PURPOSE OF THE STUDY.
Sickle cell disease (SCD) is caused by a single nucleotide mutation (A to T) in the β-globin (HBB) gene leading to a Glu6Val amino acid change. The purpose of this study is to show that this gene can be effectively edited in human hematopoietic stem and progenitor cells (HSPCs) using the CRISPR/Cas9 system and to develop a method to enable efficient engraftment of edited human cells in mice.
HSPCs from mobilized peripheral blood of healthy donors and SCD patients were used for gene editing ex vivo. Immune-deficient NSG mice were used as recipients of edited HSPCs.
Cas9 protein can generate double-strand breaks in DNA that can then be repaired by nonhomologous end joining or homologous recombination (HR) if a donor strand is provided. HSPCs were electroporated with Cas9 protein combined with sgRNA (Cas9 RNP) targeting the HBB gene. Cells were then infected with recombinant adeno-associated viral vectors of serotype 6 (rAAV6) carrying a donor sequence with a single nucleotide mutation (A to T) in the HBB gene and a green fluorescent protein (GFP) sequence or truncated nerve growth factor receptor (tNGFR) expression cassette. tNGFR is expressed at the surface of transfected cells and allows for enrichment of transduced cells by utilizing magnetic bead–based separation technology. Gene-edited HSPCs enriched for high expression levels of GFP or tNGFR were transplanted into NSG mice and reconstitution of human hematopoietic cells was evaluated. Glu6Val mutation was corrected in SCD patients’ HSPCs by using this same method.
On average, 29% of HSPCs that were electroporated with Cas9 RNP and provided with a GFP-tagged donor strand were positive for GFP expression. Mice transplanted with GFPhigh cells had a median of 90% GFP+ human cells at week 16 after transplant, with a proper distribution within the myeloid and lymphoid compartments. On-target integration was confirmed by sequencing GFP+ cells. Mice transplanted with bulk tNGFR+ cells showed 7.5% of edited cells 16 weeks after transplant, while mice transplanted with enriched tNGFR+ cells showed 10% to 75% of edited cells. HBB-targeted sickle cell patients’ HSPCs reverted an average of 50% of disease-causing variant alleles to wild type. When using an antisickling HBB cDNA-EF1α-tNGFR donor, 11% of tNGFR-positive cells were achieved. These cells were able to differentiate into erythroid cells in vitro. Expression of corrected β-globin was assessed by RT-qPCR. Erythrocytes differentiated from bulk tNGFR+ expressed 20% of corrected (HbAS3) out of total β-globin mRNA (HbAS3), and those differentiated from tNGFRhigh expressed 70% HbAS3 mRNA.
The HBB gene can be targeted by using CRISPR/Cas9 system to correct SCD-causing mutations in HPSCs. Introducing a tNGFR cassette allowed the enrichment of corrected cells. The methodology used for enrichment allowed a fivefold increase in the engraftment of gene-edited cells. SCD patients’ HSPCs can be corrected using this strategy, and edited cells are able to differentiate into erythrocytes that express adult β-globin.
This is a preclinical study in which the authors show effective gene editing of the HBB gene in HSPCs and efficient engraftment of these cells in a mouse model. CRISPR/Cas9 gene editing of HSPCs enables the replacement of a disease-causing mutation with a wild-type sequence integrated in the genome under the physiologic promoter, therefore avoiding complications of other methods for gene therapy. The strategy presented for enrichment and expansion of edited HSPCs is particularly exciting, as these strategies may be required to optimize gene editing as a therapeutic option. This study not only sets the ground for gene therapy for SCD but also for other congenital hematologic and immune diseases.
- Copyright © 2017 by the American Academy of Pediatrics