Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRIS
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RESEARCH
Open Access
Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRISPR genome editing Jianguo Wen1, Wenjing Tao2, Suyang Hao1 and Youli Zu1*
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. Keywords: CRISPR/Cas9 genome editing, Hematopoietic stem/progenitor cell colonies, Sickle cell disease
Background Sickle cell disease (SCD) is an inherited blood disorder caused by the point mutation 69A>T in the hemoglobin beta chain gene (HBB), encoding the beta globin subunit of hemoglobin-A (HbA) in normal red blood cells (RBCs). This genomic mutation of HBB results in expression of abnormal hemoglobin-S (HbS). RBCs of SCD patients produce HbS and lack HbA because they inherit two alleles of HbS gene. Cellular HbS molecules at high concentration tend to stick together and form polymers under stress conditions including hypoxia, high altitude, dehydration, and temperature changes. Polymerization of * Correspondence: [email protected] 1 Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston Methodist Research Institute, Houston, TX 77030, USA Full list of author information is available at the end of the article
abnormal cellular HbS causes deformation of RBCs rendering them rigid and sickle- or crescent-shaped. The resulting sickle-shaped RBCs can stick in small vessel walls and break down prematurely, which induces anemia, bacterial infections, and stroke [1, 2]. Currently, allogeneic bone marrow transplant is the only potential approach to cure SCD [3, 4]. However, in clinical practice, locating a suitable donor is difficult and the allogeneic marrow transplant procedure has serious risks, including patient death [4, 5].
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