149 results on '"Alejandro Garanto"'
Search Results
2. Generation of hiPSC lines from four glutaric aciduria type I (GA1) patients carrying pathogenic biallelic variants in GCDH
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Imke M.E. Schuurmans, Clara D.M. van Karnebeek, Anita D.M. Hoogendoorn, Antonia Ribes, Nael Nadif Kasri, and Alejandro Garanto
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Biology (General) ,QH301-705.5 - Abstract
GCDH encodes for the enzyme catalyzing the sixth step of the lysine degradation pathway. Autosomal recessive variants in GCDH are associated with glutaric aciduria type I (GA1), of which a wide genotypic spectrum of pathogenic variants have been described. In this study, hiPSC lines derived from four GA1 patients with different genotypes were generated and fully characterized. Two patients carry compound heterozygous variants in GCDH, while the other two patients carry a variant in homozygosis. These hiPSC lines can significantly contribute to better understand the molecular mechanism underlying GA1 and provide excellent models for the development of new therapeutic strategies.
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- 2024
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3. Lipopeptide-mediated Cas9 RNP delivery: A promising broad therapeutic strategy for safely removing deep-intronic variants in ABCA4
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Irene Vázquez-Domínguez, Mert Öktem, Florian A. Winkelaar, Thai Hoang Nguyen, Anita D.M. Hoogendoorn, Eleonora Roschi, Galuh D.N. Astuti, Raoul Timmermans, Nuria Suárez-Herrera, Ilaria Bruno, Albert Ruiz-Llombart, Joseph Brealey, Olivier G. de Jong, Rob W.J. Collin, Enrico Mastrobattista, and Alejandro Garanto
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MT: RNA/DNA Editing ,peptide-mediated delivery ,CRISPR-Cas9 genome editing ,lipopeptide ,intron removal ,ABCA4 deep-intronic variants ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Deep-intronic (DI) variants represent approximately 10%–12% of disease-causing genetic defects in ABCA4-associated Stargardt disease (STGD1). Although many of these DI variants are amenable to antisense oligonucleotide-based splicing-modulation therapy, no treatment is currently available. These molecules are mostly variant specific, limiting their applicability to a broader patient population. In this study, we investigated the therapeutic potential of the CRISPR-Cas9 system combined with the amphipathic lipopeptide C18:1-LAH5 for intracellular delivery to correct splicing defects caused by different DI variants within the same intron. The combination of these components facilitated efficient editing of two target introns (introns 30 and 36) of ABCA4 in which several recurrent DI variants are found. The partial removal of these introns did not affect ABCA4 splicing or its expression levels when assessed in two different human cellular models: fibroblasts and induced pluripotent stem cell-derived photoreceptor precursor cells (PPCs). Furthermore, the DNA editing in STGD1 patient-derived PPCs led to a ∼50% reduction of the pseudoexon-containing transcripts resulting from the c.4539+2001G>A variant in intron 30. Overall, we provide proof-of-concept evidence of the use of C18:1-LAH5 as a delivery system for therapeutic genome editing for ABCA4-associated DI variants, offering new opportunities for clinical translation.
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- 2024
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4. CRISPR-Cas9 correction of a nonsense mutation in LCA5 rescues lebercilin expression and localization in human retinal organoids
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Tess A.V. Afanasyeva, Dimitra Athanasiou, Pedro R.L. Perdigao, Kae R. Whiting, Lonneke Duijkers, Galuh D.N. Astuti, Jean Bennett, Alejandro Garanto, Jacqueline van der Spuy, Ronald Roepman, Michael E. Cheetham, and Rob W.J. Collin
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retinal organoid ,retinal disease ,Leber congenital amaurosis ,CRISPR-Cas9 ,isogenic ,lebercilin ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Mutations in the lebercilin-encoding gene LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report on the generation of a patient-specific cellular model to study LCA5-associated retinal disease. CRISPR-Cas9 technology was used to correct a homozygous nonsense variant in LCA5 (c.835C>T; p.Q279∗) in patient-derived induced pluripotent stem cells (iPSCs). The absence of off-target editing in gene-corrected (isogenic) control iPSCs was demonstrated by whole-genome sequencing. We differentiated the patient, gene-corrected, and unrelated control iPSCs into three-dimensional retina-like cells, so-called retinal organoids. We observed opsin and rhodopsin mislocalization to the outer nuclear layer in patient-derived but not in the gene-corrected or unrelated control organoids. We also confirmed the rescue of lebercilin expression and localization along the ciliary axoneme within the gene-corrected organoids. Here, we show the potential of combining precise single-nucleotide gene editing with the iPSC-derived retinal organoid system for the generation of a cellular model of early-onset retinal disease.
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- 2023
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5. Generation of an iPSC line (RMCGENi020-A) from a patient with Stargardt disease harboring the recurrent intronic ABCA4 variant c.4253+43G>A
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Nuria Suárez-Herrera, Nico Leijsten, Silvia Albert, Nathalie M. Bax, Carel B. Hoyng, Frans P.M. Cremers, Alejandro Garanto, and Rob W.J. Collin
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Biology (General) ,QH301-705.5 - Abstract
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.
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- 2023
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6. Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi
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Alessia Cavazza, Ayal Hendel, Rasmus O. Bak, Paula Rio, Marc Güell, Duško Lainšček, Virginia Arechavala-Gomeza, Ling Peng, Fatma Zehra Hapil, Joshua Harvey, Francisco G. Ortega, Coral Gonzalez-Martinez, Carsten W. Lederer, Kasper Mikkelsen, Giedrius Gasiunas, Nechama Kalter, Manuel A.F.V. Gonçalves, Julie Petersen, Alejandro Garanto, Lluis Montoliu, Marcello Maresca, Stefan E. Seemann, Jan Gorodkin, Loubna Mazini, Rosario Sanchez, Juan R. Rodriguez-Madoz, Noelia Maldonado-Pérez, Torella Laura, Michael Schmueck-Henneresse, Cristina Maccalli, Julian Grünewald, Gloria Carmona, Neli Kachamakova-Trojanowska, Annarita Miccio, Francisco Martin, Giandomenico Turchiano, Toni Cathomen, Yonglun Luo, Shengdar Q. Tsai, and Karim Benabdellah
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MT: RNA/DNA Editing ,European Cooperation in Science and Technology ,COST ,GenE-HumDi ,genome editing ,base editors ,Therapeutics. Pharmacology ,RM1-950 - Abstract
The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the ''Genome Editing to treat Human Diseases'' (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi’s primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing’s potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.
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- 2023
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7. Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease
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Nuria Suárez-Herrera, Catherina H. Z. Li, Nico Leijsten, Dyah W. Karjosukarso, Zelia Corradi, Femke Bukkems, Lonneke Duijkers, Frans P. M. Cremers, Carel B. Hoyng, Alejandro Garanto, and Rob W. J. Collin
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N-of-1 ,antisense oligonucleotide ,splicing modulation ,pseudoexon ,RNA therapy ,ABCA4 ,Cytology ,QH573-671 - Abstract
Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient.
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- 2024
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8. Generation of an induced pluripotent stem cell line carrying biallelic deletions (SCTCi019-B) in ALDH7A1 using CRISPR/Cas9
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Imke M.E. Schuurmans, Ka M. Wu, Clara D.M. van Karnebeek, Nael Nadif Kasri, and Alejandro Garanto
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Biology (General) ,QH301-705.5 - Abstract
Biallelic pathogenic variants in ALDH7A1 are associated with pyridoxine-dependent epilepsy (PDE). ALDH7A1 encodes for the third enzyme of the lysine catabolism pathway. In this study a human isogenic ALDH7A1 knock-out iPSC line was created using CRISPR/Cas9 technology. One clone (SCTCi019-B) with biallelic deletions in ALDH7A1 was obtained and fully characterized, showing expression of pluripotency markers, a normal karyotype and no off-targets. Human-based models derived from this iPSC line will contribute to gain insights in the molecular mechanism of disease underlying PDE.
