HBB gene-editing for treating sickle cell disease

HBB 基因编辑治疗镰状细胞病

• 批准号: 10392986
• 负责人: Gang Bao
• 金额: $ 60.91万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-17 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392986
• 关键词: Address; Adoption; Adverse effects; Affect; Alleles; Allogenic; American; Animals; Benefits and Risks; Blood Transfusion; CD34 gene; CRISPR/Cas technology; Cell Differentiation process; Cell Line; Cells; Chromosomal Rearrangement; Chromosomal translocation; Chromosome Deletion; Chromosome inversion; Chronic; Chronic Disease; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cooley' s anemia; Differentiated Gene; Engraftment; Erythrocytes; Erythroid; Erythroid Progenitor Cells; Event; Fetal Hemoglobin; Gene-Modified; Genes; Genetic Diseases; Goals; Guide RNA; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobin; Hemoglobin A; Hemoglobin concentration result; Hypoxia; Injections; Life Expectancy; Measures; Minor; Modification; Morbidity - disease rate; Mus; Mutation; Nonhomologous DNA End Joining; Oligonucleotides; Organ; Pain; Patients; Persons; Pharmacology; Prevention; Proteins; Regimen; Research; Ribonucleoproteins; Risk; Safety; Severity of illness; Sickle Cell; Sickle Cell Anemia; Sickle Cell Trait; Sickle Hemoglobin; Site; Stroke; Testing; Translating; Translations; base editing; beta Globin; beta Thalassemia; clinical practice; clinically relevant; curative treatments; gene correction; genome editing; hydroxyurea; insertion/deletion mutation; mortality; mouse model; mutant; next generation sequencing; response; sickling; tool; treatment strategy

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.

镰状细胞疾病（SCD）是一种影响全球数百万人的遗传疾病，具有重要意义 四十多岁的发病率和中位预期寿命。尽管SCD可以通过同种异体治疗 造血干细胞移植（HSCT），该治疗策略具有重大局限性，仅是 约15％的患者可用。我们已经制定了一种基于基因组编辑的策略，用于治疗SCD 纠正患者造血茎/祖细胞（HSPCS）中β-珠蛋白（HBB）基因中的镰状突变 使用CRISPR/CAS9并校正单链寡核苷酸（SSODN）供体模板 可以校正高达约37％的突变HBB等位基因。将基因编辑的SCD HSPC注入 免疫缺陷的点头/SCID/IL-2RGNULL（NSG）小鼠显示出与临床相关的植入水平。我们进一步 证明与基因编辑的SCD HSPC区分开的细胞产生了高水平的正常水平 血红蛋白A（HBA），导致镰状血红蛋白（HBS）的量显着减少 红细胞。特别是，将Cas9/GRNA RNP递送到没有SSODN模板的SCD CD34+细胞中 （即仅使用CAS9切割HBB）导致胎儿血红蛋白（HBF）诱导大幅增加 HB的量显着减少，即使在缺氧条件下也可以预防疾病。 但是，CAS9切割诱导HBF的基础机制知之甚少，临床 在HBB攻击型切割点和染色体重排的大型删除/插入的含义需要 待确定，需要评估HBB Indels引起的β-丘脑贫血的风险。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的效率和安全性。在AIM 1研究中，我们将 通过评估CAS9切割诱导的HBF的机制，通过评估SCD HSPC中的HBF CAS9在红细胞培养中切割HBB在HSPC上，并测量对HBB的相对表达的影响 和HBG。在AIM 2中，我们将量化HBB的靶标地点的大删除，并在 SCD HSPC使用新的PCR和下一代测序工具。在AIM 3中，我们将确定 通过量化总血红蛋白蛋白，在SCD HSPC中诱导HBB基因编辑引起的β-th无质 使用我们的Sickle Hudep-2细胞系和基因编辑的SCD细胞的水平和完整的血红蛋白谱 HSPC，并在镰状小鼠模型中植入了编辑的细胞。这些研究将促进翻译 基于基因组编辑的SCD处理临床实践。