June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Lipid nanoparticles transfect multiple retinal cell types in the non-human primate.
Author Affiliations & Notes
  • Renee Christine Ryals
    Ophthalmology, Oregon Health & Science University, Portland, Oregon, United States
  • Milan Gautam
    Pharmaceutical Sciences, Oregon State University, Portland, Oregon, United States
  • Jonathan Stoddard
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
  • Rene Reynaga
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
  • Scott Shubert
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
  • Lauren Renner
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
  • Jeonghwan Kim
    Pharmaceutical Sciences, Oregon State University, Portland, Oregon, United States
  • Wayne Tschetter
    Ophthalmology, Oregon Health & Science University, Portland, Oregon, United States
  • Ian Fries
    Ophthalmology, Oregon Health & Science University, Portland, Oregon, United States
  • Andreas Lauer
    Ophthalmology, Oregon Health & Science University, Portland, Oregon, United States
  • Martha Neuringer
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
    Ophthalmology, Oregon Health & Science University, Portland, Oregon, United States
  • Gaurav Sahay
    Pharmaceutical Sciences, Oregon State University, Portland, Oregon, United States
    Ophthalmology, Oregon Health & Science University, Portland, Oregon, United States
  • Footnotes
    Commercial Relationships   Renee Ryals None; Milan Gautam None; Jonathan Stoddard None; Rene Reynaga None; Scott Shubert None; Lauren Renner None; Jeonghwan Kim None; Wayne Tschetter None; Ian Fries None; Andreas Lauer None; Martha Neuringer None; Gaurav Sahay None
  • Footnotes
    Support  ONPRC Pilot Grant, 5R21EY031066, P30 EY010572
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3475. doi:
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    • Get Citation

      Renee Christine Ryals, Milan Gautam, Jonathan Stoddard, Rene Reynaga, Scott Shubert, Lauren Renner, Jeonghwan Kim, Wayne Tschetter, Ian Fries, Andreas Lauer, Martha Neuringer, Gaurav Sahay; Lipid nanoparticles transfect multiple retinal cell types in the non-human primate.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3475.

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

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Abstract

Purpose : Our lab has generated novel lipid nanoparticles (LNPs) for delivery of gene editors to the retina and demonstrated robust transfection of the rodent retina. The goal of this study was to evaluate retinal transfection efficiency in a more clinically relevant model, the non-human primate (NHP).

Methods : Novel LNP variants were prepared via rapid microfluidic mixing of an organic phase containing the lipids (ionizable lipid, DSPC, sterols, and PEG) and aqueous phase containing GFP mRNA. LNPs were characterized for hydrodynamic radius, polydispersity index (PDI), encapsulation efficiency and zeta potential (ZP). LNPs were transfected into NHP iPSC-derived RPE cells at multiple doses ranging from 500 ng to 10 µg. Rhesus macaques received baseline optical coherence tomography (OCT), fundus autofluorescence (FAF) and ultra-wide-field imaging. Subretinal surgery was performed by a skilled retinal surgeon using an Alcon Constellation vitrectomy machine to deliver 100 µl (50 µg total) of LNPs to each bleb per eye. No systemic immune suppression was used. At 48 hours post-injection, in vivo retinal imaging was performed and eyes were then harvested for histology and immunohistochemistry (IHC). LNPs were also subretinally delivered to WT C57BL6 mice. In vivo retinal imaging and IHC of retinal sections were performed to characterize intracellular gene expression.

Results : All LNPs had a diameter <85 nm, with a PDI <0.10. The ZP of the particles varied from -1.1 to -5.3 and encapsulation efficiency ranged from 98-99%. GFP expression was observed in NHP iPSC-derived RPE cells at 48 hours post-LNP transfection at all doses tested. Baseline imaging confirmed that all macaques had normal retinal morphology prior to surgery. At 48 hours post-injection, in vivo retinal FAF imaging showed robust GFP expression in the subretinal blebs. IHC demonstrated GFP expression localized to photoreceptors, RPE and Müller glia (Figure 1). Evaluation of the same particles in WT mice on the same day confirmed GFP expression and localization.

Conclusions : LNPs successfully transfected many retinal cells in the NHP including photoreceptors, RPE and Müller glia post-subretinal delivery. These data demonstrate the translatability of LNP-mediated gene delivery to retinal cells across species and support their development as delivery systems for retinal disease therapies.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

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