Gene Therapy For Wiskott-Aldrich Syndrome Research Articles

Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome

AbstractWiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein–expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10–producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Long-Term Follow-up Study after Lentiviral Hematopoietic Stem/Progenitor Cell Gene Therapy for Wiskott - Aldrich Syndrome

▪Wiskott Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency associated with thrombocytopenia, eczema, infectious, autoimmune complications, and lymphomas. Patients lacking an HLA-matched donor may benefit from an alternative therapeutic approach based on the infusion of autologous gene corrected CD34+ cells.We previously reported a non-randomised, open-label, phase 1/2 clinical study applying a lentiviral vector based gene therapy (GT) protocol in 7 paediatric patients with severe WAS (score ≥ 3/5) (S. Hacein-Bey Abina et al, JAMA 2015). One patient died 7 months after GT because of pre-existing severe opportunistic infections, as reported. Two additional patients have been treated since that initial report, with a follow-up of at least 4 years. We here present a comprehensive long-term study on 8 patients with a follow-up from 4 to 9 years (median 7.6). The safety and efficacy of the approach is thoroughly investigated, with a particular focus on the correction of thrombocytopenia and auto-immunity.A stable engraftment of genetically and functionally corrected lymphoid and myeloid cells was reached in all patients, with no serious treatment-associated adverse events or concerning clonal expansion. Corrected lymphoid cells displayed a selective advantage over time with increasing vector copy number (VCN) level. In turn, this led to (i) sustained expression of WAS protein (WASp) in the patients' cells and (ii) clinical resolution of severe eczema and susceptibility to recurrent infections. In line with these results, T-cell function was restored after GT, as shown by the recovery of immune synapse assembly and the normalization of naïve T cell numbers. The T-cell compartment was also reconstituted in the patient treated at the age of 30 years, suggesting that GT for WAS is a treatment option in adult patients.In parallel with the robustness of T-cell reconstitution a normalized B-cell compartment was observed after GT, as shown in particular by increasing levels of WASp + switched memory B cells over time and the age-matched levels of KRECs. Five patients out of 8 were able to discontinue Ig replacement therapy while achieving normal post-vaccination antibody titers.Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. After GT, a few autoimmune manifestations were observed: the persistence of lower extremity vasculitis (P2, very severe prior to GT), the new occurrence of nephrotic syndrome (P9), and the presence of anti-platelet antibodies (P2, P4, P7). The levels of circulating autoantibodies detected before GT (including ANA and vasculitis-related autoantibodies) normalized after treatment. Following GT, platelets were found to express sub-normal levels of WASp and to only partially augment their size. Platelet function studies indicated a partial correction of the platelet compartment achieved by GT, which may be sufficient to prevent occurrence of the hemorrhagic symptoms typical of WAS.Our results suggest that lentiviral GT provides sustained clinical benefits for patients with WAS. Overall clinical remission was observed in our patients despite very severe disease scores before GT. More efficacious and more reliable transduction protocols and conditioning regimen are likely to further improve outcomes, particularly with regard to platelet recovery, where the advantages of intrinsic correction are less apparent. DisclosuresBooth: Orchard Therapeutics: Consultancy, Honoraria; SOBI: Consultancy, Honoraria; Takeda: Honoraria; GSK: Honoraria; Rocket Pharmaceuticals, Inc.: Consultancy. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cavazzana: Smart Immune: Other: co-founder.

Hematopoietic Stem Cell Therapy for Wiskott-Aldrich Syndrome: Improved Outcome and Quality of Life.

The Wiskott–Aldrich syndrome (WAS) is an X-linked disorder caused by mutations in the WAS gene resulting in congenital thrombocytopenia, eczema, recurrent infections and an increased incidence of autoimmune diseases and malignancies. Without curative therapies, affected patients have diminished life expectancy and reduced quality of life. Since WAS protein (WASP) is constitutively expressed only in hematopoietic stem cell-derived lineages, hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are well suited to correct the hematologic and immunologic defects. Advances in high-resolution HLA typing, new techniques to prevent GvHD allowing the use of haploidentical donors, and the introduction of reduced intensity conditioning regimens with myeloablative features have increased overall survival (OS) to over 90%. The development of GT for WAS has provided basic knowledge into vector selection and random integration of various viral vectors into the genome, with the possibility of inducing leukemogenesis. After trials and errors, inactivating lentiviral vectors carrying the WAS gene were successfully evaluated in clinical trials, demonstrating cure of the disease except for insufficient resolution of the platelet defect. Thus, 50 years of clinical evaluation, genetic exploration and extensive clinical trials, a lethal syndrome has turned into a curable disorder.

