Src homology 3 (SH3) domains mediate specific protein‐protein interactions crucial for signal transduction and protein subcellular localization. Upon phagocyte stimulation, two SH3 domain‐containing cytosolic components of the NADPH oxidase, p47phox and p67phox, are recruited to the membrane where they interact with flavocytochrome b558 to form an activated microbicidal oxidase. Deletion analysis of p47phox and p67phox in transfected K562 cells demonstrated multiple SH3‐mediated interactions between p47phox and the transmembrane flavocytochrome b558 and also between the cytosolic components themselves. The core region of p47phox (residues 151–284), spanning both SH3 domains, was required for flavocytochrome‐dependent translocation and oxidase activity in whole cells. Furthermore, translocation of p67phox occurred through interactions of its N‐terminal domain (residues 1–246) with p47phox SH3 domains. Both of these interactions were promoted by PMA activation of cells and were influenced by the presence of other domains in both cytosolic factors. Deletion analysis also revealed a third SH3 domain‐mediated interaction involving the C‐termini of both cytosolic factors, which also promoted p67phox membrane translocation. These data provide evidence for a central role for p47phox in regulation of oxidase assembly through several SH3 domain interactions.

BACKGROUNDSevere combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODSWe treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTSOverall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONSTreatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.

Patients lacking functional adenosine deaminase activity suffer from severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy-GT). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a Phase II clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND-ADA gamma-retroviral vector (gRV) and infused following busulfan reduced intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8-11 years. Nine of ten patients have sufficient immune reconstitution to protect against serious infections, and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of nine evaluable patients with the highest gene marking and B cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells, and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID, but risks of genotoxicity with gRVs. (Clinicaltrials.gov #NCT00794508)