Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for ß-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

CRISPR/Cas based gene editing: marking a new era in medical science.

CRISPR/Cas9 system, a bacterial adaptive immune system developed into a genome editing technology, has emerged as a powerful tool revolutionising genome engineering in all branches of biological science including agriculture, research and medicine. Rapid evolution of CRISPR/Cas9 system from the generation of double strand breaks to more advanced applications on gene regulation has made the wide-spread use of this technology possible. Medical science has benefited greatly from CRISPR/Cas9; being both a versatile and economical tool, it has brought gene therapy closer to reality. In this review, the development of CRISPR/Cas9 system, variants thereof and its application in different walks of medical science- research, diagnostics and therapy, will be discussed.

Pediatric non-urgent emergency department visits and prior care-seeking at primary care.

BACKGROUND: We aimed to examine how caregiver perceptions of primary care affects care-seeking prior to pediatric non-urgent ED visits. METHODS: We performed a cross-sectional survey of caregivers of children presenting to a pediatric ED during weekday business hours and triaged as low acuity. We first compared caregiver sociodemographic characteristics, perceptions of primary care, and stated preference in care sites (ED vs PCP) for caregivers who had sought care from their child's PCP office versus had not sought care from their child's PCP office prior to their ED visit. We then examined odds of having sought care from their PCP office prior to their ED visit using multivariable logistic regression models sequentially including caregiver primary care perceptions and stated care site preferences along with caregiver sociodemographic characteristics. RESULTS: Of 140 respondents, 64 (46%) sought care from their child's PCP office prior to presenting to the ED. In unadjusted analysis, children insured by Medicaid or CHIP, caregivers identifying as Black, and caregivers with lower educational attainment were less likely to have sought PCP care before presenting to the ED (p < 0.005, each). Caregivers who had sought PCP care were more likely to prefer their PCP relative to the ED in terms of ease of travel, cost, and wait times (p < 0.001, all). When including these stated preferences in a multivariable model, child insurance, caregiver race, and caregiver education were no longer significantly associated with odds of having sought PCP care prior to their ED visit. CONCLUSIONS: Differential access to primary care may underlie observed demographic differences in non-urgent pediatric ED utilization.

Inadequate Hydration, BMI, and Obesity Among US Adults: NHANES 2009-2012.

PURPOSE: Improving hydration is a strategy commonly used by clinicians to prevent overeating with the goal of promoting a healthy weight among patients. The relationship between weight status and hydration, however, is unclear. Our objective was to assess the relationship between inadequate hydration and BMI and inadequate hydration and obesity among adults in the United States. METHODS: Our study used a nationally representative sample from the National Health and Nutrition Examination Survey (NHANES) 2009 to 2012, and included adults aged 18 to 64 years. The primary outcome of interest was body mass index (BMI), measured in continuous values and also categorized as obese (BMI ≥30) or not (BMI <30). Individuals with urine osmolality values of 800 mOsm/kg or greater were considered to be inadequately hydrated. Linear and logistic regressions were performed with continuous BMI and obesity status as the outcomes, respectively. Models were adjusted for known confounders including age, race/ethnicity, sex, and income-to-poverty ratio. RESULTS: In this nationally representative sample (n = 9,528; weighted n = 193.7 million), 50.8% were women, 64.5% were non-Hispanic white, and the mean age was 41 years. Mean urine osmolality was 631.4 mOsm/kg (SD = 236.2 mOsm/kg); 32.6% of the sample was inadequately hydrated. In adjusted models, adults who were inadequately hydrated had higher BMIs (1.32 kg/m(2); 95% CI, 0.85-1.79; P <.001) and higher odds of being obese (OR = 1.59; 95% CI, 1.35-1.88; P <.001) compared with hydrated adults. CONCLUSION: We found a significant association between inadequate hydration and elevated BMI and inadequate hydration and obesity, even after controlling for confounders. This relationship has not previously been shown on a population level and suggests that water, an essential nutrient, may deserve greater focus in weight management research and clinical strategies.

Trends in childhood pneumococcal vaccine coverage in Shanghai, China, 2005-2011: a retrospective cohort study.

BACKGROUND: In China, the pneumococcal conjugate vaccine (PCV7) and the pneumococcal polysaccharide vaccine (PPSV23) are not offered under the government's Expanded Program on Immunization and are instead administered for a fee. PCV7 is more effective and covers more serotypes associated with invasive disease in children, but is also more expensive, than PPSV23. Because of their expense, there is concern that these vaccines, especially PCV7, have low uptake particularly among non-locals, migrants from outside of Shanghai. This paper characterizes the differential coverage of PCV7 and PPSV23 between locals and non-locals in Shanghai, and illustrates coverage trends over time. METHODS: In this retrospective cohort study, children born between 2005 and 2011 were sampled from the Shanghai Immunization Program Information System. Bivariate and multivariable analyses examined the relationships between demographic characteristics, residency status (non-locals vs locals), and vaccination coverage. RESULTS: PPSV23 coverage (29.8 %) among children over 2 years of age was higher than PCV7 coverage (10.1 %) for locals and non-locals. Uptake of PCV7 increased substantially after children were 2 years of age. Overall, non-local populations had higher PPSV23 coverage (OR: 1.34; 98 % CI: 1.22, 1.46) but lower PCV7 coverage (OR: 0.617, 98 % CI: 0.547, 0.695) than locals. CONCLUSIONS: There is a need for increasing overall pneumococcal coverage in Shanghai children, particularly with the more effective PCV7 vaccine. Morbidity and mortality due to invasive pneumococcal disease for children <1 year of age are unlikely to be mitigated if the current age-related vaccination patterns are not improved.

