Aaron B. Clark

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

XMEN disease, defined as "X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus infection and N-linked glycosylation defect," is a recently described primary immunodeficiency marked by defective T cells and natural killer (NK) cells. Unfortunately, a potentially curative hematopoietic stem cell transplantation is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for patients with XMEN using a CRISPR/Cas9 adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9 AAV-targeted gene editing (GE) is hampered by low engraftable gene-edited hematopoietic stem and progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1 glycosylation function in human NK and CD8+ T cells restored NK group 2 member D (NKG2D) expression and function in XMEN lymphocytes for potential treatment of infections, and it corrected HSPCs for long-term gene therapy, thus offering 2 efficient therapeutic options for XMEN poised for clinical translation.

Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation using a scalable, Good Manufacturing Practice-compliant system to restore protein expression and NADPH oxidase function. Dose-escalating clinical-scale in vivo studies in a nonhuman primate model verify the feasibility, safety, and persistence in peripheral blood of infusions of mRNA-transfected autologous granulocyte-enriched apheresis cells, supporting this novel therapeutic approach as a potential nonalloimmunizing adjunct treatment of intractable infections in CGD patients.