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. NGS=Next generation sequencing; SD-OCT= Spectral domain-optical coherence tomography.

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 ∼3 Log2FC increased RHO mRNA levels in patient (RM) organoids at both the time-points of rod differentiation and maturation. No significant change was observed in other photoreceptor genes except for a small ∼1 Log2FC 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. 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. 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 OTX2 displaying the proper RHO localization (arrowheads) in the outer segments and RHO mislocalization (arrowheads) in the cell body of photoreceptors within the control (RC; top) and patient (RM; bottom) organoids respectively at two time-points, D200, and >D250. Occasional outer segments with proper RHO localization in the patient (RM) organoids were seen at >D250 time-point (C) SAG expression (red) followed a similar trajectory as RHO, with increased labelling in patient organoids (bottom) over control (top). DAPI (blue) labels nuclei. OS=outer segment; ONL=outer nuclear layer. Scale bar = 25 µm. (D-E) 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. 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 acts 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. The most appropriate RHO localization to OS is seen at 0.25 µM PR3 (arrowheads). DAPI (blue) labels nuclei. OS=outer segment; ONL=outer nuclear layer. Scale bar =25 µm. (C) qRT-PCR analysis shows ∼2-5 Log2FC 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 PR3 treatment in D300+ patient organoids with cutoffs at p<0.01 and ∼1log2FC. (B) 3D principal component analysis (PCA) plot showing the tightly clustered independent biological replicates. Additionally, 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. (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 organoids. N=3 independent experiments and 12-15 organoids per experiment. Log2FC=Log2 Fold change.