Clonal tracking in gene therapy patients reveals a diversity of human hematopoietic differentiation programs

AbstractIn gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied 5 cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or β hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2000 to 50 000) was correlated with the number of HSPCs per kilogram infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of sparse sampling of the various cell types during the analytical procedure, contamination during cell isolation, and different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant, and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs and provided methods for analyzing these complex data.

Outcome of Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich Syndrome

Background Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and lymphoma. Gene therapy (GT) using autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplant for patients who lack well matched donors, avoiding graft-versus-host-disease. An initial experience with gene therapy using a γ-retroviral vector showed correction of hematological defects in 9/10 patients, but was aggravated by development of leukemia in 7 of them. We report the outcomes of a phase I/II clinical trial in which 5 WAS patients underwent GT using a self-inactivating lentiviral (SIN-LV) vector expressing the human WAS cDNA under the control of a 1.6kB fragment of the human WAS promoter. Subjects and Methods Five patients with severe WAS (clinical score 3-5) were enrolled at a median age of 1.8 years (1.4 - 8 years) at a single pediatric tertiary care center. WAS protein (WASP) was absent or markedly decreased in 2 and 3 subjects, respectively. Purified CD34+ cells from mobilized peripheral blood (n = 4) or both mobilized peripheral blood and bone marrow (n = 1) were transduced ex-vivo with the SIN-LV vector and re-infused after conditioning with busulfan (target AUC of 70-80 mg*h/L) and fludarabine (120mg/m2). The median dose of CD34+ cells infused was 9.8 x 106 cells/kg (6.3 - 24.9 x 106 cells/kg) with a mean vector copy number (VCN) of 1.7 copies/cell in CD34+ cells (0.54 - 3.37). In addition to eczema, thrombocytopenia and WAS-related infections in all patients, two subjects also had autoimmunity pre-GT, manifested as skin vasculitis and autoimmune cytopenias. Results All 5 subjects were alive and well at median follow-up of 4.8 years (2.5 - 5.9 years). Multi-lineage vector gene marking was sustained over time. All subjects had improvement or resolution of eczema and none have had any intercurrent severe infectious events. WASP expression measured by flow cytometry in T cells was increased over baseline in all patients, but remained below normal levels and correlated with VCN and cell dose received. Proliferation of T cells in response to anti-CD3, which was initially defective in 4/5 patients, improved post-GT. Humoral immune deficiency was also ameliorated, as evidenced by independence from Ig replacement and vaccine responses in those tested. All subjects remained platelet transfusion-free and none have had severe bleeding events. Platelet levels increased to >50 x 103 cells/uL in two patients with a VCN ≥2 in transduced stem cells and myeloid VCN ~1 copy/cell in neutrophils; the other 3 subjects sustained platelet counts <50 x 103 cells/uL. Cytoskeleton function was highly abnormal in myeloid cells pre-GT, as shown by the near absence of podosome formation in monocyte-derived dendritic cells. At 12 months post-GT, the % of podosome-forming cells was improved in all subjects, and reached the level of healthy controls in the 2 patients with highest VCN in myeloid cells. Both subjects with pre-existing autoimmunity had post-GT autoimmunity: patient 4 had a flare of autoimmune cytopenias at 18 months post-GT, and patient 5 developed refractory autoimmune hepatitis and hemolytic anemia at 8 months post-GT. While all subjects had WASP expression in lymphocytes, those with autoimmunity had poor recovery of T cells, Tregs, and transitional B cells at the time of clinical symptoms. IL-10 producing regulatory B cells were deficient pre-GT and recovered to varying degrees in all subjects. No severe GT-related adverse events have occurred to date. Replication-competent lentivirus was not detected. Analysis of integration site distributions in five subjects showed reconstitution to be highly polyclonal, with no clones expanded to >20% of the transgene-marked cell population. To date, there have been no malignancies reported, either related to GT or WAS itself. Conclusion In summary, our data confirm and extend the safety and efficacy of GT in correcting disease manifestations associated with WAS, as seen in other studies using SIN-LV. Higher VCN in the drug product and in transduced stem cells correlated with better reconstitution of platelets and myeloid function. In contrast to other groups, we found in our study that patients with poor lymphocyte reconstitution post-GT may be at risk of ongoing autoimmunity despite high-level gene marking. Disclosures London: ArQule, Inc: Consultancy; United Therapeutics: Consultancy. Despotovic:Novartis: Research Funding; Amgen: Research Funding; Dova: Honoraria. Forbes:Takeda: Consultancy. Galy:Genethon: Employment. Williams:Novartis: Membership on an entity's Board of Directors or advisory committees; bluebird bio: Other: License of certain IP relevant to hemoglobinopathies. Potential for future royalty/milestone income. Received payment in past through BCH institutional licensing agreement., Research Funding; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, potential for future royalty/milestone income, Research Funding; Alerion Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. OffLabel Disclosure: CliniMACS technology for CD34+ cell selection

