65. Long-Term Effects of Hematopoietic Stem Cell Gene Therapy in the Murine Model of Wiskott-Aldrich Syndrome: Persistence of Functional Correction of T Cells and Lack of Malignant Trasformation

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|[minus]|/|[minus]|), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupr|[eacute]|, 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|[minus]|/|[minus]| mice

WASP regulates suppressor activity of human and murine CD4+CD25+FOXP3+ natural regulatory T cells

A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4+CD25+FOXP3+ natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS−/− mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS−/− nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS−/− nTreg cells failed to proliferate and to produce transforming growth factor β upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS−/− nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS−/− nTreg cells showed reduced in vitro suppressor activity on both WT and WAS−/− effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4+ effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients

280 lentiviral mediated gene therapy restores b cell homeostasis and tolerance in wiskott aldrich syndrome patients

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, naive 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 naive 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

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 CD21low

Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34+ cells

<p>Abstract</p> <p>Background</p> <p>Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting.</p> <p>Objective</p> <p>To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method.</p> <p>Methods</p> <p>We cultured myeloma-positive CD34⁺PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34⁺cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, ΔNGFR). We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture.</p> <p>Results</p> <p>Overall recovery of CD34⁺cells after culture was 128.5%; ΔNGFR transduction rate was 28.8% for CD34⁺cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34⁺cells after ΔNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7–1.4 logs in tumor load after the CD34⁺cell selection, and up to 2.3 logs after culture and ΔNGFR selection.</p> <p>Conclusion</p> <p>We conclude that <it>ex-vivo </it>culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.</p

Wiskott-Aldrich Syndrome protein deficiency perturbs the homeostasis of B-cell compartment in humans

Wiskott-Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott-Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B-cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1α, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21low B-cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B-cell homeostasis thus adding a new layer of

Novel lentiviral vectors for gene therapy of sickle cell disease combining gene addition and gene silencing strategies

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