Modeling PRPF31 retinitis pigmentosa using retinal pigment epithelium and organoids combined with gene augmentation rescue.

"data availability the raw rna sequencing data are deposited in the gene expression omnibus (geo) database under accession code gse206529. we also excluded potential off-target effects (supplementary data 1 ).; these 6 clusters encompassed 337 degs including several adhesion molecules such as seven members of the protocadherin/cadherin family which were found among the most differentially expressed in irpe cells carrying a prpf31 mutation (supplementary fig 8 and supplementary data 2 ).; hierarchical clustering based on z score allowed the selection of 7 clusters in which the 322 identified degs showed similar expression levels between cys247x-iso and control as well as cys247x-as but a different one in cys247x (fig 9b and supplementary data 3 ).; hierarchical clustering based on z score allowed the selection of five clusters in which the 84 identified degs showed a similar expression level between cys247x-iso and control as well as cys247x-as but different in cys247x (supplementary fig 11 and supplementary data 3 ).; such approach led to the identification of 230 splicing events altering a total of 152 unique genes (supplementary data 4 ).; as such we identified 1049 differential splicing events corresponding to 600 unique genes (fig 11a and supplementary data 5 ).; using the same filtering parameter applied for d100 we identified 513 differential splicing events corresponding to 307 unique genes comparing d130 organoids derived from cys247x and cys247x-iso ipscs (supplementary fig 10c d and supplementary data 5 ).; after 24 h media were aspirated and cells were cultured with appropriate stemdiff tm trilineage medium for 5 (mesoderm and endoderm lineages) or 7 days (ectoderm lineage) with a daily medium change before cell fixation and assessment of differentiation for each germ layer by immunofluorescence using specific markers (supplementary data 6 ).; pcr amplification flanking prpf31 mutations in exons4 or 8 (supplementary data 6 ) was performed using hot fire pol dna polymerase (solis biodyne).; the sgrna (supplementary data 6 ) which targets only the mutated sequence and is predicted to have low off-targets was chosen and synthetized using alt-r guide rna modifications (integrated dna technologies).; the ssodn template with wild-type prpf31 sequence was designed manually with 91-bp homology arms on each side of the mutation region (supplementary data 6 ).; the genotype was finally visualized after migration of the dna in a 3% agarose gel (supplementary data 6 ).; the pcr products were then sequenced to check the off-target effects of sgrna (supplementary data 6 ).; incubation with the primary antibody (supplementary data 6 ) appropriately diluted in blocking solution was performed alternatively for 1 h at rt or overnight at 4 ?; after transfer membranes were saturated using a blocking solution (pbs0 1% tween-20 with 5% non-fat powder milk) for 1 h at rt and incubated with the primary antibody (supplementary data 6 ) overnight at 4 ?; uncropped blots are shown in supplementary data 7 .; all primers and mgb probes labeled with fam for amplification were purchased from thermo fisher scientific (supplementary data 6 ). data availability the raw rna sequencing data are deposited in the gene expression omnibus (geo) database under accession code gse206529"

"Competing interests A.S.-B., S.R., J.-A.S., and O.G. are inventors on patents on hiPSC retinal differentiation and on the use of hiPSC retinal derivatives to treat retinal degeneration, licensed to Gamut Cell Tx. L.C.B. is an inventor of patent applications for AAV capsid variants and AAV screening methods, and she is a founder of Avista Therapeutics and Vegavect."

"We are grateful to Drs. S. Mohand-Said and D. Dagostinoz (CIC1423, Hôpital des Quinze-Vingts) for their help in patients recruitment and M. Cailleret (IStem, Corbeli-Essones, France) for his help in CRISPR/Cas9 strategies. We acknowledge V. Bazin and G. Toutirais from the electron microscopy facility (Sorbonne Université) for performing SEM and TEM analysis, C. Condroyer for sequencing analysis, and E. Vanoni for her help with the phagocytosis assay. We thank N. Oudrhiri and A. Bennaceur (GHU Paris-Sud APHP) for the conventional cytogenetic analysis, Dr. O. Feraud and professor F. Griscelli (ESTeam Paris-Sud/U935 INGESTEM ANR Program Investissement d’Avenir) for the teratoma assay, Dr. Edwin Hernandez-Garzon (Institut de la Vision) for his help with RPE shape analysis and Dr. C. Craft (Mary D. Allen Laboratory for Vision Research, USC ROSKI Eye Institute) for the cone arrestin antibody. This study was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Sorbonne Université, LABEX LifeSenses (ANR-10-LABX-65), IHU FOReSIGHT (ANR-18-IAHU-01), ANR grants (ANR-15-RHUS-001 and ANR-17-CE18-0004-01), Paris Ile-de-France Region (DIM Gene Therapy), Fondation Maladies Rares, Retina France association and by the Foundation Fighting Blindness (Individual Investigator Award to LCB). The funding organizations had no role in the design or conduct of this research."