Foetal haemoglobin elevation, unfavourable prognosis, and protective role of genetic variants HBG2 rs7482144, HBS1L-MYB rs9399137 and BCL11A rs4671393 in children with ALL.

In acute lymphoblastic leukaemia (ALL), elevated foetal haemoglobin (HbF) levels have been associated with the prognosis of patients. Genetic variants in HbF regulatory genes: BAF chromatin remodelling complex subunit (BCL11A), HBS1L-MYB transcriptional GTPase intergenic region (HBS1L-MYB), Krüppel-like factor 1 (KLF1), haemoglobin gamma subunit 2 (HBG2), haemoglobin gamma subunit 1 (HBG1), and haemoglobin subunit beta pseudogene 1 (HBBP1) are often associatedwith elevatedHbF concentration. This study investigated the association of genetic variants in HbF regulatory genes with HbF concentration, unfavourable prognosis, and outcome in children with ALL.We quantified HbF concentration and genotyped 17 genetic variants in 48 patients with ALL and 64 children without ALL as a reference group. HbF concentrationwas higher in patients than in the reference group (4.4%vs 1.4%), and 75%(n = 36) of thepatientshadHbF>2.5%.Unfavourable prognosis ALL was established in 68.8% (n = 33) of the patients. Variant HBG2 rs7482144 was associated with high HbF concentration (P = 0.015); while HBS1L-MYB rs9399137 (P = 0.001), HBG2 rs7482144 (P = 0.001) and the ß-globin genes HBG2, HBG1, and HBPP1 haplotypeTGC(P = 0.017) with unfavourable prognosisALL.Additionally, variantBCL11A rs4671393 showed a protective role (P = 0.0001). In conclusion, variants HBG2 rs7482144, HBS1L-MYB rs9399137 and BCL11A rs4671393 may play a significant role in ALL.

[Advance of research on the role of BCL11A in the occurrence and treatment of ß-Thalassemia].

ß-Thalassemia is a single-gene disease caused by mutations in ß-globin and has a distinct geographical characteristics. Current treatment for patients with moderate to severe thalassemia has mainly relied on long-term blood transfusion and/or hematopoietic stem cell transplantation. B cell lymphoma/leukemia 11A (BCL11A) as a transcriptional repressor plays a vital role in monitoring γ/ß hemoglobin switching, maintaining the normal function of hematopoietic stem cells, and regulating erythrocyte differentiation and lymphocyte development. With the rapid progress in gene editing technology, the BCL11A as a therapeutic target for ß-thalassemia has shown promising results. This article has systematically summarized the regulatory mechanism and therapeutic potential of the BCL11A, with an aim to provide new ideas for the treatment of ß-thalassemia.

Genetic Modifiers of Sickle Cell Anemia Phenotype in a Cohort of Angolan Children.

The aim of this study was to identify genetic markers in the HBB Cluster; HBS1L-MYB intergenic region; and BCL11A, KLF1, FOX3, and ZBTB7A genes associated with the heterogeneous phenotypes of Sickle Cell Anemia (SCA) using next-generation sequencing, as well as to assess their influence and prevalence in an Angolan population. Hematological, biochemical, and clinical data were considered to determine patients' severity phenotypes. Samples from 192 patients were sequenced, and 5,019,378 variants of high quality were registered. A catalog of candidate modifier genes that clustered in pathophysiological pathways important for SCA was generated, and candidate genes associated with increasing vaso-occlusive crises (VOC) and with lower fetal hemoglobin (HbF) were identified. These data support the polygenic view of the genetic architecture of SCA phenotypic variability. Two single nucleotide polymorphisms in the intronic region of 2q16.1, harboring the BCL11A gene, are genome-wide and significantly associated with decreasing HbF. A set of variants was identified to nominally be associated with increasing VOC and are potential genetic modifiers harboring phenotypic variation among patients. To the best of our knowledge, this is the first investigation of clinical variation in SCA in Angola using a well-customized and targeted sequencing approach.

Pharmacogenomics of Drugs Used in ß-Thalassemia and Sickle-Cell Disease: From Basic Research to Clinical Applications.

