Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4+ T-cells for the treatment of Hyper IgM1.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

Circulating hematopoietic stem/progenitor cell subsets contribute to human hematopoietic homeostasis.

ABSTRACT: In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPCs) are present in the peripheral blood, but their contribution to human hematopoiesis remain unsolved. By integrating advanced immunophenotyping, single-cell transcriptional and functional profiling, and integration site (IS) clonal tracking, we unveiled the biological properties and the transcriptional features of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPCs reduced in cell count over aging and are enriched for primitive, lymphoid, and erythroid subpopulations, showing preactivated transcriptional and functional state. Moreover, cHSPCs have low expression of multiple BM-retention molecules but maintain their homing potential after xenotransplantation. By generating a comprehensive human organ-resident HSPC data set based on single-cell RNA sequencing data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Notably, circulating multi-lymphoid progenitors are primed for seeding the thymus and actively contribute to T-cell production. Human clonal tracking data from patients receiving gene therapy (GT) also showed that cHSPCs connect distant BM niches and participate in steady-state hematopoietic production, with primitive cHSPCs having the highest recirculation capability to travel in and out of the BM. Finally, in case of hematopoietic impairment, cHSPCs composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis. GT patients' clinical trials were registered at ClinicalTrials.gov (NCT01515462 and NCT03837483) and EudraCT (2009-017346-32 and 2018-003842-18).

Wiskott-Aldrich syndrome: a study of 577 patients defines the genotype as a biomarker for disease severity and survival.

ABSTRACT: Wiskott-Aldrich syndrome (WAS) is a multifaceted monogenic disorder with a broad disease spectrum and variable disease severity and a variety of treatment options including allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT). No reliable biomarker exists to predict disease course and outcome for individual patients. A total of 577 patients with a WAS variant from 26 countries and a median follow-up of 8.9 years (range, 0.3-71.1), totaling 6118 patient-years, were included in this international retrospective study. Overall survival (OS) of the cohort (censored at HSCT or GT) was 82% (95% confidence interval, 78-87) at age 15 years and 70% (61-80) at 30 years. The type of variant was predictive of outcome: patients with a missense variant in exons 1 or 2 or with the intronic hot spot variant c.559+5G>A (class I variants) had a 15-year OS of 93% (89-98) and a 30-year OS of 91% (86-97), compared with 71% (62-81) and 48% (34-68) in patients with any other variant (class II; P < .0001). The cumulative incidence rates of disease-related complications such as severe bleeding (P = .007), life-threatening infection (P < .0001), and autoimmunity (P = .004) occurred significantly later in patients with a class I variant. The cumulative incidence of malignancy (P = .6) was not different between classes I and II. It confirms the spectrum of disease severity and quantifies the risk for specific disease-related complications. The class of the variant is a biomarker to predict the outcome for patients with WAS.

Hematopoietic Stem- and Progenitor-Cell Gene Therapy for Hurler Syndrome.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. METHODS: We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. RESULTS: We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. CONCLUSIONS: The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.).

Lentiviral haematopoietic stem-cell gene therapy for early-onset metachromatic leukodystrophy: long-term results from a non-randomised, open-label, phase 1/2 trial and expanded access.

BACKGROUND: Effective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD. METHODS: This study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182. FINDINGS: At the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64-7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3-42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6-12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9-82·3]) and early-juvenile MLD (42% [12·3-71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes. INTERPRETATION: Treatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy. FUNDING: Orchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline.

Follicular helper T cell signature of replicative exhaustion, apoptosis, and senescence in common variable immunodeficiency.

Common variable immunodeficiency (CVID) is the most frequent primary antibody deficiency whereby follicular helper T (Tfh) cells fail to establish productive responses with B cells in germinal centers. Here, we analyzed the frequency, phenotype, transcriptome, and function of circulating Tfh (cTfh) cells in CVID patients displaying autoimmunity as an additional phenotype. A group of patients showed a high frequency of cTfh1 cells and a prominent expression of PD-1 and ICOS as well as a cTfh mRNA signature consistent with highly activated, but exhausted, senescent, and apoptotic cells. Plasmatic CXCL13 levels were elevated in this group and positively correlated with cTfh1 cell frequency and PD-1 levels. Monoallelic variants in RTEL1, a telomere length- and DNA repair-related gene, were identified in four patients belonging to this group. Their blood lymphocytes showed shortened telomeres, while their cTfh were more prone to apoptosis. These data point toward a novel pathogenetic mechanism in CVID, whereby alterations in DNA repair and telomere elongation might predispose to antibody deficiency. A Th1, highly activated but exhausted and apoptotic cTfh phenotype was associated with this form of CVID.

