Malech, Harry L.

Abstract Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening infections and inflammatory conditions. Hematopoietic cell transplantation (HCT) is the definitive treatment for CGD, but questions remain regarding patient selection and impact of active disease on transplant outcomes. We performed a multi-institutional retrospective and prospective study of 391 patients with CGD treated either conventionally (non-HCT) enrolled from 2004 to 2018 or with HCT from 1996 to 2018. Median follow-up after HCT was 3.7 years with a 3-year overall survival of 82% and event-free survival of 69%. In a multivariate analysis, a Lansky/Karnofsky score <90 and use of HLA-mismatched donors negatively affected survival. Age, genotype, and oxidase status did not affect outcomes. Before HCT, patients had higher infection density, higher frequency of noninfectious lung and liver diseases, and more steroid use than conventionally treated patients; however, these issues did not adversely affect HCT survival. Presence of pre-HCT inflammatory conditions was associated with chronic graft-versus-host disease. Graft failure or receipt of a second HCT occurred in 17.6% of the patients and was associated with melphalan-based conditioning and/or early mixed chimerism. At 3 to 5 years after HCT, patients had improved growth and nutrition, resolved infections and inflammatory disease, and lower rates of antimicrobial prophylaxis or corticosteroid use compared with both their baseline and those of conventionally treated patients. HCT leads to durable resolution of CGD symptoms and lowers the burden of the disease. Patients with active infection or inflammation are candidates for transplants; HCT should be considered before the development of comorbidities that could affect performance status. This trial was registered at www.clinicaltrials.gov as #NCT02082353.

Abstract X-linked chronic granulomatous disease (X-CGD) is an immune deficiency that results from mutations in the gp91phox subunit of the phagocyte NADPH-oxidase. We have developed a modified RD114-pseudotyped simian immunodeficiency virus (SIVmac) vector encoding human gp91phox and report here that this vector efficiently targets peripheral blood-mobilized CD34+ hematopoietic stem cells (PBSCs) from X-CGD patients, correcting the oxidase deficient phenotype. The majority of lentiviral gene transfer studies have relied on human immunodeficiency virus (HIV) 1-derived vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope. Because HIV is a human pathogen, there are theoretical benefits to considering other types of lentivectors engineered from viruses less pathogenic for humans such as SIV-based vectors. In addition, the VSV-G envelope has some toxicity for cells, limiting the titers of vector that can be used. RD114/TR-pseudotyped SIV gp91phox encoding vector particles were transiently produced following transfection of 293T cells and concentrated by high-speed centrifugation achieving stock titers of 1.5 × 10E7 infectious units/ml. PBSCs from two patients with X-CGD (P1 and P2) were cultured and transduced (multiplicity of infection 2–3) overnight four times before transplantation into sublethally irradiated NOD/SCID mice on day 5 of culture. Ex vivo transduction efficiency was 40.5% and 46%, respectively. Cells maintained in culture showed a progressive decrease in marking over the first 9 days, after which marking stabilized at 22.7% (P1) and 27.1% (P2) out to day 26 of culture with an averaged copy number of 2.2–2.4 per transduced cell at that time. Compared to healthy control, 18–19% of myeloid colonies derived from patient CD34+ cells stained positive in the nitroblue tetrazolium (NBT) test. Liquid cell cultures at 20 days were analyzed for production of reactive oxidative species using the flow cytometry dihydrorhodamine 123 (DHR) assay. Cells from P1 demonstrated 15.3% and from P2 9.1% of the activity of healthy cells. At 6 weeks after transplant, analysis of mouse bone marrow (mBM) samples revealed human cell engraftment levels of 36.8–74% (CD45+ cells). Compared to analysis of mice transplanted with healthy human PBSCs, 10.5% (P1) and 7.3% (P2) of the myeloid cells (CD13+) developing from X-CGD CD34+ cells in the chimeric mBM expressed gp91phox transgene. Human CD34+ cells were isolated from mBM and grown in liquid culture or plated for colony assays. 2.1% (P1) and 1.2% (P2) of cultured cells were gp91phox positive at day 14 with an averaged copy number of 2.3–5.2 per marked cell. 4.8% and 3.6% of colonies derived from the same cells were positive in the NBT test. DHR assay at day 21 of culture demonstrated 2.7% (P1) and 2.0% (P2) of the activity of healthy cells. Linear amplification-mediated PCR retrieved multiple unique vector insertion sites in CD34+ cells from both patients isolated from chimeric mBM, without predominance of any one clone suggesting polyclonal marking. In conclusion, these data suggest that modified RD114-pseudotyped SIVmac-based vectors may be suitable for hematopoietic gene therapy for CGD and represent an alternative to HIV-1 based vectors. Studies are ongoing to further improve vector production titers to achieve higher transduction levels of PBSCs.

