RESUMEN
The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.
Asunto(s)
Cromosomas Humanos Par 17/química , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Retinitis Pigmentosa/genética , Factores de Transcripción/genética , Adulto , Secuencia de Aminoácidos , Diferenciación Celular , Reprogramación Celular , Niño , Mapeo Cromosómico , Estudios de Cohortes , Elementos de Facilitación Genéticos , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Genes Dominantes , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Proteínas Nucleares/metabolismo , Organoides/metabolismo , Organoides/patología , Hidrolasas Diéster Fosfóricas/metabolismo , Polimorfismo Genético , Cultivo Primario de Células , Células Fotorreceptoras Retinianas Conos/patología , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Factores de Transcripción/metabolismo , Secuenciación Completa del GenomaRESUMEN
Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Retinitis Pigmentosa/tratamiento farmacológico , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Exones , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Modelos Moleculares , Oligonucleótidos Antisentido/farmacología , Retina/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Sequence analysis of the coding regions and splice site sequences in inherited retinal diseases is not able to uncover â¼40% of the causal variants. Whole-genome sequencing can identify most of the non-coding variants, but their interpretation is still very challenging, in particular when the relevant gene is expressed in a tissue-specific manner. Deep-intronic variants in ABCA4 have been associated with autosomal-recessive Stargardt disease (STGD1), but the exact pathogenic mechanism is unknown. By generating photoreceptor precursor cells (PPCs) from fibroblasts obtained from individuals with STGD1, we demonstrated that two neighboring deep-intronic ABCA4 variants (c.4539+2001G>A and c.4539+2028C>T) result in a retina-specific 345-nt pseudoexon insertion (predicted protein change: p.Arg1514Leufs∗36), likely due to the creation of exonic enhancers. Administration of antisense oligonucleotides (AONs) targeting the 345-nt pseudoexon can significantly rescue the splicing defect observed in PPCs of two individuals with these mutations. Intriguingly, an AON that is complementary to c.4539+2001G>A rescued the splicing defect only in PPCs derived from an individual with STGD1 with this but not the other mutation, demonstrating the high specificity of AONs. In addition, a single AON molecule rescued splicing defects associated with different neighboring mutations, thereby providing new strategies for the treatment of persons with STGD1. As many genes associated with human genetic conditions are expressed in specific tissues and pre-mRNA splicing may also rely on organ-specific factors, our approach to investigate and treat splicing variants using differentiated cells derived from individuals with STGD1 can be applied to any tissue of interest.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Intrones/genética , Degeneración Macular/congénito , Mutación/genética , Sitios de Empalme de ARN/genética , Alelos , Secuencia de Bases , Simulación por Computador , Exones/genética , Humanos , Degeneración Macular/genética , Oligonucleótidos Antisentido/farmacología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Enfermedad de StargardtRESUMEN
Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on ABCA4 pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in ABCA4 allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Degeneración Macular/congénito , Precursores del ARN/genética , Sitios de Empalme de ARN , Empalme del ARN , Transportadoras de Casetes de Unión a ATP/biosíntesis , Adulto , Femenino , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Masculino , Precursores del ARN/metabolismo , Enfermedad de StargardtRESUMEN
PURPOSE: Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability. METHODS: Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects. RESULTS: In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects. CONCLUSION: Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Oligonucleótidos Antisentido/genética , Isoformas de Proteínas/genética , Enfermedad de Stargardt/genética , Adolescente , Adulto , Anciano , Niño , Exones/genética , Células HEK293 , Humanos , Intrones/genética , Persona de Mediana Edad , Mutación/genética , Oligonucleótidos Antisentido/farmacología , Linaje , Polimorfismo de Nucleótido Simple/genética , Empalme del ARN/genética , Enfermedad de Stargardt/patología , Adulto JovenRESUMEN
Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional.
