Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.

Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease.

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.

Interpersonal sensitivity and childhood trauma in patients with major depressive disorder, bipolar I, and II disorder.

Childhood trauma and interpersonal sensitivity impact the development of mood disorders. In this study, we investigate the association between childhood trauma and interpersonal sensitivity in patients with mood disorders. A total 775 patients (major depressive disorder [MDD, n = 241], bipolar I disorder [BD I, n = 119], and bipolar II disorder [BD II, n = 415]) and 734 controls. For evaluation, we used the Childhood Trauma Questionnaire-Short Form (CTQ) and Interpersonal Sensitivity Measure (IPSM). We examined between-group differences for each subscale in the CTQ and IPSM. Patients with BD II had significantly higher IPSM total scores than patients with MDD, BD I, or controls. The CTQ total score was related to the IPSM total score in all participants and subgroups. Among the CTQ subscales, emotional abuse showed the highest correlation with the IPSM total score, while separation anxiety and fragile inner self showed higher positive correlations with CTQ than the other subscales of IPSM in all patient groups and the control group, respectively. The findings reveal that childhood trauma and interpersonal sensitivity are positively correlated among patients with MDD, BD I, and BD II, and that interpersonal sensitivity is higher in patients with BD II than those with BD I or MDD. Childhood trauma is associated with interpersonal sensitivity, and each trauma type has a different impact on mood disorders. We expect that this study will encourage future research on interpersonal sensitivity and childhood trauma in mood disorders to improve treatment approaches.

Relationship between early traumatic experiences and emotional regulation skills in major depressive and bipolar disorders.

Childhood trauma can cause deficits in emotional regulation. However, few studies have investigated childhood trauma and emotional regulation skills in patients with mood disorders. We aimed to investigate how childhood trauma and Emotion Regulation Skills Questionnaire (ERSQ) scores are associated with mood disorders.This study included 779 patients with mood disorders (major depressive disorder [MDD, n = 240], bipolar I disorder [BDI, n = 121], and bipolar II disorder [BDII, n = 418]). We used their Childhood Trauma Questionnaire-Short Form (CTQ) and ERSQ scores for the evaluation.The between-group differences in CTQ and ERSQ scores were examined. The CTQ and ERSQ total scores were negatively correlated. Among the CTQ subscales, emotional neglect showed a significant correlation with the ERSQ total score, whereas acceptance and tolerance showed higher negative correlations with the CTQ than with the other ERSQ subscales. The negative relationship between emotional regulation and childhood trauma varied significantly depending on the group, with the BDI group showing a more prominent association than the other groups.Based on various mood disorders, we observed associations between childhood trauma and emotional regulation skills. Consequently, our study offers notable insights for future research on the impact of childhood trauma on ERSQ.

Overexpression of Arabidopsis nucleolar GTP-binding 1 (NOG1) proteins confers drought tolerance in rice.

As a major adverse environmental factor in most parts of the world, drought causes substantial crop yield losses. Rice (Oryza sativa) is one of the staple foods for more than one-half of the world's population. Rice plants are sensitive to even mild drought stress and need almost twice the amount of water compared to wheat (Triticum aestivum) or maize (Zea mays). Arabidopsis (Arabidopsis thaliana) small GTPase Nucleolar GTP-binding protein 1 (AtNOG1) plays a role in biotic stress tolerance. Here, we created transgenic rice lines constitutively overexpressing AtNOG1-1 or AtNOG1-2. We also developed rice RNA interference (RNAi) lines that show downregulation of OsNOG1. AtNOG1-1 and AtNOG1-2 overexpressors showed enhanced drought tolerance without compromising grain yield, whereas OsNOG1-RNAi was more susceptible to drought when compared to wild-type plants. Analysis of physiological parameters showed increased cell sap osmolality, relative water content, and abscisic acid (ABA) level, but decreased leaf water loss in AtNOG1-1 or AtNOG1-2 overexpressor lines compared to the control. We found upregulation of several genes involved in ABA and jasmonic acid (JA) signaling, stomata regulation, osmotic potential maintenance, stress protection, and disease resistance in AtNOG1-1 and AtNOG1-2 overexpressor lines compared to the control. We elucidated the role of NOG1-2 and NOG1-1 in regulation of silica body formation around stomata to prevent transpirational water loss. These results provide an avenue to confer drought tolerance in rice.

