Machine learning to optimize automated RH genotyping using whole-exome sequencing data.

Rh phenotype matching reduces but does not eliminate alloimmunization in patients with sickle cell disease (SCD) due to RH genetic diversity that is not distinguishable by serological typing. RH genotype matching can potentially mitigate Rh alloimmunization, but comprehensive and accessible genotyping methods are needed. We developed RHtyper as an automated algorithm to predict RH genotypes using whole-genome sequencing (WGS) data with high accuracy. Here, we adapted RHtyper for whole-exome sequencing (WES) data which are more affordable but challenged by uneven sequencing coverage and exacerbated sequencing read misalignment, resulting in uncertain prediction for 1) RHD zygosity and hybrid alleles, 2) RHCE*C versus RHCE*c alleles, 3) RHD c.1136C>T zygosity, and 4) RHCE c.48G>C zygosity. We optimized RHtyper to accurately predict RHD and RHCE genotypes using WES data by leveraging machine learning models and improved the concordance of WES with WGS predictions from 90.8% to 97.2% for RHD and 96.3 to 98.2% for RHCE among 396 patients in the Sickle Cell Clinical Research and Intervention Program (SCCRIP). In a second validation cohort with 3030 cancer survivors (15.2% Black or African Americans) from the St. Jude Lifetime Cohort Study (SJLIFE), the optimized RHtyper reached concordance rates between WES and WGS predications to 96.3% for RHD, and 94.6% for RHCE. In conclusion, machine learning improved the accuracy of RH predication from WES data. RHtyper has the potential, once implemented, to provide a precision medicine-based approach to facilitate RH genotype-matched transfusion and improve transfusion safety for patients with SCD.

Hematopoietic stem cell collection for sickle cell disease gene therapy.

PURPOSE OF REVIEW: Gene therapy for sickle cell disease (SCD) is advancing rapidly, with two transformative products recently approved by the US Food and Drug Administration and numerous others under study. All current gene therapy protocols require ex vivo modification of autologous hematopoietic stem cells (HSCs). However, several SCD-related problems impair HSC collection, including a stressed and damaged bone marrow, potential cytotoxicity by the major therapeutic drug hydroxyurea, and inability to use granulocyte colony stimulating factor, which can precipitate severe vaso-occlusive events. RECENT FINDINGS: Peripheral blood mobilization of HSCs using the CXCR4 antagonist plerixafor followed by apheresis collection was recently shown to be safe and effective for most SCD patients and is the current strategy for mobilizing HSCs. However, exceptionally large numbers of HSCs are required to manufacture an adequate cellular product, responses to plerixafor are variable, and most patients require multiple mobilization cycles, increasing the risk for adverse events. For some, gene therapy is prohibited by the failure to obtain adequate numbers of HSCs. SUMMARY: Here we review the current knowledge on HSC collection from individuals with SCD and potential improvements that may enhance the safety, efficacy, and availability of gene therapy for this disorder.

Metabolic regulation of erythrocyte development and disorders.

The formation of new red blood cells (RBC) (erythropoiesis) has served as a paradigm for understanding cellular differentiation and developmental control of gene expression. The metabolic regulation of this complex, coordinated process remains poorly understood. Each step of erythropoiesis, including lineage specification of hematopoietic stem cells, proliferation, differentiation, and terminal maturation into highly specialized oxygen-carrying cells, has unique metabolic requirements. Developing erythrocytes in mammals are also characterized by unique metabolic events such as loss of mitochondria with switch to glycolysis, ejection of nucleus and organelles, high-level heme and hemoglobin synthesis, and antioxidant requirement to protect hemoglobin molecules. Genetic defects in metabolic enzymes, including pyruvate kinase and glucose-6-phosphate dehydrogenase, cause common erythrocyte disorders, whereas other inherited disorders such as sickle cell disease and ß-thalassemia display metabolic abnormalities associated with disease pathophysiology. Here we describe recent discoveries on the metabolic control of RBC formation and function, highlight emerging concepts in understanding the erythroid metabolome, and discuss potential therapeutic benefits of targeting metabolism for RBC disorders.

Interspecies regulatory landscapes and elements revealed by novel joint systematic integration of human and mouse blood cell epigenomes.

Knowledge of locations and activities of cis -regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our V al i dated S ystematic I ntegrati on (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

Potent and uniform fetal hemoglobin induction via base editing.

