ALBERT

Description: Key Points NRF2 knockout inhibits fetal hemoglobin expression during gestational erythropoiesis in SCD mice. Loss of the cellular antioxidant response mediated by NRF2 exacerbates spleen damage, inflammation, and oxidative stress in SCD mice.

Description: Background: Severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) is a rare disorder caused by ADA gene mutations, leading to lymphotoxic build-up of purine metabolites and profound immunodeficiency. Historically, enzyme replacement therapy (ERT) has been used as a bridge therapy until patients can receive an allogeneic hematopoietic stem cell transplantation (HSCT), ideally from a matched related donor (MRD) or, if none is identified, a non-matched and/or unrelated donor. We developed a self-inactivating lentiviral vector (LV), denoted EFS-ADA LV, encoding the human ADA cDNA sequence under the control of a shortened human elongation factor 1α gene promoter. A fresh or cryopreserved formulation of a drug product (OTL-101), composed of autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with EFS-ADA LV, was evaluated in 2 prospective, non-randomized Phase I/II clinical trials at 2 USA centers. We report on safety and efficacy of OTL-101 in 30 ADA-SCID pediatric gene therapy (GT) subjects treated from 2013-2017 with a median follow up (FU) of 24 months (mo; range 12-26 mo), compared to a historical cohort of 26 ADA-SCID patients treated with HSCT. Methods: UCLA Fresh Study (NCT01852071): Autologous CD34+ HSPCs were isolated from bone marrow and pre-stimulated with cytokines before transduction with EFS-ADA LV to yield OTL-101, which was infused as a fresh formulation in 20 subjects (9 male, 11 female; aged 4 mo-4.3 yrs). Single dose busulfan (4 mg/kg) was administered prior to infusion of OTL-101. Subjects were followed for 24 mo. UCLA Cryo Study (NCT02999984): 10 subjects (4 male, 6 female; aged 5-15 mo) received a cryopreserved formulation of OTL-101, which allowed for an extended shelf-life and full quality control prior to infusion. Busulfan was administered in 2 doses, the first at 3 mg/kg and the second adjusted to target a total area under the curve of 4,900 µM*min (20 ng/mL*hr). At the time of analysis, all subjects reached 12 mo FU (except 1 subject who was withdrawn from the study due to lack of engraftment); 7 subjects reached 18 mo of FU. Historical Control Group: 26 patients (aged 0.2 mo-9.8 yrs) were treated with allogeneic HSCT (MRDs n=12, non-MRDs n=14) at Great Ormond Street Hospital, UK (n=16) or Duke University Children's Hospital, USA (n=10) from 2000-2016. Results: Sustained engraftment of genetically modified HSPCs was observed in 29/30 GT subjects by 6-8 mo and persisted through FU in both studies, based on vector gene marking in granulocytes and CD3+ T cell reconstitution (Figure). Subjects who engrafted maintained long-term metabolic detoxification from deoxyadenosine nucleotides after stopping ERT approximately 1 mo post-GT. At last FU (median 24 mo; range 12-24 mo) in the GT group, overall survival (OS) was 30/30 (100%) and event-free survival (survival in the absence of ERT reinstitution or rescue allogeneic HSCT; EvFS) was 29/30 (97%). OS and EvFS were higher in the GT group at last FU compared with HSCT controls (with or without an MRD) at 2 years (Table). One of 30 OTL-101 subjects (3%) did not engraft and was restarted on ERT; the subject was withdrawn from the study at 5.9 mo and subsequently received a rescue HSCT, whereas 42% of HSCT patients required rescue HSCT, PEG-ADA ERT or died. Among the 20 OTL-101 subjects in the UCLA Fresh Study who reached 2 years FU, 18 (90%) stopped immunoglobin replacement therapy (IgRT), compared to 52% of HSCT patients. Preliminary results were observed in 5/7 (71%) OTL-101 subjects in the UCLA Cryo Study with more limited (18 mo) FU. Twelve OTL-101 subjects experienced one or more serious adverse events, most frequently infections and gastrointestinal events; only 1 of which was considered treatment-related (bacteremia due to product contamination). In the GT group, there were no events of autoimmunity with ≤24 mo FU. Due to the autologous nature of OTL-101, there was no incidence of graft vs host disease (GvHD); in contrast, 8 HSCT patients experienced GvHD events (5 acute, 3 chronic events), 1 of which resulted in death. Conclusions: Based on sustained gene correction and restoration of immune function in all subjects who engrafted, treatment of ADA-SCID with OTL-101 has a favorable benefit-risk profile. Key correlates of engraftment were consistent across the expanded cohort. Importantly, higher rates of OS and EvFS compared with HSCT (with or without an MRD) were observed. Disclosures Kohn: Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor on IP licensed from UC Regents to Orchard Therapeutics. Future royalties may occur., Research Funding; NIH: Research Funding. Shaw:Orchard Therapeutics: Consultancy, Other: Personal fees and non-financial support; NIH: Research Funding. Carbonaro-Sarracino:NIH: Other: Salary while working on project at UCLA 2013-2016, Research Funding; Orchard Therapeutics: Consultancy, Employment. De Oliveira:National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding. Terrazas:California Institute for Regenerative Medicine: Research Funding; Gene Therapy Resource Program, NHLBI/NIH: Research Funding. Hollis:Curative Therapeutics: Consultancy, Other: Personal fees. Trevisan:Orchard Therapeutics: Research Funding. Arduini:Orchard Therapeutics: Employment, Equity Ownership. Lynn:Orchard Therapeutics: Employment, Equity Ownership. Kudari:Orchard Therapeutics: Employment, Equity Ownership. Spezzi:Orchard Therapeutics: Employment, Equity Ownership. Buckley:Duke University: Research Funding. Booth:SOBI: Consultancy; GSK: Honoraria; NovImmune: Consultancy. Thrasher:Generation Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 4BIOCapital: 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. Gaspar:Orchard Therapeutics: Employment, Equity Ownership, Patents & Royalties: Lentiviral vector for gene therapy of ADA-SCID.

