Rapid Variant Pathogenicity Analysis by CRISPR Activation of CRB1 Gene Expression in Patient-Derived Fibroblasts.

Inherited retinal diseases (IRDs) are a heterogeneous group of blinding genetic disorders caused by pathogenic variants in genes expressed in the retina. In this study, we sought to develop a method for rapid evaluation of IRD gene variant pathogenicity by inducing expression of retinal genes in patient-derived fibroblasts using CRISPR-activation (CRISPRa). We demonstrate CRISPRa of CRB1 expression in fibroblasts derived from patients with retinitis pigmentosa, enabling investigation of pathogenic mechanisms associated with specific variants. We show the CRB1 c.4005 + 1G>A variant caused exon 11 skipping in CRISPR-activated fibroblasts and retinal organoids (ROs) derived from the same RP12 patient. The c.652 + 5G>C variant was shown to enhance exon 2 skipping in CRISPR-activated fibroblasts and differentially affected CRB1 isoform expression in fibroblasts and ROs. Our study demonstrates an accessible platform for transcript screening of IRD gene variants in patient-derived fibroblasts, which can potentially be applied for rapid pathogenicity assessments of any gene variant.

Mitochondrial Dysfunction and Impaired Antioxidant Responses in Retinal Pigment Epithelial Cells Derived from a Patient with RCBTB1-Associated Retinopathy.

Mutations in the RCBTB1 gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we investigated the effect of RCBTB1 deficiency on mitochondria and oxidative stress responses in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells from control subjects and a patient with RCBTB1-associated retinopathy. Oxidative stress was induced with tert-butyl hydroperoxide (tBHP). RPE cells were characterized by immunostaining, transmission electron microscopy (TEM), CellROX assay, MitoTracker assay, quantitative PCR and immunoprecipitation assay. Patient-derived RPE cells displayed abnormal mitochondrial ultrastructure and reduced MitoTracker fluorescence compared with controls. Patient RPE cells displayed increased levels of reactive oxygen species (ROS) and were more sensitive to tBHP-induced ROS generation than control RPE. Control RPE upregulated RCBTB1 and NFE2L2 expression in response to tBHP treatment; however, this response was highly attenuated in patient RPE. RCBTB1 was co-immunoprecipitated from control RPE protein lysates by antibodies for either UBE2E3 or CUL3. Together, these results demonstrate that RCBTB1 deficiency in patient-derived RPE cells is associated with mitochondrial damage, increased oxidative stress and an attenuated oxidative stress response.

Characterising splicing defects of ABCA4 variants within exons 13-50 in patient-derived fibroblasts.

The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T>C; 5603A>T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13-50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction.

Anti-retinal IgG antibodies in patients with early and advanced type 2 macular telangiectasia.

Type 2 idiopathic macular telangiectasia (MacTel-2) is a progressive adult-onset macular disease associated with bilateral perifoveal vascular changes, Muller cell degeneration and increased blood-retinal barrier permeability. The pathophysiological mechanisms of MacTel-2 remain unclear, however it was previously reported that anti-retinal antibodies in MacTel-2 patients are a significant feature of the disease. In this study, we aimed to compare the prevalence of anti-retinal antibodies in patients MacTel-2, healthy controls and patients with other retinal diseases. MacTel-2 patients diagnosed with multimodal imaging were enrolled and their disease severities were graded using spectral-domain optical coherence tomography. For comparison, patients with age-related macular degeneration (AMD), inherited retinal diseases (IRDs) or no retinal disease (healthy controls) were recruited as controls. Blood serum samples were screened for immunoglobulin G anti-retinal antibodies by western blotting, followed by densitometry analysis. Odds ratios (OR) with 95% confidence intervals (CI) were calculated and p < 0.05 considered statistically significant. Overall, anti-retinal antibody-positive cases were older (64 ± 15 vs 53 ± 17 years, p < 0.001) and females were more likely to develop anti-retinal antibodies (OR: 2.41, CI: 1.12-5.18). The frequency of anti-retinal antibody detection in MacTel-2 patients (n = 42, 36%) was not significantly different from healthy controls (n = 52, 25%) or IRD patients (n = 18, 25%) and the majority of MacTel-2 patients had no anti-retinal antibodies. In contrast, the frequency of anti-retinal antibody detection was significantly higher in patients with AMD (n = 15, 73%, p < 0.001). The lack of a greater anti-retinal antibody frequency or specificity in the MacTel-2 cohort suggests that antibody mediated immunological mechanisms may play a less significant role in MacTel-2 disease pathogenesis.

