Additional file 1 of Stargardt disease-associated in-frame ABCA4 exon 17 skipping results in significant ABCA4 function
Creators: name: Kaltak, Melita
name: Blanco-Garavito, Rocio
name: Molday, Laurie L.
name: Dhaenens, Claire-Marie
name: Souied, Eric E.
name: Platenburg, Gerard
name: Swildens, Jim
name: Molday, Robert S.
name: Cremers, Frans P. M.
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Science Policy

description: Additional file 1: Figure S1. Proband’s baseline full field electroretinography (ffERG) results (ISCEV protocol; MetroVision, Perenchies, France). A Moderately subnormal scotopic rod (dark adapted) responses for both left (OS, oculus sinister) and right eyes (OD, oculus dexter). B Moderately subnormal mixed response (dark adapted), of which the right eye shows a more diminished response. C Subnormal oscillatory responses (dark adapted) for both eyes (OP, oscillatory potential). D Very diminished cone responses (light adapted) in both eyes. E Subnormal flicker response (light adapted) for both eyes. Figure S2. Expression of ABCA4 constructs in HEK293T (A, C) and ARPE-19 (B, D, E) cells. A Coomassie blue stained gel of affinity purified wild-type (WT) and ΔAsp864_Gly885 proteins. B Immunoblot of wild-type (WT) and ΔAsp864_Gly885 proteins from ARPE-19 protein lysates. Vinculin (VCL) was used as loading control. C, D Quantitation showed that ABCA4 ΔAsp864_Gly885 expressed at levels similar to those measured for the WT ABCA4 in both HEK293T and ARPE-19 cells, respectively. Data are shown as mean±SEM. E ARPE-19 cells were transfected with the ABCA4 plasmids, and the localization of ABCA4 was detected using the ABCA4 3F4 clone (yellow). The ER, detected with anti-calnexin (CNX), is shown in magenta. ΔAsp864_Gly885 is associated with intracellular vesicles, that resembles the localization of the WT. Part of the truncated protein is retained in the ER, which was not identified with WT protein. Scale 20 μM. Figure S3. Percentages of ABCA4 Δexon 17 transcripts upon treatment with AONs in the first screening. The AONs were administered to WERI-Rb-1 cells gymnotically and the treatment lasted 48 h. Data are shown as mean±SEM, **p≤0.01, ****p≤0.0001 vs. untreated. Figure S4. Binding location of antisense oligonucleotides in first and second screening. Capital letters display ABCA4 exon 17. Colored horizontal lines represent the binding sites for RNA-splicing proteins (adapted from Alamut Visual Plus). The AONs in red represent the best candidates that were used in treatment of retinal organoids. Figure S5. The expression of retinal markers in retinal organoids (ROs) treated with AONs. All markers show similar expressions between all groups of ROs, except for OPN1MW that showed significant differences in ROs treated with AON24 and AON25 vs. scrambled group. Data are shown as mean±SEM. Asterisks indicated the significant differences in expression vs. scrambled, **p≤0.01, ***p≤0.001. Table S1. List of antisense oligonucleotides. Table S2. List of primers and probes used in dPCR assays.