lombardi, None; E. Vendramin, None; A. Calistri, None; S. Piermarocchi, None; C. Parolin, None; G. Prosdocimo, None.
  • Footnotes
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Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1685. doi:https://doi.org/
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    • Get Citation

      G. Lo Giudice, G. Lombardi, E. Vendramin, A. Calistri, S. Piermarocchi, C. Parolin, G. Prosdocimo; Lentiviral-Mediated Delivery of Short Hairpin RNA Targeting VEGF in Human RPE. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1685. doi: https://doi.org/.

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

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Abstract

Purpose:: Choroidal NeoVascularization (CNV) is the leading cause of blindness in Age-related Macular Degeneration (AMD). Several lines of evidence implicate increased levels of Vascular Endothelial Growth Factor (VEGF) in Retinal Pigment Epithelium (RPE) from patients with AMD. Current approaches to attenuate VEGF or its receptors show significant promise, but still have limited efficacy and require repeat administrations. The aim of this study was to develop a strategy for long-term endogenous expression of a short hairpin RNA (shRNA) that would significantly attenuate VEGF and hence act as a potential therapy for AMD. We decided to employ a Human Immunodeficiency Virus type 1 (HIV-1)-based vector in order to exploit the innate peculiarity of lentiviruses to efficiently and stably infect post-mitotic target cells as RPE.

Methods:: An shRNA against VEGF was cloned into HIV-1-derived Self-Inactivating (SIN) vector and packaged into VSVG protein pseudotyped recombinant infectious particles (shRNA-VEGF- LVTHM). Lentiviral vectors were used to transduce human RPE cell cultures in normoxic and in hypoxic condition in order to mimic AMD in vitro. The efficiency of transduction of target cells was quantified by FACS analysis thanks to the enhanced Green Fluorescent Protein (eGFP) present as reporter gene in the lentiviral vector. At different times post-transduction VEGF expression levels were analyzed by Western Blotting. In addition, the amount of released VEGF was determined by ELISA.

Results:: Our preliminary data indicate that more than 90% of human RPE cells were successfully transduced by shRNA-VEGF- LVTHM with the reporter gene expression lasting for more than 10 days. A reduction in VEGF release, compared with not-transduced cells, both in normoxic and hypoxic conditions, was observed.

Conclusions:: Taking into account limitations of current therapies for AMD, our approach seems a new effective anti-VEGF therapy. Further in vitro and in vivo study will be necessary to confirm this hypothesis.

Keywords: age-related macular degeneration • choroid: neovascularization • gene transfer/gene therapy 
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