Abstract
Purpose :
Age-related macular degeneration (AMD) is a degenerative eye disease, which is a leading cause of blindness worldwide. Yet, effective treatments for dry AMD remain unavailable. One canonical feature of AMD is the formation of drusen, which are comprised of proteins, lipids, and trace elements. Previously, laboratory models that truly replicate such dry AMD characteristics were lacking. This made the development and testing of new therapies targeted at drusen pathobiology challenging. However, recent scientific advances have made it possible to generate drusen-like deposits in a culture dish using primary porcine RPE cells.
Methods :
Fresh pig eyes were obtained from a local slaughterhouse and RPE cells were retrieved by serial trypsinization and plated on T-25 flasks in 1xDMEM, 10% FBS then switched to 1xDMEM, 1% FBS, with nicotinamide (NIC) and THT after 1 week. Cells were then trypsinized and plated on transwells. After 1 week, NIC was removed and the cells were maintained and aged. We longitudinally assessed cellular autofluorescence, transepithelial electrical resistance (TEER), LDH release, hydroxyapatite, and mineral/lipid deposition (BoneTag680 and BODIPY488) in live cells. EM was used for end-stage confirmation of sub-RPE deposits. Six different AAV serotypes (1, 2, 4, 6, 7m8, and 10) expressing GFP were also tested for their potential to infect primary cells effectively without compromising RPE health.
Results :
We successfully cultured RPE cells on 12-, 24-, and 96-well transwells that exhibit high TEERs (500-900 ohm/cm2) as well as RPE markers (RPE65, ZO-1 etc). We also monitored RPE health and sub-RPE changes in live cells and followed their progression (deposits appear at 12 weeks). EM imaging shows healthy RPE at 1 month of age while substantial deposit formation at 3 and 10 months. Out of the six AAVs tested, AAV6 was the most efficient in infecting cells with long-term and stable expression at titers 5E3 and 1E4 without any adverse effects on RPE health.
Conclusions :
Through our study, we were able to recapitulate and validate porcine RPE cultures as a model to study drusen-associated RPE pathobiology and demonstrate efficient genetic manipulation by using an AAV6 vector. We also show that effective utilization of these non-terminal, multiplexed analyses will serve as a platform for testing therapeutics in HTS.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.