Inducing fetal hemoglobin (HbF) in red blood cells can alleviate ß-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease or adenine base editors. The most potent modification was adenine base editor generation of γ-globin -175A>G. Homozygous -175A>G edited erythroid colonies expressed 81 ± 7% HbF versus 17 ± 11% in unedited controls, whereas HbF levels were lower and more variable for two Cas9 strategies targeting a BCL11A binding motif in the γ-globin promoter or a BCL11A erythroid enhancer. The -175A>G base edit also induced HbF more potently than a Cas9 approach in red blood cells generated after transplantation of CD34+ hematopoietic stem and progenitor cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 can cause unexpected phenotypic variation that can be circumvented by base editing.

ETV6 represses inflammatory response genes and regulates HSPC function during stress hematopoiesis in mice.

ETS variant 6 (ETV6) encodes a transcriptional repressor expressed in hematopoietic stem and progenitor cells (HSPCs), where it is required for adult hematopoiesis. Heterozygous pathogenic germline ETV6 variants are associated with thrombocytopenia 5 (T5), a poorly understood genetic condition resulting in thrombocytopenia and predisposition to hematologic malignancies. To elucidate how germline ETV6 variants affect HSPCs and contribute to disease, we generated a mouse model harboring an Etv6R355X loss-of-function variant, equivalent to the T5-associated variant ETV6R359X. Under homeostatic conditions, all HSPC subpopulations are present in the bone marrow (BM) of Etv6R355X/+ mice; however, these animals display shifts in the proportions and/or numbers of progenitor subtypes. To examine whether the Etv6R355X/+ mutation affects HSPC function, we performed serial competitive transplantation and observed that Etv6R355X/+ lineage-sca1+cKit+ (LSK) cells exhibit impaired reconstitution, with near complete failure to repopulate irradiated recipients by the tertiary transplant. Mechanistic studies incorporating cleavage under target and release under nuclease assay, assay for transposase accessible chromatin sequencing, and high-throughput chromosome conformation capture identify ETV6 binding at inflammatory gene loci, including multiple genes within the tumor necrosis factor (TNF) signaling pathway in ETV6-sufficient mouse and human HSPCs. Furthermore, single-cell RNA sequencing of BM cells isolated after transplantation reveals upregulation of inflammatory genes in Etv6R355X/+ progenitors when compared to Etv6+/+ counterparts. Corroborating these findings, Etv6R355X/+ HSPCs produce significantly more TNF than Etv6+/+ cells post-transplantation. We conclude that ETV6 is required to repress inflammatory gene expression in HSPCs under conditions of hematopoietic stress, and this mechanism may be critical to sustain HSPC function.

Loss of miR-144/451 alleviates ß-thalassemia by stimulating ULK1-mediated autophagy of free α-globin.

Most cells can eliminate unstable or misfolded proteins through quality control mechanisms. In the inherited red blood cell disorder ß-thalassemia, mutations in the ß-globin gene (HBB) lead to a reduction in the corresponding protein and the accumulation of cytotoxic free α-globin, which causes maturation arrest and apoptosis of erythroid precursors and reductions in the lifespan of circulating red blood cells. We showed previously that excess α-globin is eliminated by Unc-51-like autophagy activating kinase 1 (ULK1)-dependent autophagy and that stimulating this pathway by systemic mammalian target of rapamycin complex 1 (mTORC1) inhibition alleviates ß-thalassemia pathologies. We show here that disrupting the bicistronic microRNA gene miR-144/451 alleviates ß-thalassemia by reducing mTORC1 activity and stimulating ULK1-mediated autophagy of free α-globin through 2 mechanisms. Loss of miR-451 upregulated its target messenger RNA, Cab39, which encodes a cofactor for LKB1, a serine-threonine kinase that phosphorylates and activates the central metabolic sensor adenosine monophosphate-activated protein kinase (AMPK). The resultant enhancement of LKB1 activity stimulated AMPK and its downstream effects, including repression of mTORC1 and direct activation of ULK1. In addition, loss of miR-144/451 inhibited the expression of erythroblast transferrin receptor 1, causing intracellular iron restriction, which has been shown to inhibit mTORC1, reduce free α-globin precipitates, and improve hematological indices in ß-thalassemia. The beneficial effects of miR-144/451 loss in ß-thalassemia were inhibited by the disruption of Cab39 or Ulk1 genes. Together, our findings link the severity of ß-thalassemia to a highly expressed erythroid microRNA locus and a fundamental, metabolically regulated protein quality control pathway that is amenable to therapeutic manipulation.

