Retinal cells derived from patients with DRAM2-dependent CORD21 dystrophy exhibit key lysosomal enzyme deficiency and lysosomal content accumulation

Summary Biallelic mutations in DRAM2 lead to an autosomal recessive cone-rod dystrophy known as CORD21, which typically presents between the third and sixth decades of life. Although DRAM2 localizes to the lysosomes of photoreceptor and retinal pigment epithelium (RPE) cells, its specific role in retinal degeneration has not been fully elucidated. In this study, we generated and characterized retinal organoids (ROs) and RPE cells from induced pluripotent stem cells (iPSCs) derived from two CORD21 patients. Our investigation revealed that CORD21-ROs and RPE cells exhibit abnormalities in lipid metabolism, defects in autophagic flux, accumulation of aberrant lysosomal content, and reduced lysosomal enzyme activity. We identified potential interactions of DRAM2 with vesicular trafficking proteins, suggesting its involvement in this cellular process. These findings collectively suggest that DRAM2 plays a crucial role in maintaining the integrity of photoreceptors and RPE cells by regulating lysosomal function, autophagy, and potentially vesicular trafficking.


iPSC reprogramming
Dermal fibroblasts were transduced at a density of 30,000 cells/cm 2 using the CytoTune™-iPS 2.0 Reprogramming Kit (Life Technologies, A16517) following the manufacturer's instruc�ons.The established pa�ent iPSCs were assessed for pluripotency and clearance of Sendai virus-associated transgenes using primers shown in Table S1.The DRAM2 gene muta�ons were corroborated by PCR using primer pairs shown in Table S1, followed by Sanger sequencing.In addi�on, iPSCs derived from dermal fibroblasts from two age-matched individuals (WT1 and WT3, collec�vely named WT) with no history of re�nal disease were used as healthy controls as outlined in Buskin et al. 2018.Further informa�on about the cell lines is available in Table S1.iPSC culture iPSCs were maintained at standard cell culture incubator condi�ons at 95% humidity, 5% C02 and 37°C.

Off-target sequencing
The online pla�orm htp://www.rgenome.net/cas-offinder/was used to iden�fy genomic regions bearing high sequence homology to the sites of CRISPR/Cas9 correc�on.Query sequences were allowed a maximum of up to three mismatches.Top 10 sequences exhibi�ng the highest sequence homology were selected and primers enclosing the respec�ve genomic regions were designed for offtarget sequencing as shown in Table S1.

Immunofluorescence analysis of ROs
The organoid sec�ons were le� to dry at RT for an hour and washed 3x5 minutes with PBS (Thermo Fisher Scien�fic) to remove any remaining OCT.To minimize non-specific binding, sec�ons were blocked for 1 hour at RT with a solu�on of PBS containing 10% goat serum and 0.3% Triton-X100.
Sec�ons were then double stained overnight at 4°C in a humidifier chamber using primary an�bodies diluted in AD (An�body diluent) buffer (0.001% BSA-PBS and 0.3% Triton-X, Sigma Aldrich, USA) (Table S1).Following incuba�on overnight, RO sec�ons were washed 3x 10 minutes with AD buffer.Secondary an�bodies diluted in PBS (1:1000) (Table S1) were applied to the sec�ons and incubated for 2 hours at RT. Finally, the sec�ons were washed three �mes with PBS for 15 minutes.Nuclei were counterstained with a 1:1000 dilu�on of Hoechst (cat. No. 33342,Thermo Fisher Scien�fic) in Vectashield (cat.No. H-1000-10, Vector Laboratories, CA).The slides were then covered with 24x60 mm coverslips, sealed on each side with nail polish, and stored at 4°C in the short term.For quality control, sec�ons stained only with a secondary an�body were analysed for each staining to ensure the specificity of the fluorescent an�body signal.High-resolu�on fluorescence imaging was facilitated by op�cal sec�oning on an Axio Imager microscope (Zeiss, Apotome, 10-15 Z-stacks).Images were acquired as maximum intensity projec�ons on Zen (Zeiss).Quan�fica�on of immunostained RO sec�ons were carried out on MATLAB (Mathworks) based on a protocol by Dorgau et al. (2019).