Gene replacement therapy restores RCBTB1 expression and cilium length in patient-derived retinal pigment epithelium.

Biallelic mutations in the RCBTB1 gene cause retinal dystrophy. Here, we characterized the effects of RCBTB1 gene deficiency in retinal pigment epithelial (RPE) cells derived from a patient with RCBTB1-associated retinopathy and restored RCBTB1 expression in these cells using adeno-associated viral (AAV) vectors. Induced pluripotent stem cells derived from a patient with compound heterozygous RCBTB1 mutations (c.170delG and c.707delA) and healthy control subjects were differentiated into RPE cells. RPE cells were treated with AAV vectors carrying a RCBTB1 transgene. Patient-derived RPE cells showed reduced expression of RCBTB1. Expression of NFE2L2 showed a non-significant reduction in patient RPE cells compared with controls, while expression of its target genes (RXRA, IDH1 and SLC25A25) was significantly reduced. Trans-epithelial electrical resistance, surface microvillus densities and primary cilium lengths were reduced in patient-derived RPE cells, compared with controls. Treatment of patient RPE with AAV vectors significantly increased RCBTB1, NFE2L2 and RXRA expression and cilium lengths. Our study provides the first report examining the phenotype of RPE cells derived from a patient with RCBTB1-associated retinopathy. Furthermore, treatment of patient-derived RPE with AAV-RCBTB1 vectors corrected deficits in gene expression and RPE ultrastructure, supporting the use of gene replacement therapy for treating this inherited retinal disease.

Determinants of Disease Penetrance in PRPF31-Associated Retinopathy.

Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.

Generation of an induced pluripotent stem cell line from a patient with Stargardt disease caused by biallelic c.[5461-10T>C;5603A>T];[6077T>C] mutations in the ABCA4 gene.

Mutations in ABCA4 gene are causative for autosomal recessive Stargardt disease (STGD1), the most common inherited retinal dystrophy. Here, we report the generation of an induced pluripotent stem cell (iPSC) line from a STGD1 patient carrying biallelic c.[5461-10T>C;5603A>T];[6077T>C] mutations in the ABCA4 gene. Episomes carrying OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed for the reprogramming of patient-derived fibroblasts. This iPSC line expressed comparable pluripotency markers as in a commercially available human iPSC line, displayed normal karyotype and potential for trilineage differentiation, and were negative for both reprogramming episomes and mycoplasma test.

Generation of two induced pluripotent stem cell lines from a patient with Stargardt disease caused by compound heterozygous mutations in the ABCA4 gene.

Stargardt disease (STGD1) is the most common inherited retinal dystrophy and ABCA4 c.546--10 T>C is the most commonly reported splice mutation. Here, we generated and characterized two induced pluripotent stem cell (iPSC) lines from a STGD1 patient with compound heterozygous mutations in ABCA4 (c.[5461-10 T > C;5603A > T];[4163 T > C;455G > A]). Episomal vectors containing OCT4, SOX2, KLF4, L-MYC, LIN28 and mp53DD were employed to conduct the reprogramming of patient-derived fibroblasts. Both lines had a normal karyotype, displayed iPSC morphology, expressed pluripotency markers and showed trilineage differentiation potential. These lines can provide a powerful platform for further investigating the pathophysiological consequences of mutations in ABCA4.

