June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Optimizing lipid nanoparticle-mediated photoreceptor transfection in the nonhuman primate.
Author Affiliations & Notes
  • Renee Christine Ryals
    Ophthalmology, Oregon Health & Science University Casey Eye Institute, Portland, Oregon, United States
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
  • Milan Gautam
    Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, United States
  • Rene Reynaga
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
  • Antony Jozic
    Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, United States
  • Jeonghwan Kim
    Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, United States
  • Elissa Bloom
    Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, United States
  • Rachel Spears
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
  • Jonathan Stoddard
    Neuroscience, Oregon Health & Science University Oregon National Primate Research Center, Beaverton, Oregon, United States
  • Andreas K Lauer
    Ophthalmology, Oregon Health & Science University Casey Eye Institute, 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 Casey Eye Institute, Portland, Oregon, United States
  • Gaurav Sahay
    Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, United States
    Ophthalmology, Oregon Health & Science University Casey Eye Institute, Portland, Oregon, United States
  • Footnotes
    Commercial Relationships   Renee Ryals None; Milan Gautam None; Rene Reynaga None; Antony Jozic None; Jeonghwan Kim None; Elissa Bloom None; Rachel Spears None; Jonathan Stoddard None; Andreas Lauer None; Martha Neuringer None; Gaurav Sahay EnterX Bio, Code O (Owner)
  • Footnotes
    Support  ONPRC Pilot Grant, NEI 1R21EY031066, NEI R01EY033423, ONPRC Core Grant P51 OD011092 and S10RR024585, CEI Core Grant P30 EY010572 from the NIH, and unrestricted departmental funding from Research to Prevent Blindness.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 785. doi:
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    • Get Citation

      Renee Christine Ryals, Milan Gautam, Rene Reynaga, Antony Jozic, Jeonghwan Kim, Elissa Bloom, Rachel Spears, Jonathan Stoddard, Andreas K Lauer, Martha Neuringer, Gaurav Sahay; Optimizing lipid nanoparticle-mediated photoreceptor transfection in the nonhuman primate.. Invest. Ophthalmol. Vis. Sci. 2023;64(8):785.

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

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Abstract

Purpose : We have demonstrated that novel lipid nanoparticles (LNPs) can deliver mRNA to multiple cell types in the non-human primate (NHP) retina. The goal of this study was to measure the photoreceptor transfection efficiency associated with LNP dose and immune suppression in the NHP.

Methods : A novel LNP encapsulating GFP mRNA was prepared via microfluidic mixing. This LNP was characterized for hydrodynamic radius, polydispersity index (PDI), encapsulation efficiency (EE), zeta potential (ZP) and endotoxin. Two adult rhesus macaques received baseline optical coherence tomography (OCT), fundus autofluorescence (FAF) and ultra-wide-field imaging. In right eyes, two 50 µl subretinal blebs (superior and inferior) were generated with BSS. In left eyes, two 50 µl subretinal blebs were generated with 2.5 µg LNP-GFP (superior) and 25 µg LNP-GFP (inferior). One animal received 1 mg/kg oral prednisone as well as triamcinolone acetonide periocularly. At 48 hours post-injection, in-vivo retinal imaging was performed and eyes were harvested for immunofluorescence (IF). GFP expression was quantified in FAF images. Photoreceptor transfection efficiency was quantified by counting GFP positive nuclei in 10 retinal cross sections that spanned the treated retina.

Results : The generated LNP had a diameter of 137 nm, with a PDI of 0.13. The ZP of the particle was 4.2 mV, EE was 95%, and endotoxin was 7.1 EU/mL. In-vivo FAF imaging showed robust GFP expression in the LNP-injected subretinal blebs. Quantification of FAF images demonstrated that high dose injections had significantly increased GFP expression compared to low dose injections. For both doses, GFP expression was significantly increased in the animal that received systemic and local immune suppression. OCT images were unremarkable after BSS and low dose injections. In the high dose blebs, subretinal accumulation of debris and detachments were visualized by OCT. Photoreceptor counts supported FAF quantification, with high dose blebs having significantly more GFP positive nuclei than low dose blebs. The addition of immune suppression significantly increased the number of transfected photoreceptors.

Conclusions : With immune suppression, a low dose of 2.5 µg LNP-mRNA mediates robust photoreceptor transfection throughout the treated retina, which may prove to be safe and effective for future gene therapies.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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