Replication Competent Lentivirus Research Articles

Gene Therapy Expressing Platelet-Derived Factor VIII for Correction of Hemophilia Α with a History of Inhibitors

Introduction: Platelet targeted gene therapy with human factor VIII has established hemostasis in murine and canine models with severe hemophilia A withouteliciting inhibitory antibodies. Here-in we describe feasibility, safety and efficacy of the first subject treated on a first-in-human phase 1 trial that targets factor FVIII synthesis and storage within platelet α-granules for delivery at the site of vascular injury for hemophilia A (NCT03818763). Method: Eligible are severe hemophilia A patients ≥18 years with a history of inhibitors to factor VIII (≥0.6 Bethesda Units [BU]/ml). Following mobilization, autologous CD 34+ cells (≥6 x 10 6/kg) are collected and transduced with a lentiviral vector encoding the ITGA2B gene promoter for ectopic expression of human B-domain-deleted factor VIII within the megakaryocyte lineage. The transduced cells are infused after reduced intensity cytoreduction with fludarabine (120 mg/m 2) and melphalan (120 mg/m 2) followed by a washout period of ≥24 hours. The primary endpoints of feasibility and safety are defined as: 1) feasibility of the cell manufacturing procedure by the availability of ≥4 x 10 6/kg transduced clinical grade CD34+cells, cell viability ≥70% and undetectable microbial contamination and 2) safety defined as hematopoietic recovery ≤28 days of infusion and absence of ≥ grade 3 toxicity (CTCAE version 5.0). Outcome: Subject 1 is a 29-year-old male with severe hemophilia A who developed inhibitors to factor VIII (2.6 BU/ml) in his first year of life. Immune tolerance to factor VIII had been established with a non-detectable inhibitor titer at enrollment. Hemostatic prophylaxis was maintained with emicizumab weekly and recombinant factor VIII for breakthrough bleeding. His annualized bleeding rate was 16 in the 12-month period preceding infusion. The cell product was the result of 1 day of collection yielding 6.73 x 10 6 /kg total viable CD34+ cells post-transduction and vector copy number by qPCR of 1.16 copies/cell. Megakaryocyte factor VIII:C levels were 0.00 mU/10 6 before transduction and 101.32 mU/10 6 after transduction. The cell product satisfied release criteria. Neutrophil recovery (≥0.5 x 10 9/L) and platelet transfusion independence (≥50 x 10 9/L) was achieved 15 days post-infusion and sustained for >12 months. The duration of hospitalization was 21 days with no re-admissions. There was no breakthrough bleeding during collection and hospitalization. During and post-infusion there was no unexpected toxicity ≥ grade 1. He has not required immune suppression. Emicizumab was discontinued 3.6 months post-infusion after demonstration of whole blood vector copy number at 1 and 3-months. There has been no spontaneous bleeding or a need for “on demand” factor VIII after discontinuing emicizumab. Secondary outcomes are summarized in Table 1. No replication competent lentivirus was detected through month 12 post-infusion. Integration site analysis was carried out, and none of the samples tested through 12 months contained cell clones which exceeded 20% relative abundance. Transduced cells were highly polyclonal. Integration site analysis showed no enrichment of integration near cancer-associated genes in the cell product nor in whole blood cell lineages post-infusion. Conclusion: We report feasibility, safety and efficacy in the first subject with severe hemophilia A and a history of inhibitors who received lentiviral vector gene therapy directed to induce megakaryocytes to synthesize and store factor VIII within platelets. These findings extend the potential to treat severe hemophilia A patients who are not eligible for valoctcogene roxaoarvovec (AAV5-hFVIII-SQ).

Sustained Efficacy, Safety, and Improved Quality of Life in Adult and Pediatric Patients with Transfusion-Dependent β-Thalassemia up to 9 Years Post Treatment with Betibeglogene Autotemcel (Beti-cel)

Introduction: Beti-cel gene therapy addresses the underlying cause of transfusion-dependent β-thalassemia (TDT) by adding functional copies of a modified β-globin gene to autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) via a third-generation, self-inactivating lentiviral vector (LVV), BB305, which produces functional adult hemoglobin (Hb), HbA T87Q, in red blood cells (RBCs). Here, we report efficacy, safety, and quality of life (QOL) data from adult and pediatric patients treated with beti-cel who were followed for up to 9 years after treatment. These outcomes may inform patient selection for real-world treatment with beti-cel. Methods: Patients with TDT who completed either a phase 1/2 (HGB-204 [NCT01745120]; HGB-205 [NCT02151526]) or phase 3 (HGB-207 [NCT02906202]; HGB-212 [NCT03207009]) beti-cel parent study and subsequently participated in the long-term, 13-year follow-up study LTF-303 (NCT02633943) were included in this analysis. Analyses were performed with data collected through January 30, 2023. Efficacy (including transfusion independence [TI], defined as a weighted average Hb ≥9 g/dL without packed RBC transfusions for ≥12 months) and safety are reported through last follow-up. QOL data are reported for studies HGB-204, HGB-207, and HGB-212 through month 36. Statistical analyses were conducted to examine key outcomes in subgroups based on patient age at enrollment (pediatric: <18 years; adult: ≥18 years). Results:As of January 30, 2023, 63 patients (median [range] age: 17 [4-35] years) had received beti-cel in a phase 1/2 or 3 study and enrolled in LTF-303, with a median (range) follow-up of 60.1 (23.8-109.5) months. Phase 3 studies used the commercial drug product manufacturing process. Ninety percent (37/41) of phase 3 patients achieved and maintained TI through last follow-up (up to 6 years; Table 1). TI rates by study, genotype, and age for phase 3 patients are also presented in Table 1. In phase 1/2 studies that used an older drug manufacturing process, 68.2% (15/22) of patients achieved TI; 14 of these patients sustained TI through last follow-up (up to 9 years). One patient no longer meets protocol-defined TI as a result of Hb level <9g/dL at year 6 due to acute health events unrelated to β-thalassemia, which were not attributed to loss of beti-cel treatment effect. Approximately 80% of pediatric and adult patients required only one mobilization cycle to achieve the drug product dose. The median percentage of drug product cells transduced with the BB305 LVV was comparable between adult and pediatric populations (78% and 80%, respectively), as were the month 6 median peripheral blood vector copy number (1.4 c/dg and 1.1 c/dg) and HbA T87Q (9.4 g/dL and 8.3 g/dL). Adult patients reported improvements in QOL up to month 36 as assessed by the Short Form-36 Health Survey Questionnaire mental and physical component summary scores, Functional Assessment of Cancer Therapy, and EuroQol ( Table 2). Pediatric Quality of Life Inventory scores will be reported at the time of presentation. Overall, 19% (12/63) of patients experienced ≥1 beti-cel-related adverse event (AE); the most common beti-cel-related AEs (occurring in ≥3 patients) were abdominal pain (experienced in 5/63 [7.9%] patients) and thrombocytopenia (3/63 [4.8%] patients). Five patients experienced serious veno-occlusive liver disease; all 5 received defibrotide and recovered. No malignancies, insertional oncogenesis, or vector-derived replication-competent lentivirus were reported. Age subgroup safety analysis as of last follow-up will be reported in the final presentation. Conclusion: Beti-cel is a potentially curative gene therapy for patients with TDT across ages and genotypes through achievement of TI and normal or near-normal Hb. These data will inform real-world beti-cel treatment decisions for patients with TDT and providers.

