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lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) comprises autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified β-globin gene (βA-T87Q ) to produce anti-sickling hemoglobin (HbAT87Q ). The efficacy and safety of lovo-cel for SCD are being evaluated in the ongoing phase 1/2 HGB-206 study (ClinicalTrials.gov: NCT02140554). The treatment process evolved over time, using learnings from outcomes in the initial patients to optimize lovo-cel's benefit-risk profile. Following modest expression of HbAT87Q in the initial patients (Group A, n = 7), alterations were made to the treatment process for patients subsequently enrolled in Group B (n = 2, patients B1 and B2), including improvements to cell collection and lovo-cel manufacturing. After 6 months, median Group A peripheral blood vector copy number (≥0.08 c/dg) and HbAT87Q levels (≥0.46 g/dL) were inadequate for substantial clinical effect but stable and sustained over 5.5 years; both markedly improved in Group B (patient B1: ≥0.53 c/dg and ≥2.69 g/dL; patient B2: ≥2.14 c/dg and ≥6.40 g/dL, respectively) and generated improved biologic and clinical efficacy in Group B, including higher total hemoglobin and decreased hemolysis. The safety of the lovo-cel for SCD treatment regimen largely reflected the known side effects of HSPC collection, busulfan conditioning regimen, and underlying SCD; acute myeloid leukemia was observed in two patients in Group A and deemed unlikely related to insertional oncogenesis. Changes made during development of the lovo-cel treatment process were associated with improved outcomes and provide lessons for future SCD GT studies.

β-Thalassemia pathology is due not only to loss of β-globin (HBB), but also to erythrotoxic accumulation and aggregation of the β-globin-binding partner, α-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in β-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize β-globin:α-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing β-thalassemia.

Ability to accurately attribute adverse events post–gene therapy is required to describe the benefit-risk of these novel treatments. A SCD patient developed myelodysplastic syndrome post-LentiGlobin treatment; we show how insertional oncogenesis was excluded as the cause.

BackgroundDonors other than matched siblings and low-intensity conditioning regimens are increasingly used in haematopoietic stem cell transplantation. We aimed to compare the relative risk of donor type and conditioning regimen intensity on the transplantation outcomes of in patients with sickle cell disease.MethodsFor this retrospective cohort study, we collected data from 90 US centres reported to the Center for International Blood and Marrow Transplant Research. Eligible patients were younger than 50 years, had genetically confirmed sickle cell disease (Hb SS) or sickle beta thalassemia (Hb Sβ), and underwent allogeneic haematopoietic cell transplantation between Jan 15, 2008, and Dec 28, 2017. We considered transplants from donor-recipient pairs matched at the allele-level (HLA-A, HLA-B, HLA-C, and HLA-DRB1), including HLA-matched sibling donors, haploidentical related donors, matched unrelated donors, or mismatched unrelated donors. The main outcome was event-free survival. The effect of donor type, conditioning regimen intensity (myeloablative, non-myeloablative, and reduced-intensity regimens), age (≤12 or 13-49 years), sex, performance score, comorbidity index, recipient cytomegalovirus serostatus, graft type (bone marrow, peripheral blood, or umbilical cord blood), and transplantation period (2008-12 and 2013-17) on outcomes was studied using Cox regression models.FindingsOf 996 patients with sickle cell disease and who underwent transplantation in 2008-17, 910 (91%) were included (558 [61%] patients had HLA-matched sibling donors, 137 [15%] haploidentical related donors, 111 [12%] matched unrelated donors, and 104 [11%] mismatched unrelated donors). The median follow-up was 36 months (IQR 18-60) after transplantation from HLA-matched siblings, 25 months (12-48) after transplantation from haploidentical related donors, 37 months (23-60) after transplantation from HLA-matched unrelated donors, and 47 months (24-72) after transplantation from mismatched unrelated donors. Event-free survival was worse in recipients aged 13 years or older than in those younger than 13 years (hazard ratio 1·74, 95% CI 1·24-2·45; p=0·0014) and in those who received a transplant from haploidentical related donors (5·30, 3·17-8·86; p

