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，影响全球数百万人， 发病率很高，平均寿命在45岁左右。虽然SCD可以通过同种异体移植治愈， 造血干细胞移植（alloHSCT），这种治疗策略具有很大的局限性， 仅适用于约15%的患者。用于治疗SCD的基于CRISPR/Cas9的基因组编辑策略已经被广泛应用。 通过纠正β-珠蛋白（HBB）基因中的镰状突变或破坏BCL 11 A红系细胞而开发 在患者的造血干细胞和祖细胞（HSPC）中的增强子。使用基因的多项临床试验 编辑策略已获得FDA批准，Vertex的1期临床试验（NCT 03745287） 药物和CRISPR治疗已经显示出希望。我们最近发现， CRISPR/Cas9基因组编辑可以诱导非预期的大基因修饰，如大缺失， 插入和复杂的局部重排，在Cas9靶向切割位点。我们的研究结果表明， 在Cas9靶向切割位点处/附近以高频率发生多达几千个碱基的缺失 HBB（11.7-35.4%）、HBG（14.3%）和BCL 11 A（13.2%）基因分别在来自HSPC的患者中表达。 SCD。然而，这些大的基因修饰的持久性和生物学后果在很大程度上是 由于未知，产生大的缺失和插入的机制仍然难以捉摸，并且没有方法 可用于减少不必要的大基因修饰。有一个未满足的需求，以确定临床 基因编辑的SCD HSPC中非预期的大基因修饰的影响。的中心假设 建议的研究是，一个很好的理解的持久性和功能的后果， 非预期的大基因修饰和控制它们的能力将增加药物的有效性和安全性。 基因编辑治疗SCD在目标1研究中，我们将确定无效成熟和HbF 通过进行SMRT-seq和单细胞在基因编辑的SCD HSPC中由于大基因修饰引起的诱导 RNA分析在目标2中，我们将确定HBB和BCL 11 A中大基因修饰的持久性 将基因编辑的SCD HSPC植入小鼠和接受CRISPR/Cas9基因治疗的患者后的等位基因。 基于SCD的临床试验编辑。在目标3中，我们将制定策略，以尽量减少有害的大缺失 通过更好地理解不同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。这些研究将解决一个未得到满足的 需要在治疗性基因组编辑领域和促进基于SCD的基因组编辑的翻译 治疗进入临床实践。