In this short review we have presented and discussed studies on pharmacogenomics (also termed pharmacogenetics) of the drugs employed in the treatment of ß-thalassemia or Sickle-cell disease (SCD). This field of investigation is relevant, since it is expected to help clinicians select the appropriate drug and the correct dosage for each patient. We first discussed the search for DNA polymorphisms associated with a high expression of γ-globin genes and identified this using GWAS studies and CRISPR-based gene editing approaches. We then presented validated DNA polymorphisms associated with a high HbF production (including, but not limited to the HBG2 XmnI polymorphism and those related to the BCL11A, MYB, KLF-1, and LYAR genes). The expression of microRNAs involved in the regulation of γ-globin genes was also presented in the context of pharmacomiRNomics. Then, the pharmacogenomics of validated fetal hemoglobin inducers (hydroxyurea, butyrate and butyrate analogues, thalidomide, and sirolimus), of iron chelators, and of analgesics in the pain management of SCD patients were considered. Finally, we discuss current clinical trials, as well as international research networks focusing on clinical issues related to pharmacogenomics in hematological diseases.

Exagamglogene Autotemcel for Transfusion-Dependent ß-Thalassemia.

BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis through ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of the erythroid-specific enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). METHODS: We conducted an open-label, single-group, phase 3 study of exa-cel in patients 12 to 35 years of age with transfusion-dependent ß-thalassemia and a ß0/ß0, ß0/ß0-like, or non-ß0/ß0-like genotype. CD34+ HSPCs were edited by means of CRISPR-Cas9 with a guide mRNA. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was transfusion independence, defined as a weighted average hemoglobin level of 9 g per deciliter or higher without red-cell transfusion for at least 12 consecutive months. Total and fetal hemoglobin concentrations and safety were also assessed. RESULTS: A total of 52 patients with transfusion-dependent ß-thalassemia received exa-cel and were included in this prespecified interim analysis; the median follow-up was 20.4 months (range, 2.1 to 48.1). Neutrophils and platelets engrafted in each patient. Among the 35 patients with sufficient follow-up data for evaluation, transfusion independence occurred in 32 (91%; 95% confidence interval, 77 to 98; P<0.001 against the null hypothesis of a 50% response). During transfusion independence, the mean total hemoglobin level was 13.1 g per deciliter and the mean fetal hemoglobin level was 11.9 g per deciliter, and fetal hemoglobin had a pancellular distribution (≥94% of red cells). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No deaths or cancers occurred. CONCLUSIONS: Treatment with exa-cel, preceded by myeloablation, resulted in transfusion independence in 91% of patients with transfusion-dependent ß-thalassemia. (Supported by Vertex Pharmaceuticals and CRISPR Therapeutics; CLIMB THAL-111 ClinicalTrials.gov number, NCT03655678.).

Exagamglogene Autotemcel for Severe Sickle Cell Disease.

BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A. METHODS: We conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed. RESULTS: A total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred. CONCLUSIONS: Treatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.).

Revolutionising healing: Gene Editing's breakthrough against sickle cell disease.

Recent advancements in gene editing illuminate new potential therapeutic approaches for Sickle Cell Disease (SCD), a debilitating monogenic disorder caused by a point mutation in the ß-globin gene. Despite the availability of several FDA-approved medications for symptomatic relief, allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole curative option, underscoring a persistent need for novel treatments. This review delves into the growing field of gene editing, particularly the extensive research focused on curing haemoglobinopathies like SCD. We examine the use of techniques such as CRISPR-Cas9 and homology-directed repair, base editing, and prime editing to either correct the pathogenic variant into a non-pathogenic or wild-type one or augment fetal haemoglobin (HbF) production. The article elucidates ways to optimize these tools for efficacious gene editing with minimal off-target effects and offers insights into their effective delivery into cells. Furthermore, we explore clinical trials involving alternative SCD treatment strategies, such as LentiGlobin therapy and autologous HSCT, distilling the current findings. This review consolidates vital information for the clinical translation of gene editing for SCD, providing strategic insights for investigators eager to further the development of gene editing for SCD.

Fetal anemia causes placental and maternal cellular damage: a lesson from fetal hemoglobin Bart's disease.

