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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)
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.
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: 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.
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.
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