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Abstract We aimed to investigate the efficacy of romosozumab treatment compared with that of denosumab in especially male osteoporosis patients. This retrospective cohort study included 174 Japanese male patients receiving either denosumab or romosozumab for 12 months. Propensity score matching extracted 50 patients per treatment group for standardization of group characteristics. The endpoints include the rate of change in the bone mineral density (BMD) of the lumbar spine, total hip, and femoral neck after 12 months of treatment as well as the changes in serum bone metabolism markers. The mean 12-month percentage increase in the lumbar spine BMD from baseline was significantly greater with romosozumab (13.0% ±1.7%) than with denosumab (4.5%±0.6%) (P

Abstract Inherited non-hemolytic anemia is a group of rare bone marrow disorders characterized by erythroid defects. Although concerted efforts have been made to explore the underlying pathogenetic mechanisms of these diseases, the understanding of the causative mutations are still incomplete. Here we identify in a diseased pedigree that a gain-of-function mutation in toll-like receptor 8 (TLR8) is implicated in inherited non-hemolytic anemia. TLR8 is expressed in erythroid lineage and erythropoiesis is impaired by TLR8 activation whereas enhanced by TLR8 inhibition from erythroid progenitor stage. Mechanistically, TLR8 activation blocks annexin A2 (ANXA2)-mediated plasma membrane localization of STAT5 and disrupts EPO signaling in HuDEP2 cells. TLR8 inhibition improves erythropoiesis in RPS19 +/− HuDEP2 cells and CD34+ cells from healthy donors and inherited non-hemolytic anemic patients. Collectively, we identify a gene implicated in inherited anemia and a previously undescribed role for TLR8 in erythropoiesis, which could potentially be explored for therapeutic benefit in inherited anemia.

Objective: Previous studies suggest older patients with multiple health conditions and medications may experience adverse interactions, leading to negative outcomes. However, there’s limited research on this in older adults receiving home medical care. This study assesses whether polypharmacy is linked to falls or other clinical outcomes. Methods: The study population included 217 participants, aged ≥65 years, receiving home medical care, who consented to participate in the Osaka Home Care Registry (OHCARE) study in Japan. The survey examined the association between polypharmacy and clinical outcomes. We defined “polypharmacy” as six or more medications taken regularly. Results: Of the participants, 135 (62.6%) had polypharmacy and were significantly more likely to have hypertension or diabetes. Common medications included those for hypertension, diabetes, and mental disorders. Participants with polypharmacy experienced significantly more falls. Multivariate analysis showed an association between polypharmacy and falls (odds ratio: 2.81, 95% confidence interval [1.34, 5.92]). Conclusion: Even in older patients receiving home health care, the use of six or more medications poses a risk of falls. Careful observations and life support by medical stuffs are necessary to prevent falls in older patients with polypharmacy receiving home medical care.

Lysine succinylation (Ksu) has recently emerged as a protein modification that regulates diverse functions in various biological processes. However, the systemically and precise role of lysine succinylation in erythropoiesis remains to be fully elucidated. In this study, we noted a prominent increase of succinyl-CoA and lysine succinylation during human erythroid differentiation. To explore the functional significance of succinylation, we inhibited succinylation by either knock downing key succinyltransferases or overexpressing desuccinylases. Succinylation inhibition led to suppressed cell proliferation, increased apoptosis, and disrupted erythroid differentiation. In vivo overexpression of the desuccinylases SIRT5 delayed erythroid differentiation. Furthermore, integrative proteome and succinylome analysis identifies 939 succinylated proteins with 3,562 Ksu sites, distributed across various cellular compartments and involved in multiple cellular processes. Significantly, inconsistencies between protein expression levels and succinylation levels were observed, indicating that the succinylation of certain proteins may function independently of expression. Mechanistically, we implicated KAT2A-mediated succinylation of histone H3 K79, leading to chromatin remodeling and subsequently erythropoiesis regulation. Specially, we identified CYCS as a key regulator of erythropoiesis, which depends on its succinylation sites K28/K40. Taken together, our comprehensive investigation of the succinylation landscape during erythropoiesis provides valuable insights into its regulatory role and offer potential implications for erythroid-related diseases.

Myelin basic protein (MBP) is a major component of the myelin sheaths of oligodendrocytes in the central nervous system and Schwann cells of the peripheral nervous system. Here we generated heterozygous fluorescent reporter of MBP gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock in fused tdTomato fluorescent protein and EF1 alpha promoter-driven Bleomycin (Zeocin) resistance gene to the translational MBP C-terminal region. The resulting line, MBP-TEZ, showed tdTomato fluorescence upon oligodendrocyte differentiation. This reporter hiPSC line provides a precedential opportunity for monitoring human myelin formation and degeneration and purifying MBP-expressing cell lineages.