Efficacy, Safety, and Health-Related Quality of Life (HRQOL) in Patients with Sickle Cell Disease (SCD) Who Have Received Lovotibeglogene Autotemcel (Lovo-cel) Gene Therapy: Up to 60 Months of Follow-up

Introduction: Lovo-cel gene therapy uses autologous transplantation of hematopoietic stem and progenitor cells (HSPC) transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an anti-sickling hemoglobin (Hb), HbA T87Q. The phase 1/2 HGB-206 (NCT02140554) and phase 3 HGB-210 (NCT04293185) studies of lovo-cel are the largest clinical trials of gene therapy in SCD to date. We report efficacy and safety from these studies, including the first data from adult and pediatric patients (age 12 to <18 years) in HGB-210, and HRQOL data from HGB-206. Methods: This analysis includes patients from HGB-206 Group C and HGB-210 who received lovo-cel using the current HSPC mobilization and manufacturing process. Patients with SCD and recurrent severe vaso-occlusive events (VOEs) or history of overt stroke underwent plerixafor mobilization and apheresis followed by myeloablative busulfan conditioning and lovo-cel infusion. After 24 months of follow-up post lovo-cel infusion, patients enrolled in the long-term study LTF-307 (NCT04628585). Lab evaluations, SCD-related outcomes (eg, resolution of VOEs), globin response (a composite endpoint evaluating HbA T87Q percentage and total Hb), and safety are reported up to 60 months. An independent Event Adjudication Committee confirmed VOEs met protocol criteria. HRQOL data from Patient-Reported Outcomes Measurement Information System (PROMIS)-57 domains of pain interference, fatigue, and pain intensity are reported up to 48 months. Results:This analysis includes47 patients (HGB-206 Group C, n=36; HGB-210, n=11; male, 59.6%; median [range] age, 23 y [12-38]) who received a lovo-cel infusion as of Feb 13, 2023. Median (range) follow-up was 35.5 months (0.3-61.0). Median (range) time to neutrophil and platelet engraftment was 20 days (12-35) and 35 days (19-136). Peripheral blood vector copy number remained stable (median >1 c/dg through follow-up). Median HbA T87Q levels were ≥4.5 g/dL from 6 months post infusion to last study visit. Median (range) total Hb level increased from 8.7 g/dL (6.1-12.5) at relative baseline to 11.8 g/dL (8.4-15.0) at last visit; median percent HbA T87Q of nontransfused total Hb was ≈40% or more ( Figure). Of 33 evaluable patients (ie, ≥18 months follow-up and ≥4 VOEs in the 2 years before enrollment), 30 (90.9%) and 32 (97.0%) had complete resolution of VOEs and severe VOEs during the 6-18 months post infusion, vs a median (range) of 3.5 (1.5-16.5) and 3.0 (0.5-13.0) events/year in the 2 years before enrollment. Additional efficacy analyses in subgroups (eg, stroke history, α-globin genotype) will be presented. Among patients who had globin response or who had ≥18 months follow-up (n=38), 33 (86.8%) achieved globin response. Hemolysis markers (eg, reticulocytes, total bilirubin, lactate dehydrogenase) approached normal levels. Among all 47 patients, 44 (93.6%) had ≥1 AE of grade ≥3 after lovo-cel infusion; the most common of which were stomatitis (33 [70.2%] patients) and thrombocytopenia (28 [59.6%]). Serious AEs were reported for 26 (55.3%) patients, the most common of which was chronic pain/acute exacerbation of chronic pain (3 [6.4%]; 2 chronic neuropathic pain and 1 chronic pain associated with anxiety). One patient with -α3.7/-α3.7 genotype was diagnosed with myelodysplastic syndrome with stable complete blood counts 30 months post infusion; updated data to be presented. No veno-occlusive liver disease, graft failure, replication-competent lentivirus, or vector-mediated insertional oncogenesis was observed. Lovo-cel treatment regimen largely reflected known side effects of HSPC collection and busulfan conditioning regimen. A total of 25 patients (all from HGB-206 Group C; age ≥18 y) had evaluable HRQOL data. Mean scores for PROMIS-57 domains of pain intensity, pain interference, and fatigue improved (ie, decreased) over time up to 48 months ( Table). Conclusion: One-time treatment with lovo-cel resulted in sustained HbA T87Q production and near-complete resolution of VOEs and severe VOEs up to 18 months post treatment; the safety profile was consistent with underlying SCD and myeloablative conditioning. Patients reported sustained improvements in pain intensity, pain interference, and fatigue. Ongoing long-term follow-up will continue to provide important information on efficacy, safety, and patient experience post lovo-cel treatment.

Three-Year Follow-up on Efficacy and Safety Results from Phase 1 Lummicar Study 1 of Zevorcabtagene Autoleucel in Chinese Patients with Relapsed or Refractory Multiple Myeloma