Lentiviral Hematopoietic Stem and Progenitor Cell Gene Therapy for Wiskott-Aldrich Syndrome (WAS): Up to 8 Years of Follow up in 17 Subjects Treated Since 2010

Lentiviral Hematopoietic Stem and Progenitor Cell Gene Therapy for Wiskott-Aldrich Syndrome (WAS): Up to 8 Years of Follow up in 17 Subjects Treated Since 2010

Gene therapy for Wiskott-Aldrich syndrome: here to stay

Gene therapy for Wiskott-Aldrich syndrome: here to stay

Gene therapy for Wiskott-Aldrich syndrome in a severely affected adult

AbstractUntil recently, hematopoietic stem cell transplantation was the only curative option for Wiskott-Aldrich syndrome (WAS). The first attempts at gene therapy for WAS using a ϒ-retroviral vector improved immunological parameters substantially but were complicated by acute leukemia as a result of insertional mutagenesis in a high proportion of patients. More recently, treatment of children with a state-of-the-art self-inactivating lentiviral vector (LV-w1.6 WASp) has resulted in significant clinical benefit without inducing selection of clones harboring integrations near oncogenes. Here, we describe a case of a presplenectomized 30-year-old patient with severe WAS manifesting as cutaneous vasculitis, inflammatory arthropathy, intermittent polyclonal lymphoproliferation, and significant chronic kidney disease and requiring long-term immunosuppressive treatment. Following reduced-intensity conditioning, there was rapid engraftment and expansion of a polyclonal pool of transgene-positive functional T cells and sustained gene marking in myeloid and B-cell lineages up to 20 months of observation. The patient was able to discontinue immunosuppression and exogenous immunoglobulin support, with improvement in vasculitic disease and proinflammatory markers. Autologous gene therapy using a lentiviral vector is a viable strategy for adult WAS patients with severe chronic disease complications and for whom an allogeneic procedure could present an unacceptable risk. This trial was registered at www.clinicaltrials.gov as #NCT01347242.

Safety and Clinical Benefit of Lentiviral Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich Syndrome

Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, recurrent infections, eczema, autoimmunity and increased susceptibility to malignancies. Allogeneic hematopoietic stem cell transplantation (HSCT) is a recognized curative treatment for WAS, but is still associated with transplant-related complications and long-term morbidity, particularly in the absence of fully matched donors. In April 2010, we initiated a phase I/II clinical trial with hematopoietic stem cell (HSC) gene therapy (GT) for WAS. The investigational medicinal product (IMP) consists of autologous CD34+ HSC engineered with a lentiviral vector (LV) driving the expression of WAS cDNA from an endogenous 1.6 kb human WAS promoter (LV-WAS), infused after a reduced intensity conditioning (RIC) based on anti-CD20 mAb, targeted busulfan and fludarabine. We previously reported early follow up (FU) results from the first 3 patients (Aiuti et al., Science 2013). Seven patients (Zhu score ≥3) have now been treated at a median age of 1.9 years (1.1 - 11.1). As of May 2015, all patients are alive with a median FU of 3.2 years (0.7 - 5.0). CD34+ cell source was bone marrow (BM) (n=5), mobilized peripheral blood (MPB) (n=1) or both (n=1). IMP dose ranged between 7.0 and 14.1 x106 CD34+/kg, containing on average 94.4 ± 3.5% transduced clonogenic progenitors and a mean vector copy number (VCN)/genome in bulk CD34+ cells of 2.7 ± 0.8. No adverse reactions were observed after IMP infusion and RIC was well tolerated. Median duration of severe neutropenia was 19 days; granulocyte-colony stimulating factor was administered to 1 patient.In the first 6 treated patients with FU >2 years, we observed robust and persistent engraftment of gene corrected cells. At the most recent FU, transduced BM progenitors ranged between 20.7 and 59.7%, and LV-transduced cells were detected in multiple lineages, including PB granulocytes (VCN 0.34 - 0.93) and lymphocytes (VCN 1.18 - 2.73). WAS protein expression, measured by flow-cytometry, was detected in the majority of PB platelets [mean ± standard deviation (SD), 71.4 ± 14.0%], monocytes (63.3 ± 18.5%) and lymphocytes (78.9 ± 14.9%). Lymphocyte subset counts were normal in most patients and proliferative response to anti-CD3 mAb was in the normal range in all 6 patients.After immune reconstitution, a marked reduction in the annualized estimated rate of severe infections was observed, as compared with baseline (figure 1A). The first 6 treated patients discontinued anti-infective prophylaxis and no longer require a protected environment. Four patients stopped immunoglobulin supplementation and 2 of them developed specific antibodies after vaccination. Eczema resolved in 4 patients and remains mild in 2. No clinical manifestations of autoimmunity were observed ≥1 year after GT in accordance with improved B-cell development and decreased autoantibody production. All patients became platelet transfusion independent at a median of 4 months after GT (range: 1.0 - 8.7). Mean platelet counts progressively increased after treatment (mean ± SD: before GT, 13.4 ± 7.8 x109/l; 24-30 month FU, 45.8 ± 22.0 x109/l; 36-42 month FU, 57.0 ± 18.7 x109/l).The frequency and the severity of bleeding events decreased after the 1st year of FU. No severe bleedings were recorded after treatment (figure 1B).Quality of life improved in all patients after GT. From the 2nd year of FU, the number of hospitalizations for infections decreased and no hospitalizations due to bleeding were observed after treatment.The seventh patient treated, who received MPB derived CD34+ cells only, showed the fastest platelet recovery with the highest level of transduced myeloid cell engraftment, and is clinically well.No Serious Adverse Events (SAE) related to the IMP were observed. The most frequent SAE were related to infections (85%), occuring mainly during the 1st year of FU.Importantly, no evidence of abnormal clonal proliferations emerged after GT and the LV integration profile show a polyclonal pattern, with no skewing for proto-oncogenes.In conclusion, this updated report in 7 WAS patients show that GT is well tolerated and leads to a sustained clinical benefit. The high level of gene transfer obtained with LV-WAS results in robust engraftment of transduced HSC, even when combined with RIC. Prolonged FU will provide additional information on the long-term safety and clinical efficacy of this treatment. [Display omitted] DisclosuresVilla:Fondazione Telethon: Research Funding. Dott:GlaxoSmithKline: Consultancy. van Rossem:GlaxoSmithKline: Employment. Naldini:Salk Institute: Patents & Royalties: Lentiviral vectors; San Raffaele Telethon Institute: Patents & Royalties: Lentiviral vector technology; GlaxoSmithKline: Other: GSK licensed gene therapies developed at my Institute and the Institute receives milestone payments; Sangamo Biosciences: Research Funding; Biogen: Research Funding; Genenta Sciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Aiuti:GlaxoSmithKline (GSK): Other: PI of clinical trial which is financially sponsored by GSK; Fondazione Telethon: Research Funding.