Abstract The identification of regulatory networks contributing to fetal/adult gene expression switches is a major challenge in developmental biology and key to understand the aberrant proliferation of cancer cells, which often reactivate fetal oncogenes. One key example is represented by the developmental gene LIN28B, whose aberrant reactivation in adult tissues promotes tumor initiation and progression. Despite the prominent role of LIN28B in development and cancer, the mechanisms of its transcriptional regulation are largely unknown. Here, by using quantitative RT-PCR and single cell RNA sequencing data, we show that in erythropoiesis the expression of the transcription factor SOX6 matched a sharp decline of LIN28B mRNA during human embryo/fetal to adult globin switching. SOX6 overexpression repressed LIN28B not only in a panel of fetal-like erythroid cells (K562, HEL and HUDEP1; ≈92% p

Abstract K-562 is a well-known in vitro cellular model that represents human leukemia cell lines. Although the K-562 cells have been extensively characterized, there are inconsistencies in the data across publications, showing the presence of multiple K-562 cell lines. This suggests that analyzing a single K-562 cell line is insufficient to provide reliable reference data. In this study, we compared three K-562 cell lines with different IDs (RCB0027, RCB1635, and RCB1897) to investigate the fundamental characteristics of K-562 cells. Amplifications of the BCR-ABL1 fusion gene and at 13q31 were detected in all three cell lines, whereas each genome exhibited distinctive features of sequence variants and loss of heterozygosity. This implies that each K-562 cell line can be characterized by common and unique features through a comparison of multiple K-562 cell lines. Variations in transcriptome profiles and hemoglobin synthesis were also observed among the three cell lines, indicating that they should be considered sublines that have diverged from the common ancestral K-562 despite no changes from the original cell name. This leads to unintentional differences in genotypes and/or phenotypes among cell lines that share the same name. These data show that characterizing a single K-562 cell line does not necessarily provide data that are applicable to other K-562 cells. In this context, it is essential to modify cell names in accordance with changes in characteristics during cell culture. Furthermore, our data could serve as a reference for evaluating other K-562 sublines, facilitating the discovery of new K-562 sublines with distinct characteristics. This approach results in the accumulation of K-562 sublines with diverged characteristics and expands the options available, which may help in selecting the most suitable K-562 subline for each experiment.

Abstract Background Undernutrition and underweight are osteoporosis risk factors. Therefore, improving the health of underweight young women in Japan is an important medical issue. However, few studies have evaluated the association between being preconception underweight and postnatal osteoporotic fractures in young women. Methods This retrospective cohort study used a Japanese nationwide claims database (JMDC Inc.) to evaluate the effect of preconception underweight on the incidence of osteoporotic fracture within two years after delivery. Data from 16,684 mothers who delivered their first singleton babies between January 2006 and December 2020 were analysed. The combination of disease codes of fractures at sites associated with osteoporosis and medical procedures for fractures was defined as the incidence of osteoporotic fractures, whereas the body mass index (BMI) recorded 12–36 months before delivery was used as the exposure. We estimated the incidence of osteoporotic fractures by BMI category using a Kaplan–Meier curve and examined the fracture risk using Cox hazard regression analyses. Results Fifty-one women (0.31%) were affected by osteoporotic fractures within two years of delivery. More than 80% of these were rib fractures, and approximately 65% of fractures occurred after the first year postpartum. Preconception underweight (BMI

Glucose transporter 1 deficiency syndrome (GLUT1DS), caused by impaired glucose transport at the blood–brain barriers, leads to various central nervous system dysfunctions. A comprehensive understanding of the underlying disease pathogenesis is still lacking. In this study, we have generated GLUT1DS-specific human induced pluripotent stem cells (hiPSCs) derived from two patients. These established GLUT1DS-specific hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift or missense mutations in the responsible SLC2A1 gene. These novel cell resources provide new avenues for understanding disease mechanisms and developing new therapies for GLUT1DS.

Abstract Reactivation of fetal hemoglobin expression alleviates the symptoms associated with β‐globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal‐to‐adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ‐globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR‐Cas9‐mediated knock‐in, a bioluminescent tag was integrated at the HBG1 gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high‐throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.

β-thalassemia/HbE results from mutations in the β-globin locus that impede the production of functional adult hemoglobin. Base editors (BEs) could facilitate the correction of the point mutations with minimal or no indel creation, but its efficiency and bystander editing for the correction of β-thalassemia mutations in coding and non-coding regions remains unexplored. Here, we screened BE variants in HUDEP-2 cells for their ability to correct a spectrum of β-thalassemia mutations that were integrated into the genome as fragments of HBB. The identified targets were introduced into their endogenous genomic location using BEs and Cas9/homology-directed repair (HDR) to create cellular models with β-thalassemia/HbE. These β-thalassemia/HbE models were then used to assess the efficiency of correction in the native locus and functional β-globin restoration. Most bystander edits produced near target sites did not interfere with adult hemoglobin expression and are not predicted to be pathogenic. Further, the effectiveness of BE was validated for the correction of the pathogenic HbE variant in severe β0/βE-thalassaemia patient cells. Overall, our study establishes a novel platform to screen and select optimal BE tools for therapeutic genome editing by demonstrating the precise, efficient, and scarless correction of pathogenic point mutations spanning multiple regions of HBB including the promoter, intron, and exons.

Rett syndrome is characterized by severe global developmental impairments with autistic features and loss of purposeful hand skills. Here we show that human induced pluripotent stem cell (hiPSC) lines derived from four Japanese female patients with Rett syndrome are generated from peripheral blood mononuclear cells using Sendai virus vectors. The generated hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift, missense, or nonsense mutations in the MECP2 gene. Since the molecular pathogenesis caused by MECP2 dysfunction remains unclear, these cell resources are useful tools to establish disease models and develop new therapies for Rett syndrome.