INTRODUCTION: The studies about effect of fetal anemia on placental and maternal molecular changes have rarely been published. This study aimed to compare oxidative stress levels and mitochondrial function in the placenta and maternal peripheral blood mononuclear cell (PMBCs) between anemic fetuses (using fetal Hb Bart's disease as a study model) and non-anemic fetuses. METHODS: A cross-sectional study was conducted on pregnancies affected by Hb Bart's disease and non-anemic fetuses between 16 and 22 weeks of gestation. Placental tissue and maternal blood for PBMCs were collected after pregnancy termination for determination of oxidative stress and mitochondrial function. RESULTS: A total of 18 pregnancies affected by Hb Bart's disease and 12 non-anemic fetuses were enrolled. Placental thickness was significantly greater (p-value <0.001) in the affected pregnancies, whereas all Doppler indices of uteroplacental blood flow were comparable. Mitochondrial dysfunction was significantly increased (p-value <0.001) in the placenta of the affected fetuses. In the mothers of affected fetuses, there was an increase in mitochondrial oxidative stress levels with a significant increase in mitochondrial dysfunction in isolated PBMCs (p-value <0.001). DISCUSSION: In the presence of normal uteroplacental Doppler studies, fetal anemia can induce a significant increase in oxidative stress and mitochondrial dysfunction in the placentas and mothers. The findings support that the placenta can be a source of oxidative stress agents which are released into systemic circulation prior to development of maternal adverse outcomes, and may explain pathophysiology of subsequent preeclampsia in late gestation, as commonly seen in pregnancies affected by fetal Hb Bart's disease, if pregnancy is not terminated.

Compound heterozygosity for Southeast Asian hereditary persistence of fetal hemoglobin and ß0-thalassemia results in thalassemia intermedia: Pedigree analysis and genetic research in a family from South China. A case report.

RATIONALE: Compound heterozygotes for deletional ß-thalassemia can be difficult to diagnose due to its diverse clinical presentations and no routine screenings. This can lead to disease progression and delay in treatment. PATIENT CONCERNS: We reported pedigree analysis and genetic research in a family with rare ß-thalassemia. DIAGNOSIS: Pedigree analysis and genetic research demonstrated that the patient was a compound heterozygote for ß-thalassemia CD17/Southeast Asian hereditary persistence of fetal hemoglobin deletion, inherited from the parents. Magnetic resonance imaging T2* examination revealed severe iron deposition in the liver. Echocardiography revealed endocardial cushion defect. INTERVENTIONS: The patient was treated with Deferasirox after receiving the final molecular genetic diagnosis. The initial once-daily dose of Deferasirox was 20 mg/kg/d. OUTCOMES: The patient discontinued the medication three months after the first visit. Two years later, the patient visited the Department of Hepatobiliary and Pancreatic Diseases. He was recommended to undergo splenectomy after surgical repair of the congenital heart disease. However, the patient refused surgical treatment because of the economic burden. LESSONS: We report that fetal hemoglobin is a sensitive indicator for screening large deletions of the ß-globin gene, which can be effectively confirmed by the multiplex ligation-dependent probe amplification assay. In non-transfusion-dependent thalassemia patients, iron status assessment should be regularly performed, and iron chelation treatment should be initiated early. This case will provide insights for the diagnosis of rare genotypes of ß-thalassemia and has important implications for genetic counseling.

let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching.

ABSTRACT: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (ß-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).

Development of pathophysiologically relevant models of sickle cell disease and ß-thalassemia for therapeutic studies.

Ex vivo cellular system that accurately replicates sickle cell disease and ß-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and ß-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications.

Hydroxyurea Adherence for Personal Best in Sickle Cell Treatment (HABIT) efficacy trial: Community health worker support may increase hydroxyurea adherence of youth with sickle cell disease.

