Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome

Introduction Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency characterized by eczema, thrombocytopenia, infections, and a high risk of developing autoimmunity and cancer. In a recent clinical trial, a γ-retroviral vector was used to introduce a functional WAS gene into autologous hematopoietic stem/progenitor cells (HSCs), followed by reinfusion of the gene-corrected HSCs into the patients. This strategy provided clinical benefit but resulted in expansion and malignant transformation of hematopoietic clones carrying vector insertions near oncogenes, thus increasing leukemia risk. We have developed a clinical protocol for WAS based on lentiviral vector (LV) gene transfer into HSCs. Analysis of common insertion sites (CIS) in gene therapy trials using lentiviral versus γ-retroviral vectors. Word clouds show the intensity of insertion sites clustering in each of the CIS genes (the larger the gene name, the larger the number of insertion sites within or in the proximity of that gene). The names of the CIS genes detected in both gene therapy trials are reported at the intersection between the circles. Analysis of common insertion sites (CIS) in gene therapy trials using lentiviral versus γ-retroviral vectors. Word clouds show the intensity of insertion sites clustering in each of the CIS genes (the larger the gene name, the larger the number of insertion sites within or in the proximity of that gene). The names of the CIS genes detected in both gene therapy trials are reported at the intersection between the circles. Methods Three patients with WAS were treated in a phase I/II clinical trial with gene-corrected HSCs after pretreatment with a reduced-intensity myeloablative regimen. Autologous CD34+ cells were transduced with an optimized LV carrying the WAS gene under the control of its endogenous promoter. Patients were monitored for up to 2.5 years after gene therapy by molecular, immunological, and clinical tests. We also investigated the genomic distribution of LV integration sites in the patients’ bone marrow and peripheral blood cell lineages. Results Administration of autologous HSCs transduced with LV at high efficiency (>90%) resulted in robust (25 to 50%), stable, and long-term engraftment of gene-corrected HSCs in the patients’ bone marrow. WAS protein expression was detected in myeloid cells at similar rates and in nearly all circulating platelets and lymphoid cells. In vitro T cell proliferative responses, natural killer cell cytotoxic activity, immune synapsis formation, and suppressive function of T regulatory cells were normalized. In all three patients, we observed improved platelet counts, protection from bleeding and severe infections, and resolution of eczema. Vector integration analyses on >35,000 unique insertion sites showed distinct waves of HSC clonal output, resulting in highly polyclonal multilineage hematopoietic reconstitution. In contrast to ©-retroviral gene therapy, our LV-based therapy did not induce in vivo selection of clones carrying integrations near oncogenes. Consistent with this, we did not see evidence of clonal expansions in the patients for up to 20 to 32 months after gene therapy. Discussion Our gene transfer protocol provided efficient stem cell transduction in vitro, resulting in robust and stable in vivo gene marking. WAS expression was restored to near-physiological levels in the patients, resulting in immunological and hematological improvement and clinical benefit. Clonal tracking of stem cell dynamics by vector insertions showed details of hematopoietic reconstitution after gene therapy. Comparison with clinical data from ©-retroviral gene therapy in the same disease setting strongly suggests that LV gene therapy offers safety advantages, but a longer follow-up time is needed for validation. Collectively, our findings support the use of LV gene therapy to treat patients with WAS and other hematological disorders. 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. Lentivirus-mediated gene therapy produces encouraging results in three children with a rare immunodeficiency disorder. [Also see Perspective by Verma] 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.