Objectives: SARS-CoV-2 transmission and epidemic potential is related to the population's immunity levels. As such, assessing different regions’ preexisting immune responses to SARS-CoV-2 is important to understand the transmission potential of emerging SARS-CoV-2 variants. Design: In 975 serum samples from Vietnam (2014 to 2019), anti-SARS-CoV-2 Immunoglobulin G levels were determined by enzyme-linked immunosorbent assay. Plaque reduction neutralization test (PRNT) was performed using Wuhan strain and variants of concern (VOCs). Cross-reactivity was confirmed by analyzing B-cell receptor (BCR) repertoire sequences and identifying BCR repertoire sequences-derived T-cell epitopes. Results: Overall, 20.9% (n = 76/364) and 9.2% (n = 7) demonstrated SARS-CoV-2 neutralizing activity (PRNT50) against the Wuhan and Alpha strain, respectively. Neutralizing activity against Beta, Gamma, and Delta strains was absent (PRNT50

Considering the fundamental mechanism causing singularity phenomena, we performed the following abduction: Assuming that a multicellular system is driven by spontaneous fluctuation of each cell and dynamic interaction of the cells, state transition of the system would be experimentally predictable from cellular heterogeneity. This study evaluates the abductive hypothesis by analyzing cellular heterogeneity to distinguish pre-state of state transition of differentiating cells with Raman spectroscopy and human induced pluripotent stem cells (hiPSCs) technique. Herein, we investigated the time development of cellular heterogeneity in Raman spectra during cardiomyogenesis of six hiPSC lines and tested two types of analyses for heterogeneity. As expected, some spectral peaks, possibly attributed to glycogen, correctively exhibited higher heterogeneity, prior to intensity changes of the spectrum in the both analyses in the all cell-lines tested. The combination of spectral data and heterogeneity-based analysis will be an approach to the arrival of biology that uses not only signal intensity but also heterogeneity as a biological index.

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

Abstract Background Crosstalk between the aryl hydrocarbon receptor (AhR) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling is called the “AhR–Nrf2 gene battery”, which works synergistically in detoxification to support cell survival. Nrf2-dependent phase II gene promoters are controlled by coordinated recruitment of the AhR to adjacent dioxin responsive element (DRE) and Nrf2 recruitment to the antioxidative response element (ARE). The molecular interaction between AhR and Nrf2 members, and the regulation of each target, including phase I and II gene complexes, and their mediators are poorly understood. Methods Knockdown and forced expression of AhR–Nrf2 battery members were used to examine the molecular interactions between the AhR–Nrf2 axis and AhR promoter activation. Sequential immunoprecipitation, chromatin immunoprecipitation, and histology were used to identify each protein complex recruited to their respective cis-elements in the AhR promoter. Actin fiber distribution, cell spreading, and invasion were examined to identify functional differences in the AhR–Jdp2 axis between wild-type and Jdp2 knockout cells. The possible tumorigenic role of Jdp2 in the AhR–Nrf2 axis was examined in mutant Kras-Trp53-driven pancreatic tumors. Results Crosstalk between AhR and Nrf2 was evident at the transcriptional level. The AhR promoter was activated by phase I ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the AhR–Jdp2–Nrf2 axis in a time- and spatial transcription-dependent manner. Jdp2 was a bifunctional activator of DRE- and ARE-mediated transcription in response to TCDD. After TCDD exposure, Jdp2 activated the AhR promoter at the DRE and then moved to the ARE where it activated the promoter to increase reactive oxygen species (ROS)-mediated functions such as cell spreading and invasion in normal cells, and cancer regression in mutant Kras-Trp53-driven pancreatic tumor cells. Conclusions Jdp2 plays a critical role in AhR promoter activation through the AhR–Jdp2–Nrf2 axis in a spatiotemporal manner. The AhR functions to maintain ROS balance and cell spreading, invasion, and cancer regression in a mouse model of mutant Kras–Trp53 pancreatic cancer. These findings provide new insights into the roles of Jdp2 in the homeostatic regulation of oxidative stress and in the antioxidation response in detoxification, inflammation, and cancer progression.

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate β-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to β-globin exon-1 (βE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-βE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells in vivo. Furthermore, PRR-βE1 gene editing is feasible without ex vivo HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and β-thalassemia major. These findings imply that PRR-βE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for β-hemoglobinopathy gene therapy.

PurposeTo examine the hypothesis that an increase in response of postmenopausal bone to interval walking training (IWT) depends on baseline bone mineral densities (BMDs).MethodsTwo hundred and thirty-four postmenopausal women (64±5 (SD) yr) with no medication for osteoporosis performed 5-month IWT, repeating fast and slow walking at ≥70% and ~40% peak aerobic capacity, respectively, for 3 minutes each per set, ≥5 sets/day, ≥4 days/week. They were recruited from those who had performed IWT ≥6 months before participating in the study so that their physical fitness and lifestyle-related disease symptoms had almost reached a steady state at the time of their participation. We measured BMDs for the lumbar spine (LS), bilateral femoral neck (FN), and bilateral total hip (TH) by dual-energy X-ray absorptiometry (DXA) before and after the intervention. We used a multiple regression analysis to identify significant independent factors for increasing BMDs after the intervention as baseline physical characteristics, exercise intensity, and exercise time during IWT were the candidates. For any bone site where the independent factor identified was singular, we analyzed the relationship between the identified factor vs increases in BMD after the intervention (ΔBMD) by the locally weighted scatterplot smoothing (LOWESS) method.ResultsAlmost all subjects completed the designated protocol with minimal adverse events. We found that significant determinants for increasing BMDs were the baseline BMDs for all bone sites, as well as age and body mass index for TH (all, P0.3).ConclusionsIWT may be of benefit with minimal adverse events to postmenopausal women, although the effects were greater in those with lower baseline BMDs.Trial registrationUMIN000047428. https://rctportal.niph.go.jp/s/detail/um?trial_id=UMIN000047428#.