Despite disease-modifying effects of hydroxyurea on sickle cell disease (SCD), poor adherence among affected youth commonly impedes treatment impact. Following our prior feasibility trial, the "Hydroxyurea Adherence for Personal Best in Sickle Cell Treatment (HABIT)" multi-site randomized controlled efficacy trial aimed to increase hydroxyurea adherence for youth with SCD ages 10-18 years. Impaired adherence was identified primarily through flagging hydroxyurea-induced fetal hemoglobin (HbF) levels compared to prior highest treatment-related HbF. Eligible youth were enrolled as dyads with their primary caregivers for the 1-year trial. This novel semi-structured supportive, multidimensional dyad intervention led by community health workers (CHW), was augmented by daily tailored text message reminders, compared to standard care during a 6-month intervention phase, followed by a 6-month sustainability phase. Primary outcomes from the intervention phase were improved Month 6 HbF levels compared to enrollment and proportion of days covered (PDC) for hydroxyurea versus pre-trial year. The secondary outcome was sustainability of changes up to Month 12. The 2020-2021 peak coronavirus disease 2019 (COVID-19) pandemic disrupted enrollment and clinic-based procedures; CHW in-person visits shifted to virtual scheduled interactions. We enrolled 50 dyads, missing target enrollment. Compared to enrollment levels, both HbF level and PDC significantly - but not sustainably - improved within the intervention group (p = .03 and .01, respectively) with parallel increased mean corpuscular volume (MCV) (p = .05), but not within controls. No significant between-group differences were found at Months 6 or 12. These findings suggest that our community-based, multimodal support for youth-caregiver dyads had temporarily improved hydroxyurea usage. Durability of impact should be tested in a trial with longer duration of CHW-led and mobile health support.

Identification and characterization of CHD4-associated eRNA as a novel modulator of fetal hemoglobin levels in ß-thalassemia.

Fetal-to-adult hemoglobin switching is controlled by programmed silencing of γ-globin while the re-activation of fetal hemoglobin (HbF) is an effective strategy for ameliorating the clinical severity of ß-thalassemia and sickle cell disease. The identification of enhancer RNAs (eRNAs) related to the fetal (α2γ2) to adult hemoglobin (α2ß2) switching remains incomplete. In this study, the transcriptomes of GYPA+ cells from six ß-thalassemia patients with extreme HbF levels were sequenced to identify differences in patterns of noncoding RNA expression. It is interesting that an enhancer upstream of CHD4, an HbF-related core subunit of the NuRD complex, was differentially transcribed. We found a significantly positive correlation of eRNA-CHD4 enhancer-gene interaction using the public database of FANTOM5. Specifically, the eRNA-CHD4 expression was found to be significantly higher in both CD34+ HSPCs and HUDEP-2 than those in K562 cells which commonly expressed high level of HbF, suggesting a correlation between eRNA and HbF expression. Furthermore, prediction of transcription binding sites of cis-eQTLs and the CHD4 genomic region revealed a putative interaction site between rs73264846 and ZNF410, a known transcription factor regulating HbF expression. Moreover, in-vitro validation showed that the inhibition of eRNA could reduce the expression of HBG expression in HUDEP-2 cells. Taken together, the findings of this study demonstrate that a distal enhancer contributes to stage-specific silencing of γ-globin genes through direct modulation of CHD4 expression and provide insights into the epigenetic mechanisms of NuRD-mediated hemoglobin switching.

Ginsenoside Rg1 promotes fetal hemoglobin production in vitro: A potential therapeutic avenue for ß-thalassemia.

ß-thalassemia, a globally prevalent genetic disorder, urgently requires innovative treatment options. Fetal hemoglobin (HbF) induction stands as a key therapeutic approach. This investigation focused on Ginsenoside Rg1 from the Panax genus for HbF induction. Employing K562 cells and human erythroid precursor cells (ErPCs) derived from neonatal cord blood, the study tested Rg1 at different concentrations. We measured its effects on γ-globin mRNA levels and HbF expression, alongside assessments of cell proliferation and differentiation. In K562 cells, Rg1 at 400 µM significantly increased γ-globin mRNA expression by 4.24 ± 1.08-fold compared to the control. In ErPCs, the 800 µM concentration was most effective, leading to an over 80% increase in F-cells and a marked upregulation in HbF expression. Notably, Rg1 did not adversely affect cell proliferation or differentiation, with the 200 µM concentration showing an increase in γ-globin mRNA by 2.33 ± 0.58-fold, and the 800 µM concentration enhancing HbF expression by 2.59 ± 0.03-fold in K562 cells. Our results underscore Rg1's potential as an effective and safer alternative for ß-thalassemia treatment. By significantly enhancing HbF levels without cytotoxicity, Rg1 offers a notable advantage over traditional treatments like Hydroxyurea. While promising, these in vitro findings warrant further in vivo exploration to confirm Rg1's therapeutic efficacy and to unravel its underlying mechanistic pathways.

Misdiagnosis of ß-Thalassemia Major Due to Chinese Gγ+(Aγδß)0-Thalassemia Combined with ß0-Thalassemia.

