HBB gene-editing for treating sickle cell disease

HBB 基因编辑治疗镰状细胞病

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

镰状细胞病（SCD）是一种遗传性疾病，影响全世界数百万人，具有显着的影响尽管 SCD 可以通过同种异体治疗治愈，但发病率和中位预期寿命在 40 多岁。造血干细胞移植（HSCT），这种治疗策略有很大的局限性，并且只能我们已经开发出一种基于基因组编辑的策略来治疗 SCD。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 和校正单链寡核苷酸 (ssODN) 供体模板，证明高达约 37% 的突变 HBB 等位基因可以通过基因编辑的 SCD HSPC 进行基因校正。免疫缺陷的 NOD/SCID/IL-2rgnull (NSG) 小鼠显示出临床相关的植入水平。由基因编辑的 SCD HSPC 分化而来的细胞产生高水平的正常细胞血红蛋白 A (HbA)，导致体内镰状血红蛋白 (HbS) 的量显着减少特别是，在没有 ssODN 模板的情况下将 Cas9/gRNA RNP 递送到 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，并且 HbS 量显着减少，即使在缺氧条件下也能预防镰状化。然而，Cas9 切割诱导 HbF 的机制尚不清楚，临床上 HBB 靶位切割位点大缺失/插入的影响以及染色体重排的需要待确定，并且需要评估 HBB 插入缺失诱发 β-地中海贫血的风险。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 SCD HSPC 中 Cas9 切割诱导的 HbF 诱导的作用，确定机制 Cas9 对红细胞培养物中的 HSPC 进行 HBB 切割，并测量对 HBB 相对表达的影响在目标 2 中，我们将量化 HBB 靶位点的大量缺失和染色体重排。使用新的 PCR 和下一代测序工具的 SCD HSPC 在目标 3 中，我们将确定其潜力。通过量化总血红蛋白，在 SCD HSPC 中通过 HBB 基因编辑诱导 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的 SCD 细胞的血红蛋白水平和完整血红蛋白谱 HSPC 和镰状小鼠模型中的雕刻编辑细胞将有助于转化。基于基因组编辑的 SCD 治疗进入临床实践。