Description: Lymphoproliferative disease (LPD) is a recognized complication of primary immunodeficiency (PID) and immunodysregulatory syndromes. Historically, it has a very poor outcome. For patients surviving LPD, myeloablative hematopoietic stem cell transplantation (SCT) was the only cure for the underlying PID, with a high risk of developing posttransplantation complications, including recurrent lymphoproliferative disease. We describe 8 patients with a range of PID and immunodysregulatory syndromes complicated by LPD. After initial treatment of the LPD (including the use of anti-CD20 monoclonal antibody, rituximab, in 6 of the patients), all patients underwent reduced-intensity conditioning (RIC) SCT with prospective monitoring for Epstein-Barr virus (EBV) viremia. After transplantation, 3 patients received rituximab, and 3 patients received prophylactic EBV-specific cytotoxic T-lymphocytes. Only 1 patient developed recurrent LPD posttransplantation, which responded to rituximab. All patients who underwent transplantation survive free of LPD and are cured of their PID at a median follow-up of 4 years (range, 1-7 years). With careful monitoring and pre-emptive therapy, we advocate this RIC SCT approach to patients with PID who have pre-existing EBV-LPD.

Description: Key Points The outcome of HSCT in this large SCN cohort is acceptable. Given the TRM, a careful selection of HSCT candidates should be undertaken.

Description: The variety of gene therapy vectors for a multitude of different diseases has increased tremendously over the years. However, a number of patients that underwent gene therapy in different trials developed hematological malignancy caused by integration of the provirus in the vicinity of proto-oncogenes. These severe adverse advents prompted intense research efforts towards safer gene therapy, leading to the removal of the long terminal repeat enhancer elements and the use of internal promoters in retroviral vectors. Still, a bottleneck of transition from basic research to clinical application is the test for safety of integrating retro- and lentiviral vectors. Instead of laborious in vivo models with limited predictive value, in vitro assays to screen for insertional mutagenesis are strongly desirable. A decade ago, our lab developed the in vitro immortalization (IVIM) assay to quantify the genotoxic potential of viral vectors, which has been widely used to complete preclinical safety documentation of newly developed integrating vector systems. Despite general acceptance in the field of hematopoietic gene therapy, bias for insertional mutants of the myeloid lineage, a low sensitivity and a long assay run time are clear limitations. We now developed the molecular surrogate assay for genotoxicity assessment (SAGA). The new test is more robust, sensitive and biologically informative. As input we used murine lineage-negative hematopoietic stem and progenitor cells (HSPC) that were cultured as described for the IVIM assay. The murine HSPC were transduced with a number of different gammaretro- and lentiviral vectors, including vectors that have been employed in clinical trials for X-SCID and Wiskott-Aldrich Syndrome. After 14 days, whole mRNA was isolated from transduced and non-transduced samples and analyzed by Agilent custom microarrays (n=86) and qPCR from nine independent SAGA assays. We applied several Machine Learning algorithms to derive a core set of genes which distinguishes transformed from non-transformed samples in each individual SAGA assay. This set of genes from the individual analysis was further analyzed to derive a core set of genes that is able to robustly separate transformed from non-transformed samples in all assays performed. In order to account for platform-specific effects we validated all microarray results by conventional qPCR-methodology. The SAGA gene set was then cross-validated in an independent validation cohort of SAGA-assays that were not part of the SAGA-training set from which the signature was derived from. The SAGA assay was used to quantify the mutagenic potential of several benchmark vectors. It correctly assigned a high mutagenic potential to vectors (MFG.yc and CMMP.WASP) which led to serious adverse events (SAEs) in clinical trials. Most importantly, the SAGA assay reliably scored high for mutagenic vectors, even when the vector did not transform in IVIM-assays conducted in parallel, demonstrating the higher sensitivity of the SAGA-principle. In contrast, SIN lentiviral vectors with weaker internal promoters (LV.EFS.yc and LV.EFS.ADA) showed no enrichment of the SAGA-core signature and hence scored much safer in the SAGA test. We present the results for these vectors side-by-side either using IVIM or SAGA. In summary, we generated an advanced version of the currently used in vitro insertional mutagenesis screening system by integrating a molecular read-out which enhances reproducibility, sensitivity and reduces assay duration, paving the way for a better preclinical risk assessment of gene therapy vectors. Disclosures No relevant conflicts of interest to declare.

