Integration of human stem cell-derived in vitro systems and mouse preclinical models identifies complex pathophysiologic mechanisms in retinal dystrophy

**Background:** Inherited retinal dystrophies (IRDs) are heterogeneous disorders causing progressive vision loss, affecting about 1 in 3,000 people. Mutations in over 270 genes are causative, with DRAM2-associated retinopathy (cone-rod dystrophy 21) being a rare autosomal recessive form. DRAM2 encodes a lysosomal protein of unclear function, implicated in autophagy, cell death, and inflammation. The study aimed to compare human stem cell-derived in vitro systems and mouse preclinical models to decipher pathophysiologic mechanisms of DRAM2 loss in retinal dystrophy.

**Methods:** The researchers generated DRAM2 knockout human pluripotent stem cell (hPSC) lines using CRISPR/Cas9 and differentiated them into retinal organoids and RPE cells. They also created Dram2 knockout mice using CRISPR/Cas9. Single-cell RNA sequencing was performed on hPSC-retinal organoids (12 months old), mouse retinas (12 months old), and human donor eyes. In vitro assays included DRAM2 knockdown in primary human RPE cells and knockout in hPSC-RPE cells, challenged with A2E (30 µM) or sodium iodate (5 mM). In vivo, mice were subjected to constant light exposure (100,000 lux for 7 days), sodium iodate injection (20 mg/kg), or laser-induced choroidal neovascularization. Retinal thickness was measured by OCT, and cell survival was quantified.

**Key Results:** DRAM2 was ubiquitously expressed in the human eye, with lower expression in AMD retinas and RPE/choroid (p < 0.05 and p < 0.01, respectively). In hPSC-retinal organoids, DRAM2 knockout led to an extra mesenchymal cell cluster (average 67 cells per KO organoid vs. 1 per WT), expressing MGP and DCN, localized near RPE cells. In human RPE cells, DRAM2 knockdown or knockout exacerbated A2E- and NaIO3-induced cell death: median survival after A2E was 114 h (shRNA1) and 102 h (shRNA2) vs. 126 h (control); after NaIO3, 64 h and 78 h vs. 118 h. In hPSC-RPE, median survival after A2E was 42 h (KO1) and 50 h (KO2) vs. 72 h (WT); after NaIO3, 34 h vs. 86 h. In mice, Dram2 knockout caused mild age-related photoreceptor degeneration: total retinal thickness loss of 5 µm after 18 months (p < 0.05) and 7 µm after 24 months (p < 0.05), with cone loss at 4 months (p < 0.05) but no functional deficit on ERG at 21 months. No transcriptional changes were detected in photoreceptors by scRNAseq. In the CNV model, lesion size was significantly larger in Dram2 ko/ko mice (mean 256,857 µm²) vs. WT (169,340 µm², p = 0.0102). No exacerbation was seen in the NaIO3 or constant light exposure models.

**Clinical Implications:** The study demonstrates that DRAM2 loss has cell-type- and species-specific effects: it increases RPE susceptibility to stress, promotes choroidal cell proliferation, and causes mild photoreceptor degeneration. These findings align with clinical features of DRAM2-retinopathy (early cone loss, late rod degeneration, RPE disruption) and suggest that DRAM2 may also be relevant to AMD. The work emphasizes that no single model fully captures human disease; integration of multiple systems is essential to understand complex pathophysiology and guide therapeutic development.