Abstract
Purpose :
Recombinant adeno-associated virus (rAAV) is the most common viral vector adopted in clinical trials for ocular gene therapy. A major hurdle in most current gene therapy modalities is the ability to transduce target cells at high efficiencies without causing significant inflammation. This animal study experiment is designed to develop a magnetic microsphere-mediated gene delivery system to increase the efficiency of rAAV transduction.
Methods :
For ex vivo experiments, we removed the cornea and lens from freshly enucleated C57BL/6J mouse eyes and then perfused them with AAV2-CBA-GFP conjugated to magnetic microspheres (BioMag®Plus streptavidin; 1 um diameter). One eye of each pair (n=3) was placed in a magnetic field (95 mTesla field strength at surface). Immunohistochemistry staining was performed, staining nuclei with DAPI and using the intrinsic fluorescence of GFP. To assess inflammatory response in vivo, 1 uL of a ten-fold serial dilution of the magnetic beads was injected into the right eye of 12 mice, leaving the left eye as an uninjected control. Mice were divided into three treatment groups: 1) 5.0 mg/mL beads, 2) 0.5 mg/mL beads, and 3) 0.05 mg/mL beads. Color fundus photos were taken after one month.
Results :
Ex vivo results show with the application of the magnetic field there was no titration of the GFP expression (10e7 vg is as good as 10e9 vg infection). Further, there was good GFP expression from vitreous side to photoreceptor layers. Without the magnet field, there is a significant decrease in GFP fluorescence when we lowered the titer to 10e7 vg. In vivo results showed that mouse eyes receiving 5.0 mg/mL magnetic beads had significant fundus pigment changes and inflammation one month after injection. Eyes receiving 0.5 and 0.05 mg/mL dilution of the beads show normal fundus photos one month after injection.
Conclusions :
Application of magnetic microsphere-mediated gene delivery system increased the efficiency of AAV transduction through vitreous into photoreceptors, reduced viral load and is therefore a possibility to reduce inflammation from gene therapy. Further survival experiment to exam morphologic and functional change will be needed for future gene therapy of IRDs.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.