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.).

miRNA Expression Associated with HbF in Saudi Sickle Cell Anemia.

Background and Objectives: Sickle cell anemia (SCA) is a hereditary monogenic disease due to a single ß-globin gene mutation that codes for the production of sickle hemoglobin. Its phenotype is modulated by fetal hemoglobin (HbF), a product of γ-globin genes. Exploring the molecules that regulate γ-globin genes at both transcriptional and translational levels, including microRNA (miRNA), might help identify alternative therapeutic targets. Materials and Methods: Using next-generation sequencing we identified pre-miRNAs and mature miRNA expression signatures associated with different HbF levels in patients homozygous for the sickle hemoglobin gene. The involvement of identified miRNAs in potential SCD-related pathways was investigated with the DIANA TOOL and miRWalk 2.0 database. Results: miR-184 were most highly upregulated in reticulocytes. miR-3609 and miR-483-5p were most highly downregulated in sickle cell anemia with high HbF. miR-370-3p that regulates LIN28A, and miR-451a which is effective in modulating α- and ß- globin levels were also significantly upregulated. miRNA targeted gene pathway interaction identified BCL7A, BCL2L1, LIN28A, KLF6, GATA6, solute carrier family genes and ZNF genes associated with erythropoiesis, cell cycle regulation, glycosphingolipid biosynthesis, cAMP, cGMP-PKG, mTOR, MAPK and PI3K-AKT signaling pathways and cancer pathways. Conclusions: miRNA signatures and their target genes identified novel miRNAs that could regulate fetal hemoglobin production and might be exploited therapeutically.

CRISPR-Cas9 Gene Editing for Sickle Cell Disease and ß-Thalassemia.

Transfusion-dependent ß-thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells. We performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients - one with TDT and the other with SCD - received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex Pharmaceuticals; ClinicalTrials.gov numbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.).

Fetal hemoglobin in sickle cell anemia.

Fetal hemoglobin (HbF) can blunt the pathophysiology, temper the clinical course, and offer prospects for curative therapy of sickle cell disease. This review focuses on (1) HbF quantitative trait loci and the geography of ß-globin gene haplotypes, especially those found in the Middle East; (2) how HbF might differentially impact the pathophysiology and many subphenotypes of sickle cell disease; (3) clinical implications of person-to-person variation in the distribution of HbF among HbF-containing erythrocytes; and (4) reactivation of HbF gene expression using both pharmacologic and cell-based therapeutic approaches. A confluence of detailed understanding of the molecular basis of HbF gene expression, coupled with the ability to precisely target by genomic editing most areas of the genome, is producing important preliminary therapeutic results that could provide new options for cell-based therapeutics with curative intent.

Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program.

Human induced pluripotent stem cell (hiPSC) lines have previously been generated through the NHLBI sponsored NextGen program at nine individual study sites. Here, we examined the structural integrity of 506 hiPSC lines as determined by copy number variations (CNVs). We observed that 149 hiPSC lines acquired 258 CNVs relative to donor DNA. We identified six recurrent regions of CNVs on chromosomes 1, 2, 3, 16 and 20 that overlapped with cancer associated genes. Furthermore, the genes mapping to regions of acquired CNVs show an enrichment in cancer related biological processes (IL6 production) and signaling cascades (JNK cascade & NFκB cascade). The genomic region of instability on chr20 (chr20q11.2) includes transcriptomic signatures for cancer associated genes such as ID1, BCL2L1, TPX2, PDRG1 and HCK. Of these HCK shows statistically significant differential expression between carrier and non-carrier hiPSC lines. Overall, while a low level of genomic instability was observed in the NextGen generated hiPSC lines, the observation of structural instability in regions with known cancer associated genes substantiates the importance of systematic evaluation of genetic variations in hiPSCs before using them as disease/research models.

BCL2L1 is associated with γ-globin gene expression.

Fetal hemoglobin (HbF) expression is partially governed by the trans-acting quantitative trait loci BCL11A and MYB and a cis-acting locus linked to the HBB gene cluster. Our previous analysis of the Genotype-Tissue Expression database suggested that BCL2L1 was associated with HbF gene expression. In erythroid progenitors from patients with sickle cell disease, BCL2L1 messenger RNA (mRNA) levels were positively correlated with HBG mRNA and total HbF concentration (r2 = 0.72, P = .047 and r2 = 0.68, P = .01, respectively). Inhibition of BCL2L1 protein activity in HbF-expressing HUDEP-1 cells decreased HBG expression in a dose-dependent manner. Overexpression of BCL2L1 in these cells increased HBG expression fourfold (P < .05) and increased F cells by 13% (P < .05). Overexpression of BCL2L1 in erythroid progenitors derived from primary adult CD34+ cells upregulated HBG expression 11-fold (P < .05), increased F cells by 18% (P < .01), did not significantly affect cell differentiation or proliferation, and had a minor effect on survival. Although the mechanism remains unknown, our results suggest that BCL2L1 is associated with HbF gene activation.

