Deciphering unintended large gene modifications in gene editing for sickle cell disease

破译镰状细胞病基因编辑中意外的大基因修饰

• 批准号: 10720685
• 负责人: Gang Bao
• 金额: $ 66.2万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10720685
• 关键词: Address; Adoption; Affect; Alleles; Allogenic; American; Autologous; Biological; Blood Transfusion; Blood specimen; Bone Marrow; CRISPR therapeutics; CRISPR/Cas technology; Cell Separation; Cells; Chronic; Chronic Disease; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA Repair; Data; Development; Engraftment; Enhancers; Erythroid; Erythroid Cells; Erythropoiesis; Evaluation; Fetal Hemoglobin; Frequencies; Gene Modified; Genes; Genetic Diseases; Genotype; Goals; Guide RNA; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Heterogeneity; Immunodeficient Mouse; Life Expectancy; Lymphoid; Methods; Morbidity - disease rate; Mus; Mutation; Myelogenous; Oligonucleotides; Organ; Outcome; Pain; Pathway interactions; Patients; Persons; Pharmacologic Substance; Phase I Clinical Trials; Phenotype; RNA analysis; Reporting; Research; Safety; Sickle Cell Anemia; Site; Stroke; Testing; Time; Translations; base; base editing; beta Globin; clinical practice; curative treatments; design; genome editing; graft vs host disease; improved; insertion/deletion mutation; mortality; novel strategies; palliative; peripheral blood; pharmacologic; prevent; sickling; single molecule real time sequencing; single-cell RNA sequencing; therapeutic gene; therapeutic genome editing; transcriptomics; treatment strategy

Summary: 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 (alloHSCT), this treatment strategy has substantial limitations and is only available to ~15% of patients. CRISPR/Cas9 based genome-editing strategies for treating SCD have been developed by either correcting the sickle mutation in β-globin (HBB) gene or disrupting the BCL11A erythroid enhancer in patients’ hematopoietic stem and progenitor cells (HSPCs). Multiple clinical trials using gene editing strategies have received FDA approval, and the Phase 1 clinical trial (NCT03745287) by Vertex Pharmaceuticals and CRISPR Therapeutics has shown promise. We have discovered recently that CRISPR/Cas9 genome editing can induce unintended large gene modifications, such as large deletions, insertions and complex local rearrangements, at the Cas9 on-target cut-site. Our results show that large deletions of up to several thousand bases occurred with high frequencies at/near the Cas9 on-target cut-sites on the HBB (11.7-35.4%), HBG (14.3%), and BCL11A (13.2%) genes respectively in HSPCs from patients with SCD. However, the persistence and biological consequences of these large gene modifications are largely unknown, the mechanisms of generating large deletions and insertions remain elusive, and no method is available to reduce the unwanted large gene modifications. There is an unmet need to determine the clinical implications of the unintended large gene modifications in gene-edited SCD HSPCs. The central hypothesis of the proposed research is that a good understanding of the persistence and functional consequences of unintended large gene modifications and the ability to control them will increase the efficacy and safety of gene-editing based treatment of SCD. In Aim 1 studies we will determine the ineffective maturation and HbF induction due to large gene modification in gene edited SCD HSPCs by performing SMRT-seq and single-cell RNA analysis. In Aim 2 we will determine the persistence of large gene modifications in HBB and BCL11A alleles after engraftment of gene-edited SCD HSPCs into mice and patients undergoing CRISPR/Cas9 gene- editing based SCD clinical trials. In Aim 3 we will develop strategies to minimize the detrimental large deletions by establishing a better understanding of the competition between different DNA damage repair pathways and designing and optimizing ssODN templates and short gRNAs as blockers. These studies will address an unmet need in the therapeutic genome editing field and facilitate the translation of genome editing based SCD treatment into clinical practice.

摘要：镰状细胞病 (SCD) 是一种遗传性疾病，影响全世界数百万人， 发病率很高，预期寿命中位数为四十多岁。尽管SCD可以通过同种异体治愈 造血干细胞移植（alloHSCT），这种治疗策略有很大的局限性， 仅适用于约 15% 的患者。基于 CRISPR/Cas9 的基因组编辑策略用于治疗 SCD 通过纠正 β-珠蛋白 (HBB) 基因中的镰状突变或破坏 BCL11A 红细胞而开发 患者造血干细胞和祖细胞 (HSPC) 的增强剂。使用基因进行多项临床试验 编辑策略已获得 FDA 批准，Vertex 进行 1 期临床试验（NCT03745287） 制药和 CRISPR 疗法已显示出前景。我们最近发现 CRISPR/Cas9 基因组编辑可能会导致意外的大基因修饰，例如大缺失、 在 Cas9 的目标切割位点进行插入和复杂的局部重排。我们的结果表明，大 在 Cas9 目标切割位点/附近频繁发生多达数千个碱基的缺失 来自患有以下疾病的患者的 HSPC 中的 HBB (11.7-35.4%)、HBG (14.3%) 和 BCL11A (13.2%) 基因分别受到影响 SCD。然而，这些大基因修饰的持久性和生物学后果在很大程度上是 未知，产生大缺失和插入的机制仍然难以捉摸，并且没有方法 可用于减少不需要的大基因修饰。确定临床的需求尚未得到满足 基因编辑的 SCD HSPC 中意外的大基因修饰的影响。中心假设为 拟议的研究是对持久性和功能性后果的良好理解 意外的大基因修饰以及控制它们的能力将提高药物的功效和安全性 基于基因编辑的 SCD 治疗。在目标 1 研究中，我们将确定无效成熟和 HbF 通过执行 SMRT-seq 和单细胞对基因编辑的 SCD HSPC 进行大基因修饰而诱导 RNA 分析。在目标 2 中，我们将确定 HBB 和 BCL11A 中大型基因修饰的持续性 将基因编辑的 SCD HSPC 植入接受 CRISPR/Cas9 基因治疗的小鼠和患者体内后的等位基因- 基于编辑的 SCD 临床试验。在目标 3 中，我们将制定策略以尽量减少有害的大缺失 通过更好地了解不同 DNA 损伤修复途径之间的竞争 设计和优化 ssODN 模板和短 gRNA 作为阻断剂。这些研究将解决未满足的问题 治疗性基因组编辑领域的需求并促进基于基因组编辑的 SCD 的转化 治疗进入临床实践。