Abstract
Purpose :
The long-term evaluation of RPE65 gene augmentation initiated in middle-aged RPE65 mutant dogs previously uncovered notable inter-animal and intra-retinal variations in treatment efficacy. To address this, we examined gene expression changes and retinal glial cell responses in both untreated (middle-aged) and treated (advanced-age) RPE65 mutant retinas, aiming to assess the status at the time of treatment and overall outcomes.
Methods :
Immunohistochemistry utilizing cell-specific markers and qRT-PCR analysis were conducted on archival retinal sections from normal and RPE65 mutant dogs.
Results :
In untreated middle-aged RPE65 mutant dogs with varied disease severity, there was significant downregulation of 19 out of total 21 genes associated with the visual pathway, neuroprotection, immune response, and apoptosis. Changes were more pronounced in tissue sections with a thinner ONL. Furthermore, in these untreated retinas, expression levels of glial-neuronal transmitter recycling proteins GS and EAAT1 were markedly reduced in GFAP-/VIM+ Müller cell processes extending into the ONL. When compared to normal dogs, IBA1+/CD18+ microglia and GFAP+/VIM- astrocyte numbers were moderately increased while maintaining a normal localization. In four advanced-aged RPE65-mutant dogs with varied responses to mid-life-initiated AAV-RPE65 treatment, regions with the most preserved ONL were chosen for analysis. Selected retinal regions exhibited varying degrees of degeneration between animals. Representative adjacent sections were used to examine gene and protein expression. Although we found restoration of expression in the majority of the 21 genes, the pattern of expression changes showed inter-animal variability, especially in BIP, CD47, MANF, BCL2 levels. Immunolabeling of Müller glia, microglia, and astrocytes with cell-specific markers revealed no abnormalities in these cells across all middle-aged treated dogs.
Conclusions :
Data from middle-aged untreated mutant retinas indicate diminished activity in critical retinal pathways due to prolonged RPE65 deficiency. Alterations in gene expression patterns, rather than changes in glial cells, may provide more informative insights for interpreting outcomes of RPE65 gene replacement therapy.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.