July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Intravitreal Delivery of rAAV2 Vectors to the 13-Lined Ground Squirrel Retina
Author Affiliations & Notes
  • Benjamin S Sajdak
    Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Kristina J. Ertel
    Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Hanmeng Zhang
    Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Emily R. Nettesheim
    Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Dana K. Merriman
    Biology, University of Wisconsin Oshkosh, Oshkosh, Wisconsin, United States
  • Daniel M. Lipinski
    Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
    Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Joseph Carroll
    Cell Biology, Neurobiology, & Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
    Ophthalmology & Visual Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Benjamin Sajdak, None; Kristina Ertel, None; Hanmeng Zhang, None; Emily Nettesheim, None; Dana Merriman, None; Daniel Lipinski, None; Joseph Carroll, None
  • Footnotes
    Support  NIH P30EY001931, T32EY014537, and U24EY029891; FFB PPA-0617-0718-UCSF
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2896. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Benjamin S Sajdak, Kristina J. Ertel, Hanmeng Zhang, Emily R. Nettesheim, Dana K. Merriman, Daniel M. Lipinski, Joseph Carroll; Intravitreal Delivery of rAAV2 Vectors to the 13-Lined Ground Squirrel Retina. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2896.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose : To deliver rAAV2 vectors to 13-lined ground squirrel (13-LGS) cone photoreceptors via intravitreal injection and assess artefacts arising from these injections that may impact subsequent longitudinal imaging studies.

Methods : Newborn (P1; n=43) and adolescent (P70-163; n=11) 13-LGS received intravitreal injections of a capsid mutant rAAV2/2 vector containing the [7m8] peptide insertion (R588_Q589insLALGETTRPA) and five point mutations (Y272F, Y444F, T491V, Y500F, Y730F) termed herein, rAAV2[MAX], packaging an mCherry reporter gene under control of either a ubiquitously expressing CBA promoter or a photoreceptor specific GRK1 promoter. Cornea/lens damage was examined using a Bioptigen Envisu R2200 OCT. Retinal transduction of mCherry was assessed in vivo using a Heidelberg Spectralis confocal SLO, and ex vivo with immunocytochemistry. Cone transduction of mCherry was quantified with flow cytometry using dissociated 13-LGS retina incubated with antibodies to M- and S-opsin.

Results : Iris synechia was found in 65% of newborn-injected eyes, but only 1 (4%) of the adolescent-injected eyes. The outcome of iris synechia in newborn-injected 13-LGS was independent of injection substance (p = 0.29; Chi Square test), suggesting it was due to the injection procedure. Conversely, 27% of adolescent-injected eyes had hyper-reflective puncta in anterior and/or posterior chambers, but this was not seen in any newborn-injected eyes. This presumed inflammatory response was specific to rAAV2[MAX] injected eyes with the CBA promoter. Successful but variable cone transduction was seen using flow cytometry regardless of injection age or promoter used. From 0.03% to 15.1% of opsin positive cells also expressed mCherry. Newborn injections of rAAV2[MAX]-CBA resulted in strong lens expression of mCherry, and cone transduction was minimal in these animals. Preliminary flow cytometry results suggest that 13-LGS cones are transduced most efficiently with rAAV2[MAX]-CBA administered during adolescence.

Conclusions : Intravitreal injections of rAAV2[MAX] with either CBA or GRK1 promoters can effectively target cone photoreceptors in the 13-LGS. Injecting these vectors in newborn animals often results in mild iris synechia and lens damage, which in most cases does not inhibit retinal imaging with OCT. Injecting these vectors in adolescent animals can result in inflammation, which in only one instance inhibited OCT retinal imaging.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×