Description: X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the interleukin-2 receptor γ chain (IL2RG), and is characterized by lack of response to common γ-chain-dependent cytokines and profound defects in T-, B- and NK-cell functions. Previous gene therapy clinical trials based on autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically corrected with MLV-derived retroviral vectors expressing IL2RG in non-conditioned patients showed long-lasting restoration of T-cell immunity in most cases but resulted in vector-induced leukemia through enhancer-mediated insertional mutagenesis in 25% of patients. The use of an enhancer-less MLV vector to deliver the IL2RG gene caused no adverse events while retaining a significant clinical benefit. To increase the efficacy of gene therapy and reduce at the same time the probability of genotoxic insertional events, we developed a third-generation lentiviral vector carrying a codon-optimized human IL2RG cDNA under the control of the human EF1α-S promoter. The vector was VSV-G-pseudotyped and produced by transient transfection of 293T cells, followed by ion-exchange chromatography, tangential-flow filtration and formulation in HSC growth medium. Replacement of the native IL2RG open reading frame by a codon-optimized sequence resulted in a 3-fold increase in mRNA expression and a 1.5-fold increase in IL2RG protein expression per integrated vector copy. The performance of the vector was demonstrated in vitro by the restoration of a normal level of IL2RG mRNA or protein in a human IL2RG-deficient T-cell line at a VCN of 1 to 3, and by high efficiency (〉80%) transduction of human mobilized CD34+ HSPCs and transgene expression with no impact on viability or clonogenic capacity. An in vitro immortalization assay (IVIM) showed a safe genotoxic profile, while the in vivo safety and efficacy of the vector was tested it in a preclinical model of SCID-X1 gene therapy based on transplantation of genetically corrected Lin- cells from IL2rg-/- donor mice into lethally-irradiated IL2rg-/--Rag2-/- recipients. The study showed biodistribution of the transgene in hematopoietic organs only, restoration of T, B and NK cell counts in bone marrow and peripheral blood, normalization of lymphoid organs (thymus and spleen) and a low frequency of hematopoietic malignancies, comparable to that of untreated animals, six months after transplantation. An extensive insertion site analysis was carried out in bone marrow, thymus and peripheral blood of individual or groups of animals to detect the presence of any clonal abnormality or skewing, and any deviation from a normal lentiviral integration pattern. The study showed the expected genomic integration profile and no signs of clonal dominance of transduced cells. Interestingly, analysis of 〉100,000 integration sites in pre- and post-transplant murine cells showed that lentiviral vectors target at high frequency a substantially different set of genes compared to human CD34+ cells, uncovering the need for appropriate controls and at the same time the limits in the predictive power of mouse-based genotoxicity studies, particularly those addressing proto-oncogene activation and clonal dominance. These studies will enable a multicenter phase-I/II clinical trial aimed at establishing the safety and clinical efficacy of lentiviral vector-mediated gene therapy for SCID-X1. The clinical protocol is based on transplantation of autologous CD34+ HSPCs transduced with the EF1a-IL2RGco lentiviral vector in infants after non-myeloablative conditioning, and aims at stable and sustained restoration of both T- and B-cell immunity. Disclosures Williams: Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees. Gaspar:Orchard therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding.