Induced pluripotent stem cell-based mapping of ß-globin expression throughout human erythropoietic development.

Robust ß-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) increases the resolution with which red blood cell disorders such as sickle cell disease and ß thalassemia can be modeled in vitro. To better quantify efforts in augmenting ß-globin expression, we report the creation of a ß-globin reporter iPSC line that allows for the mapping of ß-globin expression throughout human erythropoietic development in real time at single-cell resolution. Coupling this tool with single-cell RNA sequencing (scRNAseq) identified features that distinguish ß-globin-expressing cells and allowed for the dissection of the developmental and maturational statuses of iPSC-derived erythroid lineage cells. Coexpression of embryonic, fetal, and adult globins in individual cells indicated that these cells correspond to a yolk sac erythromyeloid progenitor program of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identified a gradient of erythroid maturation, with ß-globin-expressing cells showing increased maturation. Compared with other cells, ß-globin-expressing cells showed a reduction in transcripts coding for ribosomal proteins, increased expression of members of the ubiquitin-proteasome system recently identified to be involved in remodeling of the erythroid proteome, and upregulation of genes involved in the dynamic translational control of red blood cell maturation. These findings emphasize that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.

Notch and Aryl Hydrocarbon Receptor Signaling Impact Definitive Hematopoiesis from Human Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult-type cells is a major barrier for iPSC-based disease modeling and drug discovery. To directly address these concerns, we have developed a chemically defined, serum and feeder-free-directed differentiation platform to generate hematopoietic stem-progenitor cells (HSPCs) and resultant adult-type progeny from iPSCs. This system allows for strict control of signaling pathways over time through growth factor and/or small molecule modulation. Through direct comparison with our previously described protocol for the production of primitive wave hematopoietic cells, we demonstrate that induced HSPCs are enhanced for erythroid and myeloid colony forming potential, and strikingly, resultant erythroid-lineage cells display enhanced expression of adult ß globin indicating definitive pathway patterning. Using this system, we demonstrate the stage-specific roles of two key signaling pathways, Notch and the aryl hydrocarbon receptor (AHR), in the derivation of definitive hematopoietic cells. We illustrate the stage-specific necessity of Notch signaling in the emergence of hematopoietic progenitors and downstream definitive, adult-type erythroblasts. We also show that genetic or small molecule inhibition of the AHR results in the increased production of CD34+ CD45+ HSPCs while conversely, activation of the same receptor results in a block of hematopoietic cell emergence. Results presented here should have broad implications for hematopoietic stem cell transplantation and future clinical translation of iPSC-derived blood cells. Stem Cells 2018;36:1004-1019.

A Mild Phenotype of Severe ß+ Thalassemia in a 16-Month-Old Boy.

ß thalassemia is characterized by a deficient production of functional ß-globin chains and a relative excess of α-globin chains. An extremely diverse clinical spectrum-asymptomatic to transfusion-dependent-is primarily due to homozygosity or compound heterozygosity for the very large number of ß-thalassemia-causing mutations, along with interacting mutations that affect the α-globin and γ-globin genes and their expression. We report a case of a 16-month-old boy who was initially diagnosed with iron deficiency anemia until he was later found to be homozygous for a severe ß-thalassemia genotype with a mild hematologic phenotype. This was likely as a result of his ability to produce high levels of fetal hemoglobin.

A long noncoding RNA from the HBS1L-MYB intergenic region on chr6q23 regulates human fetal hemoglobin expression.

