In this issue of Blood, Brault et al present exciting new data suggesting that a novel gene editing approach to the treatment of X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus (EBV) infection and N-linked glycosylation defect (XMEN) can restore magnesium transporter expression and natural killer (NK) group 2 member D (NKG2D) expression on CD8+ T cells and NK cells, thereby restoring their function.1
Patients with XMEN experience a combined immunodeficiency that leads to significant morbidity and early mortality. The syndrome is caused by mutations in the MAGT1 gene, located on the X chromosome at Xq21.1. Inherited in an X-linked recessive pattern, the syndrome is characterized by CD4 T-cell lymphopenia and associated humoral immune defects caused by poor T-cell help and viral infections, of which chronic EBV infection is the most problematic, leading to EBV lymphoproliferative disease.2 Consequently, patients often survive only into their third or fourth decade of life. Molecular defects in the magnesium transporter encoded by MAGT1 leads to a range of immunologic defects, including decreased expression of the NKG2D receptor on NK and CD8+ cytotoxic T cells. Abnormal NKG2D expression is thought to be a major contributor to the poor antiviral responses that are characteristic of the syndrome.3
A variety of approaches have been tried to control disease, enhance immune function, and improve outcomes, with only modest success. In patients with EBV lymphoproliferative disease, B-cell depletion therapy with rituximab has shown temporary efficacy, typically as a preparative step before hematopoietic cell transplantation (HCT). Immunoglobulin replacement therapy has been used to compensate for the hypogammaglobulinemia intrinsic to the disorder or induced by rituximab to decrease sinopulmonary infections, but this has had no effect on chronic EBV. Magnesium supplementation, particularly with magnesium threonate, seems to be a safe way to increase lymphocyte Mg++ levels, but NKG2D expression on CD8+ cytotoxic T cells and NK cells was not significantly improved and EBV viremia was not decreased.4 HCT has also been tried in some patients, particularly those with EBV lymphoproliferative disease, but survival has not been encouraging. At present, patients are left with few effective therapeutic options.5
Brault et al now present data on a novel gene editing approach that can restore magnesium transporter expression and NKG2D expression on CD8+ T cells and NK cells, restoring their function. The approach utilizes CRISPR to create a double-stranded DNA break within the first coding exon of the endogenous MAGT1 gene followed by insertion of a spliced, codon-optimized MAGT1 complementary DNA (cDNA) via homologous recombination using a repair template delivered by recombinant adeno-associated virus (rAAV). By placing the spliced cDNA near the initiation codon, expression of the transcript is controlled by the endogenous MAGT1 promoter, allowing appropriate tissue-specific expression. Cleverly, this approach also makes the therapy universal to virtually all patients with XMEN, regardless of the location of their mutation within the gene (excluding mutations that might affect the integrity of the gene promoter itself). The methodology also offers the potential of a 2-stage therapeutic approach where patient T cells could be edited to provide temporary control of EBV before administration of edited hematopoietic stem cells (HSCs), which will almost certainly require the use of a conditioning regimen to achieve significant levels of engraftment.
Two other advancements used by the study team enhance this approach, making it more innovative and likely to be successful therapeutically. The first improves gene editing efficiency by skewing double-strand break-repair mechanisms toward homology-directed repair. This is accomplished by transient expression of i53 to block 53BP1 accumulation at DNA breaks, thus inhibiting nonhomologous end joining. The second addresses a major problem encountered by virtually all laboratories working on gene editing in human HSCs, namely the poor engraftability of edited cells. The authors note that treatment of HSCs with the rAAV vector carrying the repair template by itself led to significant activation of the DNA damage response, reflected by increased phosphorylation of H2AX and decreased cell viability. By transiently expressing a humanized genetic suppressor element that inhibits TP53 activity during the gene editing process, they limited the DNA damage response, improved the viability of the edited cells, and achieved substantial improvements in edited HSC engraftment in a humanized murine model.
This unique combination of approaches may offer a viable opportunity for an XMEN cure that could prevent the severe outcomes and early death associated with this combined immunodeficiency syndrome.
Conflict-of-interest disclosure: The author declares no competing financial interests.