Hematopoietic cell transplantation in severe combined immunodeficiency: The SCETIDE 2006-2014 European cohort.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for patients with severe combined immunodeficiency (SCID), a group of monogenic immune disorders with an otherwise fatal outcome. OBJECTIVE: We performed a comprehensive multicenter analysis of genotype-specific HSCT outcome, including detailed analysis of immune reconstitution (IR) and the predictive value for clinical outcome. METHODS: HSCT outcome was studied in 338 patients with genetically confirmed SCID who underwent transplantation in 2006-2014 and who were registered in the SCETIDE registry. In a representative subgroup of 152 patients, data on IR and long-term clinical outcome were analyzed. RESULTS: Two-year OS was similar with matched family and unrelated donors and better than mismatched donor HSCT (P < .001). The 2-year event-free survival (EFS) was similar in matched and mismatched unrelated donor and less favorable in mismatched related donor (MMRD) HSCT (P < .001). Genetic subgroups did not differ in 2-year OS (P = .1) and EFS (P = .073). In multivariate analysis, pretransplantation infections and use of MMRDs were associated with less favorable OS and EFS. With a median follow-up of 6.2 years (range, 2.0-11.8 years), 73 of 152 patients in the IR cohort were alive and well without Ig dependency. IL-2 receptor gamma chain/Janus kinase 3/IL-7 receptor-deficient SCID, myeloablative conditioning, matched donor HSCT, and naive CD4 T lymphocytes >0.5 × 10e3/µL at +1 year were identified as independent predictors of favorable clinical and immunologic outcome. CONCLUSION: Recent advances in HSCT in SCID patients have resulted in improved OS and EFS in all genotypes and donor types. To achieve a favorable long-term outcome, treatment strategies should aim for optimal naive CD4 T lymphocyte regeneration.

A Novel Assay in Whole Blood Demonstrates Restoration of Mitochondrial Activity in Phagocytes After Successful HSCT in Hyperinflamed X-Linked Chronic Granulomatous Disease.

X-linked chronic granulomatous disease is a rare disease caused by mutations in the CYBB gene. While more extensive knowledge is available on genetics, pathogenesis, and possible therapeutic options, mitochondrial activity and its implications on patient monitoring are still not well-characterized. We have developed a novel protocol to study mitochondrial activity on whole blood of XCGD patients before and after transplantation, as well as on XCGD carriers. Here we present results of these analyses and of the restoration of mitochondrial activity in hyperinflamed X-linked Chronic Granulomatous Disease after hematopoietic stem cell transplantation. Moreover, we show a strong direct correlation between mitochondrial activity, chimerism, and DHR monitored before and after transplantation and in XCGD carriers. In conclusion, based on these findings, we suggest testing this new ready-to-use marker to better characterize patients before and after treatment and to investigate disease expression in carriers.

Emapalumab treatment in an ADA-SCID patient with refractory hemophagocytic lymphohistiocytosis-related graft failure and disseminated bacillus Calmette-Guérin infection.

Emapalumab, a fully human anti-IFNγ monoclonal antibody, has been approved in the US as second-line treatment of primary hemophagocytic lymphohistiocytosis (HLH) patients and has shown promise in patients with graft failure (GF) requiring a second allogeneic hematopoietic stem cell transplantation (HSCT). The blockade of IFNγ activity may increase the risk of severe infections, including fatal mycobacteriosis. We report a case of secondary HLH-related GF in the context of HLA-haploidentical HSCT successfully treated with emapalumab in the presence of concomitant life-threatening infections, including disseminated tuberculosis (TB). A 4 years old girl with Adenosine Deaminase-Severe Combined Immunodeficiency complicated by disseminated TB came to our attention for ex-vivo hematopoietic stem cell-gene therapy. After engraftment failure of gene corrected cells, she received two HLA-haploidentical T-cell depleted HSCT from the father, both failed due to GF related to concomitant multiple infections and secondary HLH. Emapalumab administration allowed to control HLH, as well as to prevent GF after a third haplo-HSCT from the mother. Remarkably, all infections improved with antimicrobial medications and disseminated TB did not show any reactivation. This seminal case supports emapalumab use for treatment of secondary HLH and prevention of GF in patients undergoing haplo-HSCT even in the presence of multiple infections, including TB.

