Investigating photoreceptor outer segment phagocytosis by retinal pigment epithelium in CLN3-Batten

Abstract

Purpose : CLN3-Batten disease is a lysosomal storage disorder that leads to retinal degeneration and vision loss. Using induced pluripotent stem cell (iPSC)-based disease modeling studies, we previously showed impairment of a critical retinal pigment epithelium (RPE) cell function, phagocytosis of photoreceptor outer segments (POS) in CLN3-Batten. In this study, our goal was to investigate a direct link between CLN3 mutation and retinal degeneration. Specifically, we introduced a CLN3 mutation (966 bp deletion spanning exons 7 and 8) in an embryonic stem cell line (ESC; H9) and generated a transgenic pig model to study the impact of CLN3^Δ7/8 mutation on POS phagocytosis and retinal homeostasis.

Methods : Isogenic control and mutant ESC lines were generated by CRISPR/Cas9-mediated biallelic deletion of exons 7 and 8. Control and CLN3^Δ7/8 ESC were differentiated to RPE, and parallel cultures were evaluated for morphological and functional characteristics. Yucatan miniswine carrying the orthologous mutation were generated via rAAV-mediated CLN3 editing and somatic cell nuclear transfer; heterozygote offspring were bred to produce homozygote pigs. CLN3-Batten associated retinal phenotypes (reduced RPE lipofuscin, photoreceptor loss) were evaluated by histological analysis of CLN3^Δ7/8 and wild-type (WT) pig eyes at 6-, 36-, and 48-month timepoint. To evaluate POS phagocytosis in ESC-RPE and primary porcine RPE cultures, RPE cells were fed POS (~20 POS/RPE cell) for 2h and rhodopsin (RHO; a POS protein) level was analyzed by Western blotting.

Results : CLN3^Δ7/8 ESC-RPE did not display any disease-associated cellular phenotypes in the absence of POS feeding. However, CLN3^Δ7/8 ESC-RPE showed reduced uptake of POS compared to isogenic control ESC-RPE . Furthermore, compared to POS isolated from WT pigs, POS isolated from CLN3^Δ7/8 pigs was phagocytosed less efficiently by WT primary RPE cells. Consistent with reduced POS phagocytosis, lipofuscin/autofluorescence accumulation was decreased in CLN3^Δ7/8 pig RPE by 36 months, which was followed by significant loss of photoreceptors at 48-month timepoint.

Conclusions : Using in vitro and in vivo models of CLN3-Batten, we show that CLN3^Δ7/8 mutation (that affects ~75% patients) is independently sufficient for promoting both impaired POS phagocytosis and retinal degeneration in CLN3-Batten.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.