Network structure of symptomatology of adult attention-deficit hyperactivity disorder in patients with mood disorders.

Patients with mood disorders commonly manifest comorbid psychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD). However, few studies have evaluated ADHD symptoms in this population. The current study aimed to explore the network structure of ADHD symptomology and identify central symptoms in patients with mood disorders. The Korean version of the Adult ADHD Self-Report Scale was used to assess the overall ADHD symptoms in 1,086 individuals diagnosed with mood disorders (major depressive disorder [n = 373], bipolar I disorder [n = 314], and bipolar II disorder [n = 399]). We used exploratory graph analysis to detect the number of communities, and the network structure was analyzed using regularized partial correlation models. We identified the central ADHD symptom using centrality indices. Network comparison tests were conducted with different subgroups of patients with mood disorders, including three mood diagnosis groups, between the patients who met the diagnostic criteria for ADHD [ADHD-suspected, n = 259] in their self-report and the others [ADHD-non-suspected, n = 827], and groups with high [n = 503] versus low [n = 252] levels of depressive state. The network analysis detected four communities: disorganization, agitation/restlessness, hyperactivity/impulsivity, and inattention. The centrality indices indicated that "feeling restless" was the core ADHD symptom. The result was replicated in the subgroup analyses within our clinically diverse population of mood disorders, encompassing three presentations: Patients with suspected ADHD, patients without suspected ADHD, and patients with a high depressive state. Our findings reveal that "feeling restless" is the central ADHD symptom. The treatment intervention for "feeling restless" may thus play a pivotal role in tackling ADHD symptoms in adult patients with mood disorders.

Korean Validation of the Short Version of the TEMPS-A (Temperament Evaluation of Memphis, Pisa, Paris, and San Diego Autoquestionnaire) in Patients with Mood Disorders.

BACKGROUND AND OBJECTIVES: The Temperament Evaluation of Memphis, Pisa, Paris and San Diego Autoquestionnaire (TEMPS-A) is designed to assess affective temperaments. The short version of the TEMPS-A (TEMPS-A-SV) has been translated into various languages for use in research and clinical settings. However, no research has been conducted to validate the Korean version of the TEMPS-A-SV in patients with mood disorders. The goal of this study is to evaluate the reliability and validity of the TEMPS-A-SV in Korean mood disorder patients. MATERIALS AND METHODS: In this cross-sectional retrospective study, a total of 715 patients (267 patients with major depressive disorder, 94 patients with bipolar disorder I, and 354 patients with bipolar disorder II) completed the Korean TEMPS-A-SV. Cronbach's alpha and McDonald's omega were used to assess the reliability. Exploratory factor analysis (EFA) was also performed. Spearman's correlation coefficient was used to examine associations between the five temperaments. The difference in five temperament scores between the gender or diagnosis groups was analyzed, and the correlation between five temperament scores and age was tested. RESULTS: The Korean TEMPS-A-SV displayed good internal consistency (α = 0.65-0.88, ω = 0.66-0.9) and significant correlations between the subscales except one (the correlation between hyperthymic and anxious). Using EFA, a two-factor structure was produced: Factor I (cyclothymic, depressive, irritable, and anxious) and Factor II (hyperthymic). The cyclothymic temperament score differed by gender and the anxious temperament score was significantly correlated with age. All the temperaments, except for irritable temperament, showed significant differences between diagnosis groups. CONCLUSIONS: Overall, the results show that the TEMPS-A-SV is a reliable and valid measurement that can be used for estimating Koreans' affective temperaments. However, more research is required on affective temperaments and associated characteristics in people with mood disorders.

The relationships between cognitive control and psychological symptoms in patients with somatic symptom disorder: a pilot longitudinal study.