Consensus of the Italian Primary Immunodeficiency Network on transition management from pediatric to adult care in patients affected with childhood-onset inborn errors of immunity.

Medical advances have dramatically improved the long-term prognosis of children and adolescents with inborn errors of immunity (IEIs). Transfer of the medical care of individuals with pediatric IEIs to adult facilities is also a complex task because of the large number of distinct disorders, which requires involvement of patients and both pediatric and adult care providers. To date, there is no consensus on the optimal pathway of the transitional care process and no specific data are available in the literature regarding patients with IEIs. We aimed to develop a consensus statement on the transition process to adult health care services for patients with IEIs. Physicians from major Italian Primary Immunodeficiency Network centers formulated and answered questions after examining the currently published literature on the transition from childhood to adulthood. The authors voted on each recommendation. The most frequent IEIs sharing common main clinical problems requiring full attention during the transitional phase were categorized into different groups of clinically related disorders. For each group of clinically related disorders, physicians from major Italian Primary Immunodeficiency Network institutions focused on selected clinical issues representing the clinical hallmark during early adulthood.

Urogenital Abnormalities in Adenosine Deaminase Deficiency.

BACKGROUND: Improved survival in ADA-SCID patients is revealing new aspects of the systemic disorder. Although increasing numbers of reports describe the systemic manifestations of adenosine deaminase deficiency, currently there are no studies in the literature evaluating genital development and pubertal progress in these patients. METHODS: We collected retrospective data on urogenital system and pubertal development of 86 ADA-SCID patients followed in the period 2000-2017 at the Great Ormond Street Hospital (UK) and 5 centers in Italy. In particular, we recorded clinical history and visits, and routine blood tests and ultrasound scans were performed as part of patients' follow-up. RESULTS AND DISCUSSION: We found a higher frequency of congenital and acquired undescended testes compared with healthy children (congenital, 22% in our sample, 0.5-4% described in healthy children; acquired, 16% in our sample, 1-3% in healthy children), mostly requiring orchidopexy. No urogenital abnormalities were noted in females. Spontaneous pubertal development occurred in the majority of female and male patients with a few cases of precocious or delayed puberty; no patient presented high FSH values. Neither ADA-SCID nor treatment performed (PEG-ADA, BMT, or GT) affected pubertal development or gonadic function. CONCLUSION: In summary, this report describes a high prevalence of cryptorchidism in a cohort of male ADA-SCID patients which could represent an additional systemic manifestation of ADA-SCID. Considering the impact urogenital and pubertal abnormalities can have on patients' quality of life, we feel it is essential to include urogenital evaluation in ADA-SCID patients to detect any abnormalities, initiate early treatment, and prevent long-term complications.

Lentiviral gene therapy corrects platelet phenotype and function in patients with Wiskott-Aldrich syndrome.

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.

Hematopoietic stem cell transplantation for CD40 ligand deficiency: Results from an EBMT/ESID-IEWP-SCETIDE-PIDTC study.

