September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016

Characterization of the limitations to gene transfer and associated inflammation following intravitreal AAV injection in nonhuman primates
Author Affiliations & Notes
  • Jason Comander
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Livia S Carvalho
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Ru Xiao
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Aliete Langsdorf
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Sarah Wassmer
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Brian Hafler
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • David M Wu
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Dean Eliott
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Leo A Kim
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Luk H Vandenberghe
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Jason Comander, Massachusetts Eye and Ear Infirmary (P); Livia Carvalho, None; Ru Xiao, None; Aliete Langsdorf, None; Sarah Wassmer, None; Brian Hafler, Achillion Pharmaceuticals (C), Astellas Pharma (F), Novartis Pharmaceuticals (C); David Wu, None; Dean Eliott, Avalanche (C), Massachusetts Eye & Ear (P); Leo Kim, Massachusetts Eye and Ear Infirmary (P); Luk Vandenberghe, GenSight Biologics (I), Novartis, Eleven Bio, Jefferies, Sectoral, GenSight (C), Regeneron, Cowen, GenSight (R), UPenn, ReGenX, MEEI (P)
  • Footnotes
    Support  NIH Pioneer Award (Bennett and Vandenberghe), Henwood Fund (Vandenberghe), Grousbeck Family Foundation (Vandenberghe), Research to Prevent Blindness Career Development Award (JC), NEI K12 EY16335 (JC,LK), NEI K08-EY023993-01 (DW)
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 771. doi:
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    • Get Citation

      Jason Comander, Livia S Carvalho, Ru Xiao, Aliete Langsdorf, Sarah Wassmer, Brian Hafler, David M Wu, Dean Eliott, Leo A Kim, Luk H Vandenberghe;
      Characterization of the limitations to gene transfer and associated inflammation following intravitreal AAV injection in nonhuman primates. Invest. Ophthalmol. Vis. Sci. 2016;57(12):771.

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

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Abstract

Purpose : For gene therapy applications in the primate retina, intravitreal injections of adeno-associated virus (AAV) have been shown to transduce a limited amount of retina, mainly in the perifoveal region. The internal limiting membrane (ILM) has been previously hypothesized to be a barrier to broader transduction. Also, the inflammation associated with intravitreal injections has not been well described.

Methods : Rhesus macaques (N=13 eyes, total) were injected with AAV.CMV.GFP.WPRE via a regular intravitreal injection (N=3, 1011 particles, 2/2 or Anc80 capsids) or via a subretinal injection (N=8, 1010 particles, 2/5, 2/9, or Anc80 capsids). In two additional eyes, the ILM was first surgically removed followed by concentrated application of 1011 particles of AAV 2/2 onto the peeled area. This “peel and puddle” procedure consisted of a vitrectomy, posterior hyaloid separation, ILM staining, ILM peeling over the majority of the macula, fluid-air exchange, and deposition of concentrated virus on the peeled area with 30 minutes of supine positioning.

Results : The brightness and extent of GFP reporter gene expression was much greater with the peel/puddle technique than with a regular intravitreal injection. In the peel/puddle eyes, fluorescence was noted in an area that matched the extent of the ILM peel, as shown by imaging and histology. In all eyes, inflammation in the first 2 weeks was mild or moderate and self-resolving. At weeks 3-5, all of the regular intravitreal eyes (N=3) and the peel/puddle eyes (N=2) developed vitritis. The most severe eye also developed hyphema and cataract. In all other eyes, the inflammation was well controlled with 4mg of intravitreal Triescence. Similar amounts of late inflammation were seen between the intravitreal and peel/puddle eyes. None of the eyes injected with 1010 particles subretinally developed vitritis.

Conclusions : The vitreous, ILM, and/or physical parameters of the vector delivery significantly limit gene transfer to the retina. Surgical minimization of these barriers demonstrate highly increased levels and distribution of transduction. Importantly, our studies demonstrate intravitreal injection of AAV to potentially lead to clinically significant inflammation at doses normally tolerated in the subretina.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

GFP expression across the macula using the peel/puddle technique

GFP expression across the macula using the peel/puddle technique

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