Search

The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

Precise engineered nuclease-mediated gene correction via homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the power to transform curative therapies for monogenic diseases of the immune system. Sickle cell disease (SCD) is one of the most common monogenic diseases, affecting millions of people worldwide. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment for patients with SCD; however, immunocompatibility issues, graft-versus-host disease, and graft rejection are major roadblocks for efficacious therapy. In theory, the ideal curative strategy for SCD and most monogenic immune diseases is ex vivo gene correction of patient-derived HSPCs followed by autologous HSCT of a highly purified targeted population to avoid possible complications of competition between unedited and gene-targeted HSPCs in vivo. By supplying a homologous GFP-expressing HBB donor via recombinant adeno-associated virus serotype 6 (rAAV6) in combination with a double strand break created by the CRISPR/Cas9 system, we achieved HR frequencies of 15-49% of HSPCs, and more importantly, identified a population of HBB-targeted HSPCs (HSPCHBB) through a log-fold MFI increase in chromosomal transgene expression. Fluorescence activated cell sorting (FACS) of this population revealed consistent GFP expression in >95% of cells over two weeks in vitro in multiple CD34+ donors isolated from either bone marrow, cord blood or peripheral blood. Single-cell FACS of HSPCHBB into methylcellulose led to both myeloid and lymphoid colony formation, and on-target PCR analysis revealed >95% of these HSPCHBB clones had either a mono or biallelic-targeting event. Notably, a fraction of HSPCHBB displayed the CD34+/CD38-/CD90+/CD45RA-immunophenotype, suggesting successful targeting of long-term repopulating hematopoietic stem cells (LT-HSCs). Furthermore, HSPCHBB displayed long-term engraftment in the bone marrow of immunodeficient NSG mice at 12 weeks post-transplant, where we identified that ~70% of human cells were GFP+ and also produced both myeloid (CD33+) and lymphoid (CD19+) cell types, implying that the HSPCHBB population contains true LT-HSCs that can repopulate a functional immune system. Altogether, these proof-of-concept studies showed that by combining a homologous rAAV6 donor, the CRISPR/Cas9 system, and FACS, it is feasible to generate and enrich a highly purified population of gene-corrected HSPCs that include LT-HSC potentials.