Ex vivo prime editing of patient haematopoietic stem cells rescues sickle-cell disease phenotypes after engraftment in mice.

Sickle-cell disease (SCD) is caused by an A·T-to-T·A transversion mutation in the ß-globin gene (HBB). Here we show that prime editing can correct the SCD allele (HBBS) to wild type (HBBA) at frequencies of 15%-41% in haematopoietic stem and progenitor cells (HSPCs) from patients with SCD. Seventeen weeks after transplantation into immunodeficient mice, prime-edited SCD HSPCs maintained HBBA levels and displayed engraftment frequencies, haematopoietic differentiation and lineage maturation similar to those of unedited HSPCs from healthy donors. An average of 42% of human erythroblasts and reticulocytes isolated 17 weeks after transplantation of prime-edited HSPCs from four SCD patient donors expressed HBBA, exceeding the levels predicted for therapeutic benefit. HSPC-derived erythrocytes carried less sickle haemoglobin, contained HBBA-derived adult haemoglobin at 28%-43% of normal levels and resisted hypoxia-induced sickling. Minimal off-target editing was detected at over 100 sites nominated experimentally via unbiased genome-wide analysis. Our findings support the feasibility of a one-time prime editing SCD treatment that corrects HBBS to HBBA, does not require any viral or non-viral DNA template and minimizes undesired consequences of DNA double-strand breaks.

Multiplex Base Editing to Protect from CD33-Directed Therapy: Implications for Immune and Gene Therapy.

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesis in vivo , thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs. In vitro , dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.

Gene Therapy and Gene Editing for ß-Thalassemia.

After many years of intensive research, emerging data from clinical trials indicate that gene therapy for transfusion-dependent ß-thalassemia is now possible. Strategies for therapeutic manipulation of patient hematopoietic stem cells include lentiviral transduction of a functional erythroid-expressed ß-globin gene and genome editing to activate fetal hemoglobin production in patient red blood cells. Gene therapy for ß-thalassemia and other blood disorders will invariably improve as experience accumulates over time. The best overall approaches are not known and perhaps not yet established. Gene therapy comes at a high cost, and collaboration between multiple stakeholders is required to ensure that these new medicines are administered equitably.

Genetic regulation of fetal hemoglobin across global populations.

Human genetic variation has enabled the identification of several key regulators of fetal-to-adult hemoglobin switching, including BCL11A, resulting in therapeutic advances. However, despite the progress made, limited further insights have been obtained to provide a fuller accounting of how genetic variation contributes to the global mechanisms of fetal hemoglobin (HbF) gene regulation. Here, we have conducted a multi-ancestry genome-wide association study of 28,279 individuals from several cohorts spanning 5 continents to define the architecture of human genetic variation impacting HbF. We have identified a total of 178 conditionally independent genome-wide significant or suggestive variants across 14 genomic windows. Importantly, these new data enable us to better define the mechanisms by which HbF switching occurs in vivo. We conduct targeted perturbations to define BACH2 as a new genetically-nominated regulator of hemoglobin switching. We define putative causal variants and underlying mechanisms at the well-studied BCL11A and HBS1L-MYB loci, illuminating the complex variant-driven regulation present at these loci. We additionally show how rare large-effect deletions in the HBB locus can interact with polygenic variation to influence HbF levels. Our study paves the way for the next generation of therapies to more effectively induce HbF in sickle cell disease and ß-thalassemia.

Forced enhancer-promoter rewiring to alter gene expression in animal models.

Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the ß-globin locus (locus control region [LCR]) contacts the fetal-type (HBG) and adult-type (HBB) ß-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the ß-globin locus as a model, we provide proof of concept at the organismal level that forced enhancer rewiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPCs) from mice bearing human ß-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus macaque erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene-regulatory elements may be used to alter gene expression to treat disease.

An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity.

Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multilineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/- HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation.

