Editing the Sickle Cell Disease Mutation in Primary Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle Cell Disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the ß-globin locus (HBB). We compared the activity of ZFNs and CRISPR/Cas9 for editing and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODN), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLV) and adenovirus 5/35 serotype (Ad5/35) to transvert the base-pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base-pair transversions were significantly reduced when analyzing cells in vivo in immune-deficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODN. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high efficiency gene correction in HSPCs Overall design: To assess effects of the reagents involved in editing on gene expression profiles of CD34+ PBSCs, we performed RNA-sequencing (RNA-Seq) of total RNA isolated from cells 24 hours post-editing. Mock - Control RNP - ribonucleoprotein, Cas9 + sgRNA ssODN - DNA donor template, single-stranded oligodeoxynucleotides AAV6_BG - DNA donor template, AAV6 based vector, version 1 AAV6_BG_HRPT - DNA donor template, AAV6 based vector, version 2

自体造血干祖细胞（autologous hematopoietic stem and progenitor cells, HSPCs）中引发单基因疾病的点突变的位点特异性校正，可用于治疗血细胞遗传性疾病。镰状细胞病（Sickle Cell Disease, SCD）是研究基因编辑技术潜在应用的理想模型，用于在β-珠蛋白基因座（HBB）上对单个点突变进行碱基颠换编辑。我们比较了锌指核酸酶（zinc finger nucleases, ZFNs）与CRISPR/Cas9的编辑活性，以及以单链寡脱氧核苷酸（single-stranded oligodeoxynucleotides, ssODN）、腺相关病毒血清型6（adeno-associated virus serotype 6, AAV6）、整合酶缺陷型慢病毒载体（integrase-deficient lentiviral vectors, IDLV）和腺病毒5/35血清型（adenovirus 5/35 serotype, Ad5/35）作为同源供体模板，在原代人CD34+造血干祖细胞中编辑HBB基因中导致SCD的碱基对的效果。我们发现，ZFNs与Cas9介导的核酸酶活性频率相近。体外实验中，AAV6介导的同源定向修复（homology-directed repair, HDR）频率最高，但在免疫缺陷小鼠异种移植模型的体内细胞分析中，碱基颠换水平显著降低，此时AAV6与ssODN介导的修复频率相当。此外，AAV6会显著损害集落形成祖细胞的功能以及人源细胞的植入能力。在具备植入能力的造血干细胞中进行基因校正可能受限于细胞介导HDR的能力，这提示若要在HSPCs中实现高效基因校正，可能需要额外的操作手段。

总体实验设计：为评估编辑所用试剂对CD34+外周血造血干细胞（peripheral blood stem and progenitor cells, PBSCs）基因表达谱的影响，我们对编辑后24小时分离的细胞总RNA进行了RNA测序（RNA-sequencing, RNA-Seq）。各组设置如下：
- 空白对照组（Mock）
- 核糖核蛋白（RNP）组：Cas9 + sgRNA
- ssODN供体模板组：以单链寡脱氧核苷酸作为DNA供体模板
- AAV6_BG组：基于AAV6的DNA供体载体（版本1）
- AAV6_BG_HRPT组：基于AAV6的DNA供体载体（版本2）