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.).

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.).

An integrated therapeutic approach to sickle cell disease management beyond infancy.

Hydroxyurea, the first approved drug for sickle cell disease, decreases sickle hemoglobin polymerization by inducing fetal hemoglobin. Its effects in young children are excellent; responses in adults are variable and not curative. The goal of pharmacotherapy should not be disease "moderation" but reducing morbidity and mortality by diminishing both hemolytic anemia and vaso-occlusive events. This is best done by preventing sickle hemoglobin polymerization; if anti-polymerization treatment is insufficient, agents disrupting pathophysiologic pathways "downstream" of the sickle hemoglobin polymer should be added. We recommend that all patients should be started first on maximal doses of hydroxyurea. When the clinical and hematologic response to hydroxyurea is insufficient, as it is almost always in adults, we favor adding voxelotor, a hemoglobin-oxygen affinity-shifting agent that, likely in a pancellular distribution, decreases sickle hemoglobin polymerization. The P-selectin inhibitor crizanlizumab reduces sickle cell-endothelial interactions and can be used in patients with continued vaso-occlusive events. There is no physiologic reason that all three drugs could not be combined when the response to monotherapy or dual-drug therapy is poor. Drug therapy must be considered in the context of possibly "curative" cellular therapeutics and if needed, exchange transfusion programs.

Pharmacologic induction of PGC-1α stimulates fetal haemoglobin gene expression.

Sickle cell disease (SCD) is a genetic disorder that affects millions around the world. Enhancement of fetal γ-globin levels and fetal haemoglobin (HbF) production in SCD patients leads to diminished severity of many clinical features of the disease. We recently identified the transcriptional co-activator PGC-1α as a new protein involved in the regulation of the globin genes. Here, we report that upregulation of PGC-1α by infection with a lentivirus expressing PGC-1α or by the small-molecule PGC-1α agonist ZLN005 in human primary erythroid progenitor CD34+ cells induces both fetal γ-globin mRNA and protein expression as well as the percentage of HbF-positive cell (F cells) without significantly affecting cell proliferation and differentiation. We further found that the combination of ZLN005 and hydroxyurea (hydroxycarbamide) exhibited an additive effect on the expression of γ-globin and the generation of F cells from cultured CD34+ cells. In addition, ZLN005 induced robust expression of the murine embryonic ßh1-globin gene and to a lesser extent, human γ-globin gene expression in sickle mice. These findings suggest that activation of PGC-1α by ZLN005 might provide a new path for modulating HbF levels with potential therapeutic benefit in ß-hemoglobinopathies.

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.

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.

Prevalence and Diversity of Haplotypes of Sickle Cell Disease in the Eastern Province of Saudi Arabia.

Hb F modulates sickle cell disease. Five major haplotypes of the ß-globin gene cluster are associated with sickle cell disease. In the Eastern Province of Saudi Arabia, the Arab-Indian (AI) is most common. Single nucleotide polymorphism (SNP) genotyping (rs3834466, rs28440105, rs10128556, and rs968857) was carried out by nuclease allelic discrimination assay with target-specific forward and reverse primers, TaqMan probes, labeled with VIC and FAM. In 778 patients with sickle cell disease from the Eastern Province, a haplotype was assigned to 90.9% of all samples; 9.1% were classified as compound heterozygotes for the AI and an atypical haplotype. The distribution of haplotypes for 746 Hb S (HBB: c.20A > T) homozygotes was: 614 AI/AI, nine SEN/SEN (Senegal), 42 SEN/AI, nine CAM/CAM (Cameroon), one CAR (Central African Republic)/BEN (Benin), 71 AI/atypical. In Hb S/ß-thalassemia (Hb S/ß-thal), the distribution of Hb S haplotypes was: 22 AI/AI, one CAM/CAM, four AI/SEN, five AI/atypical. Mean Hb F in the haplotypes was: AI/AI 16.6 ± 7.5%, CAM/CAM 8.0 ± 4.1%, SEN/SEN 11.0 ± 5.1%, SEN/AI 15.1 ± 4.6%, AI/atypical 16.2 ± 6.5%. The presence of the SEN and CAM haplotypes was unexpected due to the apparent homogeneity of the population of the Eastern Province. We have successfully classified sickle cell disease haplotypes using the relatively inexpensive TaqMan assay for the first time. In addition, we have previously shown that children with AI haplotype have Hb F of 30.0% and mild disease, while in our cohort of adult AI patients, which might be the largest yet reported, Hb F was about 16.6%.

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.

Biomarker signatures of sickle cell disease severity.