Introduction: Phase 1b of LUMMICAR STUDY 1 was conducted in China (NCT03975907) for zevorcabtagene autoleucel (zevor-cel, CT053), a fully human autologous chimeric antigen receptor (CAR) T-cell therapy comprising a B-cell maturation antigen-specific single-chain variable fragment, in patients with relapsed or refractory multiple myeloma (RRMM). Previously disclosed 1-year follow-up data (ASH 2021 Abstract 2821) demonstrated a tolerable safety profile and deep and durable responses in 14 patients. Here, we present the updated results with 3 years of follow-up after the last patient was infused. Methods: Patients were eligible to be enrolled with a diagnosis of RRMM, ECOG score of 0 or 1, and they had received at least 3 prior regimens including at least one proteasome inhibitor and one immunomodulatory drug. A single infusion of zevor-cel (two dose levels, 100 × 10 6 CAR + T cells and 150 × 10 6 CAR + T cells) was administered 5-7 days after the start of lymphodepletion. Response was assessed by investigator per IMWG 2016 criteria. Bone marrow aspirates were tested for minimal residual disease (MRD) by the EuroFlow assay with a minimum sensitivity of 1 in 10 5 nucleated cells. Results: Starting July 23, 2019, 14 patients with a median age of 54 years (range 34, 62), received a single infusion of zevor-cel (100 × 10 6 CAR + T cells in 3 patients, 150 × 10 6 CAR + T cells as the recommended phase 2 dose in 11 patients). The median number of prior lines of therapy was 6. A total of 50.0% of the infused patients had high-risk cytogenetics, 14.3% had extramedullary disease, 14.3% had ISS stage III, and no patients received bridging therapy. By the data cutoff date (July 17, 2023), the median survival follow-up duration was 37.7 months (range:14.8, 44.2). Overall response rate was 100% (95% CI 76.8, 100.0), in which 11 (78.6%) patients achieved complete response (CR) or stringent complete response (sCR); 2 (14.3%) patients achieved very good partial response and 1 (7.1%) patient had partial response. All patients who achieved CR or better were MRD negative. The median progression-free survival was 25.0 months (14.9, not evaluable [NE]) for all patients and 26.9 months (15.5, NE) for patients with sCR/CR (Figure 1A). The median duration of response was 24.1 months (14.0, NE) for all patients and 26.0 months (14.6, NE) (Figure 1B) for patients with sCR/CR. At data cutoff, 5 subjects still had ongoing responses, and 7 patients progressed and were still in survival follow-up. Two patients had died at month 42.6 and 32.6, respectively, and their deaths were deemed unrelated to zevor-cel. All patients experienced treatment related adverse events and grade 3 or 4 hematologic toxicity. Thirteen patients (92.9%) had cytokine release syndrome (all grade 1 or 2). No immune effector cell-associated neurotoxicity syndrome, no second primary malignancy, and no autoimmune disease were reported. All patients have been tested negative for replication competent lentivirus to date. Conclusions: At approximately 3 years of follow-up, heavily pre-treated RRMM patients maintained deep and durable responses after receiving a single infusion of zevor-cel, which showed a well-managed safety profile in the ongoing long-term follow-up.

Tvt CAR7: Phase 1 Clinical Trial of Base-Edited "Universal” CAR7 T Cells for Paediatric Relapsed/Refractory T-ALL

Background Genome editing can overcome HLA barriers to generate 'off-the-shelf’ CAR T cell therapies. Despite the success of CAR-T cell therapies in B-cell malignancies, the expression of shared T cell antigens has constrained the development of CAR T cells targeting T-cell malignancies, due to T cell fratricide. Targeted base editing using CRISPR guided cytidine deamination mediates highly precise C→U→T conversion which can directly disrupt gene expression without DNA breaks. This Phase I study will investigate the feasibility and safety of base edited (BE) allogeneic CAR T cells against CD7, disrupted for TCR, CD7 and CD52. The cells are used to secure remission ahead of allogeneic stem cell transplantation (allo-SCT). Investigational medicinal product (IMP) Two allogeneic healthy donor derived BE-CAR7 T cell banks were manufactured from steady state apheresis harvests using a semi-automated process under compliant conditions. Cells were activated with an anti-CD3/CD28 reagent (Transact) and electroporated with codon optimised base editor (coBE) mRNA and three single guide RNAs targeting TRBC, CD52 and CD7. Subsequent lentiviral transduction delivered CAR7 (59-62% efficiency) and magnetic bead processing depleted residual TCRαβ+ T cells (residual 0.1-0.3%). Highly efficient disruption of CD7 and CD52 (98-99%) was confirmed by sequencing. Cells were cryopreserved and QC tested by flow cytometry, ddPCR for copy number (3.1-3.6) and replication competent lentivirus was excluded. Anti-leukemic potency was confirmed in vitro and in human:murine chimera experiments. Trial sponsorship & approvals The TvT CAR7 study (ISRCTN15323014) is an open label, single centre, single arm and non-randomised clinical trial. The trial is sponsored by Great Ormond Street Hospital NHS trust and is supported by MRC, Wellcome Trust and NIHR. Clinical trial authorisation was provided by MHRA and ethical approval by a gene therapy advisory committee (GTAC) ahead of site initiation. Study aims and Objectives The study aims to establish the safety and feasibility of BE-CAR7 to induce molecular remission in children with relapsed/refractory (r/r) CD7-positive T-ALL, ahead of a planned allo-SCT. Assessments include incidence of adverse events, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of BE-CAR7 cells and immune recovery after allo-SCT are monitored. Eligibility, treatment and recruitment The study opened on 14th April 2022 at Great Ormond Street Hospital recruiting children across the UK aged between 6 months and 16 years with r/r CD7+ T-cell malignancies quantifiable in bone marrow (>10-4 by flow or PCR). Exclusions include active uncontrolled infections, pre-existing GvHD or the detection of anti-HLA antibodies against the IMP. Eligible patients receive lymphodepletion with fludarabine (150 mg/sqm), cyclophosphamide (120 mg/kg) and alemtuzumab (1 mg/kg), followed by infusion of 0.2-2.0x106 BE-CAR7 T cells (maximum 5x104/kg TCRαβ T cells). Patients in molecular remission at day +28 proceed to allo-SCT, to remove BE-CAR 7 cells and promote immune reconstitution. The first subject was infused on 4th May 2022. Grade 2 CRS and grade 1 ICANS were observed, but no GvHD. Bone marrow assessment at d+28 revealed morphological remission without count recovery and with PCR-MRD <10-4 and the patient proceeded to reduced intensity allo-SCT as planned. This Phase 1 study aims to treat 10 children in the UK. Conclusion The study has demonstrated the feasibility of manufacturing "off-the-shelf” GMP-compliant base edited CAR7 T cells and is investigating their safety and efficacy against paediatric r/r T-ALL.

Lovo-cel (bb1111) Gene Therapy for Sickle Cell Disease: Updated Clinical Results and Investigations into Two Cases of Anemia from Group C of the Phase 1/2 HGB-206 Study

