Abstract

[Introduction] Inherited bleeding disorders (IBD), such as coagulation factor deficiencies, Von Willebrand disease and Glanzmann thrombasthenia, are caused by various gene abnormalities of coagulation proteins, blood vessels, and platelets. IBD have been considered to be suited for gene therapy and clinical trials are ongoing. However, the safety and effectiveness of viral vectors has not been established. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, originates from the archaeal and bacterial adaptive immunity system, provides an efficient genome-editing tool in various organisms including the mammalian genome and holds potential for gene therapy. Here, we report an application of this system to gene repair using induced pluripotent stem cells (iPSCs) derived from patients of three types of IBD.

[Case1] Hemophilia B (63-year-old male). Factor IX (FIX) activity was less than 1% (normal range (NR) 70-130%) and antigen level was 2.37μg/ml (average 5.0μg/ml). Molecular analysis of the FIX gene revealed an in-frame deletion in exon 2.

[Case2] Factor V (FV) deficiency (55-years-old female). FV activity was less than 3% (NR 70-135%) and antigen level was less than 2% (NR 60-150%). A homozygous missense mutation was detected in FV gene of exon 14.

[Case3] Factor X (FX) deficiency (4-years-old male). FX activity was less than 2.84 IU/dl (NR 50-150 IU/dl) and antigen level was 0.567 IU/dl (NR 50-150 IU/dl). A compound heterozygous missense mutation was found in FX gene of exon 6 and 8 respectively.

[Methods and results] The CRISPR/Cas system comprises of a Cas9 nuclease and a sequence-specific guide RNA (gRNA). We designed gRNAs close to gene mutations.

We transfected both expression vectors into HT-1080 or 293T cells, and assessed the editing activity by SURVEYOR nuclease assay. In order to repair the mutations by homology-directed repair (HDR), we prepared targeting constructs with homology arms (1.0 kbp in length) containing the corrected sequence. After introduction of Cas9, gRNA and targeting plasmid into each iPSCs generated from peripheral blood mononuclear cells (PBMCs) using Sendai virus vector expressing the Yamanaka 4-factor genes (Oct3/4, Klf4, Sox2 and c-Myc), we could obtain iPSC clones with corrected genes by HDR from all of three IBD patients. Successful HDR events were verified by PCR amplification using integration site- and targeting construct-specific primers. Locus-specific knock-in events were confirmed by Southern blot analysis.

[Conclusion] We observed the cleavage of the target genome by using our designed gRNAs. Furthermore, the CRISPR/Cas system induced successful gene repair of iPSCs from three IBD patients. We are preparing hepatocytes induced from repaired iPSCs to confirm corrected coagulation factor synthesis. Gene-corrected iPSCs hold great promise as a cell source for autologous cell transplantation.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.