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Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in WASp, a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients develop autoimmunity due to a breakdown in T and B cell tolerance. Moreover, excessive production of type-I interferon by plasmacytoid DCs contribute to autoimmune signs, however, the factors that triggers excessive innate activation have not been defined.

BACKGROUND: Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients. OBJECTIVE: We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction. METHODS: We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis. RESULTS: We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up. CONCLUSIONS: Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.

Background Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid-specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination-activating gene ( RAG ) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor. Objective We sought to determine the efficacy of lentiviral vector (LV)–mediated GT in the murine model of OS (Rag2 R229Q/R229Q ) in correcting immunodeficiency and autoimmunity. Methods Lineage-negative cells from mice with OS were transduced with an LV encoding the human RAG2 gene and injected into irradiated recipients with OS. Control mice underwent transplantation with wild-type or OS-untransduced lineage-negative cells. Immunophenotyping, T-dependent and T-independent antigen challenge, immune spectratyping, autoantibody detection, and detailed tissue immunohistochemical analyses were performed. Results LV-mediated GT allowed immunologic reconstitution, although it was suboptimal compared with that seen in wild-type bone marrow (BM)−transplanted OS mice in peripheral blood and hematopoietic organs, such as the BM, thymus, and spleen. We observed in vivo variability in the efficacy of GT correlating with the levels of transduction achieved. Immunoglobulin levels and T-cell repertoire normalized, and gene-corrected mice responded properly to challenges in vivo . Autoimmune manifestations, such as skin infiltration and autoantibodies, dramatically improved in GT mice with a vector copy number/genome higher than 1 in the BM and 2 in the thymus. Conclusions Our data show that LV-mediated GT for patients with OS significantly ameliorates the immunodeficiency, even in an inflammatory environment.

Wiskott–Aldrich syndrome (WAS) is a primary immunodeficiency caused by mutations in the gene encoding the hematopoietic-specific WAS protein (WASp). WAS is frequently associated with autoimmunity, indicating a critical role of WASp in maintenance of tolerance. The role of B cells in the induction of autoreactive immune responses in WAS has been investigated in several settings, but the mechanisms leading to the development of autoimmune manifestations have been difficult to evaluate in the mouse models of the disease that do not spontaneously develop autoimmunity. We performed an extensive characterization of Was−/− mice that provided evidence of the potential alteration in B cell selection, because of the presence of autoantibodies against double-stranded DNA, platelets, and tissue antigens. To uncover the mechanisms leading to the activation of the potentially autoreactive B cells in Was−/− mice, we performed in vivo chronic stimulations with toll-like receptors agonists (LPS and CpG) and apoptotic cells or infection with lymphocytic choriomeningitis virus. All treatments led to increased production of autoantibodies, increased proteinuria, and kidney tissue damage in Was−/− mice. These findings demonstrate that a lower clearance of pathogens and/or self-antigens and the resulting chronic inflammatory state could cause B cell tolerance breakdown leading to autoimmunity in WAS.

Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X‐linked hyper‐IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one‐size‐fits‐all editing strategy for effective T‐cell correction, selection, and depletion and investigated the therapeutic potential of T‐cell and HSPC therapies in the HIGM1 mouse model. Edited patients’ derived CD4 T cells restored physiologically regulated CD40L expression and contact‐dependent B‐cell helper function. Adoptive transfer of wild‐type T cells into conditioned HIGM1 mice rescued antigen‐specific IgG responses and protected mice from a disease‐relevant pathogen. We then obtained ~ 25% CD40LG editing in long‐term repopulating human HSPC. Transplanting such proportion of wild‐type HSPC in HIGM1 mice rescued immune functions similarly to T‐cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T‐cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits., Here we report a comprehensive set of preclinical studies, performed both in vitro on X‐linked hyper‐IgM syndrome (HIGM1) patient‐derived cells and in vivo in HIGM1 mice, which uncovers crucial guiding principles towards clinical translation of CD40LG targeted gene correction in T cells or hematopoietic stem cells (HSC) for the treatment of HIGM1.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by reduced or absent expression of the WAS protein (WASP). WAS patients are affected by microthrombocytopenia, recurrent infections, eczema, autoimmune diseases, and malignancies. Although immune deficiency has been proposed to play a role in tumor pathogenesis, there is little evidence on the correlation between immune cell defects and tumor susceptibility. Taking advantage of a tumor-prone model, we show that the lack of WASP induces early tumor onset because of defective immune surveillance. Consistently, the B16 melanoma model shows that tumor growth and the number of lung metastases are increased in the absence of WASP. We then investigated the in vivo contribution of Was(-/-) NK cells and DCs in controlling B16 melanoma development. We found fewer B16 metastases developed in the lungs of Was(-/-) mice that had received WT NK cells as compared with mice bearing Was(-/-) NK cells. Furthermore, we demonstrated that Was(-/-) DCs were less efficient in inducing NK-cell activation in vitro and in vivo. In summary, for the first time, we demonstrate in in vivo models that WASP deficiency affects resistance to tumor and causes impairment in the antitumor capacity of NK cells and DCs.