INTRODUCTION: The present study explored how neurocognitive function correlated with the clinical symptoms of somatic symptom disorder (SSD) by evaluating changes in cognitive abilities according to differences in relevant factors. METHODS: A total of 44 patients with SSD and 30 healthy controls completed tests assessing various neurocognitive domains, including verbal memory, psychomotor speed, executive function, working memory, and sustained and divided attention. They also completed questionnaires for psychological assessment. The same tests and questionnaires were completed by 26 SSD patients 6 months later. RESULTS: The SSD patients had significantly lower scores on the attentional and verbal memory tests than did the healthy controls. Performance on the attentional test was significantly associated with the level of somatic symptoms and anxiety. The follow-up assessment results of the SSD patients revealed improved performance on the verbal learning and fluency tests as well as improvements in somatic symptoms, anxiety, and depression. It was also observed that changes in verbal learning and attentional functions were significantly associated with improvements in somatic symptoms. CONCLUSIONS: The present study suggests that neurocognitive dysfunctions are subtle and not specific to SSD, but certain cognitive functions may be related to the clinical symptoms and improvements of patients with SSD.

Development and Validation of the Mood Instability Questionnaire-Trait (MIQ-T).

Background and objectives: Mood instability (MI) is a stable trait associated with psychiatric disorders, yet there is a lack of tools to measure MI. The purpose of this study was to develop and validate the Mood Instability Questionnaire-Trait (MIQ-T) to evaluate MI in mood disorder patients. Material and methods: Items were taken from various established questionnaires to create an initial list of MIQ-T questions. Data from 309 psychiatric patients (n = 309; 62 major depressive disorder, 58 bipolar I disorder, and 189 bipolar II disorder) were gathered from their medical records and were utilized in an exploratory factor analysis to clarify the underlying components of MI. Then, anonymous survey data from 288 individuals from the general population were included in the analysis as a comparison group. Associations between MIQ-T and other previously validated clinical instruments for mood disorders were examined to test external validity. Results: The exploratory factor analysis demonstrated that the five-factor structure (Lability, Upward Tendency, Downward Tendency, Childhood Instability, and Seasonality) of 59 items was the most appropriate with clear, cohesive features. MIQ-T exhibited high internal consistency (α = 0.96) and moderate to strong correlations with other previously validated clinical instruments, which were consistent with theoretical predictions, providing evidence of criterion validity. Short forms were also created to address the high internal consistency value, which can indicate redundancy, and to increase the approachability of the measure. We found that the patients with bipolar II disorder had higher MIQ-T scores than the patients with bipolar I disorder or major depressive disorder and the comparison group. Conclusion: Together, these findings validate the newly developed MIQ-T as an instrument of mood instability. MIQ-T can be a potential research tool for mood disorder.

Optimizing lentiviral vector transduction of hematopoietic stem cells for gene therapy.

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34+ cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34+ cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34+ cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34+ cell concentrations (2-4 × 106/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34+ cell transduction with clinically relevant LVs.

Trifloxystrobin induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HaCaT, human keratinocyte cells.

As the outermost layer of the body, the skin plays an important role in exposure to pesticides, which could have negative impacts on human health. Trifloxystrobin is a widely used fungicide of the strobilurin class, however, there is little information regarding the skin contact-associated toxic mechanism. Therefore, the present study was performed in order to identify the skin toxicity mechanism of trifloxystrobin using HaCaT (keratinocyte of human skin) cells. Following 24 or 48 h treatment, cell viability, and subsequent Annexin V-FITC/propidium iodide assay, TUNEL assay and Western blotting were performed to investigate the cell death mechanism of trifloxystrobin. Exposure to trifloxystrobin resulted in diminished viability of HaCaT cells in both a time- and concentration-dependent manner. The cell death was derived through apoptotic pathways in the HaCaT cells. Furthermore, we explored the effect of trifloxystrobin on TRAIL-mediated extrinsic apoptosis using siRNA transfection. Knockdown of death receptor 5 suppressed trifloxystrobin-provoked apoptosis. These results indicate that trifloxystrobin induces TRAIL-mediated apoptosis and has an inhibitory effect in keratinocytes that can interfere with the barrier function and integrity of the skin. This could be proposed as a mechanism of skin toxicity by trifloxystrobin and considered in the management of pesticide exposure.

