Aim, design, and setting

To elucidate the pleiotropic effects of the IRD variant c.1354dupT (p.Tyr452Leufs*13) located in exon 13 of the PROM1 gene, by identifying associated modifier genes, and to create gene-edited patient-derived iPSC. The study was structured as a transversal study and experimental research. It was carried out at the Institute for Applied Ophthalmobiology (IOBA) and at the Institute of Biomedicine and Molecular Genetics (IBGM), both affiliated with the University of Valladolid.

Patient selection and sampling

A series of eight patients previously diagnosed with PROM1-related IRD was revised (Table 1). They underwent clinical and genetic characterization, and three presenting distinct phenotypes associated with the homozygous c.1354dupT (p.Tyr452Leufs*13) mutation were initially selected for the experimental study. Furthermore, a first-degree relative of each of these target patients was included.

Table 1 Genetic background of identified patients carrying PROM1-related retinopathies

Peripheral blood samples were collected from the patients and their respective relatives for CES. Dermal biopsy samples were obtained from the three target patients and were used to generate iPSC.

Ophthalmological examination

To confirm the patient’s phenotype and establish their pedigree patterns, a comprehensive anamnesis and ophthalmological examination were performed. Visual acuity (VA) was tested using an Early Treatment Diabetic Retinopathy Study panel and recorded as the logarithmic of minimum angle of resolution (logMAR) scale. Retinography and fundus autofluorescence (FAF) were obtained with the TRC 50DX Retinograph (TOPCON Europe Medical BV, Rotterdam, the Netherlands). Sweep-source optical coherence tomography (OCT) was gathered using the PLEX® Elite 9000 OCT (Carl Zeiss AC). Visual field (VF) testing was performed when fixation was possible with the Humphrey Field Analyzer (Carl Zeiss Meditec, Dublin, CA). A full field electroretinogram (ERG) was also performed according to the regularly updated standards of the International Society for Clinical Electrophysiology of Vision (ISCEV) [46].

Clinical exome sequencing

To investigate the potential influence of other modifying genes that could account for the varied phenotypic expressions associated with the target mutation, we conducted clinical CES of three target patients and three healthy relatives. DNA was extracted from blood cells, the Roche exome capture library was prepared, and extensive CES was carried out on a NovaSeq 6000 S4 (Illumina, CA, USA) with 90 × coverage. Bioinformatic analysis of the exons and adjacent intronic regions was performed on a total of 998 genes linked to retinal diseases (Supplementary file 1). We concentrated on genes associated with IRD [47], Age-related Macular Disease (AMD) [48], and genes coding for the phenotype HP:000047, indicative of abnormal retinal morphology [49]. Subsequently, in-silico predictors were applied as filters, and the findings were validated by Sanger sequencing using the ABI Prism 3130xl genetic analyzer (Thermo Fisher, MA, USA). Only variants classified as pathogenic or likely pathogenic were considered for further analysis.

Induced pluripotent stem cells generationFibroblast isolation

A dermal biopsy was performed at the patient’s cranial gluteal area, previously disinfected with 10% povidone iodine and rinsed with saline solution, under local anesthesia injected subcutaneously (1% lidocaine), using a 3-mm biopsy punch (Stiefel, Middlesex, UK). The biopsies were immersed in IMDM medium supplemented with 10% fetal bovine serum (FBS), 3% penicillin/streptomycin (P/S), 3X Amphotericin B and 0.1 µM 2-Mercaptoetanol (Gibco, Invitrogen, Paisley, UK. Cat# 11,510,596, 26,140,079, 11,548,876, 15,290,018, 31,350,010). Biopsies were then cut into small pieces (small as possible, approximately 0.5 × 0.5 mm) and cultured in collagen-coated (Sigma-Aldrich, Saint Louis, USA. Cat# C3867-1VL) 100-mm culture dishes (Thermo Fisher, MA, USA. Cat# A30907) using “fibroblast medium”, composed by IMDM supplemented with 10% FBS, 1X P/S and 1X Amphotericin B and 0.1 µM 2-Mercaptoetanol (Gibco, Invitrogen, Paisley, UK. Cat# 11,510,596, 26,140,079, 11,548,876, 15,290,018, 31,350,010) under standard conditions (37Cº, 5% CO2 atmosphere). The fibroblast medium was refreshed every two days. Biopsies pieces were maintained in culture until fibroblasts outgrowth was observed, approximately at day 10 to 14 (Fig. 1).

