Efficient integration of a corrected gene into a patient's genome without mutating off-target sites is a major hurdle in gene therapy. In a paper published today in Genome Research, scientists have used CRISPR/Cas genome editing technology to seamlessly and efficiently correct disease-causing mutations in cells from patients with β-thalassemia.
β-thalassemia results from inherited DNA mutations in the hemoglobin beta (HBB) gene, resulting in reduced HBB expression in red blood cells and, in the most severe forms, anemia. The only established curative treatment is hematopoietic stem cell transplantation; however, this treatment requires a matched donor. Gene therapy, which delivers a corrected copy of a gene into patient cells, could bypass the need for a donor. Previous attempts using a virus to randomly insert a normal gene into the genome has been successful in one β-thalassemia patient, but the long-term effect of viral insertion is not yet known.
To correct HBB mutations directly in a patient's genome, researchers first generated induced pluripotent stem cells, or iPSCs, from skin cells of patients. The real breakthrough came when they applied CRISPR/Cas9 to precisely engineer a double strand DNA break at the HBB locus in these cells, allowing a donor plasmid with the corrected sites to be efficiently integrated, thus replacing the mutated sites. The donor plasmid also contained selectable markers to identify cells with corrected copies of the gene. These selectable markers were subsequently removed with transposase and a second round of selection, generating a seamless, corrected version of HBB in the patient's genome.