The HBS1L-MYB intergenic region (chr6q23) regulates erythroid cell proliferation, maturation, and fetal hemoglobin (HbF) expression. An enhancer element within this locus, highlighted by a 3-bp deletion polymorphism (rs66650371), is known to interact with the promoter of the neighboring gene, MYB, to increase its expression, thereby regulating HbF production. RNA polymerase II binding and a 50-bp transcript from this enhancer region reported in ENCODE datasets suggested the presence of a long noncoding RNA (lncRNA). We characterized a novel 1283bp transcript (HMI-LNCRNA; chr6:135,096,362-135,097,644; hg38) that was transcribed from the enhancer region of MYB. Within erythroid cells, HMI-LNCRNA was almost exclusively present in nucleus, and was much less abundant than the mRNA for MYB. HMI-LNCRNA expression was significantly higher in erythroblasts derived from cultured adult peripheral blood CD34+ cells which expressed more HBB, compared to erythroblasts from cultured cord blood CD34+ cells which expressed much more HBG. Down-regulation of HMI-LNCRNA in HUDEP-2 cells, which expressed mostly HBB, significantly upregulated HBG expression both at the mRNA (200-fold) and protein levels, and promoted erythroid maturation. No change was found in the expression of BCL11A and other key transcription factors known to modulate HBG expression. HMI-LNCRNA plays an important role in regulating HBG expression, and its downregulation can result in a significant increase in HbF. HMI-LNCRNA might be a potential therapeutic target for HbF induction treatment in sickle cell disease and ß-thalassemia.

A candidate transacting modulator of fetal hemoglobin gene expression in the Arab-Indian haplotype of sickle cell anemia.

Fetal hemoglobin (HbF) levels are higher in the Arab-Indian (AI) ß-globin gene haplotype of sickle cell anemia compared with African-origin haplotypes. To study genetic elements that effect HbF expression in the AI haplotype we completed whole genome sequencing in 14 Saudi AI haplotype sickle hemoglobin homozygotes-seven selected for low HbF (8.2% ± 1.3%) and seven selected for high HbF (23.5% ± 2.6%). An intronic single nucleotide polymorphism (SNP) in ANTXR1, an anthrax toxin receptor (chromosome 2p13), was associated with HbF. These results were replicated in two independent Saudi AI haplotype cohorts of 120 and 139 patients, but not in 76 Saudi Benin haplotype, 894 African origin haplotype and 44 AI haplotype patients of Indian origin, suggesting that this association is effective only in the Saudi AI haplotype background. ANTXR1 variants explained 10% of the HbF variability compared with 8% for BCL11A. These two genes had independent, additive effects on HbF and together explained about 15% of HbF variability in Saudi AI sickle cell anemia patients. ANTXR1 was expressed at mRNA and protein levels in erythroid progenitors derived from induced pluripotent stem cells (iPSCs) and CD34+ cells. As CD34+ cells matured and their HbF decreased ANTXR1 expression increased; as iPSCs differentiated and their HbF increased, ANTXR1 expression decreased. Along with elements in cis to the HbF genes, ANTXR1 contributes to the variation in HbF in Saudi AI haplotype sickle cell anemia and is the first gene in trans to HBB that is associated with HbF only in carriers of the Saudi AI haplotype. Am. J. Hematol. 91:1118-1122, 2016. © 2016 Wiley Periodicals, Inc.

The genetic basis of asymptomatic codon 8 frame-shift (HBB:c25_26delAA) ß(0) -thalassaemia homozygotes.

Two 21-year old dizygotic twin men of Iraqi descent were homozygous for HBB codon 8, deletion of two nucleotides (-AA) frame-shift ß(0) -thalassaemia mutation (FSC8; HBB:c25_26delAA). Both were clinically well, had splenomegaly, and were never transfused. They had mild microcytic anaemia (Hb 120-130 g/l) and 98% of their haemoglobin was fetal haemoglobin (HbF). Both were carriers of Hph α-thalassaemia mutation. On the three major HbF quantitative trait loci (QTL), the twins were homozygous for G>A HBG2 Xmn1 site at single nucleotide polymorphism (SNP) rs7482144, homozygous for 3-bp deletion HBS1L-MYB intergenic polymorphism (HMIP) at rs66650371, and heterozygous for the A>C BCL11A intron 2 polymorphism at rs766432. These findings were compared with those found in 22 other FSC8 homozygote patients: four presented with thalassaemia intermedia phenotype, and 18 were transfusion dependent. The inheritance of homozygosity for HMIP 3-bp deletion at rs66650371 and heterozygosity for Hph α-thalassaemia mutation was found in the twins and not found in any of the other 22 patients. Further studies are needed to uncover likely additional genetic variants that could contribute to the exceptionally high HbF levels and mild phenotype in these twins.

The genetics of hemoglobin A2 regulation in sickle cell anemia.