Background: Lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) uses autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an anti-sickling hemoglobin (Hb), HbAT87Q. The ongoing phase 1/2 HGB-206 study (NCT02140554) is the largest clinical trial of GT in SCD to date. We report updated efficacy and safety results for Group C and describe the results of additional investigations in two patients (pts) in Group C with persistent anemia. Methods: Pts (≥12-≤50 yrs of age) with SCD and recurrent severe vaso-occlusive episodes (VOEs) underwent plerixafor mobilization and apheresis followed by myeloablative busulfan conditioning and lovo-cel infusion. Lab evaluations, SCD-related outcomes, and safety were assessed. Data are median (min-max) unless otherwise stated; all timepoints are from lovo-cel infusion. Results: As of July 2021, 35 Group C pts had received a lovo-cel infusion, with a median follow-up of 20.9 months (min-max: 8.5-28.5). Neutrophil and platelet engraftment were achieved on Days 20 (12-35) and 36 (18-136), respectively. The median vector copy number in peripheral blood remained stable in all pts at ≥1 c/dg from 6 months after infusion until the last study visit, and resulted in median HbAT87Q levels of ≥5 g/dL during this period. The median total Hb level increased without transfusions from 8.5 g/dL at baseline to ≥11 g/dL from 6 months through last visit; HbAT87Q contributed at least 40% of total Hb (Fig 1A). Of the 29 pts who could be evaluated, 28 had a complete resolution of severe VOEs after lovo-cel infusion in the 2-yr parent study as of last follow-up, as compared with 3.5 events per yr (1.0-13.5) in the 2 yrs before enrollment (Fig. 1B); no severe VOEs have been reported during the long-term follow up study. Key hemolysis markers approached normal levels. Overall, 15 pts (43%) had ≥1 serious adverse event after lovo-cel infusion; the most frequently reported were abdominal pain, drug withdrawal syndrome (opiate), nausea, vomiting, and dehydration (6% each). No cases of veno-occlusive liver disease, graft failure, replication-competent lentivirus, vector-mediated insertional oncogenesis, or hematologic malignancies were observed. The lovo-cel treatment regimen generally reflected the known side effects of HSPC collection and busulfan conditioning regimen, and/or adverse events commonly seen in the population being evaluated. Further investigations were conducted as of March 2022 in two pts who presented with similar clinical and pathological findings that initially raised suspicion of myelodysplastic syndrome (MDS). Presentation features included neutropenia in one patient; both patients had persistent anemia despite adequate HbAT87Q production, bone marrow morphology showing erythroid specific dysplasia (binucleated or trinucleated cells) consistent with dyserythropoiesis as seen in other hemoglobinopathies, and low level of trisomy 8 (≤7.7%) only observed by fluorescence in situ hybridization analysis. There are no clinical symptoms suggestive of hematologic malignancies, no driver mutations, no clonal process (vector-related or otherwise) as shown by Integration Site Analysis and Next-Generation Sequencing (both subjects had >29,000 Unique Integration Sites with no integration having a relative frequency above 0.425%), and no chromosomal abnormalities on karyotyping among up to 205 metaphases. Both pts have had anemia and one maintained total Hb of 9.8 g/dL at Month 18 without transfusions, and the other required chronic transfusions to maintain 10.3 g/dL at Month 18 with otherwise normal complete blood counts. Both pts experienced a reduction or resolution of severe VOEs after lovo-cel infusion. Of note, these are the only pts in the study to have two α-globin deletions (−α3.7/−α3.7). We propose that α-globin trait may contribute to anemia and dyserythropoiesis after lovo-cel infusion. Summary: In Group C of the HGB-206 study, one-time treatment with lovo-cel resulted in sustained production of HbAT87Qand near-complete resolution of severe VOEs. Investigations to date in both patients with anemia have not confirmed MDS; thus, currently no hematologic malignancies have occurred in Group C. Ongoing long-term follow-up should be continued for all pts receiving GT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Long Term Outcomes of 63 Patients with Transfusion-Dependent β-Thalassemia (TDT) Followed up to 7 Years Post-Treatment with betibeglogene autotemcel (beti-cel) Gene Therapy and Exploratory Analysis of Predictors of Successful Treatment Outcomes in Phase 3 Trials

Background: One-time treatment with beti-cel ex vivo gene therapy (GT) adds functional copies of a modified HBB gene, βA-T87Q, to autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) through transduction with BB305 lentiviral vector (LVV), thereby addressing the underlying genetic cause of Transfusion-Dependent β-Thalassemia (TDT). Here, we evaluate the long-term efficacy and safety profile of beti-cel across phase 1/2, phase 3, and long-term follow-up (LTF) studies. We also present an exploratory analysis of predictors of successful treatment outcomes in patients from phase 3 studies. Methods: Patients with TDT underwent HSPC collection followed by pharmacokinetic-adjusted myeloablative busulfan conditioning and beti-cel infusion. Patients were followed for 2 yrs in the parent phase 1/2 (HGB-204, NCT01745120; HGB-205, NCT02151526) and phase 3 (HGB-207, NCT02906202; HGB-212, NCT03207009) studies; enrollment and all beti-cel infusions have been completed. Patients then entered a 13-yr LTF study, LTF-303 (NCT02633943). Transfusion independence (TI) was defined as weighted average hemoglobin (Hb) ≥ 9 g/dL without packed red blood cell transfusions for ≥ 12 months. An exploratory multivariate analysis was also carried out in patients enrolled in the phase 3 trials to identify predictors of clinical outcomes. Results: As of August 2021, 63 patients had received beti-cel and were followed for a median of 41.4 (9.0-87.5) months across the 4 parent studies and LTF. The median time until neutrophil and platelet engraftment was 23 (13-39) and 45 (19-191) days, respectively, and lymphocyte subsets were generally within normal range after beti-cel infusion. Peripheral blood vector copy number (VCN), levels of GT-derived adult Hb (HbAT87Q), and total Hb were stable and durable across studies and higher in phase 3 vs phase 1/2 studies following optimized beti-cel drug product (DP) manufacturing. HbAT87Q levels were stabilized by Month 6 and contributed to stable levels of total Hb. Total unsupported Hb levels ≥ 9g/dL at Month 6 were significantly associated with achieving TI (p<0.0001) (March 2021 data cut). One-time beti-cel GT was followed by durable TI across ages and genotypes with up to 7 yrs follow-up. A trend towards more patients achieving TI was observed in phase 3 (34/38; 89.5%) (Fig 1A) vs phase 1/2 (15/22; 68.2%). All patients who achieved TI (N=49) continue to remain TI as of last follow-up. Patients who achieved TI had reductions in markers of ineffective erythropoiesis and iron overload. Overall, 21/37 patients who restarted chelation stopped chelation at 24 months; 25% (12/49) of patients who achieved TI utilized phlebotomy for iron removal, including 6 patients who also received iron chelation. Overall, 18% (11/63) of patients experienced ≥1 adverse event (AE) considered related or possibly related to beti-cel; the most common DP-related AEs were abdominal pain (5/63 [8%]) and thrombocytopenia (3/63 [5%]), but none were observed beyond 2 yrs post-infusion, supporting a favorable long-term safety profile. Veno-occlusive liver disease was reported in 11% (7/63) of patients and resolved after appropriate treatment. No malignancies, insertional oncogenesis, vector-derived replication competent lentivirus, or clonal predominance was observed. The safety of the beti-cel treatment regimen largely reflected the known side effects of hematopoietic stem cell collection and the busulfan conditioning regimen. An exploratory multivariate analysis in patients from phase 3 studies (n=37; March 2021 data cut) identified that the percentage of DP cells transduced with the BB305 LVV (%LVV+ cells) was the best predictor of clinical outcomes (Fig 1B). DP VCN and %LVV+ cells are current release assays for beti-cel. Summary: beti-cel is a potentially curative GT for patients with TDT across ages and genotypes through the achievement of TI and normal or near normal Hb levels. An exploratory multivariate analysis demonstrated that DP transduction efficiency was the most predictive attribute of TI, a key endpoint for clinical outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Induction of Fetal Hemoglobin and Reduction of Clinical Manifestations in Patients with Severe Sickle Cell Disease Treated with Shmir-Based Lentiviral Gene Therapy for Post-Transcriptional Gene Editing of BCL11A: Updated Results from Pilot and Feasibility Trial

