Non-Clinical Efficacy and Safety Studies on G1XCGD, a Lentiviral Vector for Ex Vivo Gene Therapy of X-Linked Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a debilitating primary immunodeficiency affecting phagocyte function due to the absence of nicotinamide dinucleotide phosphate (NADPH) oxidase activity. The vast majority of CGD patients in the Western world have mutations within the X-linked CYBB gene encoding for gp91phox (NOX2), the redox center of the NADPH oxidase complex (XCGD). Current treatments of XCGD are not entirely satisfactory, and prior attempts at autologous gene therapy using gammaretrovirus vectors did not provide long-term curative effects. A new strategy was developed based on the use of the lentiviral vector G1XCGD expressing high levels of the gp91phox transgene in myeloid cells. As a requisite for a clinical trial approval, standardized non-clinical studies were conducted in vitro and in mice in order to evaluate the pharmacodynamics and biosafety of the vector and the biodistribution of G1XCGD-transduced cells. Transduced CD34+ cells derived from XCGD patients engrafted and differentiated similarly to their non-transduced counterparts in xenograft mouse models and generated therapeutically relevant levels of NADPH activity in myeloid cells expressing gp91phox. Expression of functional gp91phox in hematopoietic cells did not affect their homing properties, which engrafted at high levels in mice. Extensive in vitro and in vivo genotoxicity studies found no evidence for adverse mutagenesis related to vector treatment. These studies paved the way for the approval of clinical trials in Europe and in the United States for the treatment of XCGD patients with G1XCGD gene-modified autologous hematopoietic cells.

X-linked Inhibitor of Apoptosis Complicated by Granulomatous Lymphocytic Interstitial Lung Disease (GLILD) and Granulomatous Hepatitis.

The X-linked inhibitor of apoptosis (XIAP) deficiency is a primary immunodeficiency characterized by Epstein-Barr virus (EBV)-driven hemophagocytic lymphohistiocytosis (HLH), splenomegaly, and colitis. Here, we present, for the first time, granulomatous hepatitis and granulomatous and lymphocytic interstitial lung disease (GLILD) as manifestations of XIAP deficiency. We report successful treatment of GLILD in XIAP deficiency with rituximab and azathioprine and discuss the role of XIAP deficiency in immune dysregulation.

How to use immunoglobulin levels in investigating immune deficiencies.

Children are often referred to immunologists for the evaluation of reduced serum immunoglobulins. Knowledge of the immunoglobulin levels in healthy children of different ages is necessary when estimating immunological deficiency states of various kinds. After the measurement of the serum levels of the three major isotypes, examination of the capacity of the child to form antibodies to several antigens is a reasonable next step in the evaluation. We can rely on vaccine responses to make the distinction between significant primary immunodeficiency diseases and transiently low immunoglobulin levels. On the other hand, normal values of IgM, IgG and IgA are not always enough to exclude a more serious condition. Regardless of immunoglobulin concentrations, if a child's history indicates that further evaluation is warranted, a complete humoral immunity study should be carried out, including IgG subclasses, specific antibody responses and identification of B lymphocyte populations.

Stem cell transplantation in severe congenital neutropenia: an analysis from the European Society for Blood and Marrow Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment of severe congenital neutropenia (SCN), but data on outcome are scarce. We report on the outcome of 136 SCN patients who underwent HSCT between 1990 and 2012 in European and Middle East centers. The 3-year overall survival (OS) was 82%, and transplant-related mortality (TRM) was 17%. In multivariate analysis, transplants performed under the age of 10 years, in recent years, and from HLA-matched related or unrelated donors were associated with a significantly better OS. Frequency of graft failure was 10%. Cumulative incidence (day +90) of acute graft-versus-host disease (GVHD) grade 2-4 was 21%. In multivariate analysis, HLA-matched related donor and prophylaxis with cyclosporine A and methotrexate were associated with lower occurrence of acute GVHD. Cumulative incidence (1 year) of chronic GVHD was 20%. No secondary malignancies occurred after a median follow-up of 4.6 years. These data show that the outcome of HSCT for SCN from HLA-matched donors, performed in recent years, in patients younger than 10 years is acceptable. Nevertheless, given the TRM, a careful selection of HSCT candidates should be undertaken.

The extended clinical phenotype of 64 patients with dedicator of cytokinesis 8 deficiency.

BACKGROUND: Mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency (CID) also classified as autosomal recessive (AR) hyper-IgE syndrome (HIES). Recognizing patients with CID/HIES is of clinical importance because of the difference in prognosis and management. OBJECTIVES: We sought to define the clinical features that distinguish DOCK8 deficiency from other forms of HIES and CIDs, study the mutational spectrum of DOCK8 deficiency, and report on the frequency of specific clinical findings. METHODS: Eighty-two patients from 60 families with CID and the phenotype of AR-HIES with (64 patients) and without (18 patients) DOCK8 mutations were studied. Support vector machines were used to compare clinical data from 35 patients with DOCK8 deficiency with those from 10 patients with AR-HIES without a DOCK8 mutation and 64 patients with signal transducer and activator of transcription 3 (STAT3) mutations. RESULTS: DOCK8-deficient patients had median IgE levels of 5201 IU, high eosinophil levels of usually at least 800/µL (92% of patients), and low IgM levels (62%). About 20% of patients were lymphopenic, mainly because of low CD4(+) and CD8(+) T-cell counts. Fewer than half of the patients tested produced normal specific antibody responses to recall antigens. Bacterial (84%), viral (78%), and fungal (70%) infections were frequently observed. Skin abscesses (60%) and allergies (73%) were common clinical problems. In contrast to STAT3 deficiency, there were few pneumatoceles, bone fractures, and teething problems. Mortality was high (34%). A combination of 5 clinical features was helpful in distinguishing patients with DOCK8 mutations from those with STAT3 mutations. CONCLUSIONS: DOCK8 deficiency is likely in patients with severe viral infections, allergies, and/or low IgM levels who have a diagnosis of HIES plus hypereosinophilia and upper respiratory tract infections in the absence of parenchymal lung abnormalities, retained primary teeth, and minimal trauma fractures.

Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency.

BACKGROUND: Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. OBJECTIVE: We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. METHODS: We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2'-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. RESULTS: The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 µmol/L (normal value, <1.5 µmol/L) and 2'-deoxyadenosine levels of 0.7, 2.7, and 2.4 µmol/L (normal value, <0.07 µmol/L); the mean levels of adenosine and 2'-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis. CONCLUSION: Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency.