# Addressing safety issues by quantify large deletions and chromosomal rearrangements in HBB gene editing

> **NIH NIH OT2** · RICE UNIVERSITY · 2020 · $1,173,874

## Abstract

Sickle cell disease (SCD) is a devastating chronic illness marked by severe pain, end organ
damage and early mortality (1, 2). It affects ~100,000 Americans and millions more worldwide
(3, 4), but treatment options for SCD remain very limited. Pharmacological therapy with
hydroxyurea or chronic blood transfusions at best modulates the disease severity but does not
cure patients (5). Currently, the only curative therapy for sickle cell disease (SCD) outside of a
limited clinical trial is a hematopoietic stem cell transplant (HSCT), typically from a matched
related donor, which is available to only ~15% of patients (6, 7). Morbidity and mortality from
HSCT increases significantly when using matched unrelated donors (8), or haploidentical
donors (9). A recent prospective study of unrelated donor HSCT in SCD concluded that, without
modifications to existing regimens, this therapy is not safe for widespread adoption (10).
With the advancement of CRISPR/Cas9 technology, there are several possible gene editing
strategies to ameliorate SCD: (i) correction of the causative A-T point mutation in β-globin
(HBB)(11-14), (ii) induction of fetal hemoglobin (HbF)(15, 16), and (iii) gene addition of a β-
globin, γ-globin, or anti-sickling β-globin cassette (17), among which correction of the A-T
mutation or producing high enough levels of HbF could be curative. We and others recently
demonstrated that, by delivering CRISPR gRNA/Cas9 ribonucleoproteins (RNPs) together with
single-stranded oligonucleotide (ssODN) donor templates into SCD patient-derived
hematopoietic stem and progenitor cells (SCD HSPCs), up to ~37% of mutant HBB alleles can
be gene corrected (12, 14). Injection of gene-edited SCD HSPCs into immunodeficient
NOD/SCID/IL-2rgnull (NSG) mice showed a clinically relevant level of engraftment, with
detectable levels of gene correction 16-19 weeks post-transplantation (14).
We have shown that by using a high-fidelity Cas9 that maintained the same level of ontarget
gene modification, the off-target effects could be significantly reduced (14). However,
potential large deletions and insertions at the HBB on-target cut-site, and off-target effects such
as chromosomal translocation and inversion in gene-edited SCD HSPCs remain a significant
safety concern, since even a very small number of HSCs harboring these detrimental
events could clonally expand in vivo and cause a disease such as cancer. Previously, we
optimized droplet digital PCR (ddPCR) assay to quantify large deletions and inversions between
the R-66 SCD gRNA target site in HBB and a known off-target site (OT18) in gRNA/Cas9 WT
RNP-treated SCD HSPCs (14). For high throughput discovery and quantification of such large
modifications, we recently developed two next-generation sequencing (NGS) based methods
based on short-read high-throughput illumina NGS platform leveraging the high sensitivity and
cost-competitiveness of short-read NGS. The first is the LongAmp-Seq (Long-range PCR
Amplification based ...

## Key facts

- **NIH application ID:** 10087778
- **Project number:** 1OT2HL154977-01
- **Recipient organization:** RICE UNIVERSITY
- **Principal Investigator:** Gang Bao
- **Activity code:** OT2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $1,173,874
- **Award type:** 1
- **Project period:** 2020-04-25 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10087778

## Citation

> US National Institutes of Health, RePORTER application 10087778, Addressing safety issues by quantify large deletions and chromosomal rearrangements in HBB gene editing (1OT2HL154977-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10087778. Licensed CC0.

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