Background Induction of fetal hemoglobin (HbF) by repressing BCL11A could reduce or eliminate sickle cell disease (SCD) clinical manifestations. The BCH-BB694 lentiviral vector (LVV) encodes a shRNA targeting BCL11A embedded in a microRNA scaffold (shmiR) allowing erythroid-specific knockdown to induce γ-globin expression and concomitantly and coordinately repress β-sickle globin expression (Brendel et al. JCI 2016). The safety and efficacy of gene therapy (GT) with BCH-BB694-transduced autologous CD34+ cells in patients with SCD is being investigated in a pilot and feasibility trial (NCT03282656), which has now completed enrollment of all 10 planned patients. Preliminary data in the first 6 patients described robust HbF induction and a favorable safety profile (Esrick et al. NEJM 2021). Additional follow-up data including all ten pilot trial patients is discussed here with longest follow-up now > 4 years. Methods Patients with severe SCD were screened for eligibility. Autologous CD34+ cells were collected via apheresis after plerixafor mobilization (Esrick and Manis et al. Blood Adv 2018) and then transduced ex vivo with the BCH-BB694 shmiR LVV. Gene modified cells were infused into subjects who had received myeloablative busulfan conditioning. Patients were monitored for adverse events (AEs), hemoglobin (Hb), HbF fraction, percent F cells, hemolysis markers, and SCD clinical manifestations. Results Ten patients (7-25 years of age at enrollment) received GT, with median follow-up of 30.5 (2-50) months. There were no Grade 3 or 4 AEs associated with mobilization, collection or infusion. Median product VCN, % transduced cells, and CD34+ cell dose in the 10 patients were 3.5 (1.8-6.9) copies/diploid genome (c/dg), 96% (62-100%), and 5.7 (3.6-8.8) x 106 CD34+ cells/kg, respectively. Engraftment of neutrophils occurred on day 22 (18-30) and of platelets on day 32 (25-62). One patient (patient 10) has low post-GT peripheral VCN, HbF, and percent F cells; most recently 0.23 c/dg, 11.2%, and 37.3% respectively. Although improved, this was considered a GT failure and the patient has been started on new SCD-directed therapies due to ongoing SCD clinical manifestations. Subsequent data described here exclude patient 10. At the most recent study visit, the 7 untransfused patients with follow up > 6 months had mean total Hb of 10.5 (8.8-12.2) g/dL, HbF/(F+S) of 30.3% (21.4 - 42.4%), percent F cells of 70.6% (58.8-79.8%), and HbF per F cell of 12 (10.1-14.4) pg (Table 1). The lowest Hb response was seen in patient 11 who has 2-gene deletion alpha thalassemia in combination with SCD. Five patients with follow-up > 6 months (patients 4, 6, 8, 9, and 11) had frequent severe vaso-occlusive events (ACS, priapism, or pain requiring inpatient or ED opioids) as indication for eligibility, with a median of 6 (3-13) events (Table 2). From 6 months after GT through present, these 5 patients have had a median of 1 (0-1) event. Three patients with follow-up > 6 months (patients 2, 3, and 7) were on chronic transfusion regimens pre-GT. Patient 3 has continued chronic transfusions due to pre-existing moyamoya with goal of maintaining HbS at pre-GT levels. Patients 2 and 7 have received no post-GT transfusions. Patients 2, 3, and 7 have had no SCD clinical events. Mean lactate dehydrogenase and absolute reticulocyte count at last study visit were 346 (215-389) U/L and 241 (200-360) 109/L, respectively, indicating decreased but persistent mild hemolysis in all patients. There have been no instances of graft failure, replication competent lentivirus detection, or clonal dominance in any patient. Six patients with ≥2 years post GT have had bone marrow biopsies at the 2 year timepoint without evidence of myelodysplasia. Targeted sequencing of ~95 genes frequently mutated in hematologic malignancies has been performed on peripheral blood samples every 6 months since early 2021 in patients with ≥6 months from GT. One patient had a pathogenic mutation detected in the DNMT3A gene, which was present in a stored pre-GT sample and has been present at a stable VAF <4% at 6, 9, and 12 months post-GT. Summary Post-transcriptional gene editing of BCL11A appears safe with stable induction of broadly distributed HbF and significant mitigation of VOE events in severe SCD in all but one treated patient. Based on the results of this pilot study, a phase 2, multi-institutional trial is now open for enrollment using the same vector and approach (NCT05353647). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SURPASS-2 trial design: A phase 2, open-label study of ADP-A2M4CD8 SPEAR T cells in advanced esophageal or esophagogastric junction cancers.