δß-thalassemia is a rare type of thalassemia characterized by increased Hb F levels, including mainly Chinese Gγ(Aγδß)0-thalassemia, Yunnanese Gγ(Aγδß)0-thalassemia, Cantonese Gγ(Aγδß)0-thalassemia in China. Due to the low rate of δß-thalassemia carriers, there are few reports of δß-thalassemia combined with ß-thalassemia causing ß-thalassemia major. Herein, we described the combination of Chinese Gγ(Aγδß)0-thalassemia and ß-thalassemia leading to ß-thalassemia major in a Chinese patient. Hemoglobin analysis was performed by capillary electrophoresis (CE). Routine genetic analysis was carried out by gap-polymerase chain reaction (Gap-PCR) and PCR and reverse dot blot (PCR-RDB). Multiple ligation-dependent probe amplification (MLPA) was used to detect the large deletion, and Gap-PCR confirmed the deletion. A CE result showed an elevated Hb F level of 98.7% and 11.7% in the proband and her mother, but the proband was diagnosed with ßCD17M/ßCD17M using routine genetic analysis. However, her father was heterozygous for CD17 in ß-globin, and her mother was detected as SEA heterozygous. The further analysis presented that the proband had actually missed the diagnosis of Chinese Gγ(Aγδß)0-thalassemia by MLPA and PCR-RDB. Finally, the genotype of the proband was corrected from ßCD17M/ßCD17M to ßCD17M/ßGγ(Aγδß)0. This is the first report of Chinese Gγ(Aγδß)0-thalassemia combined with ß-thalassemia resulting in ß-thalassemia major in China. Screening for δß-thalassemia by Hb analysis could be an effective method.

Activation of γ-globin expression by LncRNA-mediated ERF promoter hypermethylation in ß-thalassemia.

The mechanism that drives the switch from fetal to adult hemoglobin (Hb) provides a therapeutic target for ß-thalassemia. We have previously identified that hypermethylation of transcription factor ERF promoter reactivated γ-globin expression. To uncover the mechanism underlying the hypermethylation of ERF promoter, we performed RNA sequencing in ß0/ß0-thalassemia patients and identified an upregulated long noncoding RNA (RP11-196G18.23) associated with HbF production. RP11-196G18.23 bound to the ERF promoter and recruited DNA methyltransferase 3A to promote DNA hypermethylation-mediated ERF downregulation, thereby ameliorating ERF-induced γ-globin inactivation. The identification of RP11-196G18.23 provides an epigenetic mechanism for the reactivation of fetal γ-globin expression for ß-hemoglobinopathies.

Quantitative Model-Based Assessment of Multiple Sickle Cell Disease Therapeutic Approaches Alone and in Combination.

Three sickle cell disease (SCD) treatment strategies, stabilizing oxygenated hemoglobin (oxyHb), lowering 2,3-BPG, and inducing fetal hemoglobin (HbF) expression aim to prevent red blood cell (RBC) sickling by reducing tense-state sickle hemoglobin that contributes to polymer formation. Induction of 30% HbF is seen as the gold standard because 30% endogenous expression is associated with a lack of symptoms. However, the level of intervention required to achieve equivalent polymerization protection by the other strategies is uncertain, and there is little understanding of how these approaches could work in combination. We sought to develop an oxygen saturation model that could assess polymerization protection of all three approaches alone or in combination by extending the Monod-Wymann-Changeux model to include additional mechanisms. Applying the model to monotherapies suggests 51% sickle hemoglobin (HbS) occupancy with an oxyHb stabilizer or lowering RBC 2,3 BPG concentrations to 1.8 mM would produce comparable polymerization protection as 30% HbF. The model predictions are consistent with observed clinical response to the oxyHb stabilizer voxelotor and the 2,3-BPG reducer etavopivat. The model also suggests combination therapy will have added benefit in the case of dose limitations, as is the case for voxelotor, which the model predicts could be combined with 20% HbF or 2,3-BPG reduction to 3.75 mM to reach equivalent protection as 30% HbF. The proposed model represents a unified framework that is useful in supporting decisions in preclinical and early clinical development and capable of evolving with clinical experience to gain new and increasingly confident insights into treatment strategies for SCD.

Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.