CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells

Abstract

The β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia, are caused by mutations in the β-globin gene (HBB) and affect millions of people worldwide. A curative strategy for the β-hemoglobinopathies would be ex vivo gene correction in patient-derived hematopoietic stem cells (HSCs) followed by autologous transplantation. Here we report the first CRISPR/Cas9 gene-editing platform for achieving homologous recombination (HR) at the HBB gene in HSCs by combining Cas9 ribonucleoproteins and rAAV6 HR donor delivery. Notably, we devise an enrichment paradigm to purify a population of HSPCs with >90% targeted integration. We also show efficient correction of the SCD-causing E6V mutation in patient-derived HSPCs that after differentiation into erythrocytes, express adult β-globin (HbA) mRNA, confirming intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting HSCs by HR at the HBB locus to advance the development of next generation therapies for β-hemoglobinopathies.

Evolving Gene Therapy in Primary Immunodeficiency

“…116 Although functional correction through editing has now been demonstrated in induced pluripotent stem cells (iPSCs) and human HSC/Ps, it still probably remains limited by efficiency in true HSCs, which is required for sustained clinical effect in many PIDs. 117 In contrast, efficiency of editing in mature T cells can be quite high as demonstrated for XHIGM, although the challenge here will be to engraft sufficient numbers of T cells for long-term effect. These strategies are exciting, but some careful technology development is required before they are clinically applicable.…”

A chance to cut (the genome) is a chance to cure

“…However, such approaches are challenging to perform in bulk HSC populations, where the majority of genome editing events result in gene disruption. 3,4 This has motivated the search for alternative strategies that take advantage of the high frequency of genomic disruption events. For example, an erythroid enhancer of the key regulator of HbF, BCL11A, 5 has been a target of potential genome editing-based approaches.…”

CRISPR/Cas9-Mediated Genome Nano-Surgery: An Update

“…The ex-vivo delivery can also be performed by obtaining adult stem cells or fibroblasts from patients to be cultured and reprogrammed, and then to be corrected by CRISPR/Cas9 and eventually reintroduced to the patient. This approach circumvents some difficulties in delivering these novel geneediting therapies to the right tissues, such as delivering CRISPR/Cas9 to target β-globin gene for treatment of β-thalassaemia [26].…”