The aim of the present study was to investigate the perioperative and postoperative incidence of deep vein thrombosis (DVT) and validate the effectiveness of our own preventive treatment protocol for venous thromboembolism (VTE) occurrence in lower extremity arthroplasty patients. The subjects were 1,054 patients (mean age: 74.3 years) who underwent total hip arthroplasty (THA) or total knee arthroplasty (TKA) at our institutions between April 2014 and March 2017. We examined the frequencies of pre- and post-operative DVT by lower extremity Doppler images, and the incidence rate at proximal or distal regions as well as that according to preoperative DVT status were evaluated. Preoperative DVT was detected in 6.5% (69 cases) of our cohort and those were located 1.4% (15 cases) at proximal and 5.1% (54 cases) at distal regions. A significantly higher rate of postoperative DVT development was observed in preoperative DVT+ THA patients (P = 0.0075), but not in TKA patients only with a higher tendency (P = 0.56). The overall incidence of DVT up to 2 weeks after surgeries was 27.3% (288 cases); however, the rate in proximal femur regions was suppressed to 2.8% (30 cases), and there was no symptomatic pulmonary thromboembolism (PTE) case. The results demonstrated the importance of regular Doppler examination for early detection of postoperative DVT occurrence and the following immediate treatment initiation. Our own VTE preventive treatment protocol could reduce the development of proximal DVT, and the periodic monitoring as well as prompt treatment might prevent the fatal PTE. osteoarthritis (OA), rheumatoid arthritis (RA).

ABSTRACT The association between prevalent fractures and tooth loss in postmenopausal women remains unclear. Herein, we investigated the association between prevalent vertebral and nonvertebral fractures, the number of teeth present at baseline, and the number of teeth lost during follow‐up in postmenopausal Japanese women. This cross‐sectional study enrolled 843 participants (mean age 68.3 years). The number of teeth at follow‐up was evaluated in 655 women in this longitudinal study. The participants were divided into four groups according to their prevalent fracture status: no fractures, vertebral fractures alone, nonvertebral fractures alone, and both fracture types. After adjusting for covariates, Poisson regression analyses were performed to investigate differences in the number of teeth at baseline and that lost during the follow‐up period among the four groups. Participants with prevalent vertebral fractures alone had significantly fewer teeth at baseline than those in participants without fractures or nonvertebral fractures alone (p

This study describes RK-701, an inhibitor of histone methyltransferases G9a/GLP, as a promising therapeutic candidate for sickle cell disease and a universal role of BGLT3 lncRNA in fetal hemoglobin reactivation by chemical inducers including RK-701.

Abstract The gold-standard method to evaluate a functional antiviral immune response is to titer neutralizing antibodies (NAbs) against a viral pathogen. This is historically performed using an in vitro assay of virus-mediated infection, which requires BSL-3 facilities. As these are insufficient in Latin American countries, including Mexico, scant information is obtained locally about viral pathogens NAb, using a functional assay. An alternative solution to using a BSL-3 assay with live virus is to use a BSL-2-safe assay with a non-replicative pseudovirus. Pseudoviral particles can be engineered to display a selected pathogen’s entry protein on their surface, and to deliver a reporter gene into target cells upon transduction. Here we comprehensively describe the first development of a BSL-2 safe NAbs-measuring functional assay in Mexico, based on the production of pseudotyped lentiviral particles. As proof-of-concept, the assay is based on Nanoluc luciferase-mediated luminescence measurements from target cells transduced with SARS-CoV-2 Spike-pseudotyped lentiviral particles. We applied the optimized assay in a BSL-2 facility to measure NAbs in 65 serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. Overall, this is the first report of a BSL-2 safe pseudovirus-based functional assay developed in Mexico to measure NAbs, and a cornerstone methodology necessary to measure NAbs with a functional assay in limited resources settings.

Abstract Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel lineage-specific RNA interference (RNAi) lentiviral vectors, H23B-Ery-Lin-shRNA and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for any gene, and they also provide superior knockdown of the target gene expression with a single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A, two well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34+ cells with these vectors resulted in >80% reduction in the target protein levels and up to 40% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34+ cells transduced with H23B-Ery-Lin-shBCL11A LV in immunocompromised mice showed ~ 60% reduction in BCL11A protein expression with ~ 40% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34+ cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.

Abstract Acquired sideroblastic anemia, characterized by bone marrow ring sideroblasts (RS), is predominantly associated with myelodysplastic syndrome (MDS). Although somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery, are frequently found in MDS-RS, the detailed mechanism contributing to RS formation is unknown. To explore the mechanism, we established human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells stably expressing SF3B1K700E. SF3B1K700E expressing cells showed higher proportion of RS than the control cells along with erythroid differentiation, indicating the direct contribution of mutant SF3B1 expression in erythroblasts to RS formation. In SF3B1K700E expressing cells, ABCB7 and ALAS2, known causative genes for congenital sideroblastic anemia, were downregulated. Additionally, mis-splicing of ABCB7 was observed in SF3B1K700E expressing cells. ABCB7-knockdown HUDEP-2 cells revealed an increased frequency of RS formation along with erythroid differentiation, demonstrating the direct molecular link between ABCB7 defects and RS formation. ALAS2 protein levels were obviously decreased in ABCB7-knockdown cells, indicating decreased ALAS2 translation owing to impaired Fe–S cluster export by ABCB7 defects. Finally, RNA-seq analysis of MDS clinical samples demonstrated decreased expression of ABCB7 by the SF3B1 mutation. Our findings contribute to the elucidation of the complex mechanisms of RS formation in MDS-RS.

Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34+ cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34+ cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies.Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34+ cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools.Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.

Abstract X-linked sideroblastic anemia (XLSA), the most common form of congenital sideroblastic anemia, is caused by a germline mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene. In XLSA, defective heme biosynthesis leads to ring sideroblast formation because of excess mitochondrial iron accumulation. In this study, we introduced ALAS2 missense mutations on human umbilical cord blood-derived erythroblasts; hereafter, we refer to them as XLSA clones. XLSA clones that differentiated into mature erythroblasts showed an increased frequency of ring sideroblast formation with impaired hemoglobin biosynthesis. The expression profiling revealed significant enrichment of genes involved in ferroptosis, which is a form of regulated cell death induced by iron accumulation and lipid peroxidation. Notably, treatment with erastin, a ferroptosis inducer, caused a higher proportion of cell death in XLSA clones. XLSA clones exhibited significantly higher levels of intracellular lipid peroxides and enhanced expression of BACH1, a regulator of iron metabolism and potential accelerator of ferroptosis. In XLSA clones, BACH1 repressed genes involved in iron metabolism and glutathione synthesis. Collectively, defective heme biosynthesis in XLSA clones could confer enhanced BACH1 expression, leading to increased susceptibility to ferroptosis. The results of our study provide important information for the development of novel therapeutic targets for XLSA.

Abstract Ribosomal protein dysfunction causes diverse human diseases, including Diamond–Blackfan anemia (DBA). Despite the universal need for ribosomes in all cell types, the mechanisms underlying ribosomopathies, which are characterized by tissue-specific defects, are still poorly understood. In the present study, we analyzed the transcriptomes of single purified erythroid progenitors isolated from the bone marrow of DBA patients. These patients were categorized into untreated, glucocorticoid (GC)-responsive and GC-non-responsive groups. We found that erythroid progenitors from untreated DBA patients entered S-phase of the cell cycle under considerable duress, resulting in replication stress and the activation of P53 signaling. In contrast, cell cycle progression was inhibited through induction of the type 1 interferon pathway in treated, GC-responsive patients, but not in GC-non-responsive patients. Notably, a low dose of interferon alpha treatment stimulated the production of erythrocytes derived from DBA patients. By linking the innately shorter cell cycle of erythroid progenitors to DBA pathogenesis, we demonstrated that interferon-mediated cell cycle control underlies the clinical efficacy of glucocorticoids. Our study suggests that interferon administration may constitute a new alternative therapeutic strategy for the treatment of DBA. The trial was registered at www.chictr.org.cn as ChiCTR2000038510.

Peficitinib, a pan-JAK inhibitor, is known to suppress the activation of fibroblast-like synoviocytes (FLSs) and thereby reduces joint inflammation associated with rheumatoid arthritis (RA). However, the effect on osteoporosis in RA remains to be elucidated. In this study, the effect of peficitinib or etanercept on joint inflammation, and consequently decreased bone mineral density (BMD) was evaluated in mice with collagen-induced arthritis (CIA). Additionally, the effect on RANKL production from osteoblasts differentiated from the mesenchymal stem cells of RA patients was evaluated. Administration of peficitinib for established CIA ameliorated arthritis and improved BMD in the femoral metaphysis, but not in the femoral diaphysis. Conversely, etanercept suppressed an increase in synovial inflammatory markers but did not improve arthritic conditions or the reduction of BMD in either region. All elevated bone formation and bone resorption markers were decreased with peficitinib but only partially decreased with etanercept. Furthermore, production of RANKL by human osteoblasts was suppressed by peficitinib but enhanced by etanercept. Unlike etanercept, peficitinib is thought to increase BMD by ameliorating the high bone turnover associated with RA states, resulting in improvement of bone fragility. Our data provide evidence that peficitinib would be expected to show efficacy for osteoporosis associated with RA.

Although nitric oxide (NO) is a known factor that regulates the bone physiology, few and discordant results have been obtained in human studies evaluating the effect of nitrates on bone health. We investigated for the relationship between serum NOx level and incident osteoporotic fracture rate prospectively in a cohort consisting of Japanese women. A total of 871 subjects (67.5 ± 10.8 y/o) were analyzed. During the observation period (8.8 ± 7.2 yrs), incident osteoporotic fractures occurred in 267 participants (209 vertebral fractures, 57 long-bone fractures, and 1 both types). Hazard ratio, by the Cox proportional hazards model, of serum NOx for incident fracture was 0.64 (95% confidence interval 0.53-0.78, p < 0.001) after adjustment for baseline age (1.13, 1.06-1.21, p < 0.001), lumbar bone mineral density (L-BMD; 0.85, 0.78-0.92, p < 0.001), presence of prevalent fracture (3.27, 2.49-4.32, p < 0.001), and treatment of osteoporosis (0.70, 0.53-0.92, p = 0.010). The relationships between serum level of NOx and bone-related parameters were examined by multiple regression analysis; body mass index (p < 0.001) and L-BMD (p = 0.011) were significantly associated with serum NOx level. These results suggest that the low circulating NOx is one of the independent predictors for osteoporotic fracture occurrence in postmenopausal women.