Fig. 1

Generation of patient-derived iPSC from IRD1 patient. At D0, dermal biopsy is cut into small pieces, seeded in 100-mm culture dishes, and maintained with IMDM medium. At D14, outgrowth of fibroblast is noticeable from biopsy pieces. At D28, passage 2 patient-derived fibroblast are reprogrammed using the episomal vectors through electroporation. At D38, three days after electroporation, IMDM medium is changed to mTSR-E7 medium. At D56, three weeks after electroporation, iPSC colonies have emerged between fibroblasts. iPSC colonies are picked up manually and seeded in new culture dishes using the mTeSR plus medium. At D70, passage 2 iPSC colonies displaying compact colonies with distinct borders, well-defined edges, and large nuclei. Days after electroporation*

Non-integrative fibroblast reprogramming

The patient’s fibroblasts were reprogrammed using The Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher, MA, USA. Cat# A15960) containing the reprogramming vectors pCE-hOCT3/4 (OCT4 gene), pCE-hSK (Sox2 and KlF4 genes), pCE-hUL (L-Myc and Lin28 genes) in Tube A, and the pCE-mP53DD (mp53DD gene), and pCXB-EBNA1 (EBNA1 gene) in Tube B. Passage 2 fibroblast (150,000 cells) were electroporated with 1 µL of Tubes A and B, using the 100 µL pipette tip of the Neon Transfection System (Thermo Fisher, MA, USA. Cat# MPK5000) under the following pulse conditions: 1400 V; 20 ms; two pulses. The electroporated fibroblast were then seeded in Matrigel-coated (23 µg/cm2; Corning Life Sciences, NY, USA. Cat# 11,593,620) 6-well plates (Thermo Fisher Scientific, MA, USA. Cat# 11,337,694) using fibroblast medium, being changed every 24 h until day 3. Subsequently, the medium was replaced by the TeSR™-E7™ Medium (Stem Cells Technologies, Cambridge, UK. Cat# 05914) until iPSC colonies emerged, approximately 21 days. The iPSC clones were manually picked using a 22Gx2″ hypodermic needle (Terumo, Madrid, Spain), the Leica LED2500-TL3000 ERGO microscope (Thermo Fisher, MA, USA), and transferred with a p200 Pipette (Gilson PIPETMAN; Thermo Fisher Scientific, MA, USA. Cat# 1,232,613) to a Matrigel-coated (23 µg/cm2; Corning Life Sciences, NY, USA. Cat# 11,593,620) 6-well plate with mTeSR™ Plus medium supplemented with 10 µM Rock inhibitor Y-27632 (Stem Cells Technologies, Cambridge, UK. Cat# 100–0276, 72,304). iPSC were passaged as clumps using 0.5 mM EDTA (Thermo Fisher, MA, USA. Cat# 10,135,423) every 5–7 days, and frozen using freezing medium composed by 90% FBS (Thermo Fisher, MA, USA. Cat# 26,140,079) and 10% DMSO (Sigma-Aldrich, Saint Louis, USA. Cat# 34,869) (Fig. 1).

Induced pluripotent stem cells (iPSC) characterizationAlkaline phosphatase staining

To demonstrate the characteristic upregulation of alkaline phosphatase (AP) activity in the generated iPSC lines, we performed the AP blue membrane substrate solution assay (Sigma-Aldrich, Saint Louis, USA. Cat# AB0300). On day 5, a single 6-well plate (Thermo Fisher Scientific, MA, USA. Cat# 11,337,694) was fixed with 4% paraformaldehyde (Panreac Quimica, Barcelona, Spain) for 1 min. The plate was then washed with phosphatase-buffered saline (PBS) prewarmed to 64 °C (Thermo Fisher, MA, USA. Cat# 10,010,023) and incubated for 20 min at the same temperature. Afterwards, 1.5 ml of a 1:1 mixture of kit solutions A and B was added, and the plate was incubated in the dark at room temperature (RT) for 10 min. Direct micrographs were obtained using a Nikon Eclipse TS100 inverted microscope (Nikon Instruments, NY, USA).