Transcriptome profiling of differentially expressed genes in floral buds and flowers of male sterile and fertile lines in watermelon.

BACKGROUND: Male sterility is an important mechanism for the production of hybrid seeds in watermelon. Although fruit development has been studied extensively in watermelon, there are no reports on gene expression in floral organs. In this study, RNA-sequencing (RNA-seq) was performed in two near-isogenic watermelon lines (genic male sterile [GMS] line, DAH3615-MS and male fertile line, DAH3615) to identify the differentially expressed genes (DEGs) related to male sterility. RESULTS: DEG analysis showed that 1259 genes were significantly associated with male sterility at a FDR P-value of < 0.01. Most of these genes were only expressed in the male fertile line. In addition, 11 functional clusters were identified using DAVID functional classification analysis. Of detected genes in RNA-seq analysis, 19 were successfully validated by qRT-PCR. CONCLUSIONS: In this study, we carried out a comprehensive floral transcriptome sequence comparison of a male fertile line and its near-isogenic male sterile line in watermelon. This analysis revealed essential genes responsible for stamen development, including pollen development and pollen tube elongation, and allowed their functional classification. These results provided new information on global mechanisms related to male sterility in watermelon.

Network Structure of Interpersonal Sensitivity in Patients With Mood Disorders: A Network Analysis.

OBJECTIVE: Interpersonal sensitivity, characterized by a heightened awareness of others' behavior and emotions, is linked to mood disorders. However, current literature lacks a comprehensive analysis of how some items of the Interpersonal Sensitivity Measure (IPSM) interrelate and contribute to the overall construct. This study constructed a network for interpersonal sensitivity symptomatology to identify core IPSM items in patients with mood disorders. METHODS: The IPSM, a 36-item self-report scale, was utilized to evaluate interpersonal sensitivity symptoms in 837 participants (major depressive disorder [MDD], n=265; bipolar I disorder [BD I], n=126; and bipolar II disorder [BD II], n=446). We performed exploratory graph analysis, employing regularized partial correlation models to estimate the network structure. Centrality analysis identified core IPSM symptoms for each mood disorder group. Network comparison tests assessed structural differences between the MDD and BD subgroups. RESULTS: Network analysis detected five communities. Item 10 ("I worry about being criticized for things that I have said or done") showed the highest value in strength. Multiple items on "Interpersonal Worry/Dependency" and "Low Self-Esteem" showed high strength centrality. Network structure invariance and global strength invariance test results indicated no significant differences between the MDD and BD subgroups. CONCLUSION: Our findings emphasize the importance of addressing "Interpersonal Worry/Dependency" and "Low Self-Esteem" in the IPSM network among mood disorder patients based on core items of the network. Additionally, targeted treatments and comprehensive strategies in this aspect could be crucial for managing mood disorders.

Genome-wide association analyses using machine learning-based phenotyping reveal genetic architecture of occupational creativity and overlap with psychiatric disorders.

Creativity is known to be heritable and exhibits familial aggregation with psychiatric disorders; however, the complex nature of their relationship has not been well-established. In the present study, we demonstrate that using an expanded and validated machine learning (ML)-based phenotyping of occupational creativity (OC) can allow us to further understand the trait of creativity, which was previously difficult to define and study. We conducted the largest genome-wide association study (GWAS) on OC with 241,736 participants from the UK Biobank and identified 25 lead variants that have not yet been reported and three candidate causal genes that were previously associated with educational attainment and psychiatric disorders. We found extensive genetic overlap between OC and psychiatric disorders with mixed effect direction through various post-GWAS analyses, including the bivariate causal mixture model. In addition, we discovered a strongly genetic correlation between our original GWAS and the GWAS adjusted for education years (rg = 0.95). Our GWAS analysis via ML-based phenotyping contributes to the understanding of the genetic architecture of creativity, which may inform genetic discovery and genetic prediction in human cognition and psychiatric disorders.

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

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

Validation of the Short Form of the Mood Instability Questionnaire-Trait (MIQ-T-SF) in the Korean General Population.

