# Genome sequencing for evaluating the efficacy, specificity, and safety of human genome editing

> **NIH NIH U01** · WASHINGTON UNIVERSITY · 2024 · $461,687

## Abstract

The goal of this proposal is to develop a comprehensive approach for evaluating the efficiency and
specificity of genome-edited human cells using whole-genome sequencing. Genome editing has enormous
therapeutic potential by making it possible to restore genetic defects, inactivate deleterious mutant alleles, and
augment the function of cellular therapies. Although genome editing technologies are designed for optimal
efficiency and specificity, on-target editing can be variable, and unwanted mutations in edited cells can result in
unintended functional consequences, including disruption of genes due to off-target mutations, transgene
insertions, or deletions, duplications, or structural rearrangements. As a result, current draft guidance from the
Food and Drug Administration (FDA) recommends that genome-edited cellular therapies be evaluated for both
on- and off-target mutations. However, existing approaches for performing these analyses are logistically
complicated and either use antiquated methods or involve modifications to the editing process that cannot be
applied to cellular drug products that will be used in patients. We hypothesize that whole-genome sequencing
(WGS) is an ideal platform to address FDA guidelines for genomic analysis of genome-edited cellular
products because it detects the full spectrum of mutation types and can be used to evaluate fully
manufactured ‘patient-ready’ cellular therapies. Here we propose to develop a comprehensive WGS assay
specifically designed to characterize mutations in genome-edited human cells. In Aim 1, we will modify our
recently developed clinical WGS assay for somatic mutations (ChromoSeq) to measure the efficiency and
specificity of genome editing in human cells. We will use high coverage (>250x) WGS of paired edited and
unedited control cells and joint somatic variant calling methods to quantify on-target editing efficiency and detect
transgene integration sites and unintended single nucleotide variants, insertions/deletions (indels), and
chromosomal rearrangements. We will then qualify this WGS approach using a dataset of high confidence
mutations generated in three human cell lines with CRISPR/Cas9 and multiplex pools of guide RNAs (gRNA),
which will be identified via iGUIDE and targeted, error-corrected deep sequencing. In Aim 2, we will use our
WGS assay to define the landscape of mutations in genome-edited human CAR-T cells. These will include 5
replicate experiments with reagents to common CAR-T targets, and 15 existing primary human CAR-T products
edited at a range of therapeutically relevant genes that have already been generated in our labs. We will use
these data to generate a benchmark dataset of on-target editing efficiency measurements, CAR integration sites,
and unintended mutations in human CAR-T cells that will provide valuable data for future clinical trials. Finally,
we will analyze up to 20 additional genome-edited cellular products from the Somatic Cell Genome Editing
Consortiu...

## Key facts

- **NIH application ID:** 10831059
- **Project number:** 5U01AI176465-02
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** ERIC J DUNCAVAGE
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $461,687
- **Award type:** 5
- **Project period:** 2023-04-20 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10831059

## Citation

> US National Institutes of Health, RePORTER application 10831059, Genome sequencing for evaluating the efficacy, specificity, and safety of human genome editing (5U01AI176465-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10831059. Licensed CC0.

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