BACKGROUND: CD40 ligand (CD40L) deficiency, an X-linked primary immunodeficiency, causes recurrent sinopulmonary, Pneumocystis and Cryptosporidium species infections. Long-term survival with supportive therapy is poor. Currently, the only curative treatment is hematopoietic stem cell transplantation (HSCT). OBJECTIVE: We performed an international collaborative study to improve patients' management, aiming to individualize risk factors and determine optimal HSCT characteristics. METHODS: We retrospectively collected data on 130 patients who underwent HSCT for CD40L deficiency between 1993-2015. We analyzed outcome and variables' relevance with respect to survival and cure. RESULTS: Overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) were 78.2%, 58.1%, and 72.3% 5 years after HSCT. Results were better in transplantations performed in 2000 or later and in children less than 10 years old at the time of HSCT. Pre-existing organ damage negatively influenced outcome. Sclerosing cholangitis was the most important risk factor. After 2000, superior OS was achieved with matched donors. Use of myeloablative regimens and HSCT at 2 years or less from diagnosis associated with higher OS and DFS. EFS was best with matched sibling donors, myeloablative conditioning (MAC), and bone marrow-derived stem cells. Most rejections occurred after reduced-intensity or nonmyeloablative conditioning, which associated with poor donor cell engraftment. Mortality occurred mainly early after HSCT, predominantly from infections. Among survivors who ceased immunoglobulin replacement, T-lymphocyte chimerism was 50% or greater donor in 85.2%. CONCLUSION: HSCT is curative in patients with CD40L deficiency, with improved outcome if performed before organ damage development. MAC is associated with better OS, EFS, and DFS. Prospective studies are required to compare the risks of HSCT with those of lifelong supportive therapy.

Gene Therapy for Adenosine Deaminase Deficiency: A Comprehensive Evaluation of Short- and Medium-Term Safety.

Loss of adenosine deaminase activity leads to severe combined immunodeficiency (ADA-SCID); production and function of T, B, and natural killer (NK) cells are impaired. Gene therapy (GT) with an autologous CD34+-enriched cell fraction that contains CD34+ cells transduced with a retroviral vector encoding the human ADA cDNA sequence leads to immune reconstitution in most patients. Here, we report short- and medium-term safety analyses from 18 patients enrolled as part of single-arm, open-label studies or compassionate use programs. Survival was 100% with a median of 6.9 years follow-up (range, 2.3 to 13.4 years). Adverse events were mostly grade 1 or grade 2 and were reported by all 18 patients following GT. Thirty-nine serious adverse events (SAEs) were reported by 15 of 18 patients; no SAEs were considered related to GT. The most common adverse events reported post-GT include upper respiratory tract infection, gastroenteritis, rhinitis, bronchitis, oral candidiasis, cough, neutropenia, diarrhea, and pyrexia. Incidence rates for all of these events were highest during pre-treatment, treatment, and/or 3-month follow-up and then declined over medium-term follow-up. GT did not impact the incidence of neurologic/hearing impairments. No event indicative of leukemic transformation was reported.

Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency is a rare, autosomal-recessive systemic metabolic disease characterized by severe combined immunodeficiency (SCID). The treatment of choice for ADA-deficient SCID (ADA-SCID) is hematopoietic stem cell transplant from an HLA-matched sibling donor, although <25% of patients have such a donor available. Enzyme replacement therapy (ERT) partially and temporarily relieves immunodeficiency. We investigated the medium-term outcome of gene therapy (GT) in 18 patients with ADA-SCID for whom an HLA-identical family donor was not available; most were not responding well to ERT. Patients were treated with an autologous CD34(+)-enriched cell fraction that contained CD34(+) cells transduced with a retroviral vector encoding the human ADA complementary DNA sequence (GSK2696273) as part of single-arm, open-label studies or compassionate use programs. Overall survival was 100% over 2.3 to 13.4 years (median, 6.9 years). Gene-modified cells were stably present in multiple lineages throughout follow up. GT resulted in a sustained reduction in the severe infection rate from 1.17 events per person-year to 0.17 events per person-year (n = 17, patient 1 data not available). Immune reconstitution was demonstrated by normalization of T-cell subsets (CD3(+), CD4(+), and CD8(+)), evidence of thymopoiesis, and sustained T-cell proliferative capacity. B-cell function was evidenced by immunoglobulin production, decreased intravenous immunoglobulin use, and antibody response after vaccination. All 18 patients reported infections as adverse events; infections of respiratory and gastrointestinal tracts were reported most frequently. No events indicative of leukemic transformation were reported. Trial details were registered at www.clinicaltrials.gov as #NCT00598481.

B-cell reconstitution after lentiviral vector-mediated gene therapy in patients with Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene-corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. OBJECTIVE: Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. METHODS: We evaluated B-cell counts, B-cell subset distribution, B cell-activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. RESULTS: After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19(+)CD21(-)CD35(-) and CD21(low) B cells and a reduction in B cell-activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. CONCLUSIONS: We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic improvement in patients with WAS.