HBB gene-editing for treating sickle cell disease

镰状细胞病（SCD）是一种遗传性疾病，影响全世界数百万人， 发病率和平均寿命在40多岁。虽然SCD可以通过同种异体移植治愈， 造血干细胞移植（HSCT），这种治疗策略具有很大的局限性， 约15%的患者可用。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（HSPC）中β-珠蛋白（HBB）基因的镰状突变 使用CRISPR/Cas9和校正性单链寡核苷酸（ssODN）供体模板， 高达~37%的突变HBB等位基因可以被基因校正。将基因编辑的SCD HSPC注射到 免疫缺陷型NOD/SCID/IL-2 rgnull（NSG）小鼠显示出临床相关水平的植入。我们进一步 研究表明，从基因编辑的SCD HSPC分化的细胞产生高水平的正常表达。 血红蛋白A（HbA），导致存在于血液中的镰状血红蛋白（HbS）的量显著减少。 红细胞特别地，在没有ssODN模板的情况下将Cas9/gRNA RNP递送到SCD CD 34+细胞中 (i.e.仅用HBB的Cas9切割）导致胎儿血红蛋白（HbF）诱导的大幅增加， HbS的量显著降低，导致即使在缺氧条件下也能防止镰状化。 然而，临床上对Cas9切割诱导HbF的机制知之甚少。 在HBB靶切割位点和染色体重排处的大缺失/插入的影响需要 有待确定，还需要评估HBB插入缺失诱导β-地中海贫血的风险。中央 拟议研究的假设是，对HBB基因编辑后果的定量理解 将提高基于基因编辑的SCD治疗的有效性和安全性。在目标1研究中，我们将 通过评估以下因素的作用来确定SCD HSPC中Cas9切割诱导的HbF诱导的机制： 红系培养物中HSPC上HBB的Cas9切割，并测量对HBB相对表达的影响 和HBG。在目标2中，我们将定量HBB靶位点的大缺失和染色体重排， SCD HSPC使用新的PCR和下一代测序工具。在目标3中，我们将确定 通过定量总血红蛋白蛋白在SCD HSPC中诱导由于HBB基因编辑引起的β地中海贫血 使用我们的镰状HUDEP-2细胞系和来自基因编辑SCD的细胞， HSPC和镰状小鼠模型中移植的编辑细胞。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.