Immunocytochemical characterization

Immunocytochemical characterization was performed as previously described [50]. Briefly, iPSC lines were grown on Matrigel-coated (23 µg/cm2; Corning Life Sciences, NY, USA. Cat# 11,593,620) μ-Slide 8-well culture plates (Ibidi, München, Germany. Cat# 80,826) and fixed with 10% formalin solution (Sigma-Aldrich, Saint Louis, USA. Cat# HT501128). The plates were rinsed with Tris-buffered saline (TBS; Sigma-Aldrich, Saint Louis, USA. Cat# T5912) and blocked with a mixture of TBS, 0,3% Triton X-100 and 3% donkey serum, (Sigma-Aldrich, Saint Louis, USA Cat# X100, D9663) for 60 min. Primary antibodies associated with pluripotency markers (OCT4, SSEA3, SOX2, SSEA4, TRA1-60, NANOG, TRA-1–81) and germ layers markers (β-III Tubulin, α-1 Fetoprotein, α- Smooth Muscle Actin) were applied, followed by incubation with their respective species-specific secondary antibody. Nuclei were visualized by immunostaining with DAPI (4',6-diamidino-2-phenylindole, Invitrogen, CA, USA. Cat# 10,184,322). The specific conditions for the primary and secondary antibodies are detailed in Supplementary file 2.

All immunocytochemical analyses were carried out in triplicate for each experimental condition. Controls, were primary and/or secondary antibodies were omitted, were processed concurrently. Immunofluorescence micrographs were captured using a LEICA TCS SP8 LIGHTNING confocal microscope (Leica Microsystems, Hesse, Germany) and analysed with LEICA LAS AF software (Leica Microsystems, Hesse, Germany).

Expression of pluripotency factors and silencing of reprogramming vectors

To assess the expression levels of endogenous pluripotency factors, we conducted a gene-specific primer-based quantitative reverse transcription PCR (qRT-PCR) for the genes SOX2, OCT3/4, KLF4, LMYC, LIN28. On the other hand, to probe the absence of reprogramming vectors, we performed a dual assay: a copy number qPCR, using genomic DNA (gDNA) and targeting a region common to all reprogramming vectors (within the EBNA1 gene), and a vector-specific primer-based qRT-PCR, using complementary DNA (cDNA), for each exogenous reprogramming factor delivered by the reprogramming vectors.

For qRT-PCR assays, RNA was extracted from the patient-derived iPSC lines at passage 6 using the Trizol Reagent (Invitrogen, CA, USA. Cat# 15,596,026) according to the manufacturer’s protocol. The purity and concentration of the RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA. Cat# 4,368,814), following the manufacturer’s guidelines.

For EBNA1 copy number qPCR, gDNA extraction was performed using ethanol precipitation [51]. A standard curve was generated from serial dilutions of a vector contained in the Epi5 Episomal iPSC Reprogramming Kit (Thermo Fisher, MA, USA. Cat# A15960), pCE-hOCT3/4 (Addgene #41,813). The number of copies of EBNA1 in each dilution was determined based on the length of the pCE-hOCT3/4 plasmid.

All qPCRs were carried out with SYBR Green PCR Master Mix (Applied Biosystems, CA, USA. Cat# 4,309,155) on the LightCycler 480 Instrument II System (Roche, Switzerland). The cycling conditions were as follows: an initial denaturation at 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 45 s, with a final extension at 72 °C for 5 min and a subsequent melting curve analysis. GAPDH served as the housekeeping gene for normalizing mRNA expression levels. The threshold cycle was determined for each reaction, and gene expression levels were quantified using the 2−ΔΔCt method [51]. All qPCR assays were conducted in triplicate for each experimental condition.