OBJECTIVE: Mood instability (MI) is a clinically significant trait associated with psychiatric disorders. However, there are no concise measurements to evaluate MI. The initial Mood Instability Questionnaire-Trait (MIQ-T) was developed to fill this gap. The current study aimed to create a short form of MIQ-T (MIQ-T-SF) that measures MI with high validity and reliability in the Korean general population. METHODS: Of the 59 items in the MIQ-T, 17 items were chosen for the MIQ-T-SF following the factor analysis process. In total, 540 participants completed the MIQ-T-SF. Cronbach's alpha and McDonald's omega were used to evaluate reliability. Exploratory factor analysis (EFA) and confirmatory factor analysis (CFA) were used to determine construct validity. Concurrent validity was confirmed via comparisons with Personality Assessment Inventory-Borderline Features Scale. Measurement invariance across gender and age groups was confirmed before analyzing differences in scores using Kruskal-Wallis test. RESULTS: The MIQ-T-SF displayed expected correlations and high internal consistency (α=0.71-0.90, Ωt=0.72-0.92). Using EFA and CFA, a five-factor structure was confirmed. Measurement invariance was supported, and gender differences were observed. CONCLUSION: The MIQ-T-SF is an accurate and reliable method to detect MI in the Korean general population. The study's results offer new perspectives for future studies on MI.

Relationship between childhood trauma and resilience in patients with mood disorders.

BACKGROUND: Childhood trauma has lasting negative impacts on individuals' psychological functioning. However, there is limited empirical evidence on the association between childhood trauma and resilience and none examining such relationship among diverse clinical populations. This study aimed to investigate the relationship in patients with major depressive disorder, bipolar I disorder, bipolar II disorder, and a comparison group. METHODS: In total, 787 psychiatric patients and 734 people from the general population participated in the study. The Childhood Trauma Questionnaire-Short Form and Connor-Davidson Resilience Scale were used to assess childhood trauma and resilience, respectively. RESULTS: Individuals with childhood trauma showed lower levels of resilience in all subjects; among them, those who experienced emotional abuse and emotional neglect exhibited even stronger associations than other types of childhood trauma. There was a significant difference in the negative relationship between childhood trauma and resilience by group, where the association was more prominent in the comparison group than in MDD and BD II patient groups. LIMITATIONS: The generalizability of our results may be limited due to unproportionate patient sample size. Also, we could not examine the causal relationship between childhood trauma and resilience. CONCLUSION: Childhood trauma and resilience had a significantly negative association. Our results suggest that people who have experienced emotional abuse and emotional neglect should be closely assisted to develop resilience. Interventions that promote resilience should be provided to individuals predisposed to psychological risks as a result of childhood trauma.

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

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

Potent and uniform fetal hemoglobin induction via base editing.

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

Korean Validation of the Temperament Evaluation of Memphis, Pisa, Paris, and San Diego Autoquestionnaire.

OBJECTIVE: The Temperament Evaluation of Memphis, Pisa, Paris, and San Diego Autoquestionnaire (TEMPS-A) has been validated in more than 30 languages and is noted for its broad application in research and clinical settings. This study presents the first attempt to examine the reliability and validity of the TEMPS-A in Korea. METHODS: A total of 540 non-clinical participants completed the Korean TEMPS-A, which was adapted from the original English version via a comprehensive translation procedure. Reliability was assessed using Cronbach's α, and associations between temperaments were examined using Spearman's correlation coefficient. Exploratory factor analysis (EFA) was performed, and differences in TEMPS-A scores between the gender- and age-based groups were examined using Kruskal-Wallis analysis. RESULTS: The Korean TEMPS-A exhibited excellent internal consistency (0.70-0.91) and significant correlations between subscales. EFA resulted in a two-factor structure: Factor I (depressive, cyclothymic, irritable, and anxious) and Factor II (hyperthymic). Gender and age group differences were observed. CONCLUSION: Overall, our results suggest that TEMPS-A is a reliable and valid measure of affective temperaments for the Korean population. This study opens new possibilities for further research on affective temperaments and their related traits.