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

> **NIH NIH R01** · UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH · 2024 · $526,252

## Abstract

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.

## Key facts

- **NIH application ID:** 10917269
- **Project number:** 5R01HL166805-02
- **Recipient organization:** UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH
- **Principal Investigator:** JESSE ROWLEY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $526,252
- **Award type:** 5
- **Project period:** 2023-09-01 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10917269

## Citation

> US National Institutes of Health, RePORTER application 10917269, Evaluation of genetic variants affecting platelet function with CRISPR HDR in human megakaryocytes (5R01HL166805-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10917269. Licensed CC0.

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