Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease.

Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms.

Genetic reversal of the globin switch concurrently modulates both fetal and sickle hemoglobin and reduces red cell sickling.

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects (BCLshmiR, median HbF% = 27.9) and compare them to 10 HU-treated subjects demonstrating high and comparable levels of HbF (HU High Responders, median HbF% = 27.0). All BCL11A patients achieved the primary endpoint for NCT03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 109 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCLshmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCLshmiR vs. 61% in HU High Responders (p = 0.004). BCLshmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders (p = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects.

Bidirectional processes linking social determinants of health and pediatric sickle cell anemia management: A qualitative study.

BACKGROUND: Children with sickle cell anemia (SCA) have substantial medical needs and more unmet basic needs than children with other medical conditions. Despite a recent focus on social determinants of health (SDoH), there remains an incomplete understanding of the processes linking SDoH and disease management, particularly for youth with SCA. This study elucidated these processes and identified ways to mitigate deleterious effects of adverse SDoH on SCA management. METHODS: Parents/primary caregivers (N = 27) of children with SCA (≤12 years old) participated in semi-structured interviews regarding SCA management and SDoH and completed quantitative measures of basic needs. Qualitative data were systematically coded and analyzed using applied thematic analysis. Quantitative data were presented descriptively. RESULTS: Three qualitative themes were identified. First, SCA management is bidirectionally linked with the social environment, whereby challenges of SCA management can hinder basic needs from being met, and unmet basic needs and financial hardship hinder SCA management. Second, due to limited resources, parents/caregivers are faced with difficult choices between prioritizing basic needs versus SCA management. Third, addressing material, emotional, and informational needs may improve SCA management. Quantitatively, 73% of families endorsed ≥1 basic need, including food insecurity (42%), housing instability (62%), and/or energy insecurity 19% (vs. 20%). CONCLUSION: Despite documented associations, there remains a poor understanding of the processes linking SDoH and health. Findings underscore how day-to-day conditions undermine the management of SCA treatments, symptoms, and complications, limiting treatment effectiveness. Understanding these processes may inform family-centered, health equity interventions and policies to improve living conditions, disease management, and health outcomes.

Secondary Neoplasms After Hematopoietic Cell Transplant for Sickle Cell Disease.

PURPOSE: To report the incidence and risk factors for secondary neoplasm after transplantation for sickle cell disease. METHODS: Included are 1,096 transplants for sickle cell disease between 1991 and 2016. There were 22 secondary neoplasms. Types included leukemia/myelodysplastic syndrome (MDS; n = 15) and solid tumor (n = 7). Fine-Gray regression models examined for risk factors for leukemia/MDS and any secondary neoplasm. RESULTS: The 10-year incidence of leukemia/MDS was 1.7% (95% CI, 0.90 to 2.9) and of any secondary neoplasm was 2.4% (95% CI, 1.4 to 3.8). After adjusting for other risk factors, risks for leukemia/MDS (hazard ratio, 22.69; 95% CI, 4.34 to 118.66; P = .0002) or any secondary neoplasm (hazard ratio, 7.78; 95% CI, 2.20 to 27.53; P = .0015) were higher with low-intensity (nonmyeloablative) regimens compared with more intense regimens. All low-intensity regimens included total-body irradiation (TBI 300 or 400 cGy with alemtuzumab, TBI 300 or 400 cGy with cyclophosphamide, TBI 200, 300, or 400 cGy with cyclophosphamide and fludarabine, or TBI 200 cGy with fludarabine). None of the patients receiving myeloablative and only 23% of those receiving reduced-intensity regimens received TBI. CONCLUSION: Low-intensity regimens rely on tolerance induction and establishment of mixed-donor chimerism. Persistence of host cells exposed to low-dose radiation triggering myeloid malignancy is one plausible etiology. Pre-existing myeloid mutations and prior inflammation may also contribute but could not be studied using our data source. Choosing conditioning regimens likely to result in full-donor chimerism may in part mitigate the higher risk for leukemia/MDS.

ß-Thalassemia: evolving treatment options beyond transfusion and iron chelation.

After years of reliance on transfusion alone to address anemia and suppress ineffective erythropoiesis in ß-thalassemia, many new therapies are now in development. Luspatercept, a transforming growth factor-ß inhibitor, has demonstrated efficacy in reducing ineffective erythropoiesis, improving anemia, and possibly reducing iron loading. However, many patients do not respond to luspatercept, so additional therapeutics are needed. Several medications in development aim to induce hemoglobin F (HbF): sirolimus, benserazide, and IMR-687 (a phosphodiesterase 9 inhibitor). Another group of agents seeks to ameliorate ineffective erythropoiesis and improve anemia by targeting abnormal iron metabolism in thalassemia: apotransferrin, VIT-2763 (a ferroportin inhibitor), PTG-300 (a hepcidin mimetic), and an erythroferrone antibody in early development. Mitapivat, a pyruvate kinase activator, represents a unique mechanism to mitigate ineffective erythropoiesis. Genetically modified autologous hematopoietic stem cell transplantation offers the potential for lifelong transfusion independence. Through a gene addition approach, lentiviral vectors have been used to introduce a ß-globin gene into autologous hematopoietic stem cells. One such product, betibeglogene autotemcel (beti-cel), has reached phase 3 trials with promising results. In addition, 2 gene editing techniques (CRISPR-Cas9 and zinc-finger nucleases) are under investigation as a means to silence BCL11A to induce HbF with agents designated CTX001 and ST-400, respectively. Results from the many clinical trials for these agents will yield results in the next few years, which may end the era of relying on transfusion alone as the mainstay of thalassemia therapy.

Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease.

BACKGROUND: Sickle cell disease is characterized by hemolytic anemia, pain, and progressive organ damage. A high level of erythrocyte fetal hemoglobin (HbF) comprising α- and γ-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor of γ-globin expression and HbF production in adult erythrocytes. Its down-regulation is a promising therapeutic strategy for induction of HbF. METHODS: We enrolled patients with sickle cell disease in a single-center, open-label pilot study. The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR), allowing erythroid lineage-specific knockdown. Patients were assessed for primary end points of engraftment and safety and for hematologic and clinical responses to treatment. RESULTS: As of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months (range, 7 to 29). All patients had engraftment, and adverse events were consistent with effects of the preparative chemotherapy. All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.3%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell. Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period. CONCLUSIONS: This study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk-benefit profile in sickle cell disease. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656).

Highly efficient therapeutic gene editing of human hematopoietic stem cells.

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe ß-globin disorders sickle cell disease (SCD) and ß-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with ß-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

Successful hematopoietic stem cell mobilization and apheresis collection using plerixafor alone in sickle cell patients.

Novel therapies for sickle cell disease (SCD) based on genetically engineered autologous hematopoietic stem and progenitor cells (HSPCs) are critically dependent on a safe and effective strategy for cell procurement. We sought to assess the safety and efficacy of plerixafor when used in transfused patients with SCD for HSC mobilization. Six adult patients with SCD were recruited to receive a single dose of plerixafor, tested at lower than standard (180 µg/kg) and standard (240 µg/kg) doses, followed by CD34+ cell monitoring in peripheral blood and apheresis collection. The procedures were safe and well-tolerated. Mobilization was successful, with higher peripheral CD34+ cell counts in the standard vs the low-dose group. Among our 6 donors, we improved apheresis cell collection results by using a deep collection interface and starting apheresis within 4 hours after plerixafor administration. In the subjects who received a single standard dose of plerixafor and followed the optimized collection protocol, yields of up to 24.5 × 106 CD34+ cells/kg were achieved. Interestingly, the collected CD34+ cells were enriched in immunophenotypically defined long-term HSCs and early progenitors. Thus, we demonstrate that plerixafor can be employed safely in patients with SCD to obtain sufficient HSCs for potential use in gene therapy.

Genetic therapies for sickle cell disease.

After decades with few novel therapeutic options for sickle cell disease (SCD), autologous hematopoietic stem cell (HSC) based genetic therapies including lentiviral gene therapy (GT), and genome editing (GE) now appear imminent. Lentiviral GT has advanced considerably in the past decade with promising clinical trial results in multiple disorders. For ß-hemoglobinopathies, GT strategies of gene addition and fetal hemoglobin induction through BCL11A regulation are both being evaluated in open clinical trials. GE techniques offer the possibility of a nonviral curative approach, either through sickle hemoglobin mutation repair or fetal hemoglobin elevation. Although GE currently remains at the preclinical stage, multiple clinical trials will likely open soon. In addition to reviewing current strategies for GT and GE, this review highlights important next steps toward optimization of these therapies. All autologous cell-based genetic therapies rely on safely obtaining an adequate yield of autologous HSCs for genetic modification and transplantation. HSC collection is uniquely challenging in SCD. Peripheral mobilization with plerixafor has recently emerged as a promising approach. The acute and long-term toxicities associated with myeloablative conditioning are risks that may not be acceptable to a significant number of SCD patients, highlighting the need for novel conditioning regimens. Finally, increasing availability of autologous genetic therapies will require comprehensive and collaborative discussions regarding cost and access for SCD patients, at individual centers and worldwide.

Inactivation of HDAC1 or HDAC2 induces gamma globin expression without altering cell cycle or proliferation.

Other than hydroxyurea, no pharmacologic agents are clinically available for fetal hemoglobin (HbF) induction in sickle cell disease (SCD). An optimal candidate would induce HbF without causing cell cycle inhibition and would act independently of hydroxyurea in order to yield additional HbF induction when combined. We explored whether inhibition of histone deacetylase (HDAC) 1 or HDAC2 could achieve these goals. In human erythroid progenitor cells, shRNA knockdown of the HDAC1 or HDAC2 genes induced gamma globin, without altering cellular proliferation in vitro, and without altering cell cycle phase. Treatment with hydroxyurea in combination with HDAC2 knockdown yielded a further increase in gamma globin expression. Additionally, when CD34+ cells were treated with both hydroxyurea and MS-275 (an inhibitor of HDAC 1, 2, and 3), an additive induction of relative gamma globin expression was achieved. Our findings support further clinical investigation of HDAC inhibitors in combination with hydroxyurea in SCD patients.

Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing.

Persistence of human fetal hemoglobin (HbF, α(2)γ(2)) in adults lessens the severity of sickle cell disease (SCD) and the ß-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.