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- 2023
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9. Generation of an induced pluripotent stem cell line carrying a biallelic deletion (SCTCi019-A) in GCDH using CRISPR/Cas9
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Imke M.E. Schuurmans, Ka M. Wu, Clara D.M. van Karnebeek, Nael Nadif Kasri, and Alejandro Garanto
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Biology (General) ,QH301-705.5 - Abstract
GCDH encodes for the enzyme catalyzing the sixth step of the lysine catabolism pathway. Biallelic pathogenic variants in GCDH have been associated with glutaric aciduria type 1 (GA1). In this study CRISPR/Cas9 technology was used to create an isogenic GCDH knock-out human iPSC line. One clone with a biallelic deletion (SCTCi019-A) in GCDH was obtained and fully characterized, revealing a normal karyotype, no off-targets detected and expression of pluripotency markers. This iPSC line can contribute to gain insights in the molecular mechanism of disease.
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- 2023
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10. Gene augmentation of LCA5-associated Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme
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Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Helen May-Simera, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, and Ronald Roepman
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Cell biology ,Genetics ,Medicine - Abstract
Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus–based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.
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- 2023
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11. Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction
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Janine Reurink, Nicole Weisschuh, Alejandro Garanto, Adrian Dockery, L. Ingeborgh van den Born, Isabelle Fajardy, Lonneke Haer-Wigman, Susanne Kohl, Bernd Wissinger, G. Jane Farrar, Tamar Ben-Yosef, Fatma Kivrak Pfiffner, Wolfgang Berger, Marianna E. Weener, Lubica Dudakova, Petra Liskova, Dror Sharon, Manar Salameh, Ashley Offenheim, Elise Heon, Giorgia Girotto, Paolo Gasparini, Anna Morgan, Arthur A. Bergen, Jacoline B. ten Brink, Caroline C.W. Klaver, Lisbeth Tranebjærg, Nanna D. Rendtorff, Sascha Vermeer, Jeroen J. Smits, Ronald J.E. Pennings, Marco Aben, Jaap Oostrik, Galuh D.N. Astuti, Jordi Corominas Galbany, Hester Y. Kroes, Milan Phan, Wendy A.G. van Zelst-Stams, Alberta A.H.J. Thiadens, Joke B.G.M. Verheij, Mary J. van Schooneveld, Suzanne E. de Bruijn, Catherina H.Z. Li, Carel B. Hoyng, Christian Gilissen, Lisenka E.L.M. Vissers, Frans P.M. Cremers, Hannie Kremer, Erwin van Wijk, and Susanne Roosing
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USH2A ,Usher syndrome ,retinitis pigmentosa ,usherin ,whole genome sequencing ,minigene splice assay ,Genetics ,QH426-470 - Abstract
Summary: A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis.One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells.Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment.We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.
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- 2023
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12. Comparative Clustering (CompaCt) of eukaryote complexomes identifies novel interactions and sheds light on protein complex evolution.
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Joeri van Strien, Felix Evers, Madhurya Lutikurti, Stijn L. Berendsen, Alejandro Garanto, Geert-Jan van Gemert, Alfredo Cabrera-Orefice, Richard J. Rodenburg, Ulrich Brandt, Taco W. A. Kooij, and Martijn A. Huynen
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- 2023
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13. In Vitro Skeletal Muscle Model of PGM1 Deficiency Reveals Altered Energy Homeostasis
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Federica Conte, Angel Ashikov, Rachel Mijdam, Eline G. P. van de Ven, Monique van Scherpenzeel, Raisa Veizaj, Seyed P. Mahalleh-Yousefi, Merel A. Post, Karin Huijben, Daan M. Panneman, Richard J. T. Rodenburg, Nicol C. Voermans, Alejandro Garanto, Werner J. H. Koopman, Hans J. C. T. Wessels, Marek J. Noga, and Dirk J. Lefeber
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phosphoglucomutase 1 ,PGM1 deficiency ,PGM1 congenital disorder of glycosylation ,in vitro muscle model ,muscle energy homeostasis ,muscle metabolic plasticity ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.
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- 2023
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14. Generation of an iPSC line (SCTCi014-A) and isogenic control line (SCTCi014-A-1) from an age-related macular degeneration patient carrying the variant c.355G>A in the CFI gene
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Sarah de Jong, Louet Koolen, Irene Vázquez-Domínguez, Anita de Breuk, Silvia Albert, Carel B. Hoyng, Suresh Katti, Anneke I. den Hollander, and Alejandro Garanto
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Biology (General) ,QH301-705.5 - Abstract
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.
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- 2022
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15. Generation of a patient-derived induced pluripotent cell line (SCTCi016-A) carrying a homozygous variant in RPE65
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Irene Vázquez-Domínguez, Michael Kwint, Hester Y Kroes, Silvia Albert, Luke O'Gorman, Christian Gilissen, Frans P.M. Cremers, Rob W.J. Collin, Susanne Roosing, and Alejandro Garanto
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Biology (General) ,QH301-705.5 - Abstract
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.
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- 2022
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16. Detailed Phenotyping and Therapeutic Strategies for Intronic ABCA4 Variants in Stargardt Disease
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Mubeen Khan, Gavin Arno, Ana Fakin, David A. Parfitt, Patty P.A. Dhooge, Silvia Albert, Nathalie M. Bax, Lonneke Duijkers, Michael Niblock, Kwan L. Hau, Edward Bloch, Elena R. Schiff, Davide Piccolo, Michael C. Hogden, Carel B. Hoyng, Andrew R. Webster, Frans P.M. Cremers, Michael E. Cheetham, Alejandro Garanto, and Rob W.J. Collin
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ABCA4 ,antisense oligonucleotides ,intronic mutations ,splicing ,Stargardt disease ,iPSC ,Therapeutics. Pharmacology ,RM1-950 - Abstract
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.
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- 2020
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17. Correction of the Splicing Defect Caused by a Recurrent Variant in ABCA4 (c.769-784C>T) That Underlies Stargardt Disease
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Tomasz Z. Tomkiewicz, Sara E. Nieuwenhuis, Frans P. M. Cremers, Alejandro Garanto, and Rob W. J. Collin
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antisense oligonucleotides ,ABCA4 ,Stargardt disease ,inherited retinal disease ,splicing modulation ,RNA therapy ,Cytology ,QH573-671 - Abstract
Stargardt disease is an inherited retinal disease caused by biallelic mutations in the ABCA4 gene, many of which affect ABCA4 splicing. In this study, nine antisense oligonucleotides (AONs) were designed to correct pseudoexon (PE) inclusion caused by a recurrent deep-intronic variant in ABCA4 (c.769-784C>T). First, the ability of AONs to skip the PE from the final ABCA4 mRNA transcript was assessed in two cellular models carrying the c.769-784C>T variant: a midigene assay using HEK293T cells and patient-derived fibroblasts. Based on the splicing-correcting ability of each individual AON, the three most efficacious AONs targeting independent regions of the PE were selected for a final assessment in photoreceptor precursor cells (PPCs). The final analysis in the PPC model confirmed high efficacy of AON2, -5, and -7 in promoting PE exclusion. Among the three AONs, AON2 is chosen as the lead candidate for further optimization, hereby showcasing the high potential of AONs to correct aberrant splicing events driven by deep-intronic variants.
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- 2022
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18. The Predicted Splicing Variant c.11+5G>A in RPE65 Leads to a Reduction in mRNA Expression in a Cell-Specific Manner
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Irene Vázquez-Domínguez, Lonneke Duijkers, Zeinab Fadaie, Eef C. W. Alaerds, Merel A. Post, Edwin M. van Oosten, Luke O’Gorman, Michael Kwint, Louet Koolen, Anita D. M. Hoogendoorn, Hester Y. Kroes, Christian Gilissen, Frans P. M. Cremers, Rob W. J. Collin, Susanne Roosing, and Alejandro Garanto
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inherited retinal diseases ,Leber congenital amaurosis ,RPE65 gene ,retinal pigment epithelium (RPE) ,induced pluripotent stem cells (iPSCs) ,cell-specific defects ,Cytology ,QH573-671 - Abstract
Pathogenic variants in RPE65 lead to retinal diseases, causing a vision impairment. In this work, we investigated the pathomechanism behind the frequent RPE65 variant, c.11+5G>A. Previous in silico predictions classified this change as a splice variant. Our prediction using novel software’s suggested a 124-nt exon elongation containing a premature stop codon. This elongation was validated using midigenes-based approaches. Similar results were observed in patient-derived induced pluripotent stem cells (iPSC) and photoreceptor precursor cells. However, the splicing defect in all cases was detected at low levels and thereby does not fully explain the recessive condition of the resulting disease. Long-read sequencing discarded other rearrangements or variants that could explain the diseases. Subsequently, a more relevant model was employed: iPSC-derived retinal pigment epithelium (RPE) cells. In patient-derived iPSC-RPE cells, the expression of RPE65 was strongly reduced even after inhibiting a nonsense-mediated decay, contradicting the predicted splicing defect. Additional experiments demonstrated a cell-specific gene expression reduction due to the presence of the c.11+5G>A variant. This decrease also leads to the lack of the RPE65 protein, and differences in size and pigmentation between the patient and control iPSC-RPE. Altogether, our data suggest that the c.11+5G>A variant causes a cell-specific defect in the expression of RPE65 rather than the anticipated splicing defect which was predicted in silico.
