Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy.We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32).Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy.Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available.Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.
Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34+ cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34+ cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34+ cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34+ cells gene therapy for the treatment of WAS. Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34+ cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34+ cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34+ cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34+ cells gene therapy for the treatment of WAS.
Sickle cell disease (SCD) is due to a mutation in the β-globin gene causing production of the toxic sickle hemoglobin (HbS; α2βS2). Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling β-globin (βAS) is a promising treatment; however, it is only partially effective, and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing βAS3-globin and an artificial microRNA (amiRNA) specifically downregulating βS-globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPCs by the bifunctional LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells, and effective correction of the sickling phenotype, outperforming βAS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile, and neither HSPC viability, engraftment, and multilineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD. Sickle cell disease (SCD) is due to a mutation in the β-globin gene causing production of the toxic sickle hemoglobin (HbS; α2βS2). Transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviral vectors (LVs) expressing an anti-sickling β-globin (βAS) is a promising treatment; however, it is only partially effective, and patients still present elevated HbS levels. Here, we developed a bifunctional LV expressing βAS3-globin and an artificial microRNA (amiRNA) specifically downregulating βS-globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPCs by the bifunctional LV led to a substantial decrease of βS-globin transcripts in HSPC-derived erythroid cells, a significant reduction of HbS+ red cells, and effective correction of the sickling phenotype, outperforming βAS gene addition and BCL11A gene silencing strategies. The bifunctional LV showed a standard integration profile, and neither HSPC viability, engraftment, and multilineage differentiation nor the erythroid transcriptome and miRNAome were affected by the treatment, confirming the safety of this therapeutic strategy. In conclusion, the combination of gene addition and gene silencing strategies can improve the efficacy of current LV-based therapeutic approaches without increasing the mutagenic vector load, thus representing a novel treatment for SCD.
Chronic Granulomatous Disease (CGD) is caused by defective NADPH oxidase function in phagocytes leading to increased susceptibility to fungal and bacterial infections. The majority of CGD is caused by mutations in the gp91phox gene. Gene therapy with hematopoietic stem cells (HSC) may represents a good alternative to allogenic haematopoietic stem cell transplantation. Past clinical trials for X-linked CGD with γ retroviral vectors resulted in transient clinical benefit but were limited by insertional oncogenesis and lack of persistent engraftment. To circumvent these issues we developed a strategy based on regulated, self-inactivating lentiviral vectors (LVs). We designed different gp91phox-expressing LVs: 1) PGK.gp91phox, in which gp91phox is driven by an ubiquitous cellular promoter; 2) MSP.gp91phox, to control the transgene expression at a transcriptional level using a myeloid specific promoter; 3) PGK.gp91phox_126T(2), in which we exploited the miRNA system, incorporating miR-126 target sequences, to prevent the transgene off-target expression in HSC; 4) MSP.gp91phox_126T(2), combining the posttranscriptional de-targeting with the MSP (dual regulated vector). These LVs were tested on human and murine HSC and in a mouse model of X-CGD. All vectors restored gp91phox expression and function in human X-CGD primary monocytes and differentiated myeloid cells. While unregulated LVs induced a transgene ectopic expression in CD34+ cells, transcriptionally and post-transcriptionally regulated LVs reduced this off-target expression while guaranteeing a good level of expression in differentiated cells. With the dual-regulated vector, we achieved high levels of myeloid-specific transgene expression, entirely sparing the most primitive CD34+CD38-CD90+ HSC compartment. X-CGD mice transplanted with all vectors engrafted and restored gp91phox expression, with 20-70% of granulocytes and monocytes expressing human gp91phox, persisting up to 10 months after gene therapy. MSP-driven vectors were superior in maintaining regulation during BM development. Oxidase activity in corrected granulocytes was superior using MSP-driven vectors as compared to PGK. Gene therapy-treated mice were then infected with S.aureus in order to test their ability to clear one of the most recurrent bacteria causing infections in CGD patients. All gene therapy treated mice were able to clear pulmonary infection in contrast with untreated X-CGD untreated mice. The dual-regulated LV represents a promising approach for further clinical development of gp91phox therapeutic vectors.
Background: Transfusion-dependent ß-thalassemia (TDT) is a disorder due to mutations in the gene encoding the ß-globin chain causing a reduced or absent production of haemoglobin A leading to severe anaemia and lifelong transfusion dependence. Gene therapy has now been accepted as a possible alternative cure to allogeneic bone marrow (BM) transplantation. Aims: We developed a gene therapy approach based on autologous mobilized hematopoietic stem cell transduced by lentiviral vector, expressing human ß-globin gene, administered by intrabone injection, following a myeloablative conditioning (NCT02453477). Methods and Results: Nine patients with severe TDT with different genotypes have been treated with a drug product with a median cell dose of 19.5x106 CD34+ cells/kg, a transduction efficiency from 38 to 77% and a median vector copy number/genome (VCN) in bulk CD34+ cells of 0.9 (range: 0.7–1.5). Overall, gene therapy was generally well-tolerated with no adverse events related to the investigational product. No severe infectious-related adverse events were reported, except for those related to neutropenia as expected after myeloablative conditioning. Polyclonal vector integrations profiles with no evidence of clonal dominance have been detected in all patients with the expected genomic distribution for lentiviral vectors. Clinical outcome showed a reduction of transfusion requirement both in frequency and volume in adult patients up to more than 50%. Among the pediatric patients, 4 out of 6 discontinued transfusions shortly after gene therapy and are transfusion-independent at the last follow-up (up to 75 months). A robust and persistent engraftment was observed in 7 out of 9 patients, with a marking of BM progenitors that, in engrafted patients, ranged between 25.3 and 79.8% and with a median VCN in CD34+ cells of 0.53 (range: 0.34–2.21). As a relevant target for transgene expression, BM erythroid cells were stably marked (VCN range 0.3 - 2.5). Summary and Conclusions: A longer follow-up will provide further results on long-term clinical efficacy and safety of this approach. References 1. Marktel et al, Nat Med 2019; 25(2):234