Asunto(s)
Células Madre Embrionarias/metabolismo , Células Epiteliales/metabolismo , Expresión Génica , ARN Mensajero/genética , Transfección/métodos , Transporte Activo de Núcleo Celular , Western Blotting , Diferenciación Celular/genética , Línea Celular , Núcleo Celular/metabolismo , Células Madre Embrionarias/citología , Células Epiteliales/ultraestructura , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , ARN Mensajero/metabolismo , Epitelio Pigmentado de la Retina/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/metabolismoRESUMEN
PURPOSE: To elucidate the functional effect of the ABCA4 variant c.5461-10TâC, one of the most frequent variants associated with Stargardt disease (STGD1). DESIGN: Case series. PARTICIPANTS: Seventeen persons with STGD1 carrying ABCA4 variants and 1 control participant. METHODS: Haplotype analysis of 4 homozygotes and 11 heterozygotes for c.5461-10TâC and sequence analysis of the ABCA4 gene for a homozygous proband. Fibroblasts were reprogrammed from 3 persons with STGD1 into induced pluripotent stem cells, which were differentiated into photoreceptor progenitor cells (PPCs). The effect of the c.5461-10TâC variant on RNA splicing by reverse-transcription polymerase chain reaction was analyzed using PPC mRNA. In vitro assays were performed with minigene constructs containing ABCA4 exon 39. We analyzed the natural history and ophthalmologic characteristics of 4 persons homozygous for c.5461-10TâC. MAIN OUTCOME MEASURES: Haplotype and rare variant data for ABCA4, RNA splice defects, age at diagnosis, visual acuity, fundus appearance, visual field, electroretinography (ERG) results, fluorescein angiography results, and fundus autofluorescence findings. RESULTS: The frequent ABCA4 variant c.5461-10TâC has a subtle effect on splicing based on prediction programs. A founder haplotype containing c.5461-10TâC was found to span approximately 96 kb of ABCA4 and did not contain other rare sequence variants. Patient-derived PPCs showed skipping of exon 39 or exons 39 and 40 in the mRNA. HEK293T cell transduction with minigenes carrying exon 39 showed that the splice defects were the result of the c.5461-10TâC variant. All 4 subjects carrying the c.5461-10TâC variant in a homozygous state showed a young age of STGD1 onset, with low visual acuity at presentation and abnormal cone ERG results. All 4 demonstrated severe cone-rod dystrophy before 20 years of age and were legally blind by 25 years of age. CONCLUSIONS: The ABCA4 variant c.5461-10TâC is located on a founder haplotype lacking other disease-causing rare sequence variants. In vitro studies revealed that it leads to mRNA exon skipping and ABCA4 protein truncation. Given the severe phenotype in persons homozygous for this variant, we conclude that this variant results in the absence of ABCA4 activity.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Empalme Alternativo , Exones/genética , Degeneración Macular/congénito , Células Fotorreceptoras de Vertebrados/fisiología , ARN Mensajero/genética , Células Madre/fisiología , Adulto , Análisis Mutacional de ADN , Electrorretinografía , Femenino , Fibroblastos/metabolismo , Angiografía con Fluoresceína , Células HEK293 , Haplotipos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Macular/diagnóstico , Degeneración Macular/genética , Degeneración Macular/fisiopatología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Sitios de Empalme de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedad de Stargardt , Transfección , Agudeza Visual/fisiología , Campos Visuales/fisiología , Adulto JovenRESUMEN
Intellectual disability (ID) is a diverse neurodevelopmental condition and almost half of the cases have a genetic etiology. SGIP1 acts as an endocytic protein that influences the signaling of receptors in neuronal systems related to energy homeostasis through its interaction with endophilins. This study focuses on the generation and characterization of induced pluripotent stem cells (iPSC) from two unrelated patients due to a frameshift variant (c.764dupA, NM_032291.4) and a splice donor site variant (c.74 + 1G > A, NM_032291.4) in the SGIP1 gene.
Asunto(s)
Homocigoto , Células Madre Pluripotentes Inducidas , Discapacidad Intelectual , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Masculino , Femenino , Línea Celular , NiñoRESUMEN
SGIP1 encodes a protein Src homology 3-domain growth factor receptor-bound 2-like endophilin interacting protein 1. It is involved in the regulation of clathrin-mediated endocytosis along with having a role in energy homeostasis in neuronal systems. We generated an isogenic human induced pluripotent stem cell (iPSC) line with a biallelic frameshift variant in SGIP1. This exon has been shown to be subject to alternative splicing, leading to an isoform lacking 24 amino acids that are present in the longest SGIP isoform. The newly generated iPSC line will be helpful to dissect the differential properties of the two SGIP isoforms.
