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 (NCT 03282656) 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 NCT 03282656, 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

Generating controlled molecular gradients in 3D gels

A new method for producing molecular gradients of arbitrary shape in thin three dimensional gels is described. Patterns are produced on the surface of the gel by printing with a micropump that dispenses small droplets of solution at controlled rates. The molecules in the solution rapidly diffuse into the gel and create a smooth concentration profile that is independent of depth. The pattern is relatively stable for long times, and its evolution can be accurately described by finite element modeling of the diffusion equation. As a demonstration of the method, direct measurements of protein gradients are performed by quantitative fluorescence microscopy. A complementary technique for measuring diffusion coefficients is also presented. This rapid, flexible, contactless approach to gradient generation is ideally suited for cell culture experiments to investigate the role of gradients of diffusible substances in processes such as chemotaxis, morphogenesis, and pattern formation, as well as for high-throughput screening of system responses to a wide range of chemical concentrations. (c) 2005 Wiley Periodicals, Inc

A new chemotaxis assay shows the extreme sensitivity of axons to molecular gradients

Axonal chemotaxis is believed to be important in wiring up the developing and regenerating nervous system, but little is known about how axons actually respond to molecular gradients. We report a new quantitative assay that allows the long-term response of axons to gradients of known and controllable shape to be examined in a three-dimensional gel. Using this assay, we show that axons may be nature's most-sensitive gradient detectors, but this sensitivity exists only within a narrow range of ligand concentrations. This assay should also be applicable to other biological processes that are controlled by molecular gradients, such as cell migration and morphogenesis

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 \ub5g/kg) and standard (240 \ub5g/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 7 106 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

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

Acknowledgements: This work was supported by the Bill and Melinda Gates Foundation (INV-002189 and INV-038816, D.G.K.). M.S.C. was supported by a Wellcome Clinical PhD Fellowship. Work conducted by D.A.W. and colleagues was supported by National Institutes of Health grant R01HL (137848) and NHLBI Cure Sickle grant (OT2HL 154815). Investigators at the Sanger Institute were supported by a core grant from the Wellcome Trust. This research was funded in part by the Wellcome Trust (206194 and 108413/A/15/D). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. J.N. is supported by a Cancer Research UK Fellowship. Work in the D.G.K. laboratory is supported by a European Research Council Starting Grant (ERC-2016-STG-715371) and a Cancer Research UK Programme Foundation Award (DCRPGF\100008). The authors thank the IT Support team of the Cancer, Ageing and Somatic mutation programme at the Sanger Institute for their support and V. Sankaran and L. Naldini for helpful discussion. The authors also thank the patients for donating the samples that have been used in this study.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

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

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

Acknowledgements: This work was supported by the Bill and Melinda Gates Foundation (INV-002189 and INV-038816, D.G.K.). M.S.C. was supported by a Wellcome Clinical PhD Fellowship. Work conducted by D.A.W. and colleagues was supported by National Institutes of Health grant R01HL (137848) and NHLBI Cure Sickle grant (OT2HL 154815). Investigators at the Sanger Institute were supported by a core grant from the Wellcome Trust. This research was funded in part by the Wellcome Trust (206194 and 108413/A/15/D). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. J.N. is supported by a Cancer Research UK Fellowship. Work in the D.G.K. laboratory is supported by a European Research Council Starting Grant (ERC-2016-STG-715371) and a Cancer Research UK Programme Foundation Award (DCRPGF\100008). The authors thank the IT Support team of the Cancer, Ageing and Somatic mutation programme at the Sanger Institute for their support and V. Sankaran and L. Naldini for helpful discussion. The authors also thank the patients for donating the samples that have been used in this study.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

Defining global strategies to improve outcomes in sickle cell disease ::a Lancet Haematology commission

All over the world, people with sickle cell disease (an inherited condition) have premature deaths and preventable severe chronic complications, which considerably affect their quality of life, career progression, and financial status. In addition, these people are often affected by stigmatisation or structural racism, which can contribute to stress and poor mental health. Inequalities affecting people with sickle cell disease are also reflected in the distribution of the disease—mainly in sub-Saharan Africa, India, and the Caribbean—whereas interventions, clinical trials, and funding are mostly available in North America, Europe, and the Middle East. Although some of these characteristics also affect people with other genetic diseases, the fate of people with sickle cell disease seems to be particularly unfair. Simple, effective interventions to reduce the mortality and morbidity associated with sickle cell disease are available. The main obstacle preventing better outcomes in this condition, which is a neglected disease, is associated with inequalities impacting the patient populations. The aim of this Commission is to highlight the problems associated with sickle cell disease and to identify achievable goals to improve outcomes both in the short and long term. The ambition for the management of people with sickle cell disease is that curative treatments become available to every person with the condition. Although this would have seemed unrealistic a decade ago, developments in gene therapy make this potentially achievable, albeit in the distant future. Until these curative technologies are fully developed and become widely available, health-care professionals (with the support of policy makers, funders, etc) should make sure that a minimum standard of care (including screening, prophylaxis against infection, acute medical care, safe blood transfusion, and hydroxyurea) is available to all patients. In considering what needs to be achieved to reduce the global burden of sickle cell disease and improve the quality of life of patients, this Commission focuses on five key areas: the epidemiology of sickle cell disease (Section 1); screening and prevention (Section 2); established and emerging treatments for the management of the disease (Section 3); cellular therapies with curative potential (Section 4); and training and education needs (Section 5). As clinicians, researchers, and patients, our objective to reduce the global burden of sickle cell disease aligns with wider public health aims to reduce inequalities, improve health for all, and develop personalised treatment options. We have observed in the past few years some long-awaited momentum following the development of innovative point-of-care testing devices, new approved drugs, and emerging curative options. Reducing the burden of sickle cell disease will require substantial financial and political commitment, but it will impact the lives of millions of patients and families worldwide and the lessons learned in achieving this goal would unarguably benefit society as a whole