TPS363 Background: ADP-A2M4CD8 is an autologous specific peptide enhanced affinity receptor (SPEAR) mixed CD4 and CD8 T-cell product that expresses an engineered T-cell receptor (TCR) designed to target the MAGE-A4 antigen in HLA-A*02-positive patients. These SPEAR T-cells also express wild-type CD8α co-receptors, designed to provide additional functionality to CD4 T-cells. MAGE-A4 expression has been described in several solid tumors, including esophageal and esophagogastric junction (EGJ) cancers[1]. In an ongoing phase 1 study (NCT04044859) of ADP-A2M4CD8 in patients with different tumor types, most adverse events have been consistent with those typically experienced by patients undergoing chemotherapy and/or adoptive T-cell therapies, and as of 2 August 2021, among evaluable patients with esophageal and EGJ cancers, best overall responses were 1 partial response (EGJ), 4 stable disease (2 EGJ, 2 esophageal), and 1 progressive disease (EGJ)[2]. Methods: SURPASS-2 (NCT04752358) is a phase 2, open-label, single-arm trial to assess safety and efficacy of ADP-A2M4CD8 SPEAR T-cells in HLA-A*02–positive patients with advanced esophageal or EGJ cancers that express MAGE-A4 antigen. A total of 45 patients between the ages of 18 and 75 years old will be treated across sites in North America and Europe. Key eligibility criteria include measurable disease per RECIST v1.1; ECOG performance status of 0 or 1; no active autoimmune or immune-mediated disease; no leptomeningeal disease, carcinomatous meningitis, or symptomatic CNS metastases; and ≤2 prior lines of combination or single agent systemic treatment for advanced or metastatic disease before treatment with ADP-A2M4CD8 as the next therapy. Leukapheresis can be performed before initiating or at the end of second-line treatment. Collected T-cells will be transduced with a self-inactivating lentiviral vector expressing the high affinity MAGE-A4-specific TCR and the CD8α co-receptor. Lymphodepleting chemotherapy, consisting of intravenous (IV) cyclophosphamide 600 mg/m2/day for 3 days and IV fludarabine 30 mg/m2/day for 4 days, will be given approximately 1 week prior to treatment with ADP-A2M4CD8. ADP-A2M4CD8 dose will range between 1 x 109 to 10 x 109 transduced cells administered by a single IV infusion. The primary endpoint is overall response rate per RECIST v1.1 by an independent radiological assessment committee. Safety evaluations will include adverse events (AEs); serious AEs; incidence, severity, and duration of the AEs of special interest; replication competent lentivirus; and T-cell clonality and insertional oncogenesis. All patients receiving ADP-A2M4CD8 cellular therapy will be followed for 15 years from time of last T-cell infusion for observation of delayed AEs. To date, one site is activated. [1] Ishihara et al. BMC Cancer 20, 606 (2020). [2] Hong et al. E-poster 540P: ESMO (2021). Clinical trial information: NCT04752358.

Lentiviral haematopoietic stem-cell gene therapy for early-onset metachromatic leukodystrophy: long-term results from a non-randomised, open-label, phase 1/2 trial and expanded access

SummaryBackgroundEffective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD.MethodsThis study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182.FindingsAt the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64–7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3–42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6–12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9–82·3]) and early-juvenile MLD (42% [12·3–71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes.InterpretationTreatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy.FundingOrchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline.

Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials

Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials

Lentiviral Mediated Gene Therapy for Pyruvate Kinase Deficiency: Interim Results of a Global Phase 1 Study for Adult and Pediatric Patients

▪Background: Pyruvate kinase deficiency (PKD) is a rare inherited hemolytic anemia caused by mutations in the PKLR gene resulting in decreased red cell pyruvate kinase activity and impaired erythrocyte metabolism. Manifestations include anemia, reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected individuals. PKD represents a significant unmet medical need as current treatments are palliative and limited to blood transfusions, chelation therapy, and splenectomy which are associated with significant side effects. Preclinical studies in a clinically relevant PKD murine model have demonstrated that infusion of gene-modified Lin− bone marrow (BM) cells may ameliorate PKD phenotype. Based on compelling preclinical data, a global Phase 1 clinical trial RP-L301-0119 (NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy in adult and pediatric subjects with severe PKD.Methods: Six adult and pediatric patients with severe PKD (defined as severe and/or transfusion-dependent anemia despite prior splenectomy) will be enrolled. Peripheral blood (PB) hematopoietic stem cells (HSCs) are collected on 2 consecutive days via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and plerixafor. HSCs are enriched, transduced with PGK-coRPK-WPRE lentiviral vector (LV), and cryopreserved. Following release testing of the investigational product (IP), RP-L301, myeloablative therapeutic drug monitoring (TDM) busulfan is administered over 4 days. RP-L301 is then thawed and infused. Patients are followed for safety assessments, including replication competent lentivirus (RCL) and insertion site analysis (ISA), and for efficacy parameters including PB and BM genetic correction, decrease in transfusion requirements, clinically significant improvement in anemia, and reduction of hemolysis for 2 years post-infusion.Results: As of May 2021, 2 adult patients with severe anemia have received RP-L301. Patient 1 (age 31 years) received 3.9x106 CD34+ cells/kg with mean vector copy number (VCN) of 2.73. Patient 2 (age 47 years) received 2.4x106 CD34+ cells/kg with mean VCN of 2.08. Despite baseline hemoglobin (Hb) levels in the 7.0-7.5 g/dL range, both patients displayed normal-range hemoglobin (Hb), improved hemolysis markers, and have required no red blood cell transfusions post-engraftment at 9- and 6- months follow-up. Both report improved quality of life. PB mononuclear cell VCNs for both patients were >2.0 at last evaluated timepoint (6- and 3-months post-treatment, respectively). No serious adverse events have been attributed to RP-L301. Updated safety and efficacy data will be presented.Conclusions: Hematopoietic stem cell mobilization using G-CSF and plerixafor is feasible and effective in adult PKD patients. RP-L301 was successfully manufactured to meet the required specifications for the Phase 1 clinical study and administered without short-term infusion related complications. Efficacy was demonstrated by normalized Hb associated with engraftment confirmed by PB and BM VCN. DisclosuresShah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Navarro: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company, Other: Dr. Navarro has licensed medicinal products and receives research funding and equity from Rocket Pharmaceuticals, Inc., Patents & Royalties, Research Funding. Sevilla: Miltenyi: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Sevilla is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, and may be entitled to receive financial benefits from the licensing of such patents.; SOBI: Consultancy. Glader: Agios: Consultancy. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company. Beard: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Zeini: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Choi: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Rao: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Bueren: Rocket Pharmaceuticals, Inc.: Consultancy, Other: J.Bueren is an inventor on patents on lentiviral vectors filed by CIEMAT, CIBERER and Fundación Jiménez Díaz, may be entitled to receive financial benefits from the licensing of such patents and receives funding for research., Patents & Royalties, Research Funding. Schwartz: Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding.

Restoring Iron Homeostasis in Pts Who Achieved Transfusion Independence after Treatment with Betibeglogene Autotemcel Gene Therapy: Results from up to 7 Years of Follow-up

Restoring Iron Homeostasis in Pts Who Achieved Transfusion Independence after Treatment with Betibeglogene Autotemcel Gene Therapy: Results from up to 7 Years of Follow-up

TT52CAR19: Phase 1 Trial of CRISPR/Cas9 Edited Allogeneic CAR19 T Cells for Paediatric Relapsed/Refractory B-ALL