Generation of two induced pluripotent stem cell lines from a retinitis pigmentosa patient with compound heterozygous mutations in CRB1.

Two human iPSC lines were generated from dermal fibroblasts derived from a patient with retinitis pigmentosa caused by CRB1 mutation using episomal plasmids containing OCT4, SOX2, LIN28, KLF4, L-MYC and mp53DD. These clonal iPSC lines carry compound heterozygous mutations in CRB1 (c.2555 T > C and c.3014A > T). Both lines expressed pluripotency markers, displayed a normal karyotype and demonstrated the ability to differentiate into the three primary germ layers, as well as retinal organoids.

Deep clinical phenotyping and gene expression analysis in a patient with RCBTB1-associated retinopathy.

Background: Mutations in the RCC1 and BTB domain-containing protein 1 (RCBTB1) gene have been implicated in a rare form of retinal dystrophy. Herein, we report the clinical features of a 45-year-old Singaporean-Chinese female and her presymptomatic sibling, who each possesses compound heterozygous mutations in RCBTB1. Expression of RCBTB1 in patient-derived cells was evaluated.Materials and Methods: The natural history was documented by a series of ophthalmic examinations including electroretinography, fundus autofluorescence imaging, spectral-domain optical coherence tomography, visual field, microperimetry, and adaptive optics retinal imaging. Patient DNA was genetically analysed using a 537-gene Next Generation Sequencing panel and targeted Sanger sequencing. Expression of RCBTB1 in lymphocytes, fibroblasts, and induced pluripotent stem cells (iPSC) derived from the proband and healthy controls was characterized by quantitative PCR, Sanger sequencing, and western blotting.Results: The proband presented with left visual distortion at age 40 due to extrafoveal chorioretinal atrophy. Atrophy expanded at 1.3 (OD) and 1.0 (OS) mm2/year. Total macular volume declined by 0.09 (OD) and 0.13 (OS) mm3/year. Microperimetry demonstrated enlarging scotoma in both eyes. Generalised cone dysfunction was demonstrated by electroretinography. A retinal dystrophy panel testing revealed biallelic frameshifting mutations, c.170delG (p.Gly57Glufs*12) and c.707delA (p.Asn236Thrfs*11) in RCBTB1. The level of RCBTB1 mRNA expression was reduced in patient-derived lymphocytes compared to controls. RCBTB1 protein was detected in control fibroblasts and iPSC but was absent in patient-derived cells.Conclusions: Atrophy expansion rate and macular volume change are feasible endpoints for monitoring RCBTB1-associated retinopathy. We provide further functional evidence of pathogenicity for two disease-causing variants using patient-derived iPSCs.

Generation of two induced pluripotent stem cell lines from a patient with recessive inherited retinal disease caused by compound heterozygous mutations in SNRNP200.

The human induced pluripotent stem cell (iPSC) lines LEIi015-A and LEIi015-B were derived from a patient with inherited retinal disease caused by compound heterozygous mutations in the SNRNP200 gene (c.[1792C>T];[3341T>C]). Dermal fibroblasts were transfected with episomal plasmids carrying transgenes encoding OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for P53. The clonal iPSC lines LEIi015-A and LEIi015-B expressed iPSC markers, were free from genomic alterations and demonstrated trilineage differentiation potential.

Gene correction of the CLN3 c.175G>A variant in patient-derived induced pluripotent stem cells prevents pathological changes in retinal organoids.