289. Safe and Effective Gene Therapy for Wiskott – Aldrich Syndrome Using an Insulated Lentiviral Vector

Wiskott–Aldrich Syndrome (WAS) is a severe X-linked immunodeficiency disorder characterized by recurrent infection, thrombocytopenia, and autoimmunity resulting from mutations within the WAS gene. The encoded gene product, WAS protein (WASp), is a scaffold protein important for activation of the actin cytoskeleton downstream of multiple hematopoietic receptors. Development of safe and effective hematopoietic stem cell (HSC) gene therapy for WAS would provide a valuable treatment alternative to allogeneic bone marrow transplantation. Of critical importance for such an approach is the capacity to achieve sufficient WASp expression in relevant lineages to correct the multiple deficits, while avoiding development of autoimmune disease. We previously tested a panel of self-inactivating (SIN) lentiviral (LV) vectors expressing human WAS via alternative internal promoters and observed optimal expression and rescue using the modified γ-retroviral derived LTR, MND. To improve the potential safety and stability of expression, we incorporated a 650bp chimeric insulator from the chicken β-globin locus into this LV. Using in vitro assays the insulated LV exhibited minimal LMO2 transactivation and no evidence for immortalization of cultured HSC. Next, we generated a stable high-titer producer clone expressing this candidate clinical LV, CL20-i650–MND-WAS. Here, we evaluate the efficacy and safety of the insulted vs uninsulated LV (CL20-MND-WAS) in a large cohort of primary and secondary transplanted Was/- mice. Both vectors enabled stable engraftment of WASp expressing cells in all hematopoietic lineages, positive selection of WASp+ cells, and rescued functional and developmental T and B cell defects without evidence for toxicity. Inclusion of the insulator allowed for higher expression levels at lower numbers of viral integration per cell; a feature that was further exaggerated in secondary recipients. Finally, in parallel, we evaluated CL20-i650–MND LV expressing a fluorescent marker using CD34+ autologous HSC transplants in a non-human primate model (Macaca nemestrina) and observed long term stable polyclonal engraftment of LV expressing hematopoietic cells for up to 2 yr. Based on these extensive pre-clinical and toxicity studies we anticipate that CL20-i650–MND-WAS LV will provide sustained rescue of platelet and lymphocyte development and function in vivo (facilitated, in part, via the insulator) without evidence of toxicity. We plan to directly test this hypothesis in an upcoming WAS LV clinical trial.

342. Gene Edition for Wiskott-Aldrich Syndrome Gene Therapy

Site Specific nucleases (SSN) are powerful tools for genome editing that are revolutionizing basic and applied science. However, for most therapeutic applications their efficiency and/or specificity must be still improved before translation into clinic. Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency (PID) caused by mutations in the WAS gene. An interesting characteristic of this disease is that, due the growth selective advantage of the WAS-expressing cells, a small number of corrected hematopoietic stem cells (HSCs) could be enough to achieve a complete cure of the patients. In this context, our final aim is to develop gene edition tools for WAS gene therapy with the idea to achieve safe genetic correction in a small numbers of patients’ HSCs. To study efficiency and safety of our tools, we first developed a WAS-specific GFP-reporter cellular model harboring GFP coding sequences disrupted by the intron 1 of the WAS gene. This reporter cell allowed us to have a direct measurement of the efficiency and specificity of WAS-specific nucleases to achieve genetic restoration. We constructed different CRISPR-based and ZFNs-based WAS-specific nucleases expressed by Lentiviral vectors (LV). With these set of plasmids we performed a side by side comparison of two different nucleases (ZFNs and CRISPR) and two different delivery systems (nucleofection versus Integrative deficient lentiviral vectors (IDLV)) to edit the WAS locus. We showed that CRISPR and ZFNs were equally efficient for WAS gene edition (over 60% gene disruption and 4% gene restoration) when delivered by plasmid nucleofection. However, IDLV-ZFNs were more efficient than IDLV-CRISPR, probably due to the low expression levels of Cas9 in the IDLV backbone. Our data indicates that CRISPR based systems can be as efficient as other SSN using standard delivery systems, however, their efficiency using IDLV is limited.

Clinical applications of gene therapy for primary immunodeficiencies.