Background‘Off-the-shelf’ CAR T cell therapies are being investigated as alternatives to autologous CAR therapy, and can be generated using genome editing from allogeneic donors. Strategies to address HLA barriers include disruption of T cell receptor expression to prevent GVHD and removal of cell surface HLA expression to obviate host mediated rejection, as well as removal of CD52 to create a survival advantage in the presence of Alemtuzumab. In this study, lentiviral mediated CAR19 expression is linked to CRISPR editing through the incorporation of CRISPR guide RNA sequences within the vector 3'Long terminal repeat (LTR). The trial is in progress using pre-manufactured batches of TT52CAR19 T cells.Investigational medicinal product (IMP)Three allogeneic non-HLA matched donor derived CAR19 T cell banks were manufactured from steady state apheresis harvests from registry volunteer donors, using a semi-automated process under compliant conditions. Cells were activated with anti-CD3/CD28 (Transact) reagent and transduced with a lentiviral vector, TT52CAR19, and dual guide sgRNA cassettes targeting the T cell receptor alpha chain (TRAC) and CD52 loci. Next electroporation of Cas9 mRNA elicited transient genome editing, and automated magnetic bead depletion removed remaining TCRαβ+ T cells (mean 0.7%) and enriched CAR19+ T cells (mean 92.8%). Cells were cryopreserved in aliquots suitable for dose-banding and subjected to release testing, including flow cytometry, quantification of copy number (mean 3.83) and exclusion of replication competent lentivirus. Potency of each bank was confirmed in human:murine chimera experiments.Trial sponsorship & approvalsThe study is open at a single site and is sponsored by Great Ormond Street Hospital NHS trust and is supported by the Medical Research Council and National Institute for Health Research. Clinical trial authorisation was awarded by the MHRA after expert review, and ethical approval including gene therapy advisory committee (GTAC) review, and health research authority (HRA) approval. The study is open label, single arm and non-randomised.Study aims and ObjectivesThe study aims to establish the safety and feasibility of TT52CAR19 for the induction of molecular remission in children with relapsed /refractory CD19-positive B-cell acute lymphoblastic leukaemia (B-ALL) within 28 days, ahead of planned allogeneic haematopoietic stem cell transplantation (allo-SCT). Assessments include time to remission, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of TT52CAR19 cells and tracking of immune recovery after allo-SCT is monitored. Recording of complications and toxicities, including possible genotoxic side effects from CRISPR/Cas9 modification provides safety profile information.Eligibility, infusion and outcomesThe study plans to treat 10 children aged between 6 months and < 18 years with CD19+ B-ALL quantified at >10 -4 in marrow (by flow or PCR) who are ineligible for autologous therapy. To date 2/4 children screened were found eligible and proceeded to lymphodepletion comprising Fludarabine, Cyclophosphamide and Alemtuzumab followed by a single infusion of 0.8-2.0x10 6 CAR19 T cells and a maximum of 5x10 4/kg TCRαβ T cells. In both cases, there was no GVHD or CRS >grade 1 and recovery of neutrophil counts by d28. Molecular remission was achieved in one child, where TT52CAR19 cells persistence was tracked by chimerism and copy number until further conditioning and allo-SCT. This child remains in remission >6 months later.ConclusionsFeasibility of pre-manufacturing off-the-shelf CRISPR/Cas9 edited CAR19 T cells is demonstrated and the trial has provided first in human safety data and preliminary indications of potent anti-leukaemic activity in one of two subjects dosed. DisclosuresQasim: Autolus: Current equity holder in publicly-traded company; Novartis: Honoraria; Servier: Research Funding; Tessa: Membership on an entity's Board of Directors or advisory committees.

Clinical Outcomes up to 3 Years Following Lentiglobin Gene Therapy for Transfusion-Dependent β-Thalassemia in the Northstar Hgb-204 Study

BACKGROUNDAutologous hematopoietic stem cell (HSC) gene therapy could be an effective treatment for transfusion-dependent β-thalassemia (TDT) without some of the limitations and risks associated with allogeneic HSC transplantation. LentiGlobin Drug Product (DP) contains autologous HSC (CD34+ cells) transduced ex vivo with the betibeglogene darolentivec (BB305) lentiviral vector encoding a human β-globin gene with the anti-sickling T87Q mutation (HbAT87Q). The Northstar Study(HGB-204; NCT01745120) is an international, multi-center phase 1/2 clinical trial examining the safety and efficacy of LentiGlobin investigational gene therapy in patients with TDT. As of the most recent data cut (June 2, 2017), all patients have 1 to 3 years of follow-up post DP infusion. METHODSPatients (12-35 years of age) with TDT were enrolled at participating sites in the U.S., Australia, and Thailand. Autologous CD34+ cells were collected by mobilization and apheresis and transduced with the BB305 vector in a centralized facility. Patients underwent myeloablative conditioning with intravenous busulfan prior to infusion of transduced cells. Patients were monitored for hematologic engraftment, vector copy number (VCN), levels of HbAT87Q, and red blood cell (RBC) transfusion requirements post-infusion. Safety data, including adverse events (AEs), vector integration site analysis, and surveillance for replication competent lentivirus (RCL), were evaluated post-infusion. The primary analysis period for the study is 24 months following infusion; patients then transition into a long-term study for an additional 13 years of follow-up. RESULTSEighteen patients with TDT (8 with β0/β0 and 10 with non-β0/β0 genotypes; aged 12-35 years) received LentiGlobin DP. The median DP VCN was 0.7 (range: 0.3-1.5) copies/diploid genome, the median cell dose was 8.1 x 106 (range: 5.2-18.1 x 106) CD34+ cells/kg, and the proportion of transduced CD34+ cells was 17-58%. The toxicity profile observed to date has been typical of myeloablative conditioning. During a follow-up period of up to 38.3 months post-infusion (median 25.5 months; range: 14.9-38.3 months), there have been no ≥ grade 3 DP-related AEs and no evidence of clonal dominance or RCL. Serious AEs occurring after DP infusion have been reported in 6/18 (33%) patients: venoocclusive liver disease (n=2) and 1 case each of cellulitis, diarrhea, gastroenteritis, Klebisella infection, cardiac ventricular thrombosis, device-related thrombosis, and hyperglycemia. No serious AEs were considered related to DP.Of the 10 patients with non-β0/β0 genotypes, 8 have been free of transfusions for a median of 27.1 (range 12.5-35.2) months. At their latest study visit (n=8: Months 15-36), their total Hb level ranged from 9.3-13.7 g/dL, and their HbAT87Q level was 3.6-9.6 g/dL. The peripheral VCN ranged from 0.1−1.0. Notably, of these 8 patients, 6 have achieved “transfusion independence” (TI), defined as ≥12 months without transfusions with a weighted average Hb ≥9 g/dL. The 2 patients with non-β0/β0 genotypes who still require intermittent transfusions had annual transfusion volumes reduced by 30% and 94%; both received DP with a VCN in the lower range (DP VCNs: 0.3 and 0.4).Two patients with β0/β0 genotypes have not received a transfusion in more than a year. At the patients' last study visit (Month 24/Month 12), total Hb levels were 9.0 and 10.2 g/dL, HbAT87Q levels were 8.4 and 6.8 g/dL, and peripheral VCNs were 0.9 and 0.6, respectively. Six patients with β0/β0 genotypes have continued transfusions. However, their annual transfusion volumes have decreased by a median of 63% (range: 19% to 81%), from an annualized volume of 124.4-261.3 ml/kg/year at baseline to an annualized volume of 31.4-212.6 ml/kg/year (from 6 months post-infusion to data cut). CONCLUSIONSWith up to 3 years of follow-up, the Northstar Study is the largest international gene therapy trial in TDT to date. All patients, 9 of whom have at least 2 years of follow-up, have demonstrated ongoing clinical benefit with a manageable safety profile. A total of 10 patients (8/10 with non-β0/β0, 2/10 with β0/β0 genotypes) have been able to discontinue RBC transfusions and remain clinically well. The 8/18 patients still receiving transfusions have significantly reduced annualized transfusion volumes (up to 81% and 94% in patients with β0/β0 and non-β0/β0 genotypes, respectively). DisclosuresKwiatkowski:Bluebird Bio: Research Funding; Apopharma: Research Funding; Novartis: Research Funding; Agios: Consultancy, Honoraria; Ionis: Consultancy, Honoraria. Thompson:Novartis: Consultancy, Research Funding; Baxalta: Research Funding; Celgene: Consultancy, Research Funding; bluebird bio: Consultancy, Research Funding. Rasko:Genea: Equity Ownership; IMAGO Biosciences: Consultancy, Equity Ownership; Rarecyte: Consultancy, Equity Ownership; Novartis: Other: Clinical trials, Speakers Bureau; Spark: Equity Ownership, Other: clinical trials, Speakers Bureau; President-Elect International Society for Cellular Therapy: Membership on an entity's Board of Directors or advisory committees. Schiller:bluebird bio: Research Funding; mateon therapeutics: Research Funding. von Kalle:bluebird bio: Consultancy; GeneWerk: Equity Ownership. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Deary:bluebird bio: Employment, Equity Ownership. Asmal:bluebird bio: Employment, Equity Ownership. Walters:bluebird bio: Research Funding; AllCells, Inc: Other: Medical Director; Sangamo Therapeutics: Consultancy; ViaCord Processing Lab: Other: Medical Director.

Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency

BackgroundSevere combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency.MethodsWe treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up.ResultsOverall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade.ConclusionsTreatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.)

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction

Background AimsViral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct. MethodsAssay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations. ResultsThe limit of detection was 10 copies/μL, whereas negative samples showed <1.3 copies/μL. Within and between assay imprecision coefficients of variation across the reportable range (10–10 000 copies/μL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R2 >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations). ConclusionsDDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.

A Phase 1/2 Study of Lentiviral-Mediated Ex-Vivo Gene Therapy for Pediatric Patients with Severe Leukocyte Adhesion Deficiency-I (LAD-I): Results from Phase 1

Introduction: LAD-I is a rare inherited disorder of leukocyte (primarily neutrophil) adhesion to endothelial cell surfaces, migration, and chemotaxis resulting from ITGB2 gene mutations encoding for the β2-integrin component, CD18. Severe LAD-I (i.e., CD18 expression on &amp;lt;2% of PMNs) is characterized by recurrent severe infections, impaired wound healing, and childhood mortality. Although allogeneic hematopoietic stem cell transplant (alloHSCT) is potentially curative, its utilization and efficacy are limited by HLA-matched donor availability and risk of graft-versus-host disease (GVHD). RP-L201-0318 (clinical trials.gov # NCT03812263) is a phase 1/2 open-label clinical trial evaluating the safety and efficacy of autologous CD34+ cells transduced with a lentiviral vector (LV) carrying the ITGB2 gene encoding for CD18 (Chim-CD18-WPRE) in severe LAD-I. Methods: Pediatric patients ≥ 3 months old with severe LAD-I (demonstrated by CD18 expression on &amp;lt;2% PMNs and at least 1 prior significant bacterial or fungal infection) are eligible. Peripheral blood (PB) HSCs are collected via apheresis after mobilization with granulocyte-colony stimulating factor (G-CSF) and Plerixafor. CD34+ cells are selected, transduced with Chim-CD18-WPRE LV, and cryopreserved. Myeloablative conditioning with busulfan (with therapeutic drug monitoring (TDM) to adjust dosing to enable target area under the curve (AUC)) is administered, followed by infusion of the investigational drug product (RP-L201). Patients are followed for safety assessments (i.e., replication competent lentivirus (RCL) and insertion site analysis (ISA)), and efficacy -- survival to age 2 and at least 1-year post-infusion without alloHSCT, increase in neutrophil CD18 expression to at least 10%, PB vector copy number (VCN), decrease in infections and/or hospitalizations, and resolution of skin or periodontal abnormalities. Results: Two patients (ages 9 and 3) were treated in Phase 1. Both have a history of recurrent severe infections, documented ITGB2 mutations, and baseline CD18, CD11a, and CD11b expression &amp;lt; 1%. Mobilization and apheresis procedures were performed successfully and busulfan was administered at the target AUC. Investigational product comprised of 4.2x106 CD34+ cells/kg with VCN of 3.8 copies/cell (liquid culture) for Patient 1 and 2.8x106 CD34+ cells/kg with VCN of 2.5 for Patient 2 and were infused without complications. No serious treatment-emergent adverse events were reported. Neutrophil engraftment was observed in &amp;lt; 3 weeks in both patients. For Patient 1, PB PMN CD18 expression 6 months post-treatment was 47% (sustained from 45% at 3-months, vs. &amp;lt; 1% at baseline), with PB VCN of 1.3. Skin lesions present at baseline were resolving with no new lesions. Replication competent lentiviral (RCL) testing at 3 months and 6 months post-infusion were negative. Safety and efficacy data 12-months post-treatment for Patient 1 and 6-months post-treatment for Patient 2 will be available at the time of presentation. Conclusion: Initial results from Phase 1 demonstrate preliminary safety and efficacy of RP-L201 for reversal of severe LAD-I. Enrollment of patients in the Phase 2 study is underway. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents &amp; Royalties, Research Funding. Rao:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Almarza:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Nicoletti:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Law:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Beard:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Sevilla:Novartis: Other: Advisory Board; Sobi: Other: Advisory Board; Rocket Pharma: Consultancy; Amgen: Other: Advisory Board. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents &amp; Royalties, Research Funding. Schwartz:Rocket Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company.

Long-Term Efficacy and Safety of Betibeglogene Autotemcel Gene Therapy for the Treatment of Transfusion-Dependent β-Thalassemia: Results in Patients with up to 6 Years of Follow-up

Long-Term Efficacy and Safety of Betibeglogene Autotemcel Gene Therapy for the Treatment of Transfusion-Dependent β-Thalassemia: Results in Patients with up to 6 Years of Follow-up