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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.

摘要