June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Fibrin hydrogel encapsulation of adeno-associated virus for transduction of retinal pigment epithelium
Author Affiliations & Notes
  • Brittni Scruggs
    Department of Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Francesca Zenti
    Department of Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Emma Trncic
    Department of Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Travis Knudsen
    Department of Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Raymond Iezzi
    Department of Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Alan D Marmorstein
    Department of Ophthalmology, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Footnotes
    Commercial Relationships   Brittni Scruggs Genentech, Code C (Consultant/Contractor); Francesca Zenti None; Emma Trncic None; Travis Knudsen None; Raymond Iezzi Mayo Clinic , Code P (Patent); Alan Marmorstein Mayo Clinic, Code P (Patent)
  • Footnotes
    Support  Departmental Funding, Department of Ophthalmology, Mayo Clinic
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 789. doi:
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    • Get Citation

      Brittni Scruggs, Francesca Zenti, Emma Trncic, Travis Knudsen, Raymond Iezzi, Alan D Marmorstein; Fibrin hydrogel encapsulation of adeno-associated virus for transduction of retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2023;64(8):789.

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

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Abstract

Purpose : Many ocular gene therapies, including Voretigene neparvovec-rzyl (Luxturna®) and others in clinical trials, require injection of a viral solution into the subretinal space. This technique creates a localized retinal detachment, often with detachment of the fovea, in eyes with retinal degeneration. Multiple studies have demonstrated potential negative effects of subretinal bleb formation (e.g., chorioretinal atrophy, macular hole). The purpose of this study was to determine whether adeno-associated virus (AAV) could be sequestered in a high concentration (30-40mg/ml) fibrin gel and retain infectivity.

Methods : Fibrin gels were cast using fibrinogen and thrombin with addition of Trypan Blue and AAV2-GFP (green fluorescent protein) with 6.9 x 1010 capsids/punch. Fibrin gel punches with 3mm diameter were transferred into Geltrex-coated 96-well plates with wells containing either a saline solution or confluent retinal pigment epithelium cells (ARPE-19, an RPE-derived immortalized cell line) in media. Punches were removed at various timepoints. AAV2 in solution (i.e., released from gels) and AAV2 retained in the gels (i.e., after plasmin degradation) were measured using a Progen AAV2 ELISA kit to determine diffusion rate. ARPE-19 cells were imaged for GFP positivity weekly after fibrin placement. Electron microscopy of the fibrin gels was performed to identify viral particles within the gel matrix.

Results : Maximum AAV diffusion from fibrin gel punches occurred between 48 and 96 hours. Approximately 50% of the AAV particles in the gel punches was recovered in the solution, and the remaining AAV was recovered from the degraded gels. RPE cell transduction with 1.7 x 109 viral genomes/well was determined to be optimal with no benefit of adding higher concentration of virus. One month following RPE transduction, equivalent numbers of GFP expressing cells were observed in wells with AAV solution alone compared to fibrin encapsulated-AAV. Partial degradation of the gel was observed at one month.

Conclusions : Fibrin encapsulated-AAV diffuses out of the gel and retains infectivity. RPE cells transduced by AAV released from fibrin had similar expression of the GFP transgene at 1-month compared to AAV solution. RPE transduction using fibrin patches placed directly on the retina may allow gene therapy administration without subretinal injection; pig studies are ongoing to test this hypothesis.

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

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