June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Optimization of transgene expression in the trabecular meshwork (TM) by comparing capsid mutant AAV2 vectors
Author Affiliations & Notes
  • Christine Harman
    College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States
  • Kristin Koehl
    College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States
  • Annie Oh
    College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States
    College of Veterinary Medicine, North Carolina State Univeristy, Raleigh, North Carolina, United States
  • Vince A Chiodo
    Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida, United States
  • Sanford L Boye
    Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida, United States
  • William W Hauswirth
    Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida, United States
  • Shannon Elizabeth Boye
    Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida, United States
  • Andras M Komaromy
    College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States
  • Footnotes
    Commercial Relationships   Christine Harman, None; Kristin Koehl, None; Annie Oh, None; Vince Chiodo, None; Sanford Boye, None; William Hauswirth, AGTC (I), AGTC (C), Bionic Sight (I); Shannon Boye, None; Andras Komaromy, None
  • Footnotes
    Support  The Glaucoma Research Foundation, NIH Grants TRR018411C, EY024280, P30EY021721, Research to Prevent Blindness, Foundation Fighting Blindness
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4611. doi:
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    • Get Citation

      Christine Harman, Kristin Koehl, Annie Oh, Vince A Chiodo, Sanford L Boye, William W Hauswirth, Shannon Elizabeth Boye, Andras M Komaromy; Optimization of transgene expression in the trabecular meshwork (TM) by comparing capsid mutant AAV2 vectors. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4611.

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

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Abstract

Purpose : We have previously reported successful targeting of GFP expression to the rodent TM with capsid-mutant AAV2. The purpose of our study was to optimize targeting of the canine TM with AAV2 by altering capsid mutations, pupil size, and route of intraocular vector administration.

Methods : 18 eyes of 11 adult, wild-type dogs were treated by intraocular injections of AAV2-GFP with a small chicken beta actin promoter. TM fluorescence was evaluated by gonioscopy and immunohistochemistry (IHC). First, we compared 3 capsid mutants at 2 doses (109 and 1011 vg) following intracameral (IC) injection: single (Y444F), triple (Y444, 500, 730F), and quadruple (Y444, 500, 730F + T491V) capsid amino acid changes. Once we selected AAV2-Y444F as the benchmark vector, we compared doses over a range of 3 log units (109-1012 vg), and evaluated the effect of pupil size at the time of IC injection (latanoprost-induced miosis vs. atropine-induced mydriasis) on TM transgene expression. Finally, we compared IC vs. intravitreal (IVit) vector administration. The dogs were monitored for up to 4.6 months after treatment. GFP expression within the ICA was measured by IHC using ImageJ (% GFP-stained area within iridocorneal angle in a 4x low-power field).

Results : Of the 3 capsid mutant vectors only AAV2(Y444F) resulted in detectable TM GFP expression and was used as the benchmark vector in subsequent experiments. While at the 109-vg dose GFP fluorescence was barely detectable (0.01% area), the area of transgene expression was much larger (0.35-0.4%) with 1010 and 1011 vg, respectively. GFP fluorescence was most robust with 1012 vg (4.1% area, including both TM and uveoscleral outflow pathway) with clinically detectable fluorescence by gonioscopy. The highest vector dose also resulted in uveitis at 5 weeks post injection, a likely adverse reaction to the expressed GFP. There were no differences in transgene expression between the 4 quadrants. The extent of GFP expression within the TM was only minimally affected by the pupil size at the time of IC vector injection, with the mydriatic pupil resulting in lower fluorescence (2.8 vs. 4.8%). Both IC and IVit vector administration resulted in comparable TM GFP expression; levels were slightly higher with IC injection (4.1 vs. 2.8%).

Conclusions : IC injection of AAV2(Y444F) at a dose of 1012 vg resulted in the most robust transduction of the canine TM.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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