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- 2022
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19. Exploring genotype–phenotype correlations in glutaric aciduria type 1
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Imke M. E. Schuurmans, Bianca Dimitrov, Julian Schröter, Antonia Ribes, Rubén Pérez de la Fuente, Berta Zamora, Clara D. M. van Karnebeek, Stefan Kölker, and Alejandro Garanto
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All institutes and research themes of the Radboud University Medical Center ,Genetics ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Genetics (clinical) - Abstract
Contains fulltext : 292922.pdf (Publisher’s version ) (Open Access) Glutaric aciduria type 1 (GA1) is a rare neurometabolic disease caused by pathogenic variants in the gene encoding the enzyme glutaryl-CoA dehydrogenase (GCDH). We performed an extensive literature search to collect data on GA1 patients, together with unpublished cases, to provide an up-to-date genetic landscape of GCDH pathogenic variants and to investigate potential genotype-phenotype correlation, as this is still poorly understood. From this search, 421 different GCDH pathogenic variants have been identified, including four novel variants; c.179T>C (p.Leu60Pro), c.214C>T (p.Arg72Cys), c.309G>C (p.Leu103Phe), and c.665T>C (p.Phe222Ser).The variants are mostly distributed across the entire gene; although variant frequency in GA1 patients is relatively high in the regions encoding for active domains of GCDH. To investigate potential genotype-phenotype correlations, phenotypic descriptions of 532 patients have been combined and evaluated using novel combinatorial analyses. To do so, various clinical phenotypes were determined for each pathogenic variant by combining the information of all GA1 patients reported with this pathogenic variant, and subsequently mapped onto the 2D and 3D GCDH protein structure. In addition, the predicted pathogenicity of missense variants was analyzed using different in silico prediction score models. Both analyses showed an almost similar distribution of the highly pathogenic variants across the GCDH protein, although some hotspots, including the active domain, were observed. Moreover, it was demonstrated that highly pathogenic variants are significantly correlated with lower residual enzyme activity and the most accurate estimation was achieved by the REVEL score. A clear correlation of the genotype and the clinical phenotype however is still lacking.
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- 2023
20. Delivery of oligonucleotide‐based therapeutics: challenges and opportunities
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Suzan M Hammond, Annemieke Aartsma‐Rus, Sandra Alves, Sven E Borgos, Ronald A M Buijsen, Rob W J Collin, Giuseppina Covello, Michela A Denti, Lourdes R Desviat, Lucía Echevarría, Camilla Foged, Gisela Gaina, Alejandro Garanto, Aurelie T Goyenvalle, Magdalena Guzowska, Irina Holodnuka, David R Jones, Sabine Krause, Taavi Lehto, Marisol Montolio, Willeke Van Roon‐Mom, and Virginia Arechavala‐Gomeza
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delivery ,oligonucleotides ,preclinical models ,RNA therapeutics ,safety ,Medicine (General) ,R5-920 ,Genetics ,QH426-470 - Abstract
Abstract Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.
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- 2021
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21. Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models
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Kalyan Dulla, Monica Aguila, Amelia Lane, Katarina Jovanovic, David A. Parfitt, Iris Schulkens, Hee Lam Chan, Iris Schmidt, Wouter Beumer, Lars Vorthoren, Rob W.J. Collin, Alejandro Garanto, Lonneke Duijkers, Anna Brugulat-Panes, Ma’ayan Semo, Anthony A. Vugler, Patricia Biasutto, Peter Adamson, and Michael E. Cheetham
- Subjects
Therapeutics. Pharmacology ,RM1-950 - Abstract
Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing. Keywords: retinal dystrophy, oligonucleotide, stem cell, organoid, therapy, QR-110
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- 2018
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22. Experimental Model Systems Used in the Preclinical Development of Nucleic Acid Therapeutics
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Haiyan Zhou, Virginia Arechavala-Gomeza, and Alejandro Garanto
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Drug Discovery ,Genetics ,Molecular Medicine ,Molecular Biology ,Biochemistry - Published
- 2023
23. Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4
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Tomasz Z. Tomkiewicz, Nuria Suárez-Herrera, Frans P. M. Cremers, Rob W. J. Collin, and Alejandro Garanto
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antisense oligonucleotide ,ABCA4 ,Stargardt disease ,inherited retinal diseases ,splicing modulation ,RNA therapy ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.
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- 2021
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24. Probing the sub-cellular mechanisms of LCA5-Leber Congenital Amaurosis and associated gene therapy with expansion microscopy
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Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, and Ronald Roepman
- Abstract
Leber Congenital Amaurosis (LCA) is a group of Inherited Retinal Diseases (IRDs) characterized by the early onset and rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with Ultrastructure Expansion Microscopy (U-ExM), we revealed the structural and molecular defects underlying LCA type 5 (LCA5) with unprecedented resolution. We showed thatLCA5-encoded lebercilin, together with Retinitis Pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localize at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrated that mutant mice deficient for lebercilin exhibit early axonemal defects at the bulge region and the distal OS, accompanied by reduced level of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, we probed theLCA5gene augmentation therapy strategy using U-ExM by monitoring its subcellular outcome. We found that, expression ofLCA5partially restores the bulge region, preserves OS axoneme structure and membrane disc formation, as well as photoreceptor survival.
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- 2023
25. Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings
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Rita Barone, Filippo Vairo, Bobby G. Ng, Jaak Jaeken, Gert Matthijs, James Pitt, Thierry Dupré, Lyndon Gallacher, Liesbeth Keldermans, Helen Michelakakis, Marina Ventouratou, Susan M. White, Sze Chern Lim, Melissa Baerenfaenger, Mirian C. H. Janssen, Angel Ashikov, Karin Huijben, Sandrine Vuillaumier-Barrot, Diana Ballhausen, Daisy Rymen, Agustí Rodríguez-Palmero, Blai Morales-Romero, Antonia Ribes, Peter Witters, Heidi Peters, Erika Souche, Eva Morava, Agata Fiumara, Pascale de Lonlay, Matthew P. Wilson, Dirk Lefeber, Wasantha Ranatunga, Alejandro Garanto, Hudson H. Freeze, Christian Thiel, BioAnalytical Chemistry, and AIMMS
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Male ,Mutant ,congenital disorders of glycosylation ,chemistry.chemical_compound ,0302 clinical medicine ,Catalytic Domain ,Missense mutation ,Musculoskeletal Diseases ,Genetics (clinical) ,Genes, Dominant ,chemistry.chemical_classification ,Genetics ,0303 health sciences ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Middle Aged ,Disorders of movement Donders Center for Medical Neuroscience [Radboudumc 3] ,Pedigree ,Oligosaccharyltransferase complex ,Child, Preschool ,glycosylation ,Female ,Adult ,Heterozygote ,Glycosylation ,Adolescent ,Protein subunit ,Biology ,Article ,03 medical and health sciences ,All institutes and research themes of the Radboud University Medical Center ,oligosaccharyltransferase complex ,medicine ,Humans ,dominant inheritance ,Amino Acid Sequence ,030304 developmental biology ,Sequence Homology, Amino Acid ,Oligosaccharyltransferase ,Membrane Proteins ,medicine.disease ,chemistry ,Hexosyltransferases ,Nervous System Diseases ,Glycoprotein ,Congenital disorder of glycosylation ,030217 neurology & neurosurgery - Abstract
Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs. ispartof: AMERICAN JOURNAL OF HUMAN GENETICS vol:108 issue:11 pages:2130-2144 ispartof: location:United States status: published
- Published
- 2021
26. Consensus Guidelines for the Design and
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Annemieke, Aartsma-Rus, Alejandro, Garanto, Willeke, van Roon-Mom, Erin M, McConnell, Victoria, Suslovitch, Winston X, Yan, Jonathan K, Watts, and Timothy W, Yu
- Abstract
Antisense oligonucleotides (ASOs) can modulate pre-mRNA splicing. This offers therapeutic opportunities for numerous genetic diseases, often in a mutation-specific and sometimes even individual-specific manner. Developing therapeutic ASOs for as few as even a single patient has been shown feasible with the development of Milasen for an individual with Batten disease. Efforts to develop individualized ASOs for patients with different genetic diseases are ongoing globally. The N = 1 Collaborative (N1C) is an umbrella organization dedicated to supporting the nascent field of individualized medicine. N1C recently organized a workshop to discuss and advance standards for the rigorous design and testing of splice-switching ASOs. In this study, we present guidelines resulting from that meeting and the key recommendations: (1) dissemination of standardized experimental designs, (2) use of standardized reference ASOs, and (3) a commitment to data sharing and exchange.
