Abstract
Purpose :
Viral vector-mediated Gene therapy is at the forefront of the treatment for inherited and acquired retinal disorders. The eye provides a tight blood-brain barrier, low antigenicity to the viruses, and ease of surgical access. Retinal Pigment epithelium (RPE) cells are one of the primary targets for gene therapy. A combination of the cell-specific promoter and lentiviruses, adeno-associated virus (AAV)5, or AAV7m8 successfully transduces RPE cells in vivo. We sought to evaluate RPE cells' immune response after viral gene delivery by subretinal injections.
Methods :
6 weeks old C57Bl6 wild type mice were injected with Lentivirus, AAV5, or AAV7m8 carrying GFP reporter gene driven by CMV promoter via subretinal injection. After 7 or 14 days of injection, the mice were sacrificed, and the eyes were enucleated. The RPE florets were imaged for GFP fluorescence expression using the confocal microscope to confirm the transduction. RPE cells were later dissociated, mRNA was isolated and converted to cDNA. These were subjected to qPCR using the primers for IFNγ, IL-1α, IL-1β, IL-6, CD-8, and Iba-1 immunogenic markers.
Results :
IFNγ, an activator of macrophages, and IL-1α, an inflammatory cytokine, were not detected in RPE cells after the viral gene delivery. The expression of IL-6, another pro-inflammatory cytokine, was negligible at week 1, being marginally higher in the lentiviral injected eye relative to both AAV7m8 and AAV5. The expression of IL-6 subsequently decreased at week 2. Expression of IL1β cytokine was profound in the week 1 sample. Its expression in the lentiviral injected eye was 2-fold higher than AAV7m8, or AAV5, which was abolished entirely at week 2. CD-8 transcripts were 2.7-fold higher for lentiviral and 5.2 fold higher for AAV7m8 compared to AAV5 mediated gene delivery. Its expression persisted at week 2. The expression of Iba-1, a microglia marker, was significantly higher with a 2-fold higher presence for AAVs compared to lentivirus. Its upregulation continued in the AAV7m8 injected eye at week 2. We did not detect any of the above cytokines in the PBS injected control eyes.
Conclusions :
Our results indicate that transduced RPE cells by subretinal delivery of viruses elicited inflammatory cytokines. The extent of cellular toxicity in the RPE was greater for AAVs compared to lentivirus. It might be beneficial to consider adjunct immune suppressor therapy for the viral-mediated delivery or use non-viral modalities.
This is a 2021 ARVO Annual Meeting abstract.