Zinc Finger Nucleases Targeting The β-Globin Locus Drive Efficient Correction Of The Sickle Mutation In CD34⁺ Cells

Despite major improvements in clinical care and advances in understanding of its complex pathophysiology, sickle cell disease (SCD) continues to be a significant cause of morbidity and early mortality. Allogeneic hematopoietic stem cell transplant (HSCT) can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and the immunological complications of graft rejection and graft-versus-host disease that can occur, especially for the more than 80% of patients who lack an HLA-identical sibling donor. Gene therapy could provide a way to cure SCD; however, the current approaches use integrating lentiviral vectors, and therefore carry a risk of insertional oncogenesis. An alternative approach is to use site-specific nucleases to correct the patients’ own cells, obviating the need for allogeneic HSCT and the use of randomly integrating vectors.

Zinc finger nucleases (ZFNs) offer a possible way to achieve successful gene therapy by site-specifically and permanently modifying the endogenous gene in hematopoietic stem cells (HSCs). These engineered nucleases create a site-specific, double strand break upon dimerization. If a homologous donor molecule is co-introduced which contains the normal β-globin sequence at the site of the sickle mutation, the cells may undergo homology-directed repair to correct the mutation and restore functional hemoglobin production. With this aim in mind, we have designed and tested ZFN pairs targeting the β-globin locus along with a donor template that restores the normal β-globin gene sequence while simultaneously introducing a silent base pair change that generates a restriction enzyme site for analysis. These components have led to high levels of site-specific base-pair modification in introducing the sickle mutation at the normal β-globin locus in K562 cells (upwards of 45%). Using electroporation, we delivered the ZFNs as mRNA to cord blood-derived (CB) CD34⁺ cells which resulted in up to 30% allelic disruption as measured by the Surveyor Nuclease assay. To achieve gene correction, the ZFNs were again delivered as mRNA and the donor template was delivered as an integrase defective lentiviral vector (IDLV). Based on pyrosequencing data, this delivery method resulted in up to 10% gene correction (the correct nucleotide replacing the sickle mutation in β-globin). Importantly, in the clinically relevant cell source, namely CD34⁺ cells isolated from SCD patient bone marrow, these gene modification frequencies were maintained, resulting in up to 7% correction using this multi-modal delivery strategy. These data set the stage for further investigations, including ongoing studies in a humanized mouse model. Efficient correction of the sickle mutation in HSC may provide an excellent stem cell source for autologous transplantation for SCD.