Asunto(s)
Sistemas CRISPR-Cas , Exones , Mutación del Sistema de Lectura , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes Inducidas/metabolismo , Humanos , Línea Celular , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación CelularRESUMEN
Pathogenic variants in ABCA4 are associated with Stargardt disease (STGD1), an autosomal recessive macular dystrophy characterized by bilateral central vision loss due to a progressive degeneration of retinal cells. An induced pluripotent stem cell (iPSC) line was generated from late-onset STGD1 patient-derived fibroblasts harboring bi-allelic ABCA4 variants by lentivirus-induced reprogramming. The obtained iPSC line (RMCGENi020-A) showed pluripotent features after the reprogramming process. The generation of this iPSC line facilitates its use to differentiate it into relevant retinal-like cell models, with the aim to adequately evaluate the effects of the ABCA4 variants.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Stargardt , Humanos , Transportadoras de Casetes de Unión a ATP/genética , Células Madre Pluripotentes Inducidas/patología , Degeneración Macular/genética , Degeneración Macular/patología , Mutación , Enfermedad de Stargardt/patologíaRESUMEN
The marmoset monkey is a valuable model in reproductive medicine. While previous studies have evaluated germ cell dynamics in the postnatal marmoset, the features of testicular somatic cells remain largely unknown. Therefore, the aim of this study was to establish marmoset-specific markers for Sertoli and peritubular cells (PTCs) and to compare protocols for the enrichment and culture of testicular cell types. Immunohistochemistry of Sertoli and PTC-specific markers - anti-müllerian hormone (AMH), vimentin (VIM), α-smooth muscle actin (SMA) - was performed and corresponding RNA expression profiles were established by quantitative PCR analysis (SOX9,AMH, FSHR,VIM, and SMA). For these analyses, testicular tissue from newborn (n = 4), 8-week-old (n = 4) and adult (n = 3) marmoset monkeys was used. Protocols for the enrichment and culture of testicular cell fractions from the 8-week-old marmoset monkeys (n = 3) were evaluated and cells were analyzed using germ cell- and somatic cell-specific markers. The expression of AMH, VIM and SMA reflects the proportion and differentiation status of Sertoli and PTCs at the RNA and the protein levels. While applied protocols did not support the propagation of germ cells in vitro, our analyses revealed that PTCs maintain their proliferative potential and constitute the dominant cell type after short- and long-term culture. Expression of functionally meaningful testicular somatic markers is similar in the human and the marmoset monkey, indicating that this primate can indeed be used as model for human testicular development. The PTC culture system established in this study will facilitate the identification of factors influencing male sex differentiation and spermatogenesis.
Asunto(s)
Callithrix/anatomía & histología , Células Germinativas/citología , Células de Sertoli/citología , Testículo/citología , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Inmunohistoquímica , Masculino , Testículo/embriologíaRESUMEN
CACNA1A encodes a P/Q-type voltage-gated calcium channel. Heterozygous loss-of-function variants in this gene have been associated with episodic ataxia type 2. In this study, we used CRISPR/Cas9 to generate isogenic human induced pluripotent stem cell lines with a gene-dosage dependent deficiency of CACNA1A. We obtained one clone with monoallelic (UCSFi001-A-60) and two clones with biallelic (UCSFi001-A-61; UCSFi001-A-62) frameshift variants in CACNA1A. All three lines showed expression of pluripotency markers and a normal karyotype.
Asunto(s)
Células Madre Pluripotentes Inducidas , Sistemas CRISPR-Cas/genética , Canales de Calcio/metabolismo , Células Cultivadas , Mutación del Sistema de Lectura , Humanos , Células Madre Pluripotentes Inducidas/metabolismoRESUMEN
Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. AMD is multifactorial eye disease with a strong genetic contribution. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from peripheral blood mononuclear cells of three patients with AMD carrying rare variants in the complement factor H (CFH) gene. These cell lines were generated for cellular studies investigating the disease mechanisms and developing therapeutic interventions for AMD.
Asunto(s)
Células Madre Pluripotentes Inducidas , Degeneración Macular , Anciano , Línea Celular , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Degeneración Macular/genética , Degeneración Macular/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
Age-related macular degeneration (AMD) is a common eye disease among the elderly in the Western world. AMD is a multifactorial disease, with a strong association with genetic variation in the complement system. One of the AMD-associated variants is the c.355G>A (p.Gly119Arg) variant in complement factor I (CFI), a central regulator of complement activation. Here, we report the generation of an iPSC line and its isogenic wildtype control derived from peripheral blood mononuclear cells of a male AMD-affected individual carrying the heterozygous variant c.355G>A (p.Gly119Arg). The line can be utilized to study the effects of this variant in disease-specific cell types.