Abstract Background A decrease in bone mineral density is common in patients with Williams syndrome. However, appropriate management for osteoporosis in Williams syndrome patients has not been established. We report the case of a 12-year-old female patient with Williams syndrome, who underwent denosumab treatment for osteoporosis. Case presentation A 12-year-old Japanese female patient with Williams syndrome was shown to have very low bone mineral density. Bone mineral density was evaluated before treatment and at 5, 9, 17, 23, and 29 months of treatment by dual-energy X-ray absorptiometry. After denosumab therapy for 29 months, lumbar and total hip bone mineral density values had increased by 51.6% and 37.6%, respectively. No new fractures occurred during the observation period. Conclusions To the best of our knowledge, this is the first experience with denosumab treatment in Williams syndrome patients with osteoporosis. Based on our findings, denosumab may be an effective treatment option for Williams syndrome patients with osteoporosis.

Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG > GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A > T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

Introduction: Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by defective maturation of the erythroid progenitors in the bone marrow, for which treatment involves steroids, chronic transfusions, or hematopoietic stem cells transplantation. Diamond Blackfan anemia is caused by defective ribosome biogenesis due to heterozygous pathogenic variants in one of 19 ribosomal protein (RP) genes. The decreased number of functional ribosomes leads to the activation of pro-apoptotic pathways and to the reduced translation of key genes for erythropoiesis.Results and discussion: Here we characterized the phenotype of RPS26-deficiency in a cell line derived from human umbilical cord blood erythroid progenitors (HUDEP-1 cells). This model recapitulates cellular hallmarks of Diamond Blackfan anemia including: imbalanced production of ribosomal RNAs, upregulation of pro-apoptotic genes and reduced viability, and shows increased levels of intracellular calcium. Evaluation of the expression of erythroid markers revealed the impairment of erythroid differentiation in RPS26-silenced cells compared to control cells.Conclusions: In conclusion, for the first time we assessed the effect of RPS26 deficiency in a human erythroid progenitor cell line and demonstrated that these cells can be used as a scalable model system to study aspects of DBA pathophysiology that have been refractory to detailed investigation because of the paucity of specific cell types affected in this disorder.

Developing robust methodology for the sustainable production of red blood cells in vitro is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34+ cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show that our immortalization approach is reproducible for erythroid cells differentiated from bone marrow and also from far more accessible peripheral and cord blood CD34+ cells, consistently generating lines with similar improved erythroid performance. Extensive characterization of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalization. In addition, we show that only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalization. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.

Abstract Background Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis. Results We find in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specific genes as well as a nuclear condensation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specific genes observed in the absence of H2A.X. Conclusions We relate these findings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifically suppressed.

Abstract Background The cerebellum is the sensitive region of the brain to developmental abnormalities related to the effects of oxidative stresses. Abnormal cerebellar lobe formation, found in Jun dimerization protein 2 (Jdp2)-knockout (KO) mice, is related to increased antioxidant formation and a reduction in apoptotic cell death in granule cell progenitors (GCPs). Here, we aim that Jdp2 plays a critical role of cerebellar development which is affected by the ROS regulation and redox control. Objective Jdp2-promoter-Cre transgenic mouse displayed a positive signal in the cerebellum, especially within granule cells. Jdp2-KO mice exhibited impaired development of the cerebellum compared with wild-type (WT) mice. The antioxidation controlled gene, such as cystine-glutamate transporter Slc7a11, might be critical to regulate the redox homeostasis and the development of the cerebellum. Methods We generated the Jdp2-promoter-Cre mice and Jdp2-KO mice to examine the levels of Slc7a11, ROS levels and the expressions of antioxidation related genes were examined in the mouse cerebellum using the immunohistochemistry. Results The cerebellum of Jdp2-KO mice displayed expression of the cystine-glutamate transporter Slc7a11, within the internal granule layer at postnatal day 6; in contrast, the WT cerebellum mainly displayed Sla7a11 expression in the external granule layer. Moreover, development of the cerebellar lobes in Jdp2-KO mice was altered compared with WT mice. Expression of Slc7a11, Nrf2, and p21Cip1 was higher in the cerebellum of Jdp2-KO mice than in WT mice. Conclusion Jdp2 is a critical regulator of Slc7a11 transporter during the antioxidation response, which might control the growth, apoptosis, and differentiation of GCPs in the cerebellar lobes. These observations are consistent with our previous study in vitro.

In an imaging-based screen of >3,300 compounds compounds, Soboleva et al identify HDAC inhibitors as mediators of erythroid cell enucleation. They further show that the erythroid-specific cell membrane protein, SPTA1, is downregulated in HDAC inhibited cells and that restoration of SPTA1 expression using CRISPR-activation partially rescues the fragility of cells, improving enucleation efficiency.

Abstract Denosumab and romosozumab, a recently approved new drug, are effective and widely known molecular-targeted drugs for postmenopausal osteoporosis treatment. However, no studies have directly compared their therapeutic effects or safety in postmenopausal osteoporosis. This retrospective observational registry study compared the efficacy of 12-month denosumab or romosozumab treatment in postmenopausal osteoporosis patients. The primary outcome was the change in bone mineral density (BMD) at the lumbar spine. Secondary outcomes included BMD changes at the total hip and femoral neck, changes in bone turnover markers, and adverse events. Propensity score matching was employed to assemble patient groups with similar baseline characteristics. Sixty-nine patients each received either denosumab or romosozumab for 12 months. The mean 12-month percentage change from baseline in lumbar spine BMD was 7.2% in the denosumab group and 12.5% in the romosozumab group, indicating a significant difference between the groups. The percentage changes in BMD at both the total hip and femoral neck were also significantly higher at 12 months in the romosozumab group than in the denosumab group. In denosumab patients, bone formation and bone resorption markers were significantly decreased at 6 and 12 months from baseline. In the romosozumab group, the bone formation marker was significantly increased at 6 months and then returned to baseline, while the bone resorption marker was significantly decreased at both time points. Adverse events were few and predominantly minor in both groups, with no remarkable difference in the incidence of new vertebral fractures. Romosozumab showed a higher potential for improving BMD than denosumab in this clinical study of postmenopausal osteoporosis patient treatment.