Immunofluorescence analysis of RPE cells
Immunofluorescence analyses of RPE cells were performed as qualita�ve experiments using Zen so�ware (Zeiss).In short, transwells were rinsed in PBS and incubated with 4% PFA for 30 minutes.The �ssue was subsequently washed 3x5 minutes with PBS and flat RPE sheets were cut into mul�ple pieces.Addi�onal fixa�on by methanol was performed at 4°C for 20 minutes when staining for �ght junc�ons (ZO-1) and Collagen IV.To remove melanin pigment from the RPE a bleaching procedure was carried out using a Melanin Bleach kit (cat.No 24883, Polysciences), following the manufacturer's instruc�ons.To minimize non-specific binding, RPE were then blocked and permeabilized simultaneously for 1 hour in PBS containing 10% Donkey Serum (cat.No. 7332100-LAM, Stratech) and 0.3% Triton-X-100.Primary an�bodies were diluted in PBS containing 0.1% Triton-X-100 and 1% Donkey Serum-PBS and applied at 4°C overnight.The �ssue was subsequently rinsed with PBS (3x5 minutes).
Secondary an�bodies diluted in PBS were incubated for 1 hour at RT. RPE sec�ons were washed 3x5 minutes in PBS and counterstained for nuclei using Hoechst (1:1000 in PBS) for approximately 20 minutes.A�er an addi�onal PBS wash, RPE sec�ons were mounted on slides using Vectashield®.A secondary an�body only control was included in each set of experiments.For all an�body details please refer to Table S1.

Transepithelial electrical resistance (TEER)
Transepithelial electrical resistance was rou�nely conducted as a func�onal measurement of RPE barrier func�on using a volt-ohm meter (Millipore, MERS00002).RPE cells were equilibrated to RT, and a sterilized electrode was placed on either side of the transwell membranes.To determine unit area resistance (Ω • cm2), values pertaining to inserts containing media but devoid of cells (blanks) were subtracted from sample readings.Obtained values were mul�plied by the surface area of the insert (0.33 cm 2 for a 24-well plate insert).RPE monolayer was considered mature for further analyses at TEER > 250 Ω cm 2 .Measurements were taken in triplicates every two to three weeks.

Flow cytometry analysis of phagocy�c ac�vity
FITC-labelled POS were diluted in 10% FBS containing RPE medium and incubated with RPE on transwell inserts for 4 hours at 37°C.RPE cells were rinsed with PBS and dissociated into a single-cell suspension using TrypLE™ Select Enzyme (10x).The live cell dye DRAQ5™ (Abcam, ab108410) was resuspended in flow buffer (2% FBS in PBS) at a ra�o of 1:40 and incubated with the dissociated cells for 10 minutes at 37°C.To quench any residual FITC fluorescence, cells were treated with 0.2% Trypan Blue for 10 minutes (Sigma Aldrich, 93595).Samples were centrifuged and further rinsed with 2% FBS-PBS.Flow cytometry analysis was conducted on a BD™ LSR II flow cytometer (BD Biosciences) with 10,000 events acquired for each sample.A sample kept at 4°C served as a nega�ve control.

POS treatment of RPE cells
RPE were treated with 20 POSs/cell every day for 14 days and subjected to transmission electron microscopy analysis.An untreated sample was included as an internal control for each RPE cell line.

