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Universal correction of blood coagulation factor VIII in patient-derived induced pluripotent stem cells using CRISPR/Cas9

ABSTRACT: Confirmation of the nuclease acivities at the On- and Off-target sites; Hemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII). Genome editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correct the various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein in vitro and functionally rescued the disease phenotype in vivo. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE124663 | GEO | 2019/01/05

REPOSITORIES: GEO

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Project description:Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). In addition to genetic mutations in the gene encoding FVIII (F8), there is evidence that other molecular mechanisms may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them, hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-M-CM- -vis the role of nc-RNAs in the regulation of F8 expression and the development of anti-drug antibodies to FVIII infusions used to treat HA. Whole blood samples from nine patients with hemophilia A and five controls were obtained. Among those patients with hemophilia A, three of them were patients who developed neutralizing-anti-FVIII antibodies (inhibitors) and the others are patients without FVIII inhibitors. RNA was isolated and purified from all samples using the RiboPure Blood Kit (Life Technologies) according to the manufacturerM-bM-^@M-^Ys protocol with modifications to enhance the enrichment of low molecular weight RNAs. All RNA samples were poly (A)-tailed and biotin-labeled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA). After labeling, the enzyme linked oligosorbent assays (ELOSA) were performed to confirm that the biotin labeling processes were successful. Biotin-labeled RNA samples (500 ng/sample) were hybridized on GeneChip miRNA 3.0 microarrays (Affymetrix). Microarray data were analyzed to identify ncRNAs that were differentially expressed in hemophilia A groups compared to controls.

Project description:Wiskott-Aldrich syndrome (WAS) is characterized by X-linked thrombocytopenia, eczema, immunodeficiency, recurrent infections and increased risk of autoimmunity and malignancies. WAS is caused by mutations in the WAS gene, which encodes the exclusively hematopoietic WAS protein (WASp) that is classically characterized as aÎ½ actin nucleator. However, disruption of F-actin polymerization by WAS mutations can not account for many aspects of WAS pathogenesis. Ignorance of other functions of WASP precludes in-depth understanding of the pathogenic effects of mutant WASP, and therefore hampers development of effective therapy. Here we generated induced pluripotent stem cells (iPSCs) from WAS patients (WAS-iPSC) bearing different mutations and corresponding isogenic iPSCs in which the pathogenic mutations had been corrected by targeted genome editing. Hematopoietic cells differentiated from WAS-iPSCs not only recapitulated known disease phenotypes, but also revealed novel defects of WASP deficient cells. WASP co-localized with nuclear pores, nucleoli, nuclear speckles and PML bodies by immunocytochemistry and/or serial block face scanning microscopy (SBF-SEM). MudPIT (multi-dimensional protein identification technology) analysis revealed that WASP physically interacted with nuclear body components, nuclear structural proteins, chromatin modifying complexes, and many RNA-binding proteins including major components of the spliceosome. Next-generation sequencing captured a dramatic global change of alternative splicing in WAS patient cells. WAS mutation impacted splicing of multiple genes frequently mutated in myelodysplastic syndrome and other cancers. RNA sequencing showed that WAS-iPSC derived immune cells misregulated many cell cycle regulators, tumor suppressors, immune function genes and splicing factors, and activated gene networks that drive cancer development and inflammatory diseases. Together these data uncovered previously unappreciated functions of the WASP and provided a mechanistic understanding of the pathogenesis of malignancy and autoimmunity in the most severe form of WAS. These new knowledge could help develop targeted therapy for WAS in the future. Human WAS-iPSCs (p.Phe35*) and gene corrected WAS-iPSCs (cWAS-iPSC) were differentiated into macrophages. WAS patient derived B-lymphocyte line ID00003 (p.Glu133Lys) and a wile-type B-lymphocyte line GM11518 were cultured using standard protocol. Total RNAs were extracted and been analyzed by RASL-seq.

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Universal correction of blood coagulation factor VIII in patient-derived induced pluripotent stem cells using CRISPR/Cas9

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