Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRISPR genome editing

Jianguo Wen, Wenjing Tao, Suyang Hao, Youli Zu

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Background: Sickle cell disease (SCD) is a disorder of red blood cells (RBCs) expressing abnormal hemoglobin-S (HbS) due to genetic inheritance of homologous HbS gene. However, people with the sickle cell trait (SCT) carry a single allele of HbS and do not usually suffer from SCD symptoms, thus providing a rationale to treat SCD. Methods: To validate gene therapy potential, hematopoietic stem cells were isolated from the SCD patient blood and treated with CRISPR/Cas9 approach. To precisely dissect genome-editing effects, erythroid progenitor cells were cloned from single colonies of CRISPR-treated cells and then expanded for simultaneous gene, protein, and cellular function studies. Results: Genotyping and sequencing analysis revealed that the genome-edited erythroid progenitor colonies were converted to SCT genotype from SCD genotype. HPLC protein assays confirmed reinstallation of normal hemoglobin at a similar level with HbS in the cloned genome-edited erythroid progenitor cells. For cell function evaluation, in vitro RBC differentiation of the cloned erythroid progenitor cells was induced. As expected, cell sickling assays indicated function reinstitution of the genome-edited offspring SCD RBCs, which became more resistant to sickling under hypoxia condition. Conclusions: This study is an exploration of genome editing of SCD HSPCs.

Original languageEnglish (US)
Article number119
JournalJournal of Hematology and Oncology
Volume10
Issue number1
DOIs
StatePublished - Jun 13 2017

Keywords

  • CRISPR/Cas9 genome editing
  • Hematopoietic stem/progenitor cell colonies
  • Sickle cell disease

ASJC Scopus subject areas

  • Molecular Biology
  • Hematology
  • Oncology
  • Cancer Research

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