ELISA Detection of VEGF and PEDF secretion
A 96-well microplate was coated overnight with diluted Capture An�body.Unbound an�bodies were removed by washing and plate was blocked to prevent non-specific binding using Reagent Diluent at room temperature (RT).Following a round of washing, 100 μL of basal RPE media samples and standards were applied to the wells and incubated for 2 hours at RT.Following an addi�onal washing step, the wells were treated with 100 μL Streptavidin-HRP for 20 minutes at RT in the dark.The plate was rinsed further to remove unbound HRP.To facilitate detec�on, substrate solu�on was added to the samples for 20 minutes.The reac�on was terminated by the addi�on of a Stop Solu�on and op�cal density was measured at 450 nm using a Varioskan LUX Mul�mode Microplate ELISA reader (Thermo Fisher Scien�fic).An analogous procedure was carried out to determine PEDF levels in the apical compartment of RPE cells (insert media) using a Human Serpin F1/PEDF DuoSet ELISA kit by adhering to the manual's instruc�ons (Biotechne, DY1177-05).

RNA extrac�on
RNA extrac�on from RO pellets was performed in a laminar flow hood.The hood was UV sterilized and decontaminated from RNA nucleases using RNaseZap™ (Thermo Fisher Scien�fic, AM9780).RNA was extracted using the ReliaPrep™ RNA Cell Miniprep System following the manufacturer's instruc�ons (Promega, Z6012).cDNA synthesis First strand cDNA synthesis was ini�ated by conver�ng up to 5 μg of RNA in a 5 μL reac�on containing primer Oligo(dT)15 and Nuclease-Free Water (NFW).The same amount of RNA was converted across all biological replicates based on the sample showing the lowest RNA concentra�on.Samples were placed onto a pre-heated block at 70°C for 5 minutes and subsequently pre-chilled at 4°C for another 5 minutes.Each of the samples was centrifuged to remove lid condensa�on and placed briefly on ice.
A reverse transcriptase mix was prepared using GoScript TM 5X Reac�on buffer, MgCl2, dNTPs, RNasin Ribonuclease Inhibitor and GoScript Reverse Transcriptase in accordance with the guidance manual.5 μL RNA and 15 μL of the mix were then carefully mixed and RNA was converted to cDNA at the thermocycler condi�ons outlined in the manual.cDNA was stored at -20°C un�l further use.

RT-qPCR
To determine DRAM2a isoform expression by RT-qPCR, RNA samples were cleared from any poten�al DNA contamina�on using a TURBO DNA-free TM kit (Thermo Fisher Scien�fic, AM1907).Equal amounts of cDNA template were used to set up a 384-well qPCR reac�on using a GoTaq qPCR Master mix kit (Promega, A6002) on Quantstudio TM 7 Flex Real-Time PCR system (Applied Biosystems).Data were normalized to the expression of GAPDH and gene expression was calculated using the 2^(-ΔΔCT) method.Samples were run in triplicates and data were presented as means + SEM.DRAM2 oligonucleo�de sequences are shown in Table S1.

DRAM2 siRNA knockdown
To improve siRNA uptake efficiency, day 230 WT ROs were dissected and cultured on Matrigel™ Growth Factor Reduced Basement Membrane Matrix (Corning), and poly-L-ornithine (10 μg/mL) (Sigma Aldrich, A-004-C) coated 24-well plates for a single round of passaging.siRNA transfec�on was performed at 50% confluence using a Lipofectamine TM RNAiMAX Transfec�on reagent (Thermo Fisher Scien�fic, 13778030).20μM DRAM2 Silencer Select siRNA (4392420, Assay ID: s43281) and scrambled control (Thermo Fisher Scien�fic, 4390849) were incubated with the dissected ROs in an an�bio�c and serum-free maintenance media for 72 hours and collected for qPCR and WB analysis.

BCA protein assay
Cell pellets stored at -80°C were lysed for 5 minutes at RT in PhosphoSafe TM extrac�on buffer (Merck Millipore, 71296) supplemented with protease inhibitor cocktail Complete Mini EDTA-free (Roche, 04693159001).Rigorous pipe�ng was alternated by vortexing and rest on ice for 20 minutes un�l samples were completely lysed.Samples were spun at 4°C for 10 minutes at 1000xg, and the concentra�on of the sample supernatants was determined using a Pierce TM BCA Protein Assay Kit (Thermo Fisher Scien�fic, 23225).Absorbance was measured at 562 nm on a Varioskan LUX Mul�mode Microplate Reader (Thermo Fisher Scien�fic).

