Deciphering unintended large gene modifications in gene editing for sickle cell disease

Summary: 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 (alloHSCT), this treatment strategy has substantial limitations and is
only available to ~15% of patients. CRISPR/Cas9 based genome-editing strategies for treating SCD have been
developed by either correcting the sickle mutation in β-globin (HBB) gene or disrupting the BCL11A erythroid
enhancer in patients’ hematopoietic stem and progenitor cells (HSPCs). Multiple clinical trials using gene
editing strategies have received FDA approval, and the Phase 1 clinical trial (NCT03745287) by Vertex
Pharmaceuticals and CRISPR Therapeutics has shown promise. We have discovered recently that
CRISPR/Cas9 genome editing can induce unintended large gene modifications, such as large deletions,
insertions and complex local rearrangements, at the Cas9 on-target cut-site. Our results show that large
deletions of up to several thousand bases occurred with high frequencies at/near the Cas9 on-target cut-sites
on the HBB (11.7-35.4%), HBG (14.3%), and BCL11A (13.2%) genes respectively in HSPCs from patients with
SCD. However, the persistence and biological consequences of these large gene modifications are largely
unknown, the mechanisms of generating large deletions and insertions remain elusive, and no method is
available to reduce the unwanted large gene modifications. There is an unmet need to determine the clinical
implications of the unintended large gene modifications in gene-edited SCD HSPCs. The central hypothesis of
the proposed research is that a good understanding of the persistence and functional consequences of
unintended large gene modifications and the ability to control them will increase the efficacy and safety of
gene-editing based treatment of SCD. In Aim 1 studies we will determine the ineffective maturation and HbF
induction due to large gene modification in gene edited SCD HSPCs by performing SMRT-seq and single-cell
RNA analysis. In Aim 2 we will determine the persistence of large gene modifications in HBB and BCL11A
alleles after engraftment of gene-edited SCD HSPCs into mice and patients undergoing CRISPR/Cas9 gene-
editing based SCD clinical trials. In Aim 3 we will develop strategies to minimize the detrimental large deletions
by establishing a better understanding of the competition between different DNA damage repair pathways and
designing and optimizing ssODN templates and short gRNAs as blockers. These studies will address an unmet
need in the therapeutic genome editing field and facilitate the translation of genome editing based SCD
treatment into clinical practice.

Grant Number: 5R01HL169761-03
NIH Institute/Center: NIH
Principal Investigator: Gang Bao