The development of haematopoietic stem cells into mature erythrocytes - erythropoiesis - is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed maturation stage-dependent assemblies of topologically homologous RANBP9- and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH's cognate E2 ubiquitin conjugating enzyme UBE2H increased during terminal differentiation, and UBE2H expression depended on catalytically active CTLH E3 complexes. CRISPR-Cas9-mediated inactivation of CTLH E3 assemblies or UBE2H in erythroid progenitors revealed defects, including spontaneous and accelerated erythroid maturation as well as inefficient enucleation. Thus, we propose that dynamic maturation stage-specific changes of UBE2H-CTLH E2-E3 modules control the orderly progression of human erythropoiesis.

Activation of γ-globin expression by hypoxia-inducible factor 1α.

Around birth, globin expression in human red blood cells (RBCs) shifts from γ-globin to ß-globin, which results in fetal haemoglobin (HbF, α2γ2) being gradually replaced by adult haemoglobin (HbA, α2ß2)1. This process has motivated the development of innovative approaches to treat sickle cell disease and ß-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR-Cas9 screen for ubiquitin-proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel-Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1α (HIF1α)3,4, to induce γ-globin gene transcription. Mechanistically, HIF1α-HIF1ß heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem γ-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the γ-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1α for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for ß-haemoglobinopathies.

Endothelial alpha globin is a nitrite reductase.

Resistance artery vasodilation in response to hypoxia is essential for matching tissue oxygen and demand. In hypoxia, erythrocytic hemoglobin tetramers produce nitric oxide through nitrite reduction. We hypothesized that the alpha subunit of hemoglobin expressed in endothelium also facilitates nitrite reduction proximal to smooth muscle. Here, we create two mouse strains to test this: an endothelial-specific alpha globin knockout (EC Hba1Δ/Δ) and another with an alpha globin allele mutated to prevent alpha globin's inhibitory interaction with endothelial nitric oxide synthase (Hba1WT/Δ36-39). The EC Hba1Δ/Δ mice had significantly decreased exercise capacity and intracellular nitrite consumption in hypoxic conditions, an effect absent in Hba1WT/Δ36-39 mice. Hypoxia-induced vasodilation is significantly decreased in arteries from EC Hba1Δ/Δ, but not Hba1WT/Δ36-39 mice. Hypoxia also does not lower blood pressure in EC Hba1Δ/Δ mice. We conclude the presence of alpha globin in resistance artery endothelium acts as a nitrite reductase providing local nitric oxide in response to hypoxia.

Effective therapies for sickle cell disease: are we there yet?

Sickle cell disease (SCD) is a common genetic blood disorder associated with acute and chronic pain, progressive multiorgan damage, and early mortality. Recent advances in technologies to manipulate the human genome, a century of research and the development of techniques enabling the isolation, efficient genetic modification, and reimplantation of autologous patient hematopoietic stem cells (HSCs), mean that curing most patients with SCD could soon be a reality in wealthy countries. In parallel, ongoing research is pursuing more facile treatments, such as in-vivo-delivered genetic therapies and new drugs that can eventually be administered in low- and middle-income countries where most SCD patients reside.

Limitations of mouse models for sickle cell disease conferred by their human globin transgene configurations.

We characterized the human ß-like globin transgenes in two mouse models of sickle cell disease (SCD) and tested a genome-editing strategy to induce red blood cell fetal hemoglobin (HbF; α2γ2). Berkeley SCD mice contain four to 22 randomly arranged, fragmented copies of three human transgenes (HBA1, HBG2-HBG1-HBD-HBBS and a mini-locus control region) integrated into a single site of mouse chromosome 1. Cas9 disruption of the BCL11A repressor binding motif in the γ-globin gene (HBG1 and HBG2; HBG) promoters of Berkeley mouse hematopoietic stem cells (HSCs) caused extensive death from multiple double-strand DNA breaks. Long-range sequencing of Townes SCD mice verified that the endogenous Hbb genes were replaced by single-copy segments of human HBG1 and HBBS including proximal but not some distal gene-regulatory elements. Townes mouse HSCs were viable after Cas9 disruption of the HBG1 BCL11A binding motif but failed to induce HbF to therapeutic levels, contrasting with human HSCs. Our findings provide practical information on the genomic structures of two common mouse SCD models, illustrate their limitations for analyzing therapies to induce HbF and confirm the importance of distal DNA elements in human globin regulation. This article has an associated First Person interview with the first author of the paper.

Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette.

In this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.