Vivien A. Sheehan

Human genetic studies have identified key regulators of fetal haemoglobin (HbF) expression, including BCL11A, resulting in therapeutic advances ^1–8 . Yet the mechanisms by which HbF expression is activated remain incompletely understood ⁹ . Here we conduct a large multi-ancestry genome-wide association study of HbF levels in 28,279 individuals that identifies 91 conditionally independent associations across 12 genomic regions. In one previously uncharacterized associated region, the high-HbF-linked causal variant rs1010474-C reduces BACH2 expression and elevates HbF levels. Direct perturbation or inhibition of BACH2 likewise increases HbF expression. Mechanistically, BACH2 restrains activation of the HbF-encoding γ-globin genes, while loss of BACH2 enhances NRF2 chromatin occupancy and promotes the formation of activation foci at the γ-globin genes. Although BACH2 and NRF2 binding motifs in the γ-globin promoters overlap, they can be selectively edited to activate or repress γ-globin, respectively, and do so independently of BCL11A. These findings illustrate how human genetic variation continues to advance our understanding of therapeutically relevant regulatory mechanisms underlying HbF expression.

Allogeneic hematopoietic cell transplantation (HCT) and autologous hematopoietic stem cell–based gene therapies are potentially curative for sickle cell disease (SCD). Although most patients experience resolution of overt clinical symptoms associated with SCD with donor-dominant myeloid chimerism after undergoing HCT or the induction of a substantial amount of alternative hemoglobin (Hb) after gene therapy (GT), whether the underlying subclinical physiology is normalized in these individuals is unknown. One approach to evaluating the underlying physiology is to characterize the blood rheology of patients after they receive these therapies. We assessed the rheology of 123 blood samples from 69 patients, of whom 43 underwent HCT (32 from matched related donors; 11 from haploidentical donors) and 11 received GT. The results were compared to those for patients treated with hydroxyurea with optimum fetal Hb response (HUOptimum; n = 15). Median rheology parameters of recipients of HCT from donors with sickle cell trait (HbAS) were superior to those of GT recipients (elongation index under hypoxia [EImin]: 0.6 vs 0.31; point of sickling [PoS]: 0 vs 26.21) and of the HUOptimum cohort (EImin, 0.6 vs 0.15; PoS, 0 vs 36.2; percentage of dense red blood cells, 0.9 vs 4.9; P< .05 for all comparisons). Results for the GT and HUOptimum groups were comparable (P> .05). Given the substantial number of patients with abnormal rheology and the associations between rheology and SCD-related complications, rheology tests may be included in the follow-up care for HCT and GT recipients.

First-generation genome editing therapies have largely focused on correcting or compensating for pathogenic variants. However, as these approaches enter the clinic, emerging biological constraints limit maximal therapeutic impact. Because globin genes are activated late during erythroid differentiation, genome-corrected hematopoietic stem and progenitor cells (HSPCs) gain little selective advantage in the bone marrow. Here, we establish a strategy that links therapeutic genome edits to an erythroid fitness-enhancing allele to amplify the output of clinically relevant cells. We develop a multiplex base editing strategy that couples fetal hemoglobin (HbF) reactivation with erythroid lineage expansion. Introduction of a naturally occurring erythropoietin receptor truncation ( tEPOR ) associated with benign erythrocytosis increased erythroid cell production without impairing viability or differentiation. Combinatorial editing of tEPOR together with the BCL11A erythroid enhancer and HBG1/2 promoters in healthy donor, sickle cell disease, and β-thalassemia HSPCs synergistically increased erythroid proliferation and HbF expression beyond single base-edited or Casgevy-treated controls. Multiplex base-edited HSPCs retained long-term lineage repopulation and engraftment capacity in vivo , establishing a modular strategy that pairs disease correction with lineage amplification to improve therapeutic potency.

