Lentiviral gene therapy rescues p47phox chronic granulomatous disease and the ability to fight Salmonella infection in mice

Abstract

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder characterised by recurrent and often life-threatening infections and hyperinflammation. It is caused by defects of the phagocytic NADPH oxidase, a multicomponent enzyme system responsible for effective pathogen killing. A phase I/II clinical trial of lentiviral gene therapy is underway for the most common form of CGD, X-linked, caused by mutations in the gp91phox subunit of the NADPH oxidase. We propose to use a similar strategy to tackle p47phox-deficient CGD, caused by mutations in NCF1, which encodes the p47phox cytosolic component of the enzymatic complex. We generated a pCCLCHIM-p47phox lentiviral vector, containing the chimeric Cathepsin G/FES myeloid promoter and a codon-optimised version of the human NCF1 cDNA. Here we show that transduction with the pCCLCHIM-p47phox vector efficiently restores p47phox expression and biochemical NADPH oxidase function in p47phox-deficient human and murine cells. We also tested the ability of our gene therapy approach to control infection by challenging p47phox-null mice with Salmonella Typhimurium, a leading cause of sepsis in CGD patients, and found that mice reconstituted with lentivirus-transduced hematopoietic stem cells had a reduced bacterial load compared with untreated mice. Overall, our results potentially support the clinical development of a gene therapy approach using the pCCLCHIM-p47phox vector.

Preclinical Optimization and Safety Studies of a New Lentiviral Gene Therapy for p47phox-Deficient Chronic Granulomatous Disease

“…2c ). Finally, we confirmed in this setting as previously shown 8 that the lentiviral vector drives p47 phox expression preferentially in granulocytes and monocytes ( Fig. 2d ).…”

“…The self-inactivating (SIN) lentiviral transfer plasmid pCCLCHIM-p47 phox , depicted in Supplementary Fig. S1 , is described in Schejtman et al 8 The vector was produced by the manufacturing department of Indiana University Cell and Gene Therapy Manufacturing facility (IU-CGTM) according to good manufacturing practice (GMP) and its titer was determined on HT29 cells by quantitative polymerase chain reaction (qPCR) as described in Supplementary Data .…”

“…Some variability between donors has already been reported by others possibly due to different levels of viral restriction factors (e.g., IFITM3) in CD34 + cells. 25 Based on our observations in animal models, 8 VCN between 2 and 3 resulted in >30% functional neutrophils. Of note, transduction of CD34 + HSPCs using the clinical grade LV.CHIM-p47 vector, resulted in two or more lentiviral copies per cell with all ranges of MOI in the presence of transduction enhancers.…”

“…We have previously reported on the efficacy of a lentiviral vector designed for the gene therapy of p47 phox CGD in a mouse model of p47 phox−/− upon challenge with the Salmonella Typhimurium pathogen, a prominent cause of septicemia in CGD patients. 8 The lentiviral transfer plasmid used in the study, pCCLCHIM-p47 phox , contains a myeloid internal promoter that drives expression preferentially in granulocytes mainly affected by CGD. 9 Similar lentiviral vectors, containing either the CYBB or the ITGB2 transgene are currently used in phase I/II clinical trials for the X-linked form of CGD (X-CGD) and for leukocyte adhesion deficiency type-1 (LAD-I), respectively (trial registry numbers NCT02234934/NCT01855685, NCT03812263/NCT03825783).…”

Gene therapy for primary immunodeficiency

“…71 A similar approach has been adapted to another form of CGD caused by mutations in the NCF1 gene leading to abnormal P47phox expression with proof of concept demonstrated in a murine model. 129 Clinical trials of this LV are anticipated to start in the near future.…”