Media sample collec�on and concentra�on
Media samples were collected for ROs at D220 and from RPE which had been starved for 7 days.Prior to use, Amicon 10 kDa MWCO ultrafiltra�on units (Merck, UFC801096) were washed with 4 mL sterile dis�lled water and centrifuged for 10 minutes at 3000xg. 5 mL of media was loaded onto membranes and concentrated to a final volume of 500 μL, whereby protease inhibitors cocktail was added to each sample (Complete Mini EDTA-free, Roche, 04693159001).

Gel transfer
Dry transfer was performed on an iBlot 2 Dry Blo�ng system (Thermo Fisher Scien�fic) using Blot™ 2 Transfer PVDF Stacks (Thermo Fisher Scien�fic, IB24002 and IB24001) (20V for 1 minute, 23V for 4 minutes and 25V for 2 minutes).Transfer of autophagy markers, including DRAM2, was carried out at 10V for a total of 7 minutes.

CTSD kine�c ac�vity assay
To preserve enzyma�c ac�vity the RPE and RO samples were lysed in na�ve buffer (0.2M Triethylammonium bicarbonate buffer, Sigma Aldrich, T7408) containing protease inhibitors cocktail Complete Mini EDTA-free (Roche, 04693159001).Protein concentra�on was measured using the Pierce TM BCA Protein Assay Kit (Thermo Fisher Scien�fic, 23225).50 μM MCA (7-Methoxycoumarin-4-ace�c acid) diluted in assay buffer was used to generate a standard curve (0-25 μM) (Thermo Fisher Scien�fic, 265301).The kine�c assay was performed on black clear botom 96-well plates (Thermo Fisher Scien�fic) in a total volume of 100 μL assay buffer.5 μg protein lysate was diluted in 50mM sodium acetate assay buffer pH 4.0 (Thermo Fisher Scien�fic, AM9740).Protein lysate pre-incubated with 0.2mg/mL pepsta�n inhibitor at 37°C for 15 minutes (Enzo Life Sciences) served as a nega�ve control.Immediately prior to the ini�a�on of the kine�c assay, Cathepsin D & E substrate (Enzo Life Sciences) was applied at a final concentra�on of 80μM.Fluorescence signal kine�cs were recorded every 5 minutes over the course of 20 minutes upon addi�on of the substrate at 37°C with intermitent shaking.Measurements were taken at 320nm/400nm excita�on/emission spectra using a Varioskan LUX Mul�mode Microplate Reader (Thermo Fisher Scien�fic) (Kurzawa-Akanbi et al., 2021).

Glucosylceramidase and Alpha-mannosidase ac�vity assays
The glucosylceramidase ac�vity assay (Abcam, 273339) was conducted in a total volume of 160 μL.20μM 4-Methylumbelliferone Standard (4-MU) prepared in Assay Buffer was used to generate a standard curve (0-20μM).The samples were lysed using the provided Assay Buffer solu�on and concentra�on was measured using a Pierce TM BCA Protein Assay Kit (Thermo Fisher Scien�fic, 23225).
Phase par��oning was facilitated by the addi�on of 0.1 M KCL, whereby the lower organic phase containing the lipid frac�on was retained.The lipid-enriched phase was subsequently dried in the presence of nitrogen gas prior to being recons�tuted in methanol containing 5 mM ammonium formate.Lipidomic analysis was carried out under posi�ve and nega�ve ion modes over the mass to charge (m/z) range 250-2000 at a resolu�on of 100,000 using a Thermo Exac�ve Orbitrap mass spectrometer equipped with a heated electrospray ioniza�on (HESI II) probe coupled to a Thermo Fisher Scien�fic ul�mate 3000 RSLC system.Separa�on of lipids by column chromatography was performed using a Thermo Hypersil Gold C18 column (1.9μm; 2.1 mm × 100 mm) maintained at 50°C.Mobile phase A comprised an aqueous solu�on containing 10 mM ammonium formate and 0.1% (v/v) formic acid.Mobile phase B was made up of 90:10 isopropanol/acetonitrile (ACN) containing 10 mM ammonium formate and 0.1% (v/v) formic acid.The ini�al gradient condi�ons were 65%A/35%B.An increase the gradient of phase B was applied from 35% to 65% over 4 minutes, followed by 65%-100% over 15 minutes, with a hold for 2 minutes before re-equilibra�on to the star�ng condi�ons over 6 minutes.The gradient flow occurred at a rate of 400 μL/minute.