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- 2022
27. In or Out? New Insights on Exon Recognition through Splice-Site Interdependency
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Mubeen Khan, Stéphanie S. Cornelis, Riccardo Sangermano, Iris J.M. Post, Amber Janssen Groesbeek, Jan Amsu, Christian Gilissen, Alejandro Garanto, Rob W.J. Collin, and Frans P.M. Cremers
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pre-mrna ,splicing ,5′ and 3′ splice sites ,interdependency ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Noncanonical splice-site mutations are an important cause of inherited diseases. Based on in vitro and stem-cell-based studies, some splice-site variants show a stronger splice defect than expected based on their predicted effects, suggesting that other sequence motifs influence the outcome. We investigated whether splice defects due to human-inherited-disease-associated variants in noncanonical splice-site sequences in ABCA4, DMD, and TMC1 could be rescued by strengthening the splice site on the other side of the exon. Noncanonical 5′- and 3′-splice-site variants were selected. Rescue variants were introduced based on an increase in predicted splice-site strength, and the effects of these variants were analyzed using in vitro splice assays in HEK293T cells. Exon skipping due to five variants in noncanonical splice sites of exons in ABCA4, DMD, and TMC1 could be partially or completely rescued by increasing the predicted strengths of the other splice site of the same exon. We named this mechanism “splicing interdependency”, and it is likely based on exon recognition by splicing machinery. Awareness of this interdependency is of importance in the classification of noncanonical splice-site variants associated with disease and may open new opportunities for treatments.
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- 2020
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28. Species-Dependent Splice Recognition of a Cryptic Exon Resulting from a Recurrent Intronic CEP290 Mutation that Causes Congenital Blindness
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Alejandro Garanto, Lonneke Duijkers, and Rob W. J. Collin
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CEP290 ,deep-intronic mutation ,pre-mRNA splicing ,Leber congenital amaurosis ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all CEP290 transcripts. A humanized mouse model with this mutation did not recapitulate the aberrant CEP290 splicing observed in LCA patients, suggesting differential recognition of cryptic splice sites between species. To further assess this phenomenon, we generated two CEP290 minigene constructs, with and without the intronic mutation, and transfected these in cell lines of various species. RT-PCR analysis revealed that exon X is well recognized by the splicing machinery in human and non-human primate cell lines. Intriguingly, this recognition decreases in cell lines derived from species such as dog and rodents, and it is completely absent in Drosophila. In addition, other cryptic splicing events corresponding to sequences in intron 26 of CEP290 were observed to varying degrees in the different cell lines. Together, these results highlight the complexity of splice site recognition among different species, and show that care is warranted when generating animal models to mimic splice site mutations in vivo.
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- 2015
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29. mRNA expression analysis of the SUMO pathway genes in the adult mouse retina
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Víctor Abad-Morales, Elena B. Domènech, Alejandro Garanto, and Gemma Marfany
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SUMO ,sumoylation ,In situ hybridization ,mRNA expression levels ,retina ,light cycle ,Science ,Biology (General) ,QH301-705.5 - Abstract
Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-PCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. The wide expression of the SUMO pathway genes, the transcriptional response under several light/dark conditions, and the diversity of expression patterns in different cell layers clearly support sumoylation as a relevant post-translational modification in the retina. This expression atlas intends to be a reference framework for retinal researchers and to depict a more comprehensive view of the SUMO-regulated processes in the retina.
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- 2015
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30. A look into retinal organoids: methods, analytical techniques, and applications
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Ronald Roepman, Julio C. Corral-Serrano, Alejandro Garanto, Rob W.J. Collin, Michael E. Cheetham, and Tess A. V. Afanasyeva
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Organoid ,Inherited ,Cell type ,Induced Pluripotent Stem Cells ,Omics ,Review ,Biology ,Retinal ganglion ,Retina ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,0302 clinical medicine ,Retinal Diseases ,Retinal Rod Photoreceptor Cells ,medicine ,Animals ,Humans ,Induced pluripotent stem cell ,Molecular Biology ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Retinal ,Cell Differentiation ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Cell Biology ,3. Good health ,Organoids ,medicine.anatomical_structure ,Renal disorders Radboud Institute for Molecular Life Sciences [Radboudumc 11] ,chemistry ,Degeneration ,Molecular Medicine ,Therapy ,sense organs ,Cellular model ,Neuroscience ,Muller glia ,Neuroglia ,030217 neurology & neurosurgery - Abstract
Contains fulltext : 243899.pdf (Publisher’s version ) (Open Access) Inherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs.
- Published
- 2021
31. Expression Atlas of the Deubiquitinating Enzymes in the Adult Mouse Retina, Their Evolutionary Diversification and Phenotypic Roles.
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Mariona Esquerdo, Xavier Grau-Bové, Alejandro Garanto, Vasileios Toulis, Sílvia Garcia-Monclús, Erica Millo, Ma José López-Iniesta, Víctor Abad-Morales, Iñaki Ruiz-Trillo, and Gemma Marfany
- Subjects
Medicine ,Science - Abstract
Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.
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- 2016
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32. Antisense Oligonucleotide-based Splice Correction for USH2A-associated Retinal Degeneration Caused by a Frequent Deep-intronic Mutation
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Radulfus WN Slijkerman, Christel Vaché, Margo Dona, Gema García-García, Mireille Claustres, Lisette Hetterschijt, Theo A Peters, Bas P Hartel, Ronald JE Pennings, José M Millan, Elena Aller, Alejandro Garanto, Rob WJ Collin, Hannie Kremer, Anne-Françoise Roux, and Erwin Van Wijk
- Subjects
antisense oligonucleotides ,genetic therapy ,splice redirection ,USH2A ,Usher syndrome ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.
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- 2016
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33. PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor outer segment disk formation
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Max D van Essen, Rossano Butcher, Marius Ueffing, Adem Yildirim, Rob W.J. Collin, Qin Liu, Lonneke Duijkers, Krzysztof Palczewski, Anita D M. Hoogendoorn, Nikoleta Argyrou, Sylvia E. C. van Beersum, Alejandro Garanto, Uwe Wolfrum, Julio C. Corral-Serrano, Karsten Boldt, Ronald Roepman, Renate A A Ruigrok, Ideke J.C. Lamers, Stef J.F. Letteboer, Jeroen van Reeuwijk, Sanae Sakami, and Michael E. Cheetham
- Subjects
cilium ,macromolecular substances ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,Actin-Related Protein 2-3 Complex ,chemistry.chemical_compound ,Mice ,All institutes and research themes of the Radboud University Medical Center ,retinitis pigmentosa ,Retinitis pigmentosa ,medicine ,Genetics ,Animals ,Humans ,Cilia ,RNA, Small Interfering ,Ciliary tip ,Eye Proteins ,Ciliary membrane ,Actin ,Mice, Knockout ,Multidisciplinary ,Cilium ,outer segments ,Retinal ,Biological Sciences ,medicine.disease ,Rod Cell Outer Segment ,Photoreceptor outer segment ,photoreceptor ,Actins ,Cell biology ,Wiskott-Aldrich Syndrome Protein Family ,Disease Models, Animal ,Renal disorders Radboud Institute for Molecular Life Sciences [Radboudumc 11] ,chemistry ,PNAS Plus ,Gene Expression Regulation ,Retinal Cone Photoreceptor Cells ,sense organs ,actin ,Cone-Rod Dystrophies ,Visual phototransduction - Abstract
Significance The photoreceptor outer segments are primary cilia, modified for phototransduction by incorporation of stacked opsin-loaded membrane disks that are continuously regenerated. This process is disrupted in several types of inherited retinal dystrophy, but the driving force remained unclear. We show that C2orf71/PCARE (photoreceptor cilium actin regulator), associated with inherited retinal dystrophy subtype RP54, efficiently recruits the Arp2/3 complex activator WASF3 to the cilium. This activates an actin dynamics-driven expansion of the ciliary tip, resembling membrane evagination in lamellipodia formation. Colocalization of this actin dynamics module to the base of the outer segments, and absence thereof in Pcare−/− mice, suggests PCARE-regulated actin dynamics as a critical process in outer segment disk formation., The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare−/− mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.