Dilated cardiomyopathy (DCM) is a refractory heart disease characterized by dilation of the left ventricle and systolic dysfunction. LMNA, the gene encoding lamin A/C (a nuclear envelope protein), is the second leading causative gene associated with familial DCM. LMNA-related DCM is likely to develop severe heart failure, various types of arrhythmias, and poor prognosis. We established a human induced pluripotent stem cell line, derived from a patient with DCM carrying a nonsense mutation in LMNA. This line should be a useful resource for elucidating disease mechanisms and developing fundamental treatments for LMNA-related DCM.

Abstract The aim of this study was to provide definitive reference values for bone mineral density (BMD) and bone turnover markers in the general elderly population. Registered citizens of 50 to 89 years old were targeted for this survey. After random sampling from the resident registry of Obuse town, we established eight groups based on age (50 s, 60 s, 70 s, and 80 s) and gender. A total of 411 people were enrolled. We used a dual-energy x-ray absorptiometry device to measure and evaluate BMD. The bone formation marker bone alkaline phosphatase (BAP) was measured as a bone turnover marker. Bone quality marker pentosidine, and bone resorption markers including urinary total deoxypyridinoline (DPD), tartrate-resistant acid phosphatase 5b (TRACP-5b), 25-hydroxyvitamin D (25[OH]D), and whole parathyroid hormone (PTH) were also measured as bone turnover markers. Sixty-three people (15.3%) were diagnosed as osteoporosis. BMD decreased with age in the femoral neck and total hip. On the other hand, there was no characteristic change with age in the lumber spine. As for bone markers, pentosidine and DPD increased with aging, although 25(OH)D, whole PTH, and BAP showed no characteristic associations with gender and aging. In terms of the relationship between low BMD and bone markers, there was a significant independent association between low BMD and TRACP-5b in females. In conclusions, hip BMD decreased with aging in men and women. However, there was no characteristic decline with aging in the lumbar spine. All bone markers showed no significant independent characteristics associated with age or gender in a multivariate analysis model, except for a significant association between low BMD and TRACP-5b in females. TRACP-5b was a potentially useful marker for the detection of low BMD.

T-cell recognition of antigen epitopes is a crucial step for the induction of adaptive immune responses, and the identification of such T-cell epitopes is, therefore, important for understanding diverse immune responses and controlling T-cell immunity. A number of bioinformatic tools exist that predict T-cell epitopes; however, many of these methods highly rely on evaluating conventional peptide presentation by major histocompatibility complex (MHC) molecules, but they ignore epitope sequences recognized by T-cell receptor (TCR). Immunogenic determinant idiotopes are present on the variable regions of immunoglobulin molecules expressed on and secreted by B-cells. In idiotope-driven T-cell/B-cell collaboration, B-cells present the idiotopes on MHC molecules for recognition by idiotope-specific T-cells. According to the idiotype network theory formulated by Niels Jerne, such idiotopes found on anti-idiotypic antibodies exhibit molecular mimicry of antigens. Here, by combining these concepts and defining the patterns of TCR-recognized epitope motifs (TREMs), we developed a T-cell epitope prediction method that identifies T-cell epitopes derived from antigen proteins by analyzing B-cell receptor (BCR) sequences. This method allowed us to identify T-cell epitopes that contain the same TREM patterns between BCR and viral antigen sequences in two different infectious diseases caused by dengue virus and SARS-CoV-2 infection. The identified epitopes were among the T-cell epitopes detected in previous studies, and T-cell stimulatory immunogenicity was confirmed. Thus, our data support this method as a powerful tool for the discovery of T-cell epitopes from BCR sequences.

Abstract Pentosidine (PEN) and carboxymethyl-lysine (CML) are well-recognized advanced glycation end products (AGEs). However, how these AGEs affect the pathophysiology of osteoporosis and osteoporotic fractures remains controversial. This cross-sectional study aimed to investigate the associations of PEN and CML with bone markers, bone mineral density (BMD), and osteoporotic fractures in postmenopausal women from the Nagano Cohort Study. A total of 444 Japanese postmenopausal outpatients (mean ± standard deviation age: 69.8 ± 10.2 years) were enrolled after the exclusion of patients with acute or severe illness or secondary osteoporosis. The relationships among urinary PEN and serum CML levels, various bone markers, lumbar and hip BMD, and prevalent vertebral and long-bone fractures were evaluated. PEN associated significantly with prevalent vertebral fracture after adjustment for other confounders (odds ratio [OR] 1.59, 95% confidence interval [CI] 1.22–2.07; P

DiGeorge syndrome (22q11.2 deletion syndrome, or CATCH22 syndrome), caused by hemizygous deletion of chromosome 22q11.2, results in the poor development of multiple organs. Here we have generated DiGeorge syndrome-specific human induced pluripotsnt stem cells (hiPSCs) derived from four patients. These established hiPSC lines showed self-renewal and pluripotency and carried a hemizygous deletion in 22q11.2. Since the molecular pathogenesis of DiGeorge syndrome caused by the 22q11.2 deletion is largely unknown, these cell resources will be useful for recapitulating disease phenotypes and for developing new therapies for DiGeorge syndrome.