Next-Generation Gene Therapy Few disciplines in contemporary clinical research have experienced the high expectations directed at the gene therapy field. However, gene therapy has been challenging to translate to the clinic, often because the therapeutic gene is expressed at insufficient levels in the patient or because the gene delivery vector integrates near protooncogenes, which can cause leukemia (see the Perspective by Verma ). Biffi et al. ( 1233158 , published online 11 July) and Aiuti et al. ( 1233151 ; published online 11 July) report progress on both fronts in gene therapy trials of three patients with metachromatic leukodystrophy (MLD), a neurodegenerative disorder, and three patients with Wiskott-Aldrich syndrome (WAS), an immunodeficiency disorder. Optimized lentiviral vectors were used to introduce functional MLD or WAS genes into the patients' hematopoietic stem cells (HSCs) ex vivo, and the transduced cells were then infused back into the patients, who were then monitored for up to 2 years. In both trials, the patients showed stable engraftment of the transduced HSC and high expression levels of functional MLD or WAS genes. Encouragingly, there was no evidence of lentiviral vector integration near proto-oncogenes, and the gene therapy treatment halted disease progression in most patients. A longer follow-up period will be needed to further validate efficacy and safety.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by the defective expression of the WAS protein (WASP) in hematopoietic cells. It has been shown that dendritic cells (DCs) are functionally impaired in WAS patients and was(-/-) mice. We have previously demonstrated the efficacy and safety of a murine model of WAS gene therapy (GT), using stem cells transduced with a lentiviral vector (LV). The aim of this study was to investigate whether GT can correct DC defects in was(-/-) mice. As DCs expressing WASP were detected in the secondary lymphoid organs of the treated mice, we tested the in vitro and in vivo function of bone marrow-derived DCs (BMDCs). The BMDCs showed efficient in vitro uptake of latex beads and Salmonella typhimurium. When BMDCs from the treated mice (GT BMDCs) and the was(-/-) mice were injected into wild-type hosts, we found a higher number of cells that had migrated to the draining lymph nodes compared with mice injected with was(-/-) BMDCs. Finally, we found that ovalbumin (OVA)-pulsed GT BMDCs or vaccination of GT mice with anti-DEC205 OVA fusion protein can efficiently induce antigen-specific T-cell activation in vivo. These findings show that WAS GT significantly improves DC function, thus adding new evidence of the preclinical efficacy of LV-mediated WAS GT.

Aims The suppressors of cytokine signalling (SOCS) are identified inhibitors of cytokine and growth factor signalling that act via the Janus kinase (JAK) signal transducers and activators of transcription (STAT) pathways. Aberrant JAK/STAT signalling promotes progression from hypertrophy to heart failure. Little information is available concerning the role of SOCS in the transition from hypertrophy to heart failure. To this aim, we investigated the effects of SOCS1 overexpression obtained by in vivo adeno-associated gene transfer using an aortopulmonary cross-clamping technique in a chronic pressure-overload cardiac rat model. Methods and results Rats were randomized into four groups: sham-operated ( n = 18), aortic banding (AB) ( n = 18), AB + viral vector encoding for haemoagglutinin (AB + HA, n = 16), and AB + viral vector encoding for SOCS1 (AB + SOCS1, n = 18). Echocardiographic and haemodynamic measurements were performed 15 weeks after banding. While SOCS3 was upregulated during the hypertrophic phase, SOCS1 transcript levels increased significantly between 15 and 20 weeks. Remodelling was markedly worse in AB + SOCS1, showed larger left ventricular internal dimensions (+16%), higher end-diastolic pressures (+57%) and wall stress (+45%), and reduced fractional shortening (−32%) compared with AB + HA; apoptotic rate was increased three­fold and the gp130 pathway was inhibited. Ex vivo experiments showed that mechanical stretch upregulated SOCS1 expression, which was in turn attenuated by tumour necrosis factor-α (TNF-α) inhibition. Conclusion Enhanced SOCS1 myocardial signalling is associated with accelerated transition from hypertrophy to failure in an established model of pressure overload. SOCS1 may represent an attractive target for the prevention of heart failure progression.

Summary. Background: IGF-1 is a potent mitogen for vascular smooth muscle cells, but exerts protective effects on endothelial cells that may trigger antiatherogenic mechanisms. Objectives: This study was designed to test the hypothesis that an IGF-1 excess following arterial injury prevents neointima formation and vascular stenosis. Methods: Rats were subjected to carotid balloon injury and treated with IGF-1 (1.2 mg kg−1 per die) or saline for 10 days. Results: In IGF-1 treated animals, high tissue levels of eNOS, Akt and its phosphorylated form were found, confirming activation of IGF-1-dependent signaling pathways. IGF-1 markedly reduced neointima formation and post-injury arterial stenosis. IGF-1 exerted proliferative and anti-apoptotic effects in the media of injured carotids, but inhibited mitotic activity and induced apoptosis in the neointima. Furthermore, IGF-1 stimulated mobilization of progenitor endothelial cells and re-endothelialization of the injured arteries. L-NAME administration inhibited IGF-1 vasculoprotective effects. Conclusions: IGF-1 attenuates post-injury carotid stenosis by exerting differential effects in the neointima and tunica media with regard to the key components of the response to injury. The data point to a novel role of IGF-1 as a potent vasculoprotective factor.