Ex vivo gene addition into CD34+ hematopoietic stem and progenitor cells (HSPC) followed by autologous transplantation has proven a safe and efficacious therapy for immunodeficiencies, storage diseases and hemoglobinopathies. According to xenograft models and population size estimates of vector-marked cells in gene therapy-treated patients, less than 0.01% of infused CD34+ cells drive long-term (LT) repopulation. Advances in clinical-grade cell sorting technology may make HSC-enriched CD34+ subpopulations accessible for gene therapy, with advantages in terms of lentiviral vector (LV) cost, safety (lowering of integration load) and, potentially, efficacy. By differentially marking mobilized peripheral blood (mPB) CD34 subpopulations distinguished by increasing levels of CD38 expression in order to quantitatively assess their hematopoietic output in an NSG xenograft model over 6 months, we previously mapped most (>90%) LT repopulating capacity to CD34+CD38- cells (lowest 10% CD38 staining), while CD34+CD38int cells drove short-term (ST) reconstitution during the first 2 months after transplantation. We now characterize these subpopulations in terms of CD90 expression, a marker, which has been used in conjunction with CD34 for HSC purification in past clinical trials. CD34+ mPB cells were sorted into CD38-90+ (5% of CD34+), CD38-90- (5%), CD38+90+ (30%) and CD38+90- (60%) fractions and exhaustively transduced with GFP-, OFP-, BFP- and mCherry-expressing LVs, respectively. Differentially marked subfractions were pooled maintaining their original proportions and transplanted into NSG mice. ST engraftment mainly came from CD38+ cells, with equal contribution from the CD90+ and CD90- compartment. LT engraftment was almost exclusively derived from CD34+CD38- cells, of which 70% came from CD90+ and 30% from CD90- cells. Hence, CD34+CD38- is a more sensitive and specific marker combination than CD34+CD90+ to purify LT-HSC. CD34+CD38- cells can be purified by a sequential bead-based selection (CD34 selection of CD38-depleted cells) potentially applicable to clinical practice. We show that CD34+CD38- cells can be efficiently transduced with clinical grade LVs using shortened ex vivo manipulation protocols, reaching similar gene marking levels as with the standard protocol currently used in clinical trials that comprises a double dose of LV. Transduction was stable for at least 5 months when serially measured in xenotransplanted mice, and mice showed multi-lineage hematopoiesis indistinguishable from CD34+ grafts. Based on these results, we are aiming towards clinical development of a new gene therapy protocol based on CD34+CD38-HSPC efficiently transduced with minimum ex vivo culture time (<36h). Our platform will substantially improve the efficacy, safety and feasibility of future ex vivo gene therapy studies.
Gene transfer into autologous hematopoietic stem progenitor cells (HSPCs) has the potential to cure monogenic inherited disorders caused by an altered development and/or function of the blood system, such as immune deficiencies and red blood cell and platelet disorders. Gene-corrected HSPCs and their progeny can also be exploited as cell vehicles to deliver molecules into the circulation and tissues, including the central nervous system. In this review, we focus on the progress of clinical development of medicinal products based on HSPCs engineered and modified by integrating viral vectors for the treatment of monogenic blood disorders and metabolic diseases. Two products have reached the stage of market approval in the EU, and more are foreseen to be approved in the near future. Despite these achievements, several challenges remain for HSPC gene therapy (HSPC-GT) precluding a wider application of this type of gene therapy to a wider set of diseases while gene-editing approaches are entering the clinical arena.
Wiskott-Aldrich Syndrome (WAS) is a severe X-linked primary immunodeficiency characterized by micro-thrombocytopenia, eczema and increased risk of infections, autoimmunity and tumors. Allogeneic hematopoietic stem cell (HSC) transplantation is a recognized curative treatment for WAS, but when a matched donor is not available, administration of WAS gene-corrected autologous HSCs represents a valid alternative therapeutic approach. Since alterations of WAS protein (WASp)-deficient B lymphocytes contribute to immunodeficiency and autoimmunity in WAS, we followed the B cell reconstitution in 4 WAS patients treated by lentiviral vector-gene therapy (GT) after a reduced-intensity conditioning regimen combined with anti-CD20 administration. We analyzed the B cell subset distribution in the bone marrow and peripheral blood by flow cytometry and the autoantibody profile by a high-throughput autoantigen microarray platform before and after GT. Lentiviral vector-transduced progenitor cells were able to repopulate the B cell compartment with a normal distribution of transitional, naïve and memory B cells. The reduction in the proportion of autoimmune-associated CD21low B cells and in the plasma levels of B cell-activating factor was associated with the decreased autoantibody production in WAS patients after GT. Then, we evaluated the functionality of B cell tolerance checkpoints by testing the reactivity of recombinant antibodies isolated from single B cells. Before GT, we found a decreased frequency of autoreactive new emigrant/transitional B cells in WAS patients, suggesting a hyperfunctional central B cell checkpoint in the absence of WASp. In contrast, high frequency of polyreactive and Hep2 reactive clones were found in mature naïve B cells of WAS patients, indicating a defective peripheral B cell checkpoint. Both central and peripheral B cell tolerance checkpoints were restored after GT, further supporting the qualitative efficacy of this treatment. In conclusion, WASp plays an important role in the regulation of B cell homeostasis and in the establishment of B cell tolerance in humans and lentiviral-mediated GT is able to ameliorate the functionality of B cell compartment contributing to the clinical and immunological improvement in WAS patients.
Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. Hematopoietic stem cell (HSC) transplantation from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Transplantation of genetically corrected autologous HSC could represent an alternative treatment, potentially applicable to all patients. In a murine model of WAS (WAS−/−), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupré, Marangoni, et al. Hum Gene Ther. 2006, 17]. The aim of the present study was to investigate the long-term efficacy and safety of our gene therapy approach in WAS−/− mice.Transduction of WAS−/− HSC was performed with two lentiviral vectors encoding human WASP under the control of either the human PGK promoter (pW) or the full-length (1.6Kb) human WAS autologous promoter (wW). Transduced HSC were transplanted into non-lethally irradiated WAS−/− mice. Mice were sacrificed either 7 months or 16 months after gene therapy. WASP expression was detected in approximately 50% and 40% of splenic T cells from mice treated with pW and wW, respectively. These expression levels were sufficient to fully correct TCR-driven proliferation and IL-2 production. Interestingly, the percentage of WASP-expressing cells was higher in FSChi and in CD44+ T cells, as compared to other T cell subsets. This finding suggests a selective advantage for gene corrected cells within activated and memory T cells. Additionally, WASP expression was detected in T cells, B cells and granulocytes isolated from peripheral blood, as well as in bone marrow CD45+ cells.The safety of the gene therapy treatment was evaluated by hemogram and histopathologic analysis of thymus, spleen, lymph nodes and bone marrow from gene therapy treated mice. In parallel, untransplanted age-matched WAS−/− and wild-type mice were tested as controls. Normal organ architecture and histology together with the absence of leukemias or lymphomas could be demonstrated in the gene therapy treated mice.In conclusion, we provide evidence of engraftment of WASP- expressing cells and correction of T cell defects without toxicity, up to 16 months after HSC gene therapy. Experiments aimed at investigating whether WAS gene therapy can correct the defects of platelets, B cells and dendritic cells, and restore normal in vivo immune response to pathogens are ongoing. Results from these studies will contribute to the design of a clinical trial for Wiskott- Aldrich Syndrome. Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. Hematopoietic stem cell (HSC) transplantation from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Transplantation of genetically corrected autologous HSC could represent an alternative treatment, potentially applicable to all patients. In a murine model of WAS (WAS−/−), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupré, Marangoni, et al. Hum Gene Ther. 2006, 17]. The aim of the present study was to investigate the long-term efficacy and safety of our gene therapy approach in WAS−/− mice. Transduction of WAS−/− HSC was performed with two lentiviral vectors encoding human WASP under the control of either the human PGK promoter (pW) or the full-length (1.6Kb) human WAS autologous promoter (wW). Transduced HSC were transplanted into non-lethally irradiated WAS−/− mice. Mice were sacrificed either 7 months or 16 months after gene therapy. WASP expression was detected in approximately 50% and 40% of splenic T cells from mice treated with pW and wW, respectively. These expression levels were sufficient to fully correct TCR-driven proliferation and IL-2 production. Interestingly, the percentage of WASP-expressing cells was higher in FSChi and in CD44+ T cells, as compared to other T cell subsets. This finding suggests a selective advantage for gene corrected cells within activated and memory T cells. Additionally, WASP expression was detected in T cells, B cells and granulocytes isolated from peripheral blood, as well as in bone marrow CD45+ cells. The safety of the gene therapy treatment was evaluated by hemogram and histopathologic analysis of thymus, spleen, lymph nodes and bone marrow from gene therapy treated mice. In parallel, untransplanted age-matched WAS−/− and wild-type mice were tested as controls. Normal organ architecture and histology together with the absence of leukemias or lymphomas could be demonstrated in the gene therapy treated mice. In conclusion, we provide evidence of engraftment of WASP- expressing cells and correction of T cell defects without toxicity, up to 16 months after HSC gene therapy. Experiments aimed at investigating whether WAS gene therapy can correct the defects of platelets, B cells and dendritic cells, and restore normal in vivo immune response to pathogens are ongoing. Results from these studies will contribute to the design of a clinical trial for Wiskott- Aldrich Syndrome.