BACKGROUND: Mutations in CLN3 cause Batten disease, however non-syndromic CLN3 disease, characterized by retinal-specific degeneration, has been also described. Here, we characterized an induced pluripotent stem cell (iPSC)-derived disease model derived from a patient with non-syndromic CLN3-associated retinopathy. METHODS: Patient-iPSC, carrying the 1 kb-deletion and c.175G>A variants in CLN3, coisogenic iPSC, in which the c.175G>A variant was corrected, and control iPSC were differentiated into neural retinal organoids (NRO) and cardiomyocytes. CLN3 transcripts were analyzed by Sanger sequencing. Gene expression was characterized by qPCR and western blotting. NRO were characterized by immunostaining and electron microscopy. RESULTS: Novel CLN3 transcripts were detected in adult human retina and control-NRO. The major transcript detected in patient-NRO displayed skipping of exons 2 and 4-9. Accumulation of subunit-C of mitochondrial ATPase (SCMAS) protein was demonstrated in patient-derived cells. Photoreceptor progenitor cells in patient-NRO displayed accumulation of peroxisomes and vacuolization of inner segments. Correction of the c.175G>A variant restored CLN3 mRNA and protein expression and prevented SCMAS and inner segment vacuolization. CONCLUSION: Our results demonstrate the expression of novel CLN3 transcripts in human retinal tissues. The c.175G>A variant alters splicing of the CLN3 pre-mRNA, leading to features consistent with CLN3 deficiency, which were prevented by gene correction.

Generation of three induced pluripotent stem cell lines from a patient with Usher syndrome caused by biallelic c.949C > A and c.1256G > T mutations in the USH2A gene.

Mutations in the USH2A gene are the most common cause of Usher syndrome and autosomal recessive non-syndromic retinitis pigmentosa. Here, we describe the generation of three induced pluripotent stem cell lines from dermal fibroblasts derived from a patient carrying biallelic c.949C > A and c.1256G > T variants in the USH2A gene, using episomal reprogramming plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28, mir302/367 and shRNA targeting TP53. All three lines expressed pluripotency markers, displayed unaltered karyotypes as well as trilineage differentiation potential, and were negative for reprogramming episomes and mycoplasma.

Generation of two induced pluripotent stem cell lines from a patient with Stargardt Macular Dystrophy caused by the c.768G>T and c.6079C>T mutations in ABCA4.

Autosomal recessive Stargardt disease is the most common cause of inherited retinal disease. In this report, we describe the generation and characterization of two human induced pluripotent stem cell (iPSC) lines from a patient with compound heterozygous mutations in the ABCA4 gene (c.[768G>T];[6079C>T]). Patient dermal fibroblasts were reprogrammed using episomal plasmids encoding OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for P53. The clonal iPSC lines LEIi012-A and LEIi012-B were established. Both lines had a normal karyotype, displayed iPSC morphology, expressed pluripotency genes at similar levels to control iPSC and displayed trilineage differentiation potential during embryoid body differentiation.

Phenotype-genotype correlations in a pseudodominant Stargardt disease pedigree due to a novel ABCA4 deletion-insertion variant causing a splicing defect.

BACKGROUND: Deletion-insertion (delins) variants in the retina-specific ATP-binding cassette transporter gene, subfamily A, member 4 (ABCA4) accounts for <1% in Stargardt disease. The consequences of these delins variants on splicing cannot be predicted with certainty without supporting in vitro data. METHODS: Candidate ABCA4 variants were revealed by genetic and segregation analysis of a family with pseudodominant Stargardt disease using a commercial panel and Sanger sequencing. RNA extracted from patient-derived fibroblasts was analyzed by RT-PCR to evaluate splicing behavior of the ABCA4 variants. RESULTS: Affected members carrying the novel c.6031_6044delinsAGTATTTAACCAATATTT variant in exon 44 presented with contrasting phenotypes; from early-onset cone-rod dystrophy to late-onset macular dystrophy. This variant resulted in a 56-nucleotide deletion in the mutant allele by activation of a cryptic splice acceptor site which disrupts the reading frame and results in a premature termination codon (p.Ile2003LeufsTer41). If translated, the crucial functional domains near the C-terminus would be truncated from the ABCA4 protein. CONCLUSION: This work demonstrates the intrafamilial phenotypic variability in a pseudodominant Stargardt disease pedigree and the use of patient-derived fibroblasts to evaluate the effect of a novel ABCA4 delins variant on splicing to complement in silico pathogenicity assessment.

