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.