- Published
- 2020
34. Systemic complement levels in patients with age-related macular degeneration carrying rare or low-frequency variants in the CFH gene
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Sarah de Jong, Carel B. Hoyng, Lambert P J W van den Heuvel, Anita de Breuk, Elena B. Volokhina, Yara T. E. Lechanteur, Alejandro Garanto, Sascha Fauser, Suresh Katti, Bjorn Bakker, and Anneke I. den Hollander
- Subjects
Heterozygote ,Mutation, Missense ,Biology ,Polymorphism, Single Nucleotide ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,Frameshift mutation ,Macular Degeneration ,Age related ,Genetics ,medicine ,Missense mutation ,Humans ,In patient ,Molecular Biology ,Gene ,Genetics (clinical) ,Aged ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,General Medicine ,Complement System Proteins ,Macular degeneration ,medicine.disease ,eye diseases ,Complement system ,Renal disorders Radboud Institute for Molecular Life Sciences [Radboudumc 11] ,Factor H ,Complement Factor H ,Immunology ,sense organs - Abstract
Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. Genetic variants in the complement factor H (CFH) gene are associated with AMD, but the functional consequences of many of these variants are currently unknown. In this study, we aimed to determine the effect of 64 rare and low-frequency variants in the CFH gene on systemic levels of factor H (FH) and complement activation marker C3bBbP using plasma samples of 252 carriers and 159 non-carriers. Individuals carrying a heterozygous nonsense, frameshift or missense variant in CFH presented with significantly decreased FH levels and significantly increased C3bBbP levels in plasma compared to non-carrier controls. FH and C3bBbP plasma levels were relatively stable over time in samples collected during follow-up visits. Decreased FH and increased C3bBbP concentrations were observed in carriers compared to non-carriers of CFH variants among different AMD stages, with the exception of C3bBbP levels in advanced AMD stages, which were equally high in carriers and non-carriers. In AMD families, FH levels were decreased in carriers compared to non-carriers, but C3bBbP levels did not differ. Rare variants in the CFH gene can lead to reduced FH levels or reduced FH function as measured by increased C3bBbP levels. The effects of individual variants in the CFH gene reported in this study will improve the interpretation of rare and low-frequency variants observed in AMD patients in clinical practice.
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- 2022
35. How to proceed after 'negative' exome: A review on genetic diagnostics, limitations, challenges, and emerging new multiomics techniques
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Saskia B. Wortmann, Machteld M. Oud, Mariëlle Alders, Karlien L. M. Coene, Saskia N. van der Crabben, René G. Feichtinger, Alejandro Garanto, Alex Hoischen, Mirjam Langeveld, Dirk Lefeber, Johannes A. Mayr, Charlotte W. Ockeloen, Holger Prokisch, Richard Rodenburg, Hans R. Waterham, Ron A. Wevers, Bart P. C. van de Warrenburg, Michel A. A. P. Willemsen, Nicole I. Wolf, Lisenka E. L. M. Vissers, and Clara D. M. van Karnebeek
- Subjects
Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7] ,treatment ,lnfectious Diseases and Global Health Radboud Institute for Molecular Life Sciences [Radboudumc 4] ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Genomics ,Disorders of movement Donders Center for Medical Neuroscience [Radboudumc 3] ,DNA, Mitochondrial ,genome sequencing ,Phenotype ,diagnostic yield ,Genetics ,Exome ,Genetic Testing ,exome-negative ,inborn metabolic disease ,exome sequencing ,Genetics (clinical) - Abstract
Contains fulltext : 282561.pdf (Publisher’s version ) (Open Access) Exome sequencing (ES) in the clinical setting of inborn metabolic diseases (IMDs) has created tremendous improvement in achieving an accurate and timely molecular diagnosis for a greater number of patients, but it still leaves the majority of patients without a diagnosis. In parallel, (personalized) treatment strategies are increasingly available, but this requires the availability of a molecular diagnosis. IMDs comprise an expanding field with the ongoing identification of novel disease genes and the recognition of multiple inheritance patterns, mosaicism, variable penetrance, and expressivity for known disease genes. The analysis of trio ES is preferred over singleton ES as information on the allelic origin (paternal, maternal, "de novo") reduces the number of variants that require interpretation. All ES data and interpretation strategies should be exploited including CNV and mitochondrial DNA analysis. The constant advancements in available techniques and knowledge necessitate the close exchange of clinicians and molecular geneticists about genotypes and phenotypes, as well as knowledge of the challenges and pitfalls of ES to initiate proper further diagnostic steps. Functional analyses (transcriptomics, proteomics, and metabolomics) can be applied to characterize and validate the impact of identified variants, or to guide the genomic search for a diagnosis in unsolved cases. Future diagnostic techniques (genome sequencing [GS], optical genome mapping, long-read sequencing, and epigenetic profiling) will further enhance the diagnostic yield. We provide an overview of the challenges and limitations inherent to ES followed by an outline of solutions and a clinical checklist, focused on establishing a diagnosis to eventually achieve (personalized) treatment.
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- 2022
36. Antisense Oligonucleotide-Based Splicing Correction in Individuals with Leber Congenital Amaurosis due to Compound Heterozygosity for the c.2991+1655A>G Mutation in CEP290
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Lonneke Duijkers, L. Ingeborgh van den Born, John Neidhardt, Nathalie M. Bax, Laurence H. M. Pierrache, B. Jeroen Klevering, Rob W. J. Collin, and Alejandro Garanto
- Subjects
CEP290 ,antisense oligonucleotides ,splicing correction ,compound heterozygosity ,Leber congenital amaurosis ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Leber congenital amaurosis (LCA) is a rare inherited retinal disorder affecting approximately 1:50,000 people worldwide. So far, mutations in 25 genes have been associated with LCA, with CEP290 (encoding the Centrosomal protein of 290 kDa) being the most frequently mutated gene. The most recurrent LCA-causing CEP290 mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of CEP290 transcripts. We previously demonstrated that antisense oligonucleotides (AONs) have a high therapeutic potential for patients homozygously harbouring this mutation, although to date, it is unclear whether rescuing one single allele is enough to restore CEP290 function. Here, we assessed the AON efficacy at RNA, protein and cellular levels in samples that are compound heterozygous for this mutation, together with a protein-truncating mutation in CEP290. We demonstrate that AONs can efficiently restore splicing and increase protein levels. However, due to a high variability in ciliation among the patient-derived cell lines, the efficacy of the AONs was more difficult to assess at the cellular level. This observation points towards the importance of the severity of the second allele and possibly other genetic variants present in each individual. Overall, AONs seem to be a promising tool to treat CEP290-associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant.
