Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease

Abstract

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the β-globin gene (HBB). Ex vivo β-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)–mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

A Single Cell Cloning Platform for Gene Edited Functional Murine Hematopoietic Stem Cells

“…In contrast, correction via templated repair (i.e. homology-directed repair (HDR)) among long-term (LT)-HSCs remains inefficient (Lattanzi et al, 2021; Mohrin et al, 2010). Off-target mutations may also raise concerns about genotoxicity in Cas9-edited cells (Tsai and Joung, 2016).…”

CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome

“…Long-term engraftment of the targeted HSCs in transplanted NSG mice was confirmed and they were also able to correct the defective IL2RG gene in HSCs from a patient with SCID-X1. The CRISPR/Cas system was similarly applied in HSCs for the first time to correct the mutation in the β-globin gene responsible for Sickle Cell Disease (SCD) ( Lattanzi et al, 2021 ). Here, precise correction of the disease-causing mutation was performed with similar evidence of long-term engraftment in mice and reconstitution of functional β-globin ( Mohrin et al, 2010 ).…”

“…Single base pair correction has previously been shown in CD34 + HSCs and is particularly advanced for Sickle Cell Disease ( Magis et al, 2019 ; Lattanzi et al, 2021 ) This disease is one of the most prevalent genetic disorders and is caused by a single nucleotide substitution that changes a glutamic acid into valine. A direct base pair correction approach is highly desirable since no major perturbations are made to the gene and all regulatory elements of the promoter, untranslated regions, splice elements, and introns are maintained ( Figure 5A ).…”

“…This could be used to replace single exons, multiple exons, and potentially all exons ( Figures 5B–D ). Such strategies have been used preclinically before for multiple hematopoietic diseases including β-thalassemia ( Lattanzi et al, 2021 ), X-SCID ( Cromer et al, 2018 ; Pavel-Dinu et al, 2019 ), hyper-IgM syndrome ( Hubbard et al, 2016 ; Kuo et al, 2018 ; Schiroli et al, 2019 ), and X-CGD ( De Ravin et al, 2016 ; DeWitt et al, 2016 ). Since AAV is the preferred vector for delivery of the repair template, DOCK8 represents a particular challenge due to its large ORF size of 6.3 kb.…”

Rapid genome editing by CRISPR-Cas9-POLD3 fusion

“…HDR can be promoted by increasing the local concentration of the repair template ( Savic et al, 2018 ) and by general NHEJ pathway inhibition ( Maruyama et al, 2015 , Robert et al, 2015 , Riesenberg and Maricic, 2018 ), which, however, often negatively affects the cell viability and fitness ( Gu et al, 1997 , O’Driscoll et al, 2001 ). In addition, recent studies show that providing the DNA repair template with long homology arms by a non-integrating rAAV6 can significantly increase HDR rates ( Wiebking et al, 2021 , Lattanzi et al, 2021 ). Overall, genome editing is most efficient in open chromatin ( Chen et al, 2017 ) and when Cas9 is bound to an actively transcribed DNA strand where the approaching polymerase can rapidly remove the complex ( Clarke et al, 2018 , Richardson et al, 2016 , Jones et al, 2017 ).…”