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）是一种遗传疾病，影响了全球数百万的人， 四十多岁的发病率和预期寿命中位数。尽管SCD可以通过同种异体治疗 造血干细胞移植（AlloHSCT），该治疗策略具有很大的局限性，IS 仅约15％的患者可用。基于CRISPR/CAS9治疗SCD的基因组编辑策略已经 通过纠正β-珠蛋白（HBB）基因中的镰刀突变或破坏Bcl11a红细胞性侵蚀性而开发 患者造血干和祖细胞（HSPC）的增强子。使用基因的多次临床试验 编辑策略已获得FDA批准，并获得了Vertex的1期临床试验（NCT03745287） 药品和CRISPR疗法表现出了希望。我们最近发现 CRISPR/CAS9基因组编辑可以诱导意外的大基因修饰，例如大删除， 在CAS9靶向切割站点上的插入和复杂的本地重排。我们的结果表明很大 删除多达数千个基础的删除在Cas9 target切割点附近的高频发生了 HBB（11.7-35.4％），HBG（14.3％）和BCL11A（13.2％）基因的HSPC患者的患者的基因分别 scd。但是，这些大基因修饰的持久性和生物学后果在很大程度上是 未知的是，产生大删除和插入的机制仍然难以捉摸，没有方法是 可用于减少多余的大基因修饰。确定临床有未满足的需求 基因编辑的SCD HSPC中意外大基因修饰的含义。中心假设 拟议的研究是，对持久性和功能后果的良好理解 意外的大基因修饰和控制它们的能力将提高 基于基因编辑的SCD治疗。在AIM 1研究中，我们将确定无效的成熟和HBF 通过进行SMRT-SEQ和单细胞的基因修饰引起的诱导基因修饰了SCD HSPC RNA分析。在AIM 2中，我们将确定HBB和BCL11A中大基因修饰的持久性 基因编辑的SCD HSPC植入小鼠和接受CRISPR/CAS9基因的患者的等位基因 - 基于编辑的SCD临床试验。在AIM 3中，我们将制定策略以最大程度地减少有害的大删除 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模板和简短的GRNA作为阻滞剂。这些研究将解决一项未定的 在治疗基因组编辑领域的需求，并促进基于基因组编辑的SCD的翻译 治疗临床实践。