Asunto(s)
Células Madre Pluripotentes Inducidas , Degeneración Macular , Anciano , Humanos , Masculino , Factor I de Complemento/genética , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Degeneración Macular/genética , Degeneración Macular/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
Age-related macular degeneration (AMD) is a common eye disease among the elderly in the Western world. AMD is a multifactorial disease, with a strong association with genetic variation in the complement system. One of the AMD-associated variants is the c.355G>A (p.Gly119Arg) variant in complement factor I (CFI), a central regulator of complement activation. Here, we report the generation of an iPSC line and its isogenic wildtype control derived from peripheral blood mononuclear cells of a female AMD-affected individual carrying the heterozygous variant c.355G>A (p.Gly119Arg). This line can be utilized to study the effects of this variant in disease-specific cell types.
Asunto(s)
Células Madre Pluripotentes Inducidas , Degeneración Macular , Anciano , Femenino , Humanos , Factor I de Complemento/genética , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Degeneración Macular/genética , Degeneración Macular/metabolismo , Polimorfismo de Nucleótido SimpleRESUMEN
Leber congenital amaurosis (LCA) can be caused by mutations in more than 20 different genes. One of these, RPE65, encodes a protein essential for the visual cycle that is expressed in retinal pigment epithelium cells. In this work, we describe the generation and characterization of the human iPSC line SCTCi16-A. This hiPSC line was generated from peripheral blood mononuclear cells (PBMCs) from a patient affected with LCA caused by the homozygous c.11+5G>A variant in the RPE65 gene. Reprograming was conducted using episomal vectors containing OCT3/4, SOX2, KLF4, L-MYC, and LIN28.
Asunto(s)
Células Madre Pluripotentes Inducidas , Amaurosis Congénita de Leber , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/metabolismo , Leucocitos Mononucleares/metabolismo , Mutación , cis-trans-Isomerasas/genéticaRESUMEN
Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. AMD is multifactorial eye disease with a strong genetic contribution. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from peripheral blood mononuclear cells of three individuals above 70 years of age without AMD. These cell lines were generated to serve as control lines for cellular studies investigating the disease mechanisms and developing therapeutic interventions for AMD.
Asunto(s)
Células Madre Pluripotentes Inducidas , Degeneración Macular , Anciano , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Degeneración Macular/genética , Degeneración Macular/metabolismoRESUMEN
Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease.
RESUMEN
Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/genética , Inteínas , Retina/virología , Trans-Empalme/genética , Animales , Vectores Genéticos/administración & dosificación , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Organoides/ultraestructura , Organoides/virología , Fenotipo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/virología , PorcinosRESUMEN
Purpose: To investigate the role of two deep-intronic ABCA4 variants, that showed a mild splice defect in vitro and can occur on the same allele as the low penetrant c.5603A>T, in Stargardt disease (STGD1). Methods: Ophthalmic data were assessed of 18 STGD1 patients who harbored c.769-784C>T or c.4253+43G>A in combination with a severe ABCA4 variant. Subjects carrying c.[769-784C>T; 5603A>T] were clinically compared with a STGD1 cohort previously published carrying c.5603A>T noncomplex. We calculated the penetrances of the intronic variants using ABCA4 allele frequency data of the general population and investigated the effect of c.769-784C>T on splicing in photoreceptor progenitor cells (PPCs). Results: Mostly, late-onset, foveal-sparing STGD1 was observed among subjects harboring c.769-784C>T or c.4253+43G>A (median age of onset, 54.5 and 52.0 years, respectively). However, ages of onset, phenotypes in fundo, and visual acuity courses varied widely. No significant clinical differences were observed between the c.[769-784C>T; 5603A>T] cohort and the c.4253+43G>A or the c.5603A>T cohort. The penetrances of c.769-784C>T (20.5%-39.6%) and c.4253+43G>A (35.8%-43.1%) were reduced, when not considering the effect of yet unidentified or known factors in cis, such as c.5603A>T (identified in 7/7 probands with c.769-784C>T; 1/8 probands with c.4253+43G>A). Variant c.769-784C>T resulted in a pseudo-exon insertion in 15% of the total mRNA (i.e., â¼30% of the c.769-784C>T allele alone). Conclusions: Two mild intronic ABCA4 variants could further explain missing heritability in late-onset STGD1, distinguishing it from AMD. The observed clinical variability and calculated reduced penetrance urge research into modifiers within and outside of the ABCA4 gene.