Evaluation of genetic variants affecting platelet function with CRISPR HDR in human megakaryocytes

利用 CRISPR HDR 评估影响人类巨核细胞血小板功能的遗传变异

基本信息

批准号：
10737494
负责人：
JESSE ROWLEY
金额：
$ 53.7万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10737494
关键词：
Adult Affect Agonist Alpha Granule Amino Acids Applied Genetics Binding Blood Coagulation Disorders Blood Platelet Disorders Blood Platelets CD34 gene CRISPR/Cas technology Cell Differentiation process Cell Line Cells Classification Clinical Clinical Management Cloning Clustered Regularly Interspaced Short Palindromic Repeats Communities DNA Data Databases Decision Making Diagnosis Diagnostic Disease Evaluation Fibrinogen Future Gene Expression Generations Genes Genetic Genetic Variation Goals Hamsters Hemorrhage High-Throughput DNA Sequencing High-Throughput Nucleotide Sequencing Human Immunodeficient Mouse Individual Inherited Insertion Mutation Integrins Investigation Libraries Measures Mediating Megakaryocytes Methods Missense Mutation Modeling Modification Mus Mutagenesis Mutation Ovary PAC1 phosphatase Pathogenicity Patients Phenotype Physiological Physiology Point Mutation Positioning Attribute Production Proteins Reporting Research Single-Stranded DNA Sorting Specific qualifier value Surveys Testing Thrombasthenia Time Umbilical Cord Blood Validation Variant Virus Work clinical application clinically relevant disease classification exome sequencing functional mimics gene function genetic disorder diagnosis genetic variant genome sequencing genomic locus high throughput screening human DNA in vivo indexing innovation insight loss of function mouse model mutant novel overexpression platelet function platelet phenotype precision medicine repaired response targeted sequencing tool variant of unknown significance virulence gene

项目摘要

Abstract Numerous genetic variants associated with inherited platelet function disorders are classified as variants of unknown significance (VUS) because they lack functional evaluation. The functional validation of the genetic causes of platelet mediated disease has been difficult because of the inability to make precise genetic modifications in anucleate human platelets. As the precursor to platelets, megakaryocytes share many functions with platelets and are the natural choice for platelet function studies, but have been traditionally difficult to genetically modify . In this proposal, we develop a non-viral and selection free CRISPR/CAS9 approach to make precise gene edits in human cord blood and adult CD34+ cell derived megakaryocytes using homology directed repair. We apply the approach to functionally and mechanistically define VUS in ITGA2B, mutations in which can cause the bleeding disorder Glanzmann’s Thrombasthenia. Our preliminary data demonstrate the precise generation of insertions and point mutations in the gene ITGA2B in >95% of megakaryocytes. We show that megakaryocytes harboring point mutations or insertions in ITGA2B that are known to cause Glanzmann’s mimic functional responses observed for platelets from patients. In Aim 1 we generate 57 VUS or likely pathogenic variants in ITGA2B and test their effect on platelet-like functional responses in megakaryocytes towards their clinical reclassification, while at the same time defining rules for efficient homology directed repair in megakaryocytes. We further deep phenotype and mechanistically dissect select ITGA2B variants, including in human platelets generated in mice. In Aim 2, we use homology directed saturating mutagenesis in CD34+ derived megakaryocytes, followed by high throughput sequencing, to identify all functional amino acid changes across mutation hot-spots of ITGA2B. This work is innovative: we use a novel and straightforward approach to generate precise point mutations and insertions at unprecedented levels in human CD34+ cells differentiated into megakaryocytes. We use innovative methods, including the generation of gene edited human platelets in mice, and saturating mutagenesis functional screens in primary cells, to examine the effect of genetic variants on megakaryocyte/platelet functions. This work is significant because it will result in the clinical classification of genetic variants that can be directly applied for the genetic diagnosis of patients, and provide hope for future treatment options. Our studies will also provide new mechanistic insights into how variants affect ITGA2B production and function in a physiologically relevant human primary cell.

抽象的与遗传血小板功能障碍相关的许多遗传变异被归类为未知的意义（VUS）是因为它们缺乏功能评估。通用的功能验证血小板介导的疾病的原因很困难，因为无法进行精确的通用在Anugleate人血小板中进行修饰。作为血小板的前体，巨核细胞分享了许多血小板的功能是血小板功能研究的自然选择，但传统上一直是难以基因修改。在此提案中，我们开发了一个非病毒和选择免费CRISPR/CAS9 在人脐带血和成人CD34+细胞衍生的巨核细胞中进行精确基因编辑的方法使用同源性维修。我们将方法应用于功能和机械定义VUS itga2b，突变会导致出血障碍Glanzmann的血栓性疾病。我们的初步数据证明了> 95％的基因itga2b中插入和点突变的精确生成巨核细胞。我们表明，含有点突变或插入itga2b的巨核细胞是已知会导致对患者血小板观察到的Glanzmann的模拟功能反应。在AIM 1中，我们在ITGA2B中产生57个VU或可能的致病变异，并测试它们对血小板样的影响巨核细胞对其临床重分类的功能反应，同时定义有效同源性修复巨核细胞的规则。我们进一步深入表型和机理剖析选择ITGA2B变体，包括在小鼠中产生的人血小板中。在AIM 2中，我们使用同源性 CD34+衍生的巨核细胞中的定向饱和诱变，然后进行高吞吐量测序，确定跨itga2b突变热点之间的所有功能性氨基酸变化。这项工作是创新的：我们使用一种新颖而直接的方法来产生精确点突变在人CD34+细胞中前所未有水平的插入中，分化为巨核细胞。我们使用创新方法，包括基因的产生，在小鼠中编辑了人血小板，并饱和在原代细胞中的诱变功能筛选，以检查遗传变异的影响巨核细胞/血小板功能。这项工作很重要，因为它将导致可以直接用于患者遗传诊断的遗传变异，并为未来提供希望治疗选择。我们的研究还将提供有关变体如何影响itga2b的新机械见解在物理相关的人类原代细胞中的生产和功能。