Abstract Number: PB1143
Meeting: ISTH 2020 Congress
Background: We have shown that genome editing using CRISPR-Cas9 could repair the phenotype responsible for bleeding in mice with hemophilia B. Recently, because of the reduced potential risk for genotoxicity, the application of base-editing, “non-cut” genome editing, for the treatment of genetic diseases has attracted much attention. However, the requirement of protospacer adjacent motif (PAM) sequence for Cas9 recognition (NGG for SpCas9) may prevent its application to treat a variety of mutations.
Aims: This research was aimed at correcting the mutation of disease pattern responsible for human hemophilia B by base-editing system based on SpCas9-NG, an engineered Cas9 with broad PAM flexibility.
Methods: We generated induced pluripotent stem cells (iPSCs) from human peripheral blood mononuclear cells using non-integrative Sendai virus vector carrying human reprogramming factors. Sanger sequencing was performed to ensure the genetic mutation. We examined the genome editing by the surveyor assay and next generation sequencing. The differentiation to hepatocytes was achieved through the use of several combinations of cytokines under hypoxic conditions.
Results: We found that the C to T conversion in F9 gene in the patient-derived iPSCs (H002 cells) could repair the disease genotype (c.947T > C). We designed 3 gRNAs near the mutation region and confirmed the broad PAM flexibility of SpCas9-NG. Next, we developed the cytidine base editors from wild type SpCas9 or SpCas9-NG combined with APOBEC1 or PmCDA1 deaminase and the most effective gene repair was observed after treatment with PmCDA1 based on Cas9-NG. We obtained the mutation-repaired patient-derived iPS clone and both original H002 clone and the repaired clone could be differentiated into their hepatocyte lineage.
Conclusions: We showed that Cas9-NG-based cytidine base-editing could effectively repair the genetic mutation in hemophilia B iPSCs without host genome digestion. The broad PAM flexibility of Cas9-NG can be applicable to treat disease mutations by using a base-editing approach.
To cite this abstract in AMA style:Hiramoto T, Kashiwakura Y, Abe T, Kamoshita N, Hayakawa M, Inaba H, Togashi T, Nishimasu H, Hanazono Y, Nureki O, Ohmori T. Correction of Human Hemophilia B Gene in iPSCs by Base-Editing Approach Based on Engineered Cas9 with Broad PAM Flexibility [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/correction-of-human-hemophilia-b-gene-in-ipscs-by-base-editing-approach-based-on-engineered-cas9-with-broad-pam-flexibility/. Accessed September 24, 2023.
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