April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Increasing the Efficiency of Gene Therapy in Glaucoma by Selecting an Optimal scAAV serotype
Author Affiliations & Notes
  • Renekia Elliott
    University of North Carolina at Chapel Hill, Chapel Hill, NC
  • Brandon Lane
    University of North Carolina at Chapel Hill, Chapel Hill, NC
  • LaKisha Buie
    University of North Carolina at Chapel Hill, Chapel Hill, NC
  • Terete Borras
    University of North Carolina at Chapel Hill, Chapel Hill, NC
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5649. doi:
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      Renekia Elliott, Brandon Lane, LaKisha Buie, Terete Borras; Increasing the Efficiency of Gene Therapy in Glaucoma by Selecting an Optimal scAAV serotype. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5649.

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

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Abstract

Purpose: Our goal is to optimize scAAV viral gene delivery to the trabecular meshwork (TM). Our purpose was to evaluated serotypes scAAV1, scAAV2, scAAV5, scAAV6, scAAV8 and scAAV9 in the TM using postmortem perfused anterior segments (AS) of porcine eyes and primary human TM cells (HTM). To compare reporters eGFP and newest MaxFP for fluorescence sensitivity and cell toxicity in vivo and in vitro.

Methods: Recombinant scAAV-fluorescence reporter vectors were prepared at UNC Vector Core Facility. Six h postmortem porcine AS were perfused at constant flow 3-4 µl/min overnight to reach a stable outflow facility. Twenty µl of each viral vector (2-6x10^10 vg) were HPLC valve-injected and perfused for 72h. Tissue was sectioned in quadrants, fixed with 4% PARA and embedded in OCT. An equal traced TM area of 8.1x105 µm2 was used for each serotype at the same exposure (n=3 eyes; 16 images/serotype). HTM cells were infected in subconfluent 12-well plates with 3x10^10vg of each serotype and assayed at 72h. Plasmids of the same background carrying either eGFP or maxFP were calcium phosphate transfected into HEK-293 and assayed at 72h. Equal vg of viral vectors scAAV2.eGFP and scAAV2.MaxFP were infected into HTM cells for 72h and injected intracamerally into the eyes of living rats and assayed at 2 wks post-injection. Fluorescence intensity in cells and tissue was measured using MetaMorph for Olympus Imaging Software.

Results: Porcine AS perfused with serotypes #1, 8, and 9 had no visible GFP. Serotype #2 showed the greatest amount of GFP expression in the TM with 56.4 ± 1.6 fluorescence intensive units (FIU)/µm2. Serotype #6 was next, with values of 40.3 ± 1.8 FIU/µm2 followed by serotype #5 with 34.0 ± 1.9 FIU/µm2. Infected HTM cells intensity values correlated to those in perfused porcine intact TM tissue. MaxGP fluorescence was 18.9% more intense than eGFP in HEK-293 cells and 50% more in HTM cells. In vivo, initial results in rat TM and iris correlate well with the cells. MaxFP is less toxic and exhibits considerable photobleaching during measurements.

Conclusions: Findings from porcine perfused AS and HTM cells reveal the scAAV2 vector as the best serotype for gene delivery to the TM. Determining the most efficient TM serotype is key for gene therapy of glaucoma. It would allow reducing the number of viral genomes needed and increase the safety of the gene therapy treatment.

Keywords: 735 trabecular meshwork • 538 gene transfer/gene therapy  
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