Identifying sickle cell disease patients at high risk of complications could lead to personalized treatment and better prognosis but despite many advances prediction of the clinical course of these patients remains elusive. We propose a system-type approach to discover profiles of multiple, common biomarkers that correlate with morbidity and mortality in sickle cell disease. We used cluster analysis to discover 17 signatures of 17 common circulating biomarkers in 2320 participants of the Cooperative Study of Sickle Cell Disease, and evaluated the association of these signatures with risk for stroke, pain, leg ulceration, acute chest syndrome, avascular necrosis, seizure, death, and trend of fetal hemoglobin and hemolysis using longitudinally collected data. The analysis shows that some of the signatures are associated with reduced risk for complications, while others are associated with increased risk for complications. We also show that these signatures repeat in two more contemporary studies of sickle cell disease and correlate with recently discovered biomarkers of pulmonary vascular disease. With replication and further study, these biomarker signatures could become an important and affordable precision medicine tool to aid treatment and management of the disease.

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 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 phased SNP-based classification of sickle cell anemia HBB haplotypes.

BACKGROUND: Sickle cell anemia causes severe complications and premature death. Five common ß-globin gene cluster haplotypes are each associated with characteristic fetal hemoglobin (HbF) levels. As HbF is the major modulator of disease severity, classifying patients according to haplotype is useful. The first method of haplotype classification used restriction fragment length polymorphisms (RFLPs) to detect single nucleotide polymorphisms (SNPs) in the ß-globin gene cluster. This is labor intensive, and error prone. METHODS: We used genome-wide SNP data imputed to the 1000 Genomes reference panel to obtain phased data distinguishing parental alleles. RESULTS: We successfully haplotyped 813 sickle cell anemia patients previously classified by RFLPs with a concordance >98%. Four SNPs (rs3834466, rs28440105, rs10128556, and rs968857) marking four different restriction enzyme sites unequivocally defined most haplotypes. We were able to assign a haplotype to 86% of samples that were either partially or misclassified using RFLPs. CONCLUSION: Phased data using only four SNPs allowed unequivocal assignment of a haplotype that was not always possible using a larger number of RFLPs. Given the availability of genome-wide SNP data, our method is rapid and does not require high computational resources.

Fetal hemoglobin in sickle cell anemia: The Arab-Indian haplotype and new therapeutic agents.

Fetal hemoglobin (HbF) has well-known tempering effects on the symptoms of sickle cell disease and its levels vary among patients with different haplotypes of the sickle hemoglobin gene. Compared with sickle cell anemia haplotypes found in patients of African descent, HbF levels in Saudi and Indian patients with the Arab-Indian (AI) haplotype exceed that in any other haplotype by nearly twofold. Genetic association studies have identified some loci associated with high HbF in the AI haplotype but these observations require functional confirmation. Saudi patients with the Benin haplotype have HbF levels almost twice as high as African patients with this haplotype but this difference is unexplained. Hydroxyurea is still the only FDA approved drug for HbF induction in sickle cell disease. While most patients treated with hydroxyurea have an increase in HbF and some clinical improvement, 10 to 20% of adults show little response to this agent. We review the genetic basis of HbF regulation focusing on sickle cell anemia in Saudi Arabia and discuss new drugs that can induce increased levels of HbF.

SIRT1 activates the expression of fetal hemoglobin genes.

High fetal hemoglobin (HbF, α2 γ2 ) levels ameliorate the clinical manifestations of sickle cell disease and ß thalassemia. The mechanisms that repress HbF expression and silence γ-globin genes in adults are incompletely characterized and only a single HbF inducer, hydroxyurea, is approved for treatment, and only in patients with sickle cell disease. We identified SIRT1, a protein deacetylase, as a new inducer of γ-globin. SIRT1 knockdown decreased, while SIRT1 ectopic expression upregulated γ-globin gene (HBG) expression in primary human erythroid cells and in K562 cells. The small molecule SIRT1 activators SRT2104 and SRT1720 enhanced HBG expression in cord blood human erythroblasts and reactivated silenced HBG in adult human erythroblasts. Furthermore, SIRT1 binds in the ß-globin gene cluster locus control region (LCR) and HBG promoters, promotes the looping of the LCR to HBG promoter, and increases the binding of RNA polymerase II and H4K16Ac in the HBG promoter. SIRT1 suppressed the expression of the HBG suppressors BCL11A, KLF1, HDAC1 and HDAC2. Lastly, SIRT1 did not change the proliferation of human erythroid progenitor cells or the expression of differentiation marker CD235a. These data suggest that SIRT1 activates HBG expression through facilitating LCR looping to the HBG promoter, inhibiting the expression of transcriptional suppressors of HBG, and indirectly increasing histone acetylation in the HBG promoter. SIRT1 is a potential therapeutic target for γ-globin gene induction, and small molecule SIRT1 activators might serve as a lead compound for the development of new HbF inducers.