Description: Hematopoietic stem cell transplantation (HSCT) is a highly successful treatment for severe congenital immunodeficiencies. However, some studies have suggested that children may experience cognitive difficulties after HSCT. This large-scale study assessed cognitive and behavioral function for the cohort of children treated by HSCT at one center between 1979 and 2003 to determine the frequency and severity of problems and to identify risk factors. A total of 105 patients were assessed on standardized measures of cognitive and emotional and behavioral function together with a control group of unaffected siblings. The average IQ for the cohort was 85 (95% confidence interval, 81-90), significantly lower than both the population average of 100 (P 〈 .001) and unaffected siblings. Multivariate analysis indicated that the underlying genetic defect, diagnosis of adenosine deaminase-deficient severe combined immunodeficiency, and consanguinity were associated with worse outcome but that age at transplantation and chemotherapy conditioning were not. Children treated by HSCT for severe immunodeficiency have an increased risk of long-term cognitive difficulties and associated emotional and behavioral difficulties. The specific genetic diagnosis, consanguinity, and severe clinical course are associated with poor outcome. Long-term follow-up of these patients should include screening to identify and manage these problems more effectively.

Description: DiGeorge syndrome (DGS) is a primary immunodeficiency characterized by various degrees of T-cell deficiency. In partial DGS (pDGS), other risk factors could predispose to recurrent infections, autoimmunity, and allergy. The aim of this study was to assess the effect of different factors in the development of infections, autoimmunity, and/or allergy in patients with pDGS. We studied 467 pDGS patients in follow-up at Great Ormond Street Hospital. Using a multivariate approach, we observed that palatal anomalies represent a risk factor for the development of recurrent otitis media with effusion. Gastroesophageal reflux/dysphagia and asthma/rhinitis represent a risk factor for the development of recurrent upper respiratory tract infections. Allergy and autoimmunity were associated with persistently low immunoglobulin M levels and lymphopenia, respectively. Patients with autoimmunity showed lower levels of CD3+, CD3+CD4+, and naïve CD4+CD45RA+CD27+ T lymphocytes compared with pDGS patients without autoimmunity. We also observed that the physiological age-related decline of the T-cell number was slower in pDGS patients compared with age-matched controls. The age-related recovery of the T-cell number depended on a homeostatic peripheral proliferation of T cells, as suggested by an accelerated decline of the naïve T lymphocytes in pDGS as well as a more skewed T-cell repertoire in older pDGS patients. These evidences suggest that premature CD4+ T-cell aging and lymphopenia induced spontaneous peripheral T-cell proliferation might contribute to the pathogenesis of autoimmunity in patients with pDGS. Infections in these patients represent, in most of the cases, a complication of anatomical or gastroenterological anomalies rather than a feature of the underlying immunodeficiency.

Description: Key Points We describe the first successful use of gene therapy in a severely affected adult with WAS. Gene therapy is a viable strategy for adult WAS patients with severe chronic disease complications where allogeneic transplantation presents.

Description: Dysregulation of the hemostatic system in malignancy leads to a hypercoagulable state and thrombotic complications. In addition to thrombosis, growing evidence from both human and animal studies, suggests that constituents of the hemostatic system also play an important role in tumor progression and metastasis. Platelets contribute to metastasis by protecting tumor cells from immune detection and inducing epithelial-mesenchymal transition, thereby facilitating the extravasation of circulating tumor cells out of the vasculature. Moreover, platelet-tumor cell interactions have been shown to promote prostaglandin E2 (PGE2) synthesis, an important modulator of angiogenesis and proliferation. The role of von willebrand factor (VWF) in malignancy has been of particular interest as it serves as an adhesive link between platelets and endothelium, and could possibly facilitate platelet interactions with tumor cells. Yet, the absence of VWF is associated with increased metastatic potential in a B16 melanoma mouse model, suggesting that the primary contribution of VWF to the metastatic model may not be platelet mediated. We employed a combined approach using cell lines and mouse models to explore the interaction of platelets and VWF in metastasis. In vitro studies using lung and melanoma cells lines show that the interaction of resting and activated platelets with tumor cells leads to upregulation of COX-2 expression and PGE2 biosynthesis but does stimulate VEGF-A production. In an experimental metastasis model of C57BL/6 wild type and VWF null mice, the injection of B16 melanoma cells by tail vein yielded a significant increase in the number of pulmonary metastatic foci. PGE2 measurements from mouse serum showed reduced levels of prostaglandin in VWF deficient mice injected with tumor, suggesting that the absence of VWF precludes an important tumor cell-platelet interaction that facilities PGE2 production. Moreover, preliminary results show increased VEGF-A in VWF deficient mice, suggesting that increased metastasis in this model may be the result of dysregulated angiogenesis. In conclusion, platelet-tumor cell interaction yield a pro-metastatic effect, in part mediated by PGE2 production. Despite its unique role in platelet adhesion and thrombus formation, VWF does not facilitate the interaction of platelets and tumor cells to produce PGE2. Its protective effect may lie in its ability to modulate angiogenesis via VEGF-specific pathways. Disclosures No relevant conflicts of interest to declare.