Retinal imaging and next generation sequencing.

(A) Ophthalmological color fundus examination of a patient clinically diagnosed of RHO-CNV displaying bone spicule pigmentary changes. (B) SD-OCT image showing extensive loss of the outer retinal layers leaving a small intact island at the fovea. (C) Schematic illustration of NGS using a 266-gene retinal dystrophy panel showing a complex chromosome 3q22 duplication-rearrangement resulting in an inverted 48 kb triplicated region embedded within a 188 kb duplication forming three apparently intact RHO genes on one allele and a fourth, unaltered RHO on the homologous allele. Lightning bolts represent genomic breakpoints of triplication insertion. RHO was the only gene that is fully triplicated. NGS=Next generation sequencing; SD-OCT= Spectral domain-optical coherence tomography. Scale bar = 200µm in A and B.

RHO-CNV disease modeling using iPSC-derived retinal organoids showed morphological defects.

(A) Schematic representation showing the timeline of human retinal differentiation and maturation including the birth and development of rod photoreceptors. (B) Phase contrast microscopy images showing OS, long hair-like protrusions from ONL of the differentiated photoreceptors present at the apical surface of control (RC) retinal organoids at day 200, 260 and 300 (top). Conversely, retinal organoids from patient (RM) showing shorter protrusions which do not extend progressively over long-term culturing indicating maturation defects (bottom). (C) Electron microscopy images showing ultra-magnification of distinct OS, IS and CC structures of rod photoreceptors in control organoids and the absence of OS in patient organoids. CC=Connecting cilium; IS=Inner segments; ONL= outer nuclear layer; OS=Outer segments. Scale bar = 50 µm.

Transcriptomic analysis of RHO-CNV retinal organoids presented elevated RHO expression.

(A) qRT-PCR analysis shows eight fold increase in RHO mRNA levels in patient (RM) organoids compared to control (RC) at D120 and D300 of rod differentiation and maturation. No significant change was observed in other photoreceptor genes except for a small two-fold increase in rod arrestin (SAG) at D300 time-point. Log2FC=Log 2 Foldchange. Statistical two-way ANOVA analysis with Fisher’s LSD test and 95% confidence interval. * = p <0.05, and **** = p <0.0001. Blue bars, D120; Maroon bars, D300 (B) Volcano plot showing significant differentially expressed genes following bulk RNAseq analysis comparing patient to control organoids (>D300). Significantly upregulated genes are highlighted in red and significantly downregulated genes are highlighted in blue (adjusted p<0.01). (C) Dot plot showing EnrichGO analysis of biological process on the differentially expressed genes in the bulk RNAseq analysis. The size of the dot represents number of differentially expressed genes in the pathway and the X-axis represent the ratio over all genes associated with the pathway. Plot shows a defect in rods and phototransduction associated pathways as well as synaptic transmission suggesting rod dysfunction. (D) Box plot showing the data from pathway enrichment analysis of cellular component category predominantly highlighting the defect in glycosylation and Golgi/ER modification/transport. Colors in the dot and blot plots represent relative significance (calculated p-values in scale). N=3-4 independent experiments and 12-15 organoids per experiment.

Rhodopsin protein mislocalization and increased levels in RHO-CNV retinal organoids.

(A-B) Immunofluorescence staining of RHO, NRL and CRX displaying the proper RHO localization (arrowheads) in the outer segments of control (RC, top panel) organoids and RHO mislocalization (arrows) in the cell body of photoreceptors within the patient (RM; bottom) organoids at two time-points, D200, and >D250. Occasional inner/outer segments with appropriate RHO localization in the patient (RM) organoids were seen at >D250 time-point (arrowhead). (C) SAG expression (red) was also increased in the cell soma of patient organoids (bottom) compared to control organoids (top). DAPI (blue) labels nuclei. OS=outer segment; ONL=outer nuclear layer. Scale bar = 25 µm. (D-E) Western blot probed for RHO and SAG showing increased levels of 40 kDa monomer and 80 kDa dimer, RHO (D), and 48 kDa, SAG (E) in patient retinal organoids compared to controls. β-actin was used as a loading control. Densitometric analysis quantifying the relative intensity of monomeric RHO (D’), dimeric RHO (D’’), and SAG (E’) in comparisons to the control. Statistical two-tailed unpaired T-test analysis with 95% confidence level. * = p <0.05, and ** = p <0.01. N=5 independent experiment and 12-15 organoids per experiment.

Partial rescue of rhodopsin localization and expression levels in PR3 treated RHO-CNV retinal organoids.

(A) Cartoon illustration showing gene expression during the stepwise development and maturation of rod photoreceptors. Small molecule, PR3 is predicted to act on NR2E3 downregulating the expression of GNAT1, PDE6B, SAG and RHO. (B) Immunostaining of ∼300-days-old PR3-treated retinal organoid sections from patient (RM) showing the trafficking of RHO protein (arrowheads) towards outer segments at all three doses of PR3, 0.1, 0.25 and 0.5 µM. Retinas co-stained for CRX to mark photoreceptors. The most appropriate RHO localization to OS is seen at 0.25 µM PR3 (arrowheads) and much lower overall RHO expression in 0.5 µM PR3. DAPI (blue) labels nuclei. OS=outer segment; ONL=outer nuclear layer. Scale bar =25 µm. (C) qRT-PCR analysis shows eight to 30 fold decrease in RHO mRNA levels in a dose-dependent manner for PR3 treated-patient (RM) organoids. No significant change was observed in NRL, but a small decrease was observable in GNAT1, PDE6B and NR2E3 (one-way ANOVA analysis with Sidak test and 95% confidence interval). (D) qRT-PCR analysis showing a comparison of RHO mRNA levels in PR3-treated patient organoids to control (RC) organoids (unpaired t-test). Log2FC=Log2 Fold change. * = p <0.05, ** = p <0.01and *** = p <0.001. N=3-4 independent experiments and 12-15 organoids per experiment.

RNA sequencing analysis of RHO-CNV organoids following PR3 treatment.

(A) Volcano plot showing significant differentially expressed genes following 1 week of 0.25µM PR3 treatment in D300+ patient organoids compared to vehicle treated with cutoffs at p<0.01 and ∼1log2FC. (B) 3D principal component analysis (PCA) plot showing the tightly clustered independent biological replicates from the control (RC) organoids, vehicle treated patient (RM) organoids and PR3 treated patient organoids (PR3). PR3 treated patient organoids were spatially closer to control (RC) compared to patient (RM) organoids. (C) Normalized read count plots showing relative expression of RHO, GNAT1, SAG and NR2E3 in the three conditions. (D) KEGG analysis of differentially expressed genes showing dysregulation of key phototransduction pathway comparing RC with RM organoids and the recovery following PR3 treatment in RM organoids. Down- and up-regulated genes are indicated in blue and red respectively. (E, F) Box plots showing EnrichGO analysis of differentially expressed genes that are either downregulated (E) or upregulated (F) by comparing PR3 treated to vehicle treated RM organoids. N=3 independent experiments and 12-15 organoids per experiment. Log2FC=Log2 Fold change.