Untransfected fibroblasts from IRD1 patient served as the negative control for the expression of pluripotency factors essay; while fibroblasts from IRD1, transfected with the same episomal vectors as the iPSC lines, 72 h post-transfection, served as positive control. Primers used in qRT-PCR essays for endogenous pluripotency factors (gene primers) and exogenous reprogramming vectors (plasmid primers) are listed in Supplementary file 3.

In vitro differentiation into three germinal layers

To assess the differentiation potential of patient-derived iPSC lines into cells from the three germ layers, three-dimensional iPSC aggregates known as embryoid bodies (EBs) were specifically induced to differentiate into cell types characteristic of each layer. iPSC were cultured to 80% confluence on Matrigel-coated (23 µg/cm2; Corning Life Sciences, NY, USA. Cat# 11,593,620) 60 mm culture plates (Thermo Fisher, MA, USA). They were then dissociated with 0,5 mM EDTA (Thermo Fisher, MA, USA. Cat# 10,135,423), resuspended in mTeSR™ Plus medium (Stem Cells Technologies, Cambridge, UK. Cat# 100–0276) and seeded into 96-well V-bottom ultra-low attachment plates (Sigma-Aldrich, Saint Louis, USA. Cat# 10,462,012), which were centrifuged to facilitate EB formation. The EBs were cultured in ultra-low attachment 60-mm plates (Corning Life Sciences, NY, USA. Cat# 3261) for 3 days, subsequently transferred to Matrigel coated (23 µg/cm2; Corning Life Sciences, NY, USA. Cat# 11,593,620) μ-Slide 8 well culture plates (Ibidi, München, Germany. Cat# 80,826) and maintained for two to three weeks with three distinct differentiation media: endoderm medium (DMEM supplemented with 20% FBS, 2 mM Glutamax™, 100 μM non-essential amino acids, 100 μM 2-Mercaptoetanol and 1X P/S, [Gibco, Invitrogen, Paisley, UK. Cat# 13,345,364, 26,140,079, 35,050,061, 11,140,050, 11,528,926, 11548876]), mesoderm medium (endoderm medium supplemented with 100 μM ascorbic acid [Sigma-Aldrich, Saint Louis, USA. Cat# V-038]), and ectoderm medium (50% DMEM F12, 50% neurobasal medium, 2 mM Glutamax™, 1X N2 supplement, 1X B27 supplement and 1X P/S [Gibco, Invitrogen, Paisley, UK. 11,320,033, 21,103,049, 35,050,061, 17,502,048, 17,504,044, 11548876]).

Short tandem repeat analysis

To verify the genetic concordance between the patient’s fibroblasts and their respective iPSC lines, DNA fingerprinting analysis was conducted. The amplification of short tandem repeat (STR) was performed through multiple PCR using the kit GenePrint® 10 System PCR Amplification Kit (Promega, Wisconsin, USA. Cat# B9510). STR analysis at 10 loci (CSF1PO, D13S317, D16S539, D21S11, D5S818, D7S820, TH01, TPOX, vWA, and Amelogenin for sex determination) were carried out in accordance with the ASN-0002 standard established by the American Tissue Culture Collection Standards Development Organization Workgroup for cell line authentication. The amplified samples were evaluated on a 3730 DNA Analyzer (Applied Biosystems. CA. USA) using the POP-7 polymer and the ILS600 size standard. The results were analysed using the GeneMapper® software (v4.1, Applied Biosystems).

Karyotyping

To assess the genomic integrity of the generated iPSC lines, a G-banded metaphase chromosome analysis with a resolution of 300–500 bands was conducted. The patient-derived iPSC colonies at 70% of confluence were incubated in KaryoMax colcemid (Thermo Fisher, MA, USA. Cat 15,212,012) at a final concentration of 0,1 μg/mL for 3 h to induce mitotic arrest during metaphase. The cells were then trypsinized, exposed to a prewarmed 37ºC hypotonic solution (KCl 75 mM) for 15 min and fixed with methanol/glacial acetic acid (3:1) solution. A total of 20 metaphases were analysed.