Primary immunodeficiencies (PIDs) have represented a paradigmatic model for successes and pitfalls of hematopoietic stem cells gene therapy. First clinical trials performed with gamma retroviral vectors (γ-RV) for adenosine deaminase severe combined immunodeficiency (ADA-SCID), X-linked SCID (SCID-X1), and Wiskott-Aldrich syndrome (WAS) showed that gene therapy is a valid therapeutic option in patients lacking an HLA-identical donor. No insertional mutagenesis events have been observed in more than 40 ADA-SCID patients treated so far in the context of different clinical trials worldwide, suggesting a favorable risk-benefit ratio for this disease. On the other hand, the occurrence of insertional oncogenesis in SCID-X1, WAS, and chronic granulomatous disease (CGD) RV clinical trials prompted the development of safer vector construct based on self-inactivating (SIN) retroviral or lentiviral vectors (LVs). Here we present the recent results of LV-mediated gene therapy for WAS showing stable multilineage engraftment leading to hematological and immunological improvement, and discuss the differences with respect to the WAS RV trial. We also describe recent clinical results of SCID-X1 gene therapy with SIN γ-RV and the perspectives of targeted genome editing techniques, following early preclinical studies showing promising results in terms of specificity of gene correction. Finally, we provide an overview of the gene therapy approaches for other PIDs and discuss its prospects in relation to the evolving arena of allogeneic transplant.

RETRACTED: Novel and safer self-inactivating vectors for gene therapy of Wiskott-Aldrich Syndrome

The article entitled “Novel and safer self-inactivating vectors for gene therapy of Wiskott-Aldrich Syndrome”, by E. G. Coci1, T. Maetzig, D. Zychlinski, M. Rothe, J. D. Suerth, C. Klein and A.Schambach has been retracted, on the request of the authors.Kindly see Bentham Science Policy on Article retraction at the link given below:(https://www.benthamscience.com/journals/current-medical-imaging/author-guidelines/).It is a pre-requisite for authors to declare explicitly that their work is original and has not been published elsewhere. Authors are advised to properly cite the original source to avoid plagiarism and copyright violation. As such this article represents a severe abuse of the scientific publishing system. Bentham Science Publishers takes a very strong view on this matter and apologizes to the readers of the journal for any inconvenience this may cause.

Gene Therapy for Wiskott-Aldrich Syndrome—Long-Term Efficacy and Genotoxicity

Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.

A possible turning point in the hematopoietic stem cell gene therapy for primary immunodeficiency diseases? Lentiviral vectors could take the place of retroviral vectors

Evaluation of: Aiuti A, Biasco L, Scaramuzza S et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science 341(6148), 1233151 (2013).Wiskott–Aldrich syndrome (WAS), an X-linked primary immunodeficiency disease (PID) with unique and characteristic features, had been considered to be a good candidate for gene therapy. In 2010, hematopoietic stem cell (HSC) gene therapy, using a retroviral vector, was performed for WAS patients; however, concerns remain regarding the long-term safety of this therapy as several patients with PID developed myeloproliferative diseases due to insertional mutagenesis related to HSC gene therapy using retroviral vectors. Aiuti et al. first reported HSC gene therapy for WAS using a lentiviral vector and compared the safety and efficacy of the two therapies in the context of the same disease background. They undertook a detailed study of the vector integration sites and concluded that lentiviral HSC gene therapy was safer than retroviral gene therapy.

306. Lentiviral Vector-Based Gene Therapy for Wiskott-Aldrich Syndrome: Preliminary Results from the French Center

306. Lentiviral Vector-Based Gene Therapy for Wiskott-Aldrich Syndrome: Preliminary Results from the French Center

Wiskott-Aldrich syndrome protein-deficient hematopoietic cells can be efficiently mobilized by granulocyte colony-stimulating factor

The Wiskott-Aldrich syndrome protein is an essential cytoskeleton regulator found in cells of the hematopoietic lineage and controls the motility of leukocytes. The impact of WAS gene deficiency on the mobilization of hematopoietic progenitor/stem cells in circulation has remained unexplored but information would be pertinent in the context of autologous gene therapy of Wiskott-Aldrich syndrome. The response to granulocyte-colony stimulating factor mobilization was investigated in a murine WAS knock-out model of the disease, by measuring hematologic parameters, circulation and engraftment of hematopoietic progenitor/stem cells. In the steady-state, adult WAS knock-out mice have B-cell lymphopenia, marked neutrophilia, increased counts of circulating hematopoietic progenitor cells and splenomegaly, presumably caused by the retention of hematopoietic progenitor cells due to high levels of splenic CXCL12. In spite of these anomalies, the administration of granulocyte-colony-stimulating factor mobilizes progenitor/stem cells in WAS knock-out mice to the same level and with the same kinetics as in wild-type control mice. Mobilized peripheral blood cells from WAS knock-out mice can be transduced and are able to engraft into lethally-irradiated hosts reconstituting multiple lineages of cells and providing more effective radio-protection than mobilized cells from wild-type control mice. Surprisingly, the homing and the peripheral blood recovery of B lymphocytes was influenced by the background of the host. Thus, in the absence of Wiskott-Aldrich syndrome protein, effective mobilization is achieved but partial correction may occur as a result of an abnormal hematopoietic environment.

