# HBB gene-editing for treating sickle cell disease

> **NIH NIH R01** · RICE UNIVERSITY · 2021 · $612,108

## Abstract

Sickle cell disease (SCD) is a genetic disease that affects millions of people worldwide, with significant
morbidity and a median life expectancy in the mid-forties. Although SCD can be cured by allogeneic
hematopoietic stem cell transplantation (HSCT), this treatment strategy has substantial limitations and is only
available to ~15% of patients. We have developed a genome-editing based strategy for treating SCD by
correcting the sickle mutation in β-globin (HBB) gene in patient’s hematopoietic stem/progenitor cells (HSPCs)
using CRISPR/Cas9 and corrective single-stranded oligonucleotide (ssODN) donor template, demonstrated
that up to ~37% of mutant HBB alleles can be gene corrected. Injection of gene-edited SCD HSPCs into
immunodeficient NOD/SCID/IL-2rgnull (NSG) mice showed a clinically relevant level of engraftment. We further
demonstrated that cells differentiated from gene-edited SCD HSPCs produced high levels of normal
hemoglobin A (HbA), resulting in a significant reduction of the amount of sickle hemoglobin (HbS) present in
the red blood cells. In particular, delivery of Cas9/gRNA RNP into SCD CD34+ cells without ssODN template
(i.e. only with Cas9 cutting of HBB) resulted in a large increase in fetal hemoglobin (HbF) induction and
significant decrease in the amount of HbS, leading to prevention of sickling even under hypoxic conditions.
However, the mechanism underlying HbF induction by Cas9 cutting is poorly understood, the clinical
implications of large deletions/insertions at the HBB on-target cut-site and chromosomal rearrangements need
to be determined, and the risk of inducing β-thalassemia by HBB indels needs to be evaluated. The central
hypothesis of the proposed research is that a quantitative understanding of HBB gene editing consequences
will increase the efficacy and safety of gene-editing based treatment of SCD. In Aim 1 studies we will
determine the mechanism(s) of Cas9-cutting induced HbF induction in SCD HSPCs by assessing the effect of
Cas9 cutting of HBB on HSPCs in erythroid culture, and measuring the impact on relative expression of HBB
and HBG. In Aim 2 we will quantify large deletions at HBB on-target site and chromosomal rearrangements in
SCD HSPCs using new PCR and next-generation sequencing tools. In Aim 3 we will determine the potential of
inducing β-thalassemia due to HBB gene editing in SCD HSPCs by quantifying the total hemoglobin protein
levels and the complete hemoglobin profile using our sickle HUDEP-2 cell-line and cells from gene-edited SCD
HSPCs, and engrafted edited cells in a sickle mouse model. These studies will facilitate the translation of
genome editing based SCD treatment into clinical practice.

## Key facts

- **NIH application ID:** 10146476
- **Project number:** 5R01HL152314-02
- **Recipient organization:** RICE UNIVERSITY
- **Principal Investigator:** Gang Bao
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $612,108
- **Award type:** 5
- **Project period:** 2020-04-17 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10146476

## Citation

> US National Institutes of Health, RePORTER application 10146476, HBB gene-editing for treating sickle cell disease (5R01HL152314-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10146476. Licensed CC0.

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