Target mutation sequencing

The target mutation (c.1354dupT) was sequenced in the iPSC lines. gDNA extraction was performed using ethanol precipitation [52]. The region of the PROM1 gene containing the target mutation was amplified by conventional PCR. Subsequently, the PCR-amplified fragments were sequenced using Capillary Electrophoresis Sanger Sequencing on an ABI 3730xl DNA analyser (Thermo Fisher, MA, USA). The sequencing primer PROM1seqFW is listed in Supplementary file 3.

Patient-derived iPSC genetic repairgRNA designing

The gRNA were designed in silico using the CRISPOR web tool for genome editing [53], selected based on their proximity to the mutation and their specificity and efficiency scores. gRNAs were synthesized in vitro using the GeneArt™ Precision gRNA Synthesis Kit (Invitrogen, Paisley, UK. Cat# A29377) following manufacturer’s instructions. Four gRNAs (40RE, 47FW, 66FW, 77FW) and their corresponding repair oligonucleotides were generated.

The repair oligonucleotides (Alt-R HDR Donor Oligos) were engineered to include silent mutations, near the target mutation site, using the web tool Silent Mutator (https://molbiotools.com/silentmutator.php) [54] and the DNAStar Lasergene software (v.7.1, DNAstar). Two of these silent mutations were introduce to create a new restriction site for the Ssp1 enzyme (Thermo Fisher, MA, USA, Cat# ER0771), allowing the recognition of proper integration of the repair oligonucleotide. The other two silent mutations were designed to destroy the Protospacer Adjacent Motif (PAM) sequence, preventing the edited allele from being targeted repeatedly by the gRNA (Fig. 2).

Fig. 2

Design of a gene editing strategy for repairing the c.1354dupT (p.Tyr452 Leufs*13) mutation in the exon 13 of the PROM1 gene. A In silico representation of the target sequence in a WT case. The amino acid affected by target mutation “Tyrosine” is highlighted in a green circle B In silico representation of a patient affect by the c.1354dupT (p.Tyr452 Leufs*13). The mutation is indicated by a black arrow and represented by a lowercase “t”. The Leucine generated due the mutation’s frameshift is highlighted in a red circle, and the consequent stop codon is indicated by a black arrow. The localization of the four designed gRNAs (40RE, 47RE, 66FW and 77FW) are framed in orange C In silico representation of the target sequence in a genetically repaired case. Blue arrows indicate silent mutations that introduce a restriction site for the Ssp1 and destroy the Protospacer Adjacent Motif (PAM) sequence to prevent the edited allele from being targeted again by the gRNA. The amino acid affected by target mutation “Tyrosine” is highlighted in a green circle. The WT reading frame is recovered

The predicted gene-editing efficiency of the designed four gRNA guides, were tested in vitro by editing the U2OS osteosarcoma cell line (ATCC HTB-96). The U2OS cells (1 × 106 cells) were transfected with each gRNA (1 µg), the corresponding repair oligonucleotide (15µL at 10 µM), and the Cas9-protein v2 TrueCut™ (5 µg; Invitrogen, Paisley, UK. Cat# A36498) using the 100 µL pipette tip of an Neon Transfection System (Thermo Fisher, MA, USA. Cat# MPK5000), under the following pulse conditions: 1230 V; 10 Ms; 4 pulses. After 24 h of culture, gDNA from each essay was extracted, and the region containing the target mutation was amplified by conventional PCR using the PROM1seq primer. The PCR products were purified using the Wizard® PCR Preps DNA Purification System (Promega, Wisconsin, USA. Cat# A7170) and digested with the Ssp1(Thermo Fisher, MA, USA, Cat# ER0771) enzyme for 3 h at 37 °C to ascertain the most effective CRISPR/Cas9 editing. Untransfected U2OS cells served as the wild type (WT) control.