Advances in Foamy Virus Vector Technology and Disease Correction Could Speed the Path to Clinical Application

Foamy viruses (FVs) belong to the Retroviridae family, which includes the g-retrovirus murine leukemia virus (MLV) and lentiviruses (LVs) such as HIV. Whereas vectors derived from HIV and MLV have shown success in the clinic, vectors derived from FVs have yet to be tested clinically. Three articles in this issue of Molecular Therapy clear a path to greater flexibility in the engineering of this promising new class of vectors and bring clinical application of FV vectors a step closer to reality. Nasimuzzaman and Persons1 reveal a long-kept secret of FVs with the identification of an FV receptor that at least partly explains the broad tropism of these viruses and vectors derived from them. Ho et al.2 present a strategy to allow the nearly ubiquitous FV vector tropism to be flexibly modified and restricted. Both studies represent important steps toward facilitating gene therapy applications of FV-based vectors, and in this light Uchiyama et al.3 demonstrate the correction of a preclinical mouse model of Wiskott–Aldrich syndrome (WAS) with a first-generation vector based on FV. The more neutral integration pattern of FV vectors is of particular interest with respect to gene therapy of WAS because the interim analysis of the first two WAS patients treated with conventional g-retroviral vectors has revealed an accumulation of problematic common vector insertion sites that have led to serious adverse events.4

Foamy Virus Vector-mediated Gene Correction of a Mouse Model of Wiskott–Aldrich Syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, thrombocytopenia and immunodeficiency. Hematopoietic cell transplantation can cure the disease and gene therapy is being tested as an alternative treatment option. In this study, we assessed the use of foamy virus (FV) vectors as a gene transfer system for WAS, using a Was knockout (KO) mouse model. Preliminary experiments using FV vectors expressing the green fluorescent protein under the transcriptional control of the endogenous WAS promoter or a ubiquitously acting chromatin opening element allowed us to define transduction conditions resulting in high (>40%) and long-term in-vivo marking of blood cells after transplantation. In following experiments, Was KO mice were treated with FV vectors containing the human WAS complementary DNA (cDNA). Transplanted animals expressed the WAS protein (WASp) in T and B lymphocytes, as well as platelets and showed restoration of both T-cell receptor-mediated responses and B-cell migration. We also observed recovery of platelet adhesion and podosome formation in dendritic cells (DCs) of treated mice. These data demonstrate that FV vectors can be effective for hematopoietic stem cell (HSC)-directed gene correction of WAS.

Dendritic cell functional improvement in a preclinical model of lentiviral-mediated gene therapy for Wiskott–Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by the defective expression of the WAS protein (WASP) in hematopoietic cells. It has been shown that dendritic cells (DCs) are functionally impaired in WAS patients and was−/− mice. We have previously demonstrated the efficacy and safety of a murine model of WAS gene therapy (GT), using stem cells transduced with a lentiviral vector. The aim of this study was to investigate whether GT can correct DC defects in was−/− mice. As DCs expressing WASP were detected in the secondary lymphoid organs of the treated mice, we tested the in vitro and in vivo function of bone marrow-derived DCs (BMDCs). The BMDCs showed efficient in vitro uptake of latex beads and Salmonella typhimurium. When BMDCs from the treated mice (GT BMDCs) and the was−/− mice were injected into wild type hosts, we found a higher number of cells that had migrated to the draining lymph nodes compared to mice injected with was−/− BMDCs. Finally, we found that OVA-pulsed GT BMDCs or vaccination with anti-DEC205 OVA fusion protein can efficiently induce antigen-specific T cell activation in vivo. These findings show that WAS GT significantly improves DC function, thus adding new evidence of the preclinical efficacy of lentiviral vector-mediated WAS GT.