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- 2018
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37. ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice, cis-regulatory, structural, and recurrent hypomorphic variants
- Author
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Bernd Wissinger, Thomy de Ravel de l'Argentière, Frans P.M. Cremers, Jim Bauwens, Bart P. Leroy, Riccardo Sangermano, Caroline Van Cauwenbergh, Julie De Zaeytijd, Ana Fakin, Sarah De Jaegere, Toon Rosseel, Mubeen Khan, Gavin Arno, Susanne Kohl, Andrew R. Webster, Meindert De Vries, Elfride De Baere, Rob W.J. Collin, Alejandro Garanto, Irina Balikova, Keren J. Carss, Thalia Van Laethem, Miriam Bauwens, Kim De Leeneer, Marnik Vuylsteke, Sarah Naessens, Yves Sznajer, Timothy J. Cherry, Françoise Sadler, Nicole Weisschuh, Software Languages Lab, Informatics and Applied Informatics, Faculty of Sciences and Bioengineering Sciences, UCL - (SLuc) Centre de génétique médicale UCL, and UCL - SSS/IREC/SLUC - Pôle St.-Luc
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DEEP-INTRONIC VARIANTS ,Male ,ABCA4 ,PHENOTYPE ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,Cohort Studies ,0302 clinical medicine ,Gene Frequency ,Missing heritability problem ,STARGARDT-DISEASE ,Medicine and Health Sciences ,Genetics(clinical) ,Genetics (clinical) ,Genetics ,0303 health sciences ,biology ,noncoding ,deep-intronic ,Exons ,DYSTROPHY ,Middle Aged ,Phenotype ,3. Good health ,Pedigree ,Female ,Adult ,Genes, Recessive ,ANTISENSE OLIGONUCLEOTIDES ,Article ,03 medical and health sciences ,All institutes and research themes of the Radboud University Medical Center ,AON ,Retinitis pigmentosa ,RETINITIS-PIGMENTOSA ,REVEALS ,Retinal Dystrophies ,medicine ,non-coding ,Humans ,splice ,Allele ,Gene ,Alleles ,030304 developmental biology ,SPECTRUM ,TRANSPORTER GENE ABCR ,MUTATIONS ,Biology and Life Sciences ,ABCA4-associated disease ,Oligonucleotides, Antisense ,medicine.disease ,Introns ,Stargardt disease ,HEK293 Cells ,missing heritability ,Mutation ,030221 ophthalmology & optometry ,biology.protein ,ATP-Binding Cassette Transporters - Abstract
PURPOSE: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability. METHODS: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4. RESULTS: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells. CONCLUSION: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders. ispartof: GENETICS IN MEDICINE vol:21 issue:8 pages:1761-1771 ispartof: location:United States status: published
- Published
- 2019
38. Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides
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Gavin Arno, Bernhard H. F. Weber, Carel B. Hoyng, L. Ingeborgh van den Born, Nathalie M. Bax, Silvia Albert, Frans P.M. Cremers, Keren J. Carss, Stéphanie S. Cornelis, Felix Grassmann, Caroline C W Klaver, F. Lucy Raymond, Mubeen Khan, Ana Fakin, Andrew R. Webster, Muhammad Imran Khan, Claire Marie Dhaenens, Riccardo Sangermano, Elfride De Baere, Sarah Naessens, Heidi Stöhr, Rob W.J. Collin, Alberta A H J Thiadens, Jan Willem R. Pott, Esmee H. Runhart, Miriam Bauwens, Bernard Puech, Isabelle Meunier, Joke B. G. M. Verheij, Alejandro Garanto, Ophthalmology, and Epidemiology
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0301 basic medicine ,antisense oligonucleotide ,030105 genetics & heredity ,ABCA4 ,Exon ,Missing heritability problem ,Medicine and Health Sciences ,Protein Isoforms ,Genetics(clinical) ,Child ,Genetics (clinical) ,Exome sequencing ,POPULATION ,Genetics ,education.field_of_study ,Exons ,Middle Aged ,3. Good health ,Pedigree ,Stargardt disease ,RNA splicing ,Adult ,Adolescent ,RNA Splicing ,Population ,Biology ,Polymorphism, Single Nucleotide ,Article ,03 medical and health sciences ,Young Adult ,REVEALS ,medicine ,Humans ,splice ,education ,Gene ,Aged ,MUTATIONS ,deep-intronic variant ,Biology and Life Sciences ,IN-VITRO ,Oligonucleotides, Antisense ,medicine.disease ,GENE ,Introns ,030104 developmental biology ,HEK293 Cells ,Mutation ,missing heritability ,ATP-Binding Cassette Transporters - Abstract
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.
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- 2019
39. Delivery of oligonucleotide‐based therapeutics: challenges and opportunities
- Author
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Annemieke Aartsma-Rus, Marisol Montolio, Gisela Gaina, Camilla Foged, Suzan M. Hammond, Alejandro Garanto, David R. Jones, Lourdes R. Desviat, Giuseppina Covello, Sabine Krause, Magdalena Guzowska, Virginia Arechavala-Gomeza, Sven Even F. Borgos, Willeke M. C. van Roon-Mom, Taavi Lehto, Sandra Alves, Ronald A.M. Buijsen, Michela A. Denti, Aurélie Goyenvalle, Rob W.J. Collin, Irina Holodnuka, Lucia Echevarria, Handicap neuromusculaire : Physiopathologie, Biothérapie et Pharmacologies appliquées (END-ICAP), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), DFF-4184-00422 20577, 446002002 Foundation Fighting Blindness, FFB: PPA-0517-0717-RAD Fundación Ramón Areces Horizon 2020 Framework Programme, H2020: 721058, 761104 Muscular Dystrophy UK, MDUK Medical Research Council, MRC Institut National de la Santé et de la Recherche Médicale, Inserm Eesti Teadusagentuur, ETAg: PSG226 Ikerbasque, Basque Foundation for Science Prinses Beatrix Spierfonds Instituto de Salud Carlos III, ISCIII: CPII17/00004 Ministerio de Ciencia e Innovación, MICINN: PID2019-105344RB-I00 Duchenne Parent Project Hersenstichting: DR-2018-00253 Friedrich-Baur-Stiftung European Regional Development Fund, ERDF Algemene Nederlandse Vereniging ter voorkoming van Blindheid, ANVVB Landelijke Stichting voor Blinden en Slechtzienden, LSBS Stichting Retina Fonds: 2015-31, 2018-21 Stichting Blinden-Penning Ministerul Cercetării şi Inovării, MCI: 31N/2016/PN 16.22.02.05, PTDC/BBB-BMD/6301/2014, This work was supported by funding from Cooperation of Science and Technology (COST) Action CA17103 (networking grant to V.A-G). V.A-G holds a Miguel Servet Fellowship from the ISCIII [grant reference CPII17/00004] that is part-funded by the European Regional Development Fund (ERDF/FEDER) and also acknowledges funding from Ikerbasque (Basque Foundation for Science). S.M.H is funded by the Medical Research Council and Muscular Dystrophy UK. A.A-R receives funding from amongst others the Duchenne Parent Project, Spieren voor Spieren, the Prinses Beatrix Spierfonds, Duchenne UK and through Horizon2020 project BIND. A.G and R.W.J.C are supported by several foundations including the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Oogfonds Nederland, Stichting Macula Degeneratie Fonds, and Stichting Retina Nederland Fonds (who contributed through UitZicht 2015-31 and 2018-21), together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting voor Ooglijders, and Stichting Dowilvo, as well as the Foundation Fighting Blindness USA, grant no. PPA-0517-0717-RAD. R.A.M.B is supported by Hersenstichting Nederland Grant DR-2018-00253. G.G. is supported by Ministry of Research and Innovation in Romania/National Program 31N/2016/PN 16.22.02.05. S.A is supported by Project PTDC/BBB-BMD/6301/2014 (Funda??o para a Ci?ncia e a Tecnologia?MCTES, Portugal). L.R.D. is supported by Fundaci?n Ram?n Areces Grant XVII CN and Spanish Ministry of Science and Innovation (MICINN, grant PID2019-105344RB-I00). T.L is supported by Estonian Research Council grant PSG226. S.K is supported by the Friedrich-Baur-Stiftung. C.F is funded by The Danish Council for Independent Research, Technology and Production Sciences (grant number DFF-4184-00422). W.vRM is supported by ZonMw Programme Translational Research 2 [Project number 446002002], Campaign Team Huntington and AFM Telethon [Project number 20577]. S.E.B is supported by the H2020 projects B-SMART, Grant number 721058, and REFINE, Grant number 761104. A.T.G is supported by the Institut National de la sant? et la recherche m?dicale (INSERM) and the Association Monegasque contre les myopathies (AMM). L.E. is founded by the Association Monegasque contre les myopathies (AMM)., UAM. Departamento de Biología Molecular, Ministerio de Ciencia y Tecnología (España), Instituto de Salud Carlos III, Ikerbasque Basque Foundation for Science, Medical Research Council (UK), Duchenne UK, Foundation Fighting Blindness, Netherlands Brain Foundation, Ministerio de Ciencia e Innovación (España), Fundação para a Ciência e a Tecnologia (Portugal), and Fundación Ramón Areces
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safety ,0301 basic medicine ,Medicine (General) ,Computer science ,[SDV]Life Sciences [q-bio] ,Aptamer ,Gene Expression ,Review ,Computational biology ,QH426-470 ,Small Interfering ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,03 medical and health sciences ,R5-920 ,0302 clinical medicine ,Chemical Biology ,Marketed products ,Genetics ,Cost of goods ,Antisense ,RNA, Small Interfering ,Genetica Humana ,Antisense Oligonucleotides ,oligonucleotides ,Oligonucleotide ,RNA therapeutics ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Oligonucleotides, Antisense ,Biología y Biomedicina / Biología ,RNA Biology ,preclinical models ,Doenças Genéticas ,3. Good health ,030104 developmental biology ,delivery ,Nanoparticles ,Oligonucleotides ,Antisense oligonucleotides ,RNA ,Molecular Medicine ,Synthetic Biology & Biotechnology ,030217 neurology & neurosurgery - Abstract
Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics., Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.