Detection of gene-edited iPSC clones

As a screening method to identify successfully genetically repaired iPSC clones, a specific primer incorporating the silent mutations present in the selected repair oligonucleotide were designed (ALELO40RE primer) (Supplementary file 3). Due to the similarity between a WT and a gene edited sequence, amplified by the ALELO40RE primer, a gradient conventional PCR assay was conducted to determine the optimal annealing temperature to identified successfully genetically repaired iPSC clones.

To test this method, U2OS cells were transfected, under the previously described conditions, with the selected gRNA and repair oligonucleotide. After 24 h of culture, gDNA was extracted and a gradient conventional PCR was performed.

Patient-derived iPSC gene editing

After 5 days of culture, the iPSC lines derived from patients IRD1 and 2, were dissociated using Accutase (Stem Cells Technologies, Cambridge, UK. Cat# 07920) and resuspended in mTeSR™ Plus medium supplemented with CloneR™2 (Stem Cells Technologies, Cambridge, UK. Cat# 100–0276, 100–0691). A total of 1 × 106 cells were electroporated with the selected gRNA (1,5µL at 1µL/µg), the repair oligonucleotide (15 µL at 20 µM) and Cas9-protein v2 TrueCut™ (1,5µL at 5µL/µg, Thermo Fisher, MA, USA. Cat# A36498) using the Neon Transfection System (Thermo Fisher, MA, USA. Cat# MPK5000), with the following pulse conditions: 1200 V; 30 Ms; 1 pulse. The electroporated cells were plated at a density of 50 cells/cm2 on Matrigel coated (23 µg/cm2; Corning Life Sciences, NY, USA. Cat# 11,593,620) 6-well plates (Thermo Fisher Scientific, MA, USA. Cat# 11,337,694) using mTeSR™ Plus medium supplemented with CloneR™2 (Stem Cells Technologies, Cambridge, UK. Cat# 100–0276, 100–0691) for 24 h. The mTeSR™ Plus medium (Stem Cells Technologies, Cambridge, UK. Cat# 100–0276) was refreshed daily until the colonies reached a sufficient size for manual picking. iPSC clones were subsequently plated in 24-well plates (Thermo Fisher, MA, USA. Cat# 142,475), duplicated and cryopreserved.

The gDNA was extracted from 30 clones of each patient-derived iPSC lines and was amplified via conventional PCR using the PROM1seq primer (Supplementary file 3). The PCR products of the selected clones were purified with the Wizard® PCR Preps DNA Purification System (Promega, Wisconsin, USA. Cat# A7170) and digested with the Ssp1 enzyme (Thermo Fisher, MA, USA, Cat# ER0771) for 3 h at 37 °C. Proper integration of the repair oligonucleotide in the selected clones was then confirmed by Sanger sequencing using the PROM1seqFW primer (Supplementary file 3). The gene-edited clones were assessed for their ability to encode Prominin-1 through CD133 flow cytometry and western blotting.

Flow cytometry

The iPSC line derived from patient IRD1, its corresponding genetically repaired iPSC line, and the iPSC control line [FiPS] Ctrl1-Ep6F-5 were labelled with an anti-CD133 antibody conjugated to allophycocyanin (APC) (Supplementary file 2), following the manufacturer’s protocol. The flow cytometry assay was performed using the Gallios Flow Cytometer (Beckman Coulter, Indianapolis, USA) and the data were processed with Kaluza Analysis Software (Beckman Coulter, Indianapolis, USA). iPSC lines without staining served as negative controls for the assay.

Western blotting

Proteins obtained from the iPSC line derived from patient IRD2, its corresponding repaired iPSC line, and the control iPSC line [FiPS] Ctrl1-Ep6F-5 were extracted using RIPA buffer (25 mM Tris–HCl pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) with added protease inhibitors. Protein samples (20 µg each) were separated by SDS-PAGE technique and subsequently transferred to PVDF membranes. The proteins CD133 and β-actin (as a loading control) were immunodetected using specific antibodies (Supplementary file 2). Images were acquired using a GS-800 Calibrated Densitometer (Bio-Rad, Madrid, Spain) and analysed with Quantity One software (Bio-Rad, Madrid, Spain).