Wen Jianguo, Tao Wenjing, Hao Suyang, Zu Youli
Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston Methodist Research Institute, Houston, TX, 77030, USA.
Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX, 77030, USA.
J Hematol Oncol. 2017 Jun 13;10(1):119. doi: 10.1186/s13045-017-0489-9.
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.
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.
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.
This study is an exploration of genome editing of SCD HSPCs.
镰状细胞病（SCD）是一种红细胞（RBC）疾病，由于同源血红蛋白 - S（HbS）基因的遗传，红细胞表达异常的血红蛋白 - S。然而，具有镰状细胞特征（SCT）的人携带单个HbS等位基因，通常不会出现SCD症状，因此为治疗SCD提供了理论依据。
为了验证基因治疗的潜力，从SCD患者血液中分离造血干细胞，并采用CRISPR/Cas9方法进行处理。为了精确剖析基因组编辑效果，从经CRISPR处理的细胞的单个集落中克隆红系祖细胞，然后进行扩增，以同时进行基因、蛋白质和细胞功能研究。
基因分型和测序分析表明，基因组编辑的红系祖细胞集落已从SCD基因型转变为SCT基因型。高效液相色谱蛋白质分析证实，在克隆的基因组编辑红系祖细胞中，正常血红蛋白的重新安装水平与HbS相似。为了评估细胞功能，诱导克隆的红系祖细胞进行体外红细胞分化。正如预期的那样，细胞镰变试验表明基因组编辑的子代SCD红细胞的功能得以恢复，在缺氧条件下对镰变的抵抗力增强。
本研究是对SCD造血干细胞基因组编辑的探索。
