June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Evaluating capsid-labeled AAVs as a tool for monitoring ocular biodistribution
Author Affiliations & Notes
  • Tiffany Heaster
    Genentech Inc, South San Francisco, California, United States
  • Bailey Hannon
    Genentech Inc, South San Francisco, California, United States
  • Herman Gill
    Genentech Inc, South San Francisco, California, United States
  • Shawnta Yvonne Chaney
    Genentech Inc, South San Francisco, California, United States
  • Justin Elstrott
    Genentech Inc, South San Francisco, California, United States
  • Footnotes
    Commercial Relationships   Tiffany Heaster Genentech, Code E (Employment); Bailey Hannon Genentech, Code E (Employment); Herman Gill Genentech, Code E (Employment); Shawnta Chaney Genentech, Code E (Employment); Justin Elstrott Genentech, Code E (Employment)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 5024. doi:
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    • Get Citation

      Tiffany Heaster, Bailey Hannon, Herman Gill, Shawnta Yvonne Chaney, Justin Elstrott; Evaluating capsid-labeled AAVs as a tool for monitoring ocular biodistribution. Invest. Ophthalmol. Vis. Sci. 2023;64(8):5024.

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

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Abstract

Purpose : Effective retinal gene therapies rely on successful delivery of genetic material to target cells. Transgene expression can be used as a measure of delivery, but may vary based on AAV serotype or promoter. Monitoring AAV distribution independent of transgene expression may inform on pharmacokinetics of ocular gene therapies. Fluorescence-based contrast agents offer high sensitivity to retinal cell targets, thus we investigated methods of AAV labeling and evaluated the utility to monitor trafficking of functional AAVs in the eye.

Methods : Fluorescently-labeled AAVs were generated by conjugation of IRDye800-succinimidyl esters reactive to capsid amine groups. In vitro characterization of labeled AAV measured dye sensitivity, size distribution, and AAV function in HEK293T cells post-infection. Longitudinal distribution and transgene expression (IRDye/GFP fluorescence) were assessed in vivo with cSLO imaging following intravitreal (IVT) injection of C57BL6 mice with PBS, unmodified or labeled AAV (2 uL; ~1x1012 vg/mL). Pattern ERG (PERG) was measured to determine AAV effects on retinal ganglion cell (RGC) function. Collection of enucleated eyes and retro-orbital blood was used for histology and immunogenicity assays, confirming GFP expression and surveying immune tolerance for each AAV.

Results : Unmodified and labeled AAVs displayed related structural and functional properties, exhibiting similar size distribution, robust GFP expression, and low toxicity after HEK293T infection. IRDye fluorescence was detected immediately after IVT injection up to 8 weeks in the vitreous. Retinal GFP expression was significantly higher following injection of unmodified AAV compared with labeled AAV (p<0.0001). RGC function was unaffected in response to unmodified or labeled AAV (PERG amplitude, p>0.05). Both AAVs had comparable immunogenicity profiles, with corresponding Iba1+ cell infiltrate and serum neutralization capacity (p>0.05).

Conclusions : Fluorophore-conjugated AAVs allow visualization of AAV biodistribution following IVT delivery in the mouse eye. While many attributes of unmodified AAVs are conserved, labeled AAVs exhibit limited diffusion and transgene delivery in vivo, possibly due to charge or hydrophobicity of conjugated fluorophores. Additional characterization and design optimization of dye:AAV conjugates are ongoing to improve diffusion and transduction and enable translation across animal models.

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

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