Hemoglobin A2 , a tetramer of α- and δ-globin chains, comprises less than 3% of total hemoglobin in normal adults. In northern Europeans, single nucleotide polymorphisms (SNPs) in the HBS1L-MYB locus on chromosome 6q and the HBB cluster on chromosome 11p were associated with HbA2 levels. We examined the genetic basis of HbA2 variability in sickle cell anemia using genome-wide association studies. HbA2 levels were associated with SNPs in the HBS1L-MYB interval and SNPs in BCL11A. These effects are mediated by the association of these loci with γ-globin gene expression and fetal hemoglobin (HbF) levels. The association of polymorphisms downstream of the ß-globin gene (HBB) cluster on chromosome 11 with HbA2 was not mediated by HbF. In sickle cell anemia, levels of HbA2 appear to be modulated by trans-acting genes that affect HBG expression and perhaps also elements within the ß-globin gene cluster. HbA2 is expressed pancellularly and can inhibit HbS polymerization. It remains to be seen if genetic regulators of HbA2 can be exploited for therapeutic purposes.

The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation.

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.

Fetal hemoglobin in sickle cell anemia: genetic studies of the Arab-Indian haplotype.

Sickle cell anemia is common in the Middle East and India where the HbS gene is sometimes associated with the Arab-Indian (AI) ß-globin gene (HBB) cluster haplotype. In this haplotype of sickle cell anemia, fetal hemoglobin (HbF) levels are 3-4 fold higher than those found in patients with HbS haplotypes of African origin. Little is known about the genetic elements that modulate HbF in AI haplotype patients. We therefore studied Saudi HbS homozygotes with the AI haplotype (mean HbF 19.2±7.0%, range 3.6 to 39.6%) and employed targeted genotyping of polymorphic sites to explore cis- and trans- acting elements associated with high HbF expression. We also described sequences which appear to be unique to the AI haplotype for which future functional studies are needed to further define their role in HbF modulation. All cases, regardless of HbF concentration, were homozygous for AI haplotype-specific elements cis to HBB. SNPs in BCL11A and HBS1L-MYB that were associated with HbF in other populations explained only 8.8% of the variation in HbF. KLF1 polymorphisms associated previously with high HbF were not present in the 44 patients tested. More than 90% of the HbF variance in sickle cell patients with the AI haplotype remains unexplained by the genetic loci that we studied. The dispersion of HbF levels among AI haplotype patients suggests that other genetic elements modulate the effects of the known cis- and trans-acting regulators. These regulatory elements, which remain to be discovered, might be specific in the Saudi and some other populations where HbF levels are especially high.

qMRI relaxometry of mandibular bone marrow: a monomodal distribution in sickle cell disease.

PURPOSE: To identify and characterize sickle cell disease (SCD)-related changes in the composition of mandibular bone marrow using qMRI relaxometry histograms. MATERIALS AND METHODS: Thirteen SCD patients and 17 controls underwent brain MR imaging with the mixed turbo spin-echo (TSE) pulse sequence at 1.5T. The mandible was manually segmented and divided into body, angle, ramus, and condyle. T1 and T2 histograms of each mandible were modeled with Gaussian functions. The relaxation time histogram peaks were calculated, and the number of monomodal versus bimodal curves was compared. RESULTS: SCD patients exhibited monomodal distributions on both T1 and T2 histograms, consistent with a composition of predominantly red hematopoietic marrow. Eighty-eight percent of mandibles in control subjects exhibited a bimodal distribution in T1 and all showed a bimodal distribution in T2, indicating mixed but predominantly yellow marrow composition. The second peak in control subjects was shorter in T1 and longer in T2, consistent with yellow marrow composition. CONCLUSION: Instead of physiological fatty replacement, SCD patients exhibit red marrow persistence in the mandible, likely due to the increased demand for hematopoiesis. This phenomenon can be manifested by a monomodal curve in both T1 and T2 relaxometric histograms.

Severe fetal and neonatal hemolytic anemia due to a 198 kb deletion removing the complete ß-globin gene cluster.

Fetal and neonatal hemolytic anemia can be caused by (γδß)(0)-thalassemia deletions of the ß-globin gene cluster. Many of these deletions have not been well characterized, and diagnostic tests are not readily available, thus hampering carrier detection, family counseling, and antenatal diagnosis. We report and define a 198 kb deletion removing the entire ß-globin gene cluster, which was found in members of a multigeneration family of Irish/Scottish descent. The proband had life-threatening fetal and neonatal hemolytic anemia which subsided by 1 year of age.