HBB gene-editing for treating sickle cell disease

镰状细胞病(SCD)是一种遗传性疾病，影响着全球数百万人，具有显著的 发病率和预期寿命中位数为45岁左右。虽然SCD可以通过异体移植治愈 造血干细胞移植(HSCT)，这种治疗策略有很大的局限性， 可供约15%的患者使用。我们已经开发了一种基于基因组编辑的治疗SCD的策略 纠正患者造血干/祖细胞中β-珠蛋白基因的镰刀状突变 使用CRISPR/Cas9和修正的单链寡核苷酸(SsODN)供体模板 高达37%的突变HBB等位基因可以被基因纠正。基因编辑的SCD-HSPC注射入HSPC 免疫缺陷NOD/SCID/IL-2rgnull(NSG)小鼠表现出临床相关的植入水平。我们进一步 证明从经过基因编辑的SCD HSPC分化的细胞产生高水平的正常 血红蛋白A(HBA)，导致存在的镰状血红蛋白(HBS)的数量显著减少 红血球。具体地说，不以ssODN为模板将Cas9/gRNA RNP导入SCD CD34+细胞 (即仅在切断HBb的情况下)导致胎儿血红蛋白(HBF)诱导大幅增加，并 HbS的数量显著减少，导致即使在低氧条件下也不会出现镰状。 然而，临床上对Cas9切割诱导HBF的机制知之甚少。 HBB靶向切割部位的大片段缺失/插入和染色体重排需要 尚待确定，还需要评估HBb基因突变导致β-地中海贫血的风险。中环 拟议研究的假设是，对HBB基因编辑后果的量化理解 将提高以基因编辑为基础的SCD治疗的有效性和安全性。在目标1研究中，我们将 切断Cas9对SCD HSPC诱导HbF作用机制的探讨(S) 红细胞培养中HbB在HSPC上的切割及其对HbB相对表达的影响 和HBG。在目标2中，我们将量化HBB靶点上的大量缺失和染色体重排 使用新的聚合酶链式反应和下一代测序工具的SCD HSPC。在目标3中，我们将确定 用定量总血红蛋白的方法研究HBb基因编辑诱发β-地中海贫血 利用我们的镰刀HUDEP-2细胞系和来自基因编辑的SCD细胞的水平和完整的血红蛋白谱 HSPC，并将编辑后的细胞植入镰刀鼠模型中。这些研究将有助于翻译 基于基因组编辑的SCD治疗进入临床实践。

Sickle cell disease (SCD) is a genetic disease that affects millions of people worldwide, with significant morbidity and a median life expectancy in the mid-forties. Although SCD can be cured by allogeneic hematopoietic stem cell transplantation (HSCT), this treatment strategy has substantial limitations and is only available to ~15% of patients. We have developed a genome-editing based strategy for treating SCD by correcting the sickle mutation in β-globin (HBB) gene in patient’s hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 and corrective single-stranded oligonucleotide (ssODN) donor template, demonstrated that up to ~37% of mutant HBB alleles can be gene corrected. Injection of gene-edited SCD HSPCs into immunodeficient NOD/SCID/IL-2rgnull (NSG) mice showed a clinically relevant level of engraftment. We further demonstrated that cells differentiated from gene-edited SCD HSPCs produced high levels of normal hemoglobin A (HbA), resulting in a significant reduction of the amount of sickle hemoglobin (HbS) present in the red blood cells. In particular, delivery of Cas9/gRNA RNP into SCD CD34+ cells without ssODN template (i.e. only with Cas9 cutting of HBB) resulted in a large increase in fetal hemoglobin (HbF) induction and significant decrease in the amount of HbS, leading to prevention of sickling even under hypoxic conditions. However, the mechanism underlying HbF induction by Cas9 cutting is poorly understood, the clinical implications of large deletions/insertions at the HBB on-target cut-site and chromosomal rearrangements need to be determined, and the risk of inducing β-thalassemia by HBB indels needs to be evaluated. The central hypothesis of the proposed research is that a quantitative understanding of HBB gene editing consequences will increase the efficacy and safety of gene-editing based treatment of SCD. In Aim 1 studies we will determine the mechanism(s) of Cas9-cutting induced HbF induction in SCD HSPCs by assessing the effect of Cas9 cutting of HBB on HSPCs in erythroid culture, and measuring the impact on relative expression of HBB and HBG. In Aim 2 we will quantify large deletions at HBB on-target site and chromosomal rearrangements in SCD HSPCs using new PCR and next-generation sequencing tools. In Aim 3 we will determine the potential of inducing β-thalassemia due to HBB gene editing in SCD HSPCs by quantifying the total hemoglobin protein levels and the complete hemoglobin profile using our sickle HUDEP-2 cell-line and cells from gene-edited SCD HSPCs, and engrafted edited cells in a sickle mouse model. These studies will facilitate the translation of genome editing based SCD treatment into clinical practice.