Abstract Sickle cell disease (SCD) is due to a mutation in the β-globin ( HBB ) gene causing the production of the toxic sickle hemoglobin (HbS, a 2 β S 2 ). Transplantation of autologous hematopoietic stem/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 (amiR) specifically downregulating β S -globin expression with the aim of reducing HbS levels and favoring βAS3 incorporation into Hb tetramers. Efficient transduction of SCD HSPC by the bifunctional LV led to a substantial decrease of β S -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 the HSPC viability, engraftment and multi-lineage 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.
Event Abstract Back to Event Gene Therapy mediated by lentiviral vector transduced CD34+ cells for the treatment of Wiskott-Aldrich Syndrome Samantha Scaramuzza1, Francesca Ferrua2, Maria C. Castiello1, 3, Stefania Giannelli1, Luca Biasco1, Fabio Ciceri4, Maria Grazia Roncarolo1, 2, 3, Anna Villa1, 5, Luigi Naldini1, 3 and Alessandro Aiuti1, 2, 6* 1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Italy 2 Scientific Institute HS Raffaele, Pediatric Immunology, Italy 3 Università Vita-Salute San Raffaele, Italy 4 Scientific Institute HS Raffaele, Div. of Hematology, Italy 5 IRGB-CNR, Italy 6 University of Rome “Tor Vergata”, Italy Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, infections, autoimmunity and lymphomas. Gene therapy with ex vivo transduced hematopoietic stem cells could represent a valid therapeutic option to transplantation. We developed an approach based on a lentiviral vector (LV) encoding for WAS under the control of the homologous 1.6 kb WAS promoter. We designed a phase I/II clinical trial based on infusion of autologous transduced CD34+ cells derived from bone marrow (BM) or mobilized peripheral blood (PB) and reduced intensity conditioning. Transduction of clonogenic progenitors was highly efficient (94.4 ± 4.2%), with a mean vector copy number (VCN)/genome in bulk CD34+ cells of 2.7 ± 0.98. In the first 3 treated patients, at the latest follow up, robust multilineage engraftment was observed in BM (VCN in myeloid lineages: 0.36-0.78) including clonogenic progenitors (25.8-48.2%), and in PB cells (VCN range of 0.30-1.30 in myeloid cells). As expected, a selective advantage was observed in lymphoid lineages (VCN: 0.93-2.04 and WASP+ cells: 70.9 ± 4.8%). TCR repertoire, proliferative responses to anti-CD3, and NK cytotoxic ability were improved. WASP expression was also observed in the majority of platelets. All patients are currently clinically well and independent from platelet transfusions. In conclusion, the unprecedented level of gene transfer obtained with LV-WAS resulted in robust engraftment of transduced HSC even when combined to reduced intensity conditioning. The ongoing follow up will allow to establish the long-term safety and clinical efficacy of LV gene therapy for WAS. Keywords: Gene Therapy, immunodeficiency, Wiskott-Aldrich Syndrome, Lentiviral vectors, HSC Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Scaramuzza S, Ferrua F, Castiello MC, Giannelli S, Biasco L, Ciceri F, Roncarolo M, Villa A, Naldini L and Aiuti A (2013). Gene Therapy mediated by lentiviral vector transduced CD34+ cells for the treatment of Wiskott-Aldrich Syndrome. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00403 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Alessandro Aiuti, San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy, alessandro.aiuti@hsr.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Samantha Scaramuzza Francesca Ferrua Maria C Castiello Stefania Giannelli Luca Biasco Fabio Ciceri Maria Grazia Roncarolo Anna Villa Luigi Naldini Alessandro Aiuti Google Samantha Scaramuzza Francesca Ferrua Maria C Castiello Stefania Giannelli Luca Biasco Fabio Ciceri Maria Grazia Roncarolo Anna Villa Luigi Naldini Alessandro Aiuti Google Scholar Samantha Scaramuzza Francesca Ferrua Maria C Castiello Stefania Giannelli Luca Biasco Fabio Ciceri Maria Grazia Roncarolo Anna Villa Luigi Naldini Alessandro Aiuti PubMed Samantha Scaramuzza Francesca Ferrua Maria C Castiello Stefania Giannelli Luca Biasco Fabio Ciceri Maria Grazia Roncarolo Anna Villa Luigi Naldini Alessandro Aiuti Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.
Gene therapy, based on the transplantation of genetically corrected autologous hematopoietic stem cells (HSCs), has proven to be an effective therapeutic approach as an alternative to allogenic HSC transplantation for the cure of severe combined immunodeficiencies (SCID). In this article, the recent preclinical studies aiming towards gene therapy trials for the Wiskott–Aldrich syndrome (WAS), a life-threatening immunodeficiency characterized by infections, hemorrhages, autoimmune disorders and lymphomas, will be reviewed. An update of the safety and efficacy data obtained in studies performed in murine disease models and in cells from WAS patients will be presented. Based on these data and on the clinical results of the recent trials for SCID, the most critical issues regarding the implementation of a gene therapy approach for WAS will be discussed.
Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.