Transmission electron microscopy (TEM)
Samples were ini�ally fixed in 2% glutaraldehyde 0.1 M sodium cacodylate and further subjected to an addi�onal fixa�on by 2% osmium tetroxide.Samples were then dehydrated by a gradual increase.in acetone concentra�on, up to 100% then impregnated with increasing concentra�ons of epoxy resin.
A�er 2x 100% resin steps, the samples were embedded in epoxy resin and polymerised at 60 degrees for 24 hours.Epoxy resin blocks were ultra-sec�oned into slices of 70 nm thickness, collected on copper grids and further stained by uranyl acetate and lead citrate.TEM images were captured using a Hitachi HT7800 TEM microscope.TEM analyses entailed the blindfold selec�on of images to enable an unbiased evalua�on of cellular morphology findings.Image segmenta�on and processing was conducted using the open-source Microscopy Image Browser so�ware (Belevich et al., 2016).

Figure S2 .
Figure S2.CRISPR-Cas9 in situ gene edi�ng targets the DRAM2 gene without off site effects in CORD21-P1c and -P2c iPSCs.(A) Sequence in the top panel shows gRNA used in CRISPR-Cas9 experiment (muta�on site outlined by blue rectangle).Botom panel shows genomic sequence following puta�ve CRISPR-Cas9 correc�on of the c.131G>A muta�on in CORD21-P2 iPSCs (corrected muta�on outlined in blue, the Alu I and abrogated PAM sites marked in orange and magenta, respec�vely).(B) Representa�ve gel image of CRISPR-edited clones following DNA extrac�on.iPSC clones marked by blue and red asterisks were selected as primary targets for Sanger sequencing due to producing expected band paterns of approximately 216 and 192 bps following Alu I restric�on

Figure S5 .
Figure S5.DRAM2 an�body valida�on and protein detec�on in ROs by western blot.(A) WT day 230 ROs were dissected and expanded on Matrigel™, and poly-L-ornithine coated 24-well plates.Treatment with 20μM DRAM2 and scrambled siRNA was carried out for a period of 72 hours.(B) DRAM2 protein detec�on (~26kDa) in day 220 pa�ent and control ROs.TPS stands for total protein stain showing equal sample loading.Quan�ta�ve analysis (botom panel) confirms 50% reduc�on in DRAM2 protein expression.(C) Quan�ta�ve RT-PCR confirms DRAM2 knockdown in siRNA treated ROs compared to scrambled control.(B-C) Data are presented as mean + SEM (n=3 different differen�a�on experiments each consis�ng of 24 ROs/sample).**** p<0.0001.

Figure S6 .
Figure S6.CORD21-P1-ROs show increased number of branched mitochondria rela�ve to isogenic control.(A) CORD21-P1 image indicates the higher abundance of more branched mitochondria in photoreceptor cell bodies, and ISs as opposed to CORD21-P1c where less mitochondrial branching is apparent.Images from CORD21-P2 and CORD21-P2c demonstrate high levels of mitochondrial diversity, whereby both smaller and more elongated mitochondria are apparent in the photoreceptor cell bodies/inner segment.Elongated mitochondria are shown by black arrow heads and rounded mitochondria by white arrow heads, respec�vely.(B) Bar plots show no difference in mitochondrial