Rationale: Sickle cell disease (SCD) is a monogenetic condition with recurring vasoocclusive events causing lifelong pulmonary morbidity and mortality. There is increasing access to curative therapies, such as hematopoietic cell transplant (HCT), for people living with SCD. However, more information on pulmonary function in adults with SCD after HCT is needed to best guide decisions for HCT and post-HCT care. Objectives: To test the hypothesis that forced expiratory volume in 1 second (FEV1) and other pulmonary function testing (PFT) parameters remain stable 3 years after HCT. Methods: People living with SCD undergoing nonmyeloablative HCT in a prospective cohort at the NIH Clinical Center from 2004 to 2019 were evaluated for enrollment. Global Lung Function Initiative reference equations and descriptive statistics were calculated before HCT and annually for 3 years. Six-minute-walk distance (6MWD) testing was performed. Generalized estimating equations were employed to evaluate interindividual changes in PFT parameters and 6MWD. Results: Of 97 patients with SCD undergoing HCT, 41 (42%) were female with median (25th, 75th percentile) age 31.8 (24.8, 38.0) years. Each year of measurement included the following numbers of subjects available for analysis with PFTs: baseline (n = 97), Year 1 (n = 91), Year 2 (n = 72), and Year 3 (n = 55); and the following numbers of subjects available for analysis with 6MWD: baseline (n = 79), Year 1 (n = 73), Year 2 (n = 57), and Year 3 (n = 41). Pre-HCT FEV1 was median (25th, 75th percentile) 68.3% (61.3%, 80.3%) and 69.2% (60.8%, 77.7%) 3 years after HCT, and pre-HCT diffusing capacity of the lung for carbon monoxide (DlCO) was 60.5% (53.0%, 66.3%) and 64.6% (55.1%, 73.4%) 3 years after HCT. Generalized estimating equations estimated that DlCO percent predicted increased significantly by 3.7% (95% confidence interval, 1.0%, 6.3%), and the 6MWD significantly increased by 25.9 (6.6, 45.2) meters 3 years after HCT, whereas there was no significant change in percent predicted FEV1 or FVC compared with before HCT. Conclusions: Overall, PFT results remained stable and there was an improvement in DlCO and 6MWD in this predominantly adult cohort undergoing nonmyeloablative HCT for SCD. Allogeneic HCT for SCD may cease the cycle of vasoocclusive pulmonary injury and prevent continued damage. Multicenter studies are needed to evaluate the long-term lung health effects of HCT for SCD in adults and children. [ABSTRACT FROM AUTHOR]

BackgroundHaemopoietic progenitor cell (HPC) transplantation can cure sickle cell disease. Non-myeloablative conditioning typically results in donor-derived erythrocytes and stable mixed chimerism of recipient-derived and donor-derived leucocytes. Exposure to donor antigens from the HPC graft and new red cell antibodies induced by transfusion can lead to immunohaematological complications. We assessed the incidence of such complications among HPC transplant recipients with sickle cell disease.MethodsThe study population was all patients with sickle cell disease enrolled before March 31, 2015, in the three clinical trials of non-myeloablative HPC transplantation at the National Institutes of Health. We assessed formation of new red cell antibodies after transplantation and red cell incompatibility between donors and recipients.Findings61 patients were enrolled, 42 were HLA matched and 19 were haploidentical. Nine (15%) had immunohaematological complications. Before HPC transplantation, three patients had antibodies incompatible with their donors. After HPC transplantation, new red cell antibodies were seen in six patients (11 alloantibodies and two autoantibodies), among whom three developed antibodies incompatible with donor or recipient red cells and three developed compatible antibodies. The clinical course of complications was highly variable, from no severe effects attributable to antibodies, to sustained reticulocytopenia, to near-fatal haemolysis. We found no significant correlation between immunohaematological complications and graft failure, graft rejection, or death.InterpretationClinical effects ranged from seemingly not clinically important to potentially fatal. In patients with sickle cell disease, donor and recipient red cell phenotypes should be carefully assessed before transplantation to minimise and manage the risk of immunohaematological complications.FundingIntramural Research Program and National Institutes of Health.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAV51-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust [gamma]-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach., Introduction The [beta]-hemoglobin (HBB) disorders, including sickle cell disease (SCD) and [beta]-thalassemia, are the most common single-gene inherited disorders, resulting in severe morbidity and mortality worldwide. The only curative therapeutic [...]

The occurrence of clonal perturbations and leukemia in patients transplanted with gamma-retroviral (RV) vector-transduced autologous hematopoietic stem and progenitor cells (HSPCs) has stimulated extensive investigation, demonstrating that proviral insertions may perturb adjacent proto-oncogene expression. Although enhancer-deleted lentiviruses are less likely to result in insertional oncogenesis, there is evidence that they may perturb transcript splicing, and one patient with a benign clonal expansion of lentivirally transduced HPSC has been reported. The rhesus macaque model provides an opportunity for informative long-term analysis to ask whether transduction impacts on long-term HSPC properties. We used two techniques to examine whether lentivirally transduced HSPCs from eight rhesus macaques transplanted 1-13.5 years previously are perturbed at a population level, comparing telomere length as a measure of replicative history and gene expression profile of vector positive versus vector negative cells. There were no differences in telomere lengths between sorted GFP+ and GFP- blood cells, suggesting that lentiviral (LV) transduction did not globally disrupt replicative patterns. Bone marrow GFP+ and GF- CD34+ cells showed no differences in gene expression using unsupervised and principal component analysis. These studies did not uncover any global long-term perturbation of proliferation, differentiation, or other important functional parameters of transduced HSPCs in the rhesus macaque model.