- Published
- 2021
40. Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4
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Nuria Suárez-Herrera, Alejandro Garanto, Frans P.M. Cremers, Tomasz Z Tomkiewicz, and Rob W.J. Collin
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antisense oligonucleotide ,pseudoexon ,QH301-705.5 ,inherited retinal diseases ,RNA Splicing ,ABCA4 ,RNA therapy ,Locus (genetics) ,Computational biology ,Biology ,Catalysis ,Article ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,near-exon ,Inorganic Chemistry ,Exon ,All institutes and research themes of the Radboud University Medical Center ,Missing heritability problem ,medicine ,Humans ,splice ,Physical and Theoretical Chemistry ,Biology (General) ,Molecular Biology ,QD1-999 ,Spectroscopy ,Organic Chemistry ,RNA ,deep-intronic ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,General Medicine ,Oligonucleotides, Antisense ,medicine.disease ,Introns ,Computer Science Applications ,Stargardt disease ,Chemistry ,exon elongation ,RNA splicing ,biology.protein ,ATP-Binding Cassette Transporters ,splicing modulation - Abstract
The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.
- Published
- 2021
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41. Implications of genetic variation in the complement system in age-related macular degeneration
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Anita de Breuk, Sarah de Jong, Lambert P J W van den Heuvel, Yara T. E. Lechanteur, Anneke I. den Hollander, Giuliana Gagliardi, Suresh Katti, Alejandro Garanto, and Elena B. Volokhina
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0301 basic medicine ,genetic structures ,Disease ,Bioinformatics ,Systemic circulation ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,Macular Degeneration ,03 medical and health sciences ,0302 clinical medicine ,Age related ,Genetic variation ,Humans ,Medicine ,Induced pluripotent stem cell ,Complement Activation ,Aged ,business.industry ,Genetic Variation ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,Complement System Proteins ,Macular degeneration ,medicine.disease ,Sensory Systems ,eye diseases ,Complement system ,Clinical trial ,Ophthalmology ,030104 developmental biology ,Renal disorders Radboud Institute for Molecular Life Sciences [Radboudumc 11] ,030221 ophthalmology & optometry ,sense organs ,business - Abstract
Contains fulltext : 237835.pdf (Publisher’s version ) (Open Access) Age-related macular degeneration (AMD) is the main cause of vision loss among the elderly in the Western world. While AMD is a multifactorial disease, the complement system was identified as one of the main pathways contributing to disease risk. The strong link between the complement system and AMD was demonstrated by genetic associations, and by elevated complement activation in local eye tissue and in the systemic circulation of AMD patients. Several complement inhibitors have been and are being explored in clinical trials, but thus far with limited success, leaving the majority of AMD patients without treatment options to date. This indicates that there is still a gap of knowledge regarding the functional implications of the complement system in AMD pathogenesis and how to bring these towards clinical translation. Many different experimental set-ups and disease models have been used to study complement activation in vivo and in vitro, and recently emerging patient-derived induced pluripotent stem cells and genome-editing techniques open new opportunities to study AMD disease mechanisms and test new therapeutic strategies in the future. In this review we provide an extensive overview of methods employed to understand the molecular processes of complement activation in AMD pathogenesis. We discuss the findings, advantages and challenges of each approach and conclude with an outlook on how recent, exciting developments can fill in current knowledge gaps and can aid in the development of effective complement-targeting therapeutic strategies in AMD.
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- 2021
42. CLINICAL AND BIOCHEMICAL FOOTPRINTS OF INHERITED METABOLIC DISORDERS: A LESSON FROM THE KNOWLEDGEBASE
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Carlos R. Ferreira, David Cassiman, Alejandro Garanto, Georg F. Hoffmann, Gabriella A. Horvath, Diego Martinelli, Robert M. Stowe, Clara D.M. van Karnebeek, and Nenad Blau
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Endocrinology ,Endocrinology, Diabetes and Metabolism ,Genetics ,Molecular Biology ,Biochemistry - Published
- 2022
43. Unexpected CEP290 mRNA splicing in a humanized knock-in mouse model for Leber congenital amaurosis.
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Alejandro Garanto, Sylvia E C van Beersum, Theo A Peters, Ronald Roepman, Frans P M Cremers, and Rob W J Collin
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Medicine ,Science - Abstract
Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations.
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- 2013
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44. Molecular Therapies for Inherited Retinal Diseases
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Rob W.J. Collin and Alejandro Garanto
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Retinal degeneration ,Retinal pigment epithelium ,business.industry ,Retinal ,Protein degradation ,medicine.disease ,Bioinformatics ,Choroideremia ,Stargardt disease ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,RPE65 ,Retinitis pigmentosa ,medicine ,business - Published
- 2020
45. In or Out? New Insights on Exon Recognition through Splice-Site Interdependency
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Jan Amsu, Stéphanie S. Cornelis, Riccardo Sangermano, Frans P.M. Cremers, Iris J M Post, Amber Janssen Groesbeek, Rob W.J. Collin, Alejandro Garanto, Mubeen Khan, and Christian Gilissen
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RNA Splicing ,ABCA4 ,interdependency ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,Catalysis ,Article ,Inorganic Chemistry ,Dystrophin ,lcsh:Chemistry ,Pre-mRNA ,Exon ,splicing ,All institutes and research themes of the Radboud University Medical Center ,Humans ,splice ,Physical and Theoretical Chemistry ,Molecular Biology ,lcsh:QH301-705.5 ,Spectroscopy ,Genetics ,biology ,Organic Chemistry ,HEK 293 cells ,Membrane Proteins ,Metabolic Disorders Radboud Institute for Molecular Life Sciences [Radboudumc 6] ,General Medicine ,Exons ,Exon skipping ,Computer Science Applications ,HEK293 Cells ,5′ and 3′ splice sites ,lcsh:Biology (General) ,lcsh:QD1-999 ,RNA splicing ,biology.protein ,ATP-Binding Cassette Transporters ,RNA Splice Sites ,Sequence motif ,Precursor mRNA - Abstract
Noncanonical splice-site mutations are an important cause of inherited diseases. Based on in vitro and stem-cell-based studies, some splice-site variants show a stronger splice defect than expected based on their predicted effects, suggesting that other sequence motifs influence the outcome. We investigated whether splice defects due to human-inherited-disease-associated variants in noncanonical splice-site sequences in ABCA4, DMD, and TMC1 could be rescued by strengthening the splice site on the other side of the exon. Noncanonical 5&prime, and 3&prime, splice-site variants were selected. Rescue variants were introduced based on an increase in predicted splice-site strength, and the effects of these variants were analyzed using in vitro splice assays in HEK293T cells. Exon skipping due to five variants in noncanonical splice sites of exons in ABCA4, DMD, and TMC1 could be partially or completely rescued by increasing the predicted strengths of the other splice site of the same exon. We named this mechanism &ldquo, splicing interdependency&rdquo, and it is likely based on exon recognition by splicing machinery. Awareness of this interdependency is of importance in the classification of noncanonical splice-site variants associated with disease and may open new opportunities for treatments.
