April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Transgene Targeting of the Iris Pigment Epithelium (IPE) to Study and Potentially Treat Pseudoexfoliation Glaucoma
Author Affiliations & Notes
  • Terete Borras
    Department of Ophthalmology, University of North Carolina, Chapel Hill, NC
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC
  • Renekia Elliott
    Department of Ophthalmology, University of North Carolina, Chapel Hill, NC
  • LaKisha Buie
    Department of Ophthalmology, University of North Carolina, Chapel Hill, NC
  • Matthew Halton Smith
    Department of Ophthalmology, University of North Carolina, Chapel Hill, NC
  • Matthew Hirsch
    Department of Ophthalmology, University of North Carolina, Chapel Hill, NC
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC
  • Brandon Lane
    Department of Ophthalmology, University of North Carolina, Chapel Hill, NC
  • Footnotes
    Commercial Relationships Terete Borras, None; Renekia Elliott, None; LaKisha Buie, None; Matthew Smith, None; Matthew Hirsch, None; Brandon Lane, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5667. doi:
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      Terete Borras, Renekia Elliott, LaKisha Buie, Matthew Halton Smith, Matthew Hirsch, Brandon Lane; Transgene Targeting of the Iris Pigment Epithelium (IPE) to Study and Potentially Treat Pseudoexfoliation Glaucoma. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5667.

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

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Abstract

Purpose: Pseudoexfoliation (PEX) is a high pressure open angle glaucoma triggered by the deposition of PEX material (PEXm) on the eye anterior segment. The IPE is involved in the formation of PEXm, which is deposited on the lens anterior capsule and then carried to the trabecular meshwork (TM). Our goal is to deliver PEXm relevant proteins to the IPE to modulate downstream PEX-caused elevated IOP. Our purpose was to generate human primary IPE cells (hIPE), determine PEXm-related genes’ expression and investigate transduction efficiency of viral vectors serotypes. To inject the serotypes intracamerally (IC) in living rats.

Methods: A hIPE-1 primary cell line was generated from the human iris of a non-PEX, 51 y old postmortem donor. RNA was extracted at 4th passage and analyzed for the presence of LOXL-1, CLU, FBLN5, FBN1 and ELN by TaqMan PCR. Third passage subconfluent cells in 12-well plates were infected with 6X109 vg of scAAV1, scAAV2, scAAV5, scAAV 6, scAAV8 & scAAV9, all carrying the CMV promoter driving eGFP. Transduction efficiency was assessed at 72 h by fluorescent intensity of an equal area (4000 cells) with MetaMorph software, and by % of transduced cells by flow cytometry. Wistar rats (n=6) were IC injected with 3-6 109 vg and analyzed by fluorescence histochemistry at 2-12 wks postinjection.

Results: Results: hIPE-1 cells exhibit healthy epithelial morphology. All PEXm-related genes tested were highly expressed, with an 18S-normalized relative abundance ranking of FBN1, LOXL1, ELN, CLU, FBLN5. Secreted media showed IL6 levels (ELISA) of 314pg/ml, 30X the normal serum range. Serotypes #1, 8 & 9 were very inefficient, with few detectable green cells. Serotype #2 & #6 were very high and #5 was moderate. By flow cytometry, % of transduced cells were 85.7 for #2 and 89.5 for #6. In vivo, IC injection of scAAV2 targeted the IPE layer with high efficiency while the rest of iris cells were not transduced.

Conclusions: Expression of PEX relevant genes in primary hIPE cells would allow elucidation of PEX mechanisms. Because its contribution to PEXm formation and TM upstream location, targeting candidate genes to the IPE opens many avenues to study exfoliation glaucoma. IC injection of scAAV serotype 2 targeted its transgene to the IPE in rats. This targeting could deliver wild-type/mutant elastin network components to the IPE and influence the outcome of pseudoexfoliation glaucoma.

Keywords: 538 gene transfer/gene therapy • 533 gene/expression • 571 iris  
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