Optimization of silk fibroin membranes for retinal implantation.

Silk fibroin membrane displays potential for ocular tissue reconstruction as demonstrated by its ability to support a functioning retinal pigment epithelium (RPE) in vitro. Nevertheless, translation of these findings to the clinic will require the use of membranes that can be readily handled and implanted into diseased retinas, with minimal impact on the surrounding healthy tissue. To this end, we optimized the physical properties of fibroin membranes to enable surgical handling during implantation into the retina, without compromising biocompatibility or permeability. Our central hypothesis is that optimal strength and permeability can be achieved by combining the porogenic properties of poly(ethylene glycol) (PEG) with the crosslinking properties of horseradish peroxidase (HRP). Our study reveals that PEG used in conjunction with HRP enables the production of fibroin membranes with superior handling properties to conventional fibroin membranes. More specifically, the modified membranes could be more easily implanted into the retinas of rats and displayed good evidence of biocompatibility. Moreover, the modified membranes retained the ability to support construction of functional RPE derived from pluripotent stem cells. These findings pave the way for preclinical studies of RPE-implantation using the optimized fibroin membranes.

Generation of three induced pluripotent stem cell lines from an isolated inherited retinal dystrophy patient with RCBTB1 frameshifting mutations.

Variants in RCBTB1 have been implicated in inherited retinal disease (IRD). Here, we generated induced pluripotent stem cells (iPSCs) from a 45-year-old female IRD patient harbouring compound heterozygous mutations in the RCBTB1 gene. Episomal plasmids containing OCT4, SOX2, KLF4, MYCL, LIN28, shRNA for TP53 and mir302/367 microRNA were employed to conduct the reprogramming of primary dermal fibroblasts. These iPSC lines provide a useful model for further investigations on the pathophysiological role of mutations in the RCBTB1 gene in IRD.

Generation of an induced pluripotent stem cell line from a patient with retinitis pigmentosa caused by RP1 mutation.

We report the generation of the iPSC line LEIi005-B from a patient with retinitis pigmentosa caused by a dominant nonsense mutation in the RP1 gene (c.2098G>T p.E700X). Reprogramming of dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for p53 to establish the clonal iPSC line LEIi005-B. LEIi005-B expressed pluripotent stem cell markers, had a normal karyotype and differentiated into endoderm, mesoderm and ectoderm.

Generation of two induced pluripotent stem cell lines from a patient with compound heterozygous mutations in the USH2A gene.

The human iPSC lines LEIi010-A and LEIi010-B were generated from the dermal fibroblasts of a patient with Usher syndrome using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, mir302/367 microRNA and shRNA for p53. These iPSC lines carry compound heterozygous mutations (c.949C > A and c.1256G > T) in USH2A. LEIi010-A and LEIi010-B expressed pluripotent stem cell markers, had a normal karyotype and could be differentiated into endoderm, mesoderm and ectodermal lineages.

Generation of the induced pluripotent stem cell line from a patient with autosomal recessive ABCA4-mediated Stargardt Macular Dystrophy.

We report the generation of the human iPSC line LEIi007-A from a patient with autosomal recessive Stargardt disease caused by compound heterozygous mutations in the ABCA4 gene (c.[5461-10 T > C];[4139C > T]). Reprogramming of patient dermal fibroblasts was performed using episomal plasmids containing OCT4, SOX2, KLF4, L-MYC, LIN28, shRNA for p53 and mir302/367 microRNA to establish the clonal iPSC line LEIl007-A. LEIl007-A displayed normal pluripotent stem cell colony morphology, expressed pluripotent stem cell markers, displayed a normal karyotype and differentiated into ectodermal, mesodermal and endodermal germ layer lineages.