Reactivation of γ-globin expression is a promising therapeutic approach for β-hemoglobinopathies. Here, we propose a novel Cas9/AAV6-mediated genome editing strategy for the treatment of β-thalassemia: Natural HPFH mutations −113A > G, −114C > T, −117G>A, −175T > C, −195C > G, and −198T > C were introduced by homologous recombination following disruption of BCL11A binding sites in HBG1/HBG2 promoters. Precise on-target editing and significantly increased γ-globin expression during erythroid differentiation were observed in both HUDEP-2 cells and primary HSPCs from β-thalassemia major patients. Moreover, edited HSPCs maintained the capacity for long-term hematopoietic reconstitution in B-NDG hTHPO mice. This study provides evidence of the effectiveness of introducing naturally occurring HPFH mutations as a genetic therapy for β-thalassemia.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the –123 region. Base editing at –123 and –124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of –123T > C and –124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Reactivation of fetal hemoglobin (HbF, α2γ2) alleviates clinical symptoms in patients with β-thalassemia and sickle cell disease, although the regulatory mechanisms of γ-globin expression have not yet been fully elucidated. Recent studies found that interfering with the expression of the membrane protein ANTXR1 gene upregulated γ-globin levels. However, the exact mechanism by which ANTXR1 regulates γ-globin levels remains unclear. Our study showed that overexpression and knockdown of ANTXR1 in K562, cord blood CD34+, and HUDEP-2 cells decreased and increased γ-globin expression, respectively. ANTXR1 regulates the reactivation of fetal hemoglobin (HbF, α2γ2) in K562, cord blood CD34+, and adult peripheral blood CD34+ cells through interaction with LRP6 to promote the nuclear entry of β-catenin and activate the Wnt/β-catenin signaling pathway. The overexpression or knockdown of ANTXR1 on γ-globin and Wnt/β-catenin signaling in K562 cells was reversed by the inhibitor XAV939 and the activator LiCl, respectively, where XAV939 inhibits the transcription of β-catenin in the Wnt pathway, but LiCl inhibits GSK3-β. We also showed that the binding ability of the rank4 site in the transcriptional regulatory region of the SOX6 gene to c-Jun was significantly increased after overexpression of ANTXR1 in K562 cells. SOX6 protein expression was increased significantly after overexpression of the c-Jun gene, indicating that the transcription factor c-Jun initiated the transcription of SOX6, thereby silencing γ-globin. Our findings may provide a new intervention target for the treatment of β-hemoglobinopathies.

Objectives: Increasing bone mineral density (BMD) to reduce fracture risk is a primary goal of osteoporosis treatment. This prospective, observational study evaluates the effects of monthly minodronate (MIN; 50 mg) with or without eldecalcitol (ELD) addition in osteoporosis patients with rheumatoid arthritis (RA) during 18 months. Methods: The cohort was prospectively and randomly split into the MIN monotherapy group (14 cases) and MIN plus ELD group (combination group; 14 cases) due to no reports on the effectiveness and safety of MIN therapy in relation to ELD addition for comparisons of serum tartrate-resistant acid phosphatase (TRACP)-5b, bone alkaline phosphatase (BAP), and BMD of the lumbar 1–4 vertebrae (L-BMD), bilateral total hips (H-BMD; the mean value of the right and left hips), and bilateral femoral necks (FN-BMD) at baseline and at 6, 12, and 18 months of treatment. Results: Baseline values were comparable between the groups apart from a tendency for higher TRACP-5b in the combination group. Seven of 14 patients in the combination group had received previous bisphosphonate treatment. BAP was significantly more reduced in the monotherapy group at 6 months, with no other remarkable differences for TRACP5b, L-BMD, H-BMD, or FN-BMD during the observation period. Conclusions: The above findings suggest that regardless of ELD addition, MIN potentially improves BMD during 18 months in osteoporosis patients with RA. Keywords: Bone mineral density, Eldecalcitol, Minodronate, Osteoporosis, Rheumatoid arthritis

JAG1 gene encodes Jagged1 protein, which is a ligand for NOTCH receptors. JAG1 mutations cause Alagille syndrome, in which liver failure occurs caused by abnormalities in the bile ducts. In this study, we generated two homozygous JAG1 knockout iPSC lines (JAG1KO iPSC) by creating indels with CRISPR-Cas9 technology. These newly generated JAG1KO iPSC lines showed similar self-renewal and pluripotency as their original iPSC WTC11 line. These iPSC lines carried deletions around the translation start codon of JAG1 gene, causing compromised Jagged1 protein expression. These JAG1KO iPSC lines are promising bioresources to study Jagged1 function in human development and pathology.

Ex vivo expansion of hematopoietic stem cells (HSCs) is one of the most promising strategies to increase the availability of transplantable HSCs and improve bone marrow transplantation outcomes. We recently demonstrated that mouse HSCs could be efficiently expanded in polyvinyl alcohol (PVA)-containing culture medium using only recombinant stem cell factor and thrombopoietin cytokines. However, the behavior of human HSCs in these simple PVA-based media was not fully elucidated. In this study, we analyzed the compatibility of PVA of different hydrolysis rates (HR) and molecular weights (MW) to support functional human and mouse HSCs ex vivo. Human and mouse HSCs proliferated more frequently in media containing PVA with lower HR than with higher HR, but both PVA types supported HSC multilineage reconstitution potential. Importantly, human HSCs cultured in PVA-containing media engrafted not only in irradiated recipients but also in non-irradiated recipients. Our results demonstrate that human HSCs can be maintained ex vivo using PVA-based culture systems and suggest approaches for future optimization of human HSC expansion.