Abstract X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency (dysfunctional B cells; absence of T and NK cells) and early mortality, caused by mutations in the IL2RG gene encoding the common gamma chain (γc) of receptors for interleukins (IL)-2,-4,-7,-9,-15 and -21. The standard therapy for XSCID in infants is a T cell depleted bone marrow transplant with either none or very modest conditioning. While 5-yr survival is >95% with HLA-matched sibling donors, most patients receive a haploidentical graft from a parent, resulting in lower survival and less robust immune reconstitution that may be limited to only the T cell lineage. Ex vivo autologous stem cell gene therapy has emerged as an alternate treatment capable of achieving substantial to complete immune reconstitution in infants without a sibling donor. However, some children have developed lymphocytic leukemia, which appears in part to be related to vector insertional mutagenesis. The gammaretroviruses currently in use have potent enhancers in the LTR and have a predilection to insert at the 5′ end of genes. Self-inactivating (SIN) lentivirus vectors may be advantageous because they do not show this property and can be constructed with internal promotors that have less enhancer activity together with insertion of insulators. We have constructed SIN simian immunodeficiency viral vectors (SIVmac) encoding human γc (hγc), with or without a double copy chicken insulator core element in the 3′ LTR. In addition we have pseudotyped the vector with a chimeric RD114 envelope to enhance targeting of hematopoietic stem cells (HSC) and avoid the cytotoxicity of the VSV-G envelope traditionally used with lentivectors. We have previously shown that it is possible to correct a dog model of XSCID using a RD114-pseudotyped gammaretroviral vector encoding the dog γc for in vivo gene therapy. Unlike the mouse model of XSCID which lacks B cells, the dog model more closely resembles the phenotype in humans. We decided to use our in vivo dog model for preclinical testing of safety and efficacy of our SIV hγc vectors. Viral particles were produced by transient transfection of 293T cells with a 4-plasmid system and concentrated by high-speed centrifugation. 30 mls (average 2.4–3×107 viral particles in total) was injected IV into 2–5 day-old pups. Transgene marking in blood lymphocytes was detected as early as 2 wks after treatment, increased within the first 6–8 wks and became relatively stable thereafter. The absolute lymphocyte count was normalized in one dog (injected on the second day after birth) by wk 6 (4000, 76% hγc+), and improved in two other dogs (422, 29% hγc+ at 18 wks; 498, 27% hγc+, at 16 wks) which had received the same amount of virus over 2 days. Furthermore, up to 5% of the myeloid lineage showed gene marking at 10–18 wks after viral delivery, indicating that early committed progenitors or HSCs had been transduced. It is noteworthy that our finding that the hγc can improve the disease phenotype in XSCID dogs makes it an excellent large animal model for preclinical evaluation of vectors. In summary, we have demonstrated that in vivo delivery of SIV lentiviral vectors expressing hγc efficiently reconstitute the T-lymphoid compartment in the XSCID canine model. We plan to monitor these dogs closely for potential adverse events.