Vaso-occlusive episodes (VOEs) or acute pain events, involving complex interactions between sickle erythrocytes and other blood cells, are a hallmark of sickle cell disease (SCD). In this study, we analyzed changes in peripheral blood transcriptomes between steady state and VOEs in individuals with SCD. We followed a cohort of 174 individuals with SCD with or without chronic pain and collected peripheral blood at clinic visits (steady state) and during hospitalizations (VOEs). We performed RNA-Seq profiling of CD45 ⁺ leukocytes and CD71 ⁺ erythroid cells. Pathways linked to complement activation, coagulation, and IL-6/JAK/STAT3 signaling were enriched during VOEs in the CD45 ⁺ cells. Contrastingly, the CD71 ⁺ cells showed an enrichment of pathways related to the cell cycle, such as mTORC1 signaling and the G ₂ M checkpoint during VOEs. We then analyzed the expression changes of genes in patients with longitudinal data to determine potential biomarkers for VOEs. Expression of 4 genes — FAM20A , IL1B , MS4A4A , and SERPINB2 — was elevated during VOEs compared with steady state in the majority of patients. Furthermore, our results indicate that patients experiencing chronic pain exhibited 44% increased enrichment of significant pathways during VOEs when compared with patients without chronic pain.

In sickle cell disease (SCD), red blood cells (RBCs) are poorly deformable, even under normoxia (NOI). With deoxygenation, deformability of sickle RBCs is further reduced due to polymerization of hemoglobin S (HbS). Rigid, poorly deformable sickle RBCs block microvasculature, causing ischemia, pain, and organ damage. The microfluidic impedance red cell assay (MIRCA) can mechanically measure RBC deformability, providing occlusion index under NOI or hypoxia (HOI) as readouts. We analyzed RBCs from 68 adult and 34 pediatric patients with SCD using the MIRCA. Higher HOI and NOI values were positively associated with markers of inflammation, hemolysis, RBC density, older age, and severe SCD genotypes (homozygous HbSS or HbS β0-thalassemia. Each 1% higher NOI across individuals was associated with a 6.3% higher incidence of acute complications per year. Individuals with chronic complications in the past year had a 3.1% higher median NOI than those without chronic complications. Individuals on chronic transfusion therapy exhibit a subpopulation of poorly deformable RBCs captured by the MIRCA but not by a commercially available device that also measures RBC deformability, the laser assisted optical rotational cell analyzer (LoRRca). In vitro addition of voxelotor or osivelotor to samples from individuals on chronic transfusion therapy improved the deformability of these endogenous RBCs. Longitudinally collected NOI and HOI values in individuals with HbSS were stable, with a median percent point change of 13.3% and 15.7%, respectively. MIRCA can be used in combination with clinical laboratory tests to monitor RBC deformability as a biomarker of clinical status at routine clinic visits and included in clinical trials of disease-modifying agents.

Adenine base editing can convert sickle hemoglobin (HbS, βΕ6V) to G-Makassar hemoglobin (HbG, βE6A), a naturally occurring variant that is clinically asymptomatic. However, the quality and functionality of purified HbG and of mature HbGG and HbGS red blood cells (RBC) has not been assessed. Here, we develop a mouse model to characterize HbG. Purified HbG appears normal and does not polymerize under hypoxia. The topology of the hemoglobin fold with the βΕ6Α mutation is similar to HbA in the oxy and deoxy states. However, RBC containing HbGS are dehydrated, showing altered function and increased sickling under hypoxia. Blood counts and mitochondrial retention measures place HbGS RBCs as intermediate in severity between HbAS and HbSS, while organ function is comparable to HbAS. HbGG resembles HbAA for most metrics. Our results highlight the importance of functionally assessing the mature red cell environment when evaluating novel gene editing strategies for hematologic disorders.

Summary: Hydroxycarbamide (HC) is the most widely used therapeutic for individuals with sickle cell disease (SCD, including sickle cell anemia and other forms of the disease). HC's clinical benefits are primarily associated with its ability to induce foetal haemoglobin (HbF); this limited view of HC's therapeutic potential may lead to its discontinuation when a modest amount of HbF is induced. A better understanding of the HbF‐independent effects of HC on genes and pathways relevant to SCD pathophysiology is therefore needed. In this study, we performed bulk RNA‐Seq on whole blood samples collected from a cohort of 25 paediatric patients with SCD to identify genes and pathways that are affected by treatment with HC. At the maximum tolerated dose (MTD) of HC, patients showed altered expression levels of several genes and biological pathways. Pathways related to haeme metabolism, interferon‐alpha response, and interferon‐gamma response were significantly downregulated at HC MTD relative to the matched pre‐HC samples. Pathways linked with IL2‐STAT5 signalling and TNFα signalling via NF‐Kβ were observed to be up‐regulated at HC MTD. These results illustrate the range of effects exerted by HC during therapy for SCD and pave the way for an improved understanding of the HbF induction‐independent benefits of HC.