ALBERT

Beschreibung: Key PointsSurvival was 100% for 18 patients with ADA-SCID treated with genetically modified CD34+ cells (2.3-13.4 years follow up; median, 6.9 years). Long-term engraftment, immune reconstitution, and fewer severe infections were observed in 15 out of 18 patients without leukemic transformation.

Beschreibung: ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper–immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α−directed migration. Gene transfer of ARPC1B in patients’ T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

Beschreibung: A deeper understanding of T lymphocytes survival and differentiation potential in humans is paramount for the development of effective gene/cell therapies based on T-cell engineering. We here performed a comprehensive study of T-cells dynamics and plasticity in humans by a unique combination of phenotypic/functional studies and high-throughput integration sites (IS) analyses. We analyzed samples from hematopoietic stem cells (HSC) (n=10) or mature lymphocytes (PBL) gene therapy (GT) (n=4) treated ADA (adenosine deaminase) deficient-SCID patients. For comparative analyses, we also collected data from pediatric (n=19) and adult (n=52) healthy donors (HD), and from bone marrow transplanted patients (BMT) with primary immunodeficiencies (n=10, 4 with ADA-SCID). We observed that vector-positive CD62L+/CD45RA+ putative T naïve cells were detectable 12 years after last infusion of gene-corrected lymphocytes in peripheral blood of PBL-GT patients that lack the support of transduced lymphocytes precursors. We then unveiled that the vast majority of these CD62L+/CD45RA+ cells (80.3%) in PBL-GT patients could be actually classified phenotypically (CD95, IL2Rβ and IL7Rα surface expression) and functionally (IFNγ production and aCD3/aCD28 in vitro differentiation) as active long-lasting T memory stem cells (Tscm). The peculiar Tscm frequency found in PBL-GT patients was most likely due to a combinatorial in vitro and in vivo effect. Indeed, by a series of in vitro assays, we showed that Tscm relative enrichment in CD45RA+CD62L+ compartment have occurred during the in vitro manipulation of T cells before infusion. Additionally, we found higher-then-normal Tscm contribution among CD45RA+/CD62L+ cells even in ADA-SCID patients receiving HSC-GT and BMT, suggesting a role of disease background on in vivo Tscm persistence. Analyzing our cohorts of healthy donors and treated individuals we were able to further correlate Tscm contribution in vivo with age, conditioning regimen, disease background, cell source, and long-term T-cell reconstitution. One unique aspect of our study consisted in the opportunity to track Tscm clonal dynamics in vivo in humans since each gene-corrected cell infused in our GT patients is univocally and permanently tagged by a retroviral integration site.To perform in vivo molecular tracing of individual T-cell clones we sorted T naïve, Tscm, central memory and effector memory subtypes. We then collected from these subpopulations, by LAM-PCR+Illumina-Miseq sequencing, 2.584.137 integration sites (IS) sequences mapped to 1.746 unique chromosomal positions, corresponding to 910 integrations from 5 HSC-GT patients in vivo, 79 integrations from 2 PBL-GT samples of transduced cell products prior to infusion and 754 integrations from 4 PBL-GT patients in vivo. Firstly, to establish a relationship between precursors and terminally differentiated T cells we searched for the presence of identical insertion sites detected in multiple T-cell subtypes, applying stringent analytical filters for cross-contaminations. Strikingly, the level of shared integrations in each subtype was directly correlated to its stage of differentiation with Tscm, isolated from PBL-GT patients, showing the highest proportion of integration sites shared with the other T-cell subsets. Importantly, the results of the same analysis performed on HSC-GT patients were outstandingly coherent with the progressive developmental model of memory T-cell differentiation. We then assessed the survival of individual Tscm clones by performing a longitudinal IS analysis of different T-cell subtypes isolated from 3 PBL-GT patients over a 2 to 5 years timeframe up to 12 years after last infusion. We were able to formally prove the persistence of individual Tscm by re-capturing identical IS tagging specific Tscm clones in two independent timepoints in a 5- years window. Importantly, the same IS were also detected in multiple T-cell subtypes, representing the best indirect evidence that these clones were endowed with long-term precursor activity. We also documented, by IS sequencing reads, the long-term polyclonal composition of each subtype and we did not observe enrichment for IS flanking proto-oncogenes. Overall, this study validates, for the first time in humans, the safe and functional decade-long survival of engineered Tscm, paving the way for their future application in clinical settings. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Background: Wiskott-Aldrich syndrome (WAS) is a rare, X-linked, life-threatening primary immunodeficiency caused by mutations in the gene encoding the WAS protein (WASP). WASP-deficient immune cells have compromised immunological synapse formation, cell migration and cytotoxicity. Thus, WAS is characterized by development of recurrent or severe infections, eczema, and increased risk of autoimmunity and malignancies. In addition, WASP deficiency results in microthrombocytopenia, leading to severe bleeding episodes. When a suitable donor is available, WAS can be treated by hematopoietic stem cell transplant (HSCT), but HSCT can be impeded by complications such as graft versus host disease, rejection and autoimmunity. Importantly, HSCT may carry higher risks in older children (〉2-5 yrs) [Shin et al, 2012; Moratto et al, 2011]. An alternative approach is gene therapy (GT). We previously reported interim results of a Phase I/II clinical trial (NCT01515462) in 8 subjects treated with OTL-103, a drug product composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a lentiviral vector (LV) encoding human WASP cDNA under the control of the endogenous promoter [Ferrua et al, 2019]. We now report updated results on the safety and efficacy of OTL-103 in 17 subjects treated at San Raffaele Hospital as part of the same clinical trial or expanded access programs (EAP) with up to 8 yrs follow up (FU). Methods: NCT01515462: As described in Ferrua et al, 8 male subjects (mean age at GT: 4.8 yrs, range 1.1-12.4) were treated with OTL-103. The source of autologous CD34+ HSPCs was bone marrow (BM; n=5), mobilized peripheral blood (mPB; n=2) or both (n=1). As part of a reduced-intensity conditioning regimen, rituximab was given 22 days prior and busulfan + fludarabine during the week before OTL-103 infusion. At time of reporting, all subjects had ≥3 yrs FU (range: 3-8 yrs). EAP: 9 male subjects (11.2 yrs, 1.4-35.1) received identical treatment to subjects in the clinical trial; autologous CD34+ HSPCs source was mPB in all subjects. At time of reporting, subjects had a median of 1.4 yrs FU (range: 0.1-3.0 yrs) with 6/9 having ≥1 yr FU. Results: At last FU for all subjects (median: 3.0 yrs, range 0.1-8.0), overall survival was 94% (16/17). One EAP subject died 4.5 mo post-GT, due to deterioration of an underlying neurodegenerative condition considered unrelated to OTL-103 by investigator. To date, there have been no reports of insertional oncogenesis or replication-competent LV. While most subjects experienced adverse events (AEs) due to the reduced-intensity conditioning regimen (mainly mild or moderate), there were no reports of AEs related to OTL-103. Efficacy endpoints analyses were performed on surviving patients with ≥1 yr FU. Evidence of engraftment of genetically corrected HSPCs and LV+ colonies in BM was observed within 3 mo and persisted up to 8 yrs - the longest published FU of LV vector durability to date (Figure). WASP expression was restored after GT, shown by increases in the fraction of WASP+ lymphocytes and platelets (PLT) within 3 mo and maintained thereafter (Table). After GT, PLT counts improved, leading to a reduction of frequency and severity of bleeding events. Independence from PLT transfusions and absence of severe bleeding events were observed in all subjects by 9 mo FU (Table). Immune function improved; all evaluable patients discontinued immunoglobulin supplementation after GT (median time to discontinuation: 0.9 years after GT, range: 0.2-5 years). Furthermore, reduction in severe infection rate was observed post-GT, suggestive of immune reconstitution (Table). The decrease in bleeding events and severe infection rates occurred despite the integration of subjects into normal daily activities. Eczema progressively resolved or was reduced compared to baseline. Conclusions: This combined analysis of 17 subjects treated in a clinical trial or EAP with up to 8 yrs FU demonstrates that GT continues to be an effective treatment for WAS. All surviving subjects achieved high levels of multilineage engraftment, sustained restoration of WASP expression in lymphocytes and PLTs, improved PLT counts, and fewer bleeding events. A significant reduction in severe infection rate suggests reconstitution of immune function. Importantly, clinical benefit was also attained in older subjects (〉5 yrs), a group considered at higher risk when treated with allogeneic HSCT. Disclosures Jones: Orchard Therapeutics: Employment, Equity Ownership. Dott:Orchard Therapeutics: Employment, Equity Ownership. Naldini:Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

Beschreibung: Despite the recent introduction of mold-active antifungal prophylaxis (MAP), breakthrough invasive fungal infections (b-IFI) still represent a possible complication and a cause of morbidity and mortality in hematological patients and allogeneic hematopoietic stem-cell transplantation recipients (HSCT). Data on incidence and type of b-IFI are limited, although they are mainly caused by non-fumigatus Aspergillus and non-Aspergillus molds and seem to depend on specific antifungal prophylaxis and patients’ characteristics. Herein, we described the clinical presentation and management of two cases of rare b-IFI which recently occurred at our institution in patients undergoing HSCT and receiving MAP. The management of b-IFI is challenging due to the lack of data from prospective trials and high mortality rates. A thorough analysis of risk factors, ongoing antifungal prophylaxis, predisposing conditions and local epidemiology should drive the choice of antifungal treatments. Early broad-spectrum preemptive therapy with a lipid formulation of amphotericin-B, in combination with a different mold-active azole plus/minus terbinafine, is advisable. The therapy would cover against rare azole-susceptible and -resistant fungal strains, as well as atypical sites of infections. An aggressive diagnostic work-up is recommended for species identification and subsequent targeted therapy.