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- 2020
46. Preface of Special Issue 'Molecular Therapies for Inherited Retinal Diseases'
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Rob W.J. Collin and Alejandro Garanto
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genetic structures ,lcsh:QH426-470 ,business.industry ,Genetic Therapy ,macromolecular substances ,Bioinformatics ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,Food and drug administration ,Delivery methods ,lcsh:Genetics ,Editorial ,n/a ,Retinal Diseases ,Severe visual impairment ,parasitic diseases ,Genetics ,Complete Blindness ,Humans ,Incurable diseases ,Medicine ,Genetic Predisposition to Disease ,Vector (molecular biology) ,business ,Genetics (clinical) - Abstract
Contains fulltext : 218308.pdf (Publisher’s version ) (Open Access) Inherited retinal diseases (IRDs) are a group of progressive disorders that lead to severe visual impairment or even complete blindness. IRDs display a vast heterogeneity, clinically as well as genetically, with over 250 genes identified in which mutations can cause one or more clinical subtypes of IRD. Long considered incurable diseases, intense research over the last two decades, combined with major technological advancements, have enabled the development of the first therapeutic approaches for these diseases. The approval of LuxturnaTM (voretigene neparvovec), a gene augmentation therapy vector for RPE65-associated IRD, by the US Food and Drug Administration and the European Medicines Agency, is considered a true milestone in the field, and has led to the development of similar, or different therapeutic strategies for many other subtypes of IRD. Despite these major achievements, there are still many aspects that can-and need to-be improved, including more insights into the relationship between genetic variation and cellular dysfunction, optimization of the vectors and sequences used, improving delivery methods, as well as understanding and modulating the (local) immune response. In addition, the extreme rarity of some genetic subtypes of IRDs poses an enormous challenge on the development of novel therapies, in terms of e.g., costs and regulatory affairs.[...].
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- 2020
47. Detailed Phenotyping and Therapeutic Strategies for Intronic ABCA4 Variants in Stargardt Disease
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Michael C. Hogden, Rob W.J. Collin, Lonneke Duijkers, Mubeen Khan, Andrew R. Webster, David A. Parfitt, Alejandro Garanto, Davide Piccolo, Ana Fakin, Nathalie M. Bax, Frans P.M. Cremers, Gavin Arno, Kwan L. Hau, Patty P.A. Dhooge, Michael Niblock, Carel B. Hoyng, Michael E. Cheetham, Edward Bloch, Silvia Albert, and Elena R. Schiff
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0301 basic medicine ,retina ,Subfamily ,ABCA4 ,Article ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,splicing ,03 medical and health sciences ,0302 clinical medicine ,stem cells ,Drug Discovery ,medicine ,Allele ,Induced pluripotent stem cell ,Gene ,organoids ,Genetics ,iPSC ,biology ,lcsh:RM1-950 ,intronic mutations ,Intron ,photoreceptors ,medicine.disease ,3. Good health ,Stargardt disease ,lcsh:Therapeutics. Pharmacology ,030104 developmental biology ,030220 oncology & carcinogenesis ,RNA splicing ,biology.protein ,Molecular Medicine ,antisense oligonucleotides - Abstract
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., Graphical Abstract, Khan et al. report on the clinical characterization of patients harboring a recurrent deep-intronic variant in ABCA4 underlying retinal disease and demonstrate that this, and other variants close by, lead to splicing defects that can be rescued by antisense oligonucleotides.
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- 2020
48. Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature
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Jia Qi Cheng Zhang, Anita D M. Hoogendoorn, Dyah W Karjosukarso, Erwin van Wijk, Theo A. Peters, Alejandro Garanto, Lasse Jensen, Rob W.J. Collin, and Zaheer Ali
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Familial Exudative Vitreoretinopathies ,Mutant ,Mutation, Missense ,Biology ,medicine.disease_cause ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,znf408 ,03 medical and health sciences ,chemistry.chemical_compound ,All institutes and research themes of the Radboud University Medical Center ,0302 clinical medicine ,medicine ,Missense mutation ,Animals ,Vascular insufficiency ,Zebrafish ,CRISPR/Cas9 ,Mutation ,Retina ,retinal vasculature ,Biochemistry and Molecular Biology ,Retinal Vessels ,Retinal ,medicine.disease ,biology.organism_classification ,zebrafish ,DNA-Binding Proteins ,Ophthalmology ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,FEVR ,030221 ophthalmology & optometry ,Familial exudative vitreoretinopathy ,Oftalmologi - Abstract
PURPOSE. Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disease in which the retinal vasculature is affected. Patients with FEVR typically lack or have abnormal vasculature in the peripheral retina, the outcome of which can range from mild visual impairment to complete blindness. A missense mutation (p.His455Tyr) in ZNF408 was identified in an autosomal dominant FEVR family. Little, however, is known about the molecular role of ZNF408 and how its defect leads to the clinical features of FEVR. METHODS. Using CRISPR/Cas9 technology, two homozygous mutant zebrafish models with truncated znf408 were generated, as well as one heterozygous and one homozygous missense znf408 model in which the human p.His455Tyr mutation is mimicked. RESULTS. Intriguingly, all three znf408-mutant zebrafish strains demonstrated progressive retinal vascular pathology, initially characterized by a deficient hyaloid vessel development at 5 days postfertilization (dpf) leading to vascular insufficiency in the retina. The generation of stable mutant lines allowed long-term follow up studies, which showed ectopic retinal vascular hyper-sprouting at 90 dpf and extensive vascular leakage at 180 dpf. CONCLUSIONS. Together, our data demonstrate an important role for znf408 in the development and maintenance of the vascular system within the retina. Funding Agencies|Radboudumc PhD grant; Svenska Sallskapet for Medicinsk Forskning; Linkoping University; Loo och Hans Ostermans Stiftelse; Eva och Oscar Ahrens Stiftelse; Stiftelsen Sigurd och Elsa Goljes Minne; Magnus Bergvalls Stiftelse; Ogonfonden; Jeanssons Stiftelser; VetenskapsradetSwedish Research Council
- Published
- 2020
49. RNA-Based Therapeutic Strategies for Inherited Retinal Dystrophies
- Author
-
Alejandro, Garanto
- Subjects
Genetic Vectors ,Leber Congenital Amaurosis ,Retinal Dystrophies ,Humans ,RNA ,Genetic Therapy - Abstract
Inherited retinal dystrophies (IRDs) are genetic diseases affecting 1 in every 3000 individuals worldwide. Nowadays, more than 250 genes have been associated with different forms of IRD. In the last decade, it has been shown that gene therapy is a promising approach to correct the genetic defects underlying IRD. In fact, voretigene neparvovec-rzyl (Luxturna™), the first commercialized gene therapy drug to treat RPE65-associated Leber congenital amaurosis, has opened new venues. However, IRDs are highly heterogeneous at genetic level making the design of novel strategies complicated. Unfortunately, the size of several frequently mutated genes is not suitable for the approved conventional therapeutic viral vectors; therefore, there is an urgent need for the development of alternatives, such as those targeting the pre-mRNA. In this mini-review, the potential of RNA-based strategies for IRDs is discussed.
- Published
- 2019
50. Applications of antisense oligonucleotides for the treatment of inherited retinal diseases
- Author
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Rob W.J. Collin and Alejandro Garanto
- Subjects
0301 basic medicine ,genetic structures ,medicine.disease_cause ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,Genetic therapy ,03 medical and health sciences ,Exon ,chemistry.chemical_compound ,0302 clinical medicine ,Retinal Diseases ,medicine ,Animals ,Humans ,Genetics ,Mutation ,business.industry ,Retinal ,Exons ,Genetic Therapy ,General Medicine ,Oligonucleotides, Antisense ,medicine.disease ,eye diseases ,Disease Models, Animal ,Ophthalmology ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Corneal neovascularization ,Antisense oligonucleotides ,Cancer research ,sense organs ,Cytomegalovirus retinitis ,business - Abstract
Item does not contain fulltext PURPOSE OF REVIEW: Over the last years, antisense oligonucleotides (AONs) have gained attention as a therapeutic tool for the treatment of ocular diseases such as cytomegalovirus retinitis, keratitis-induced corneal neovascularization, and inherited retinal diseases (IRDs). In this review, we summarize the recent key findings, and describe the challenges and opportunities that lie ahead to translate AON-based therapies to the clinic, in particular for IRDs. RECENT FINDINGS: The efficacy of AONs to restore splice defects and cellular phenotypes associated with a common mutation in CEP290 was demonstrated in patient-derived optic cups and in a transgenic mouse model, respectively. In addition, allele-specific knockdown of a mutant RHO allele resulted in a delay of photoreceptor cell death and functional preservation of these cells in a transgenic rat model. SUMMARY: As demonstrated by several preclinical efficacy studies, AON-based therapy is moving to the clinic for the treatment of some genetic subtypes of IRD. More insights into the delivery of these molecules, the duration of the therapeutic effect, and potential off-target effects will be essential to further shape the transition to the clinic and reveal the true potential of AON-based therapy for retinal diseases.
- Published
- 2017
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