Precise correction of a spectrum of ß-thalassemia mutations in coding and non-coding regions by base editors.

ß-thalassemia/HbE results from mutations in the ß-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 ß-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 ß-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 ß-thalassemia/HbE. These ß-thalassemia/HbE models were then used to assess the efficiency of correction in the native locus and functional ß-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 ß0/ßE-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.

Protocol for arrayed gRNA screening by base editors in mammalian cell lines using lentiviral system.

Base editing, a CRISPR-based genome engineering technique, enables precise single-nucleotide modifications while minimizing double-strand breaks. Here, we present a protocol for arrayed mutagenesis using base editors to identify regulatory elements within the gamma-globin locus. We describe steps for guide RNA (gRNA) cloning into lentiviral vectors, establishing stable cell lines with base editor expression, transducing gRNAs, and assessing editing efficiency. This protocol can be applied to diverse genomic regions and cell lines for arrayed screening, facilitating genetic research, and target discovery. For complete details on the use and execution of this protocol, please refer to Ravi et al. (2022)1.

Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System.

Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.

Preferential Expansion of Human CD34+CD133+CD90+ Hematopoietic Stem Cells Enhances Gene-Modified Cell Frequency for Gene Therapy.

CD34+CD133+CD90+ hematopoietic stem cells (HSCs) are responsible for long-term multilineage hematopoiesis, and the high frequency of gene-modified HSCs is crucial for the success of hematopoietic stem and progenitor cell (HSPC) gene therapy. However, the ex vivo culture and gene manipulation steps of HSPC graft preparation significantly reduce the frequency of HSCs, thus necessitating large doses of HSPCs and reagents for the manipulation. In this study, we identified a combination of small molecules, Resveratrol, UM729, and SR1 that preferentially expands CD34+CD133+CD90+ HSCs over other subpopulations of adult HSPCs in ex vivo culture. The preferential expansion enriches the HSCs in ex vivo culture, enhances the adhesion, and results in a sixfold increase in the long-term engraftment in NSG mice. Further, the culture-enriched HSCs are more responsive to gene modification by lentiviral transduction and gene editing, increasing the frequency of gene-modified HSCs up to 10-fold in vivo. The yield of gene-modified HSCs obtained by the culture enrichment is similar to the sort-purification of HSCs and superior to Cyclosporin-H treatment. Our study addresses a critical challenge of low frequency of gene modified HSCs in HSPC graft by developing and demonstrating a facile HSPC culture condition that increases the frequency of gene-modified cells in vivo. This strategy will improve the outcome of HSPC gene therapy and also simplify the gene manipulation process.

Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2.

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.

Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.

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.

Hilariously Bad News: Medical Improv as a Novel Approach to Teach Communication Skills for Bad News Disclosure.

A targeted, improv-based exercise was developed as a novel skills training approach to bad news disclosure for medical professionals and trainees, focusing on specific characteristics that influence these conversations including nature of the bad news, implications, personal responsibility, and status differences.

A New Method for Determination of Minimum Biofilm Eradication Concentration for Accurate Antimicrobial Therapy.

Antimicrobial susceptibility testing (AST) is an important technique to find the susceptibility pattern of clinical isolates in order to administer the appropriate drug. One such technique is minimum inhibitory concentration (MIC), which not only identifies the right drug but also suggests the appropriate concentration necessary to neutralize the organisms in planktonic form. MIC can vary in case of adherent organisms since they form biofilms and activate survival mechanisms like quorum sensing. Here we have strategized a new method which used an inoculator plate, a resazurin dye, and a standard plate to identify minimum biofilm eradication concentration (MBEC) of adherent organisms.

The potential of different molecular biology methods in tracking clones of Acinetobacter baumannii in an ICU setting.

PURPOSE: This study aimed to characterize A. baumannii strains isolated from patients in an intensive care unit (ICU) setting. Molecular techniques were used to study clonal relatedness and determine a fast, efficient and cost-effective way of detecting persistent clones. METHODOLOGY: A. baumannii (n=17) were obtained in June and November 2015 from a single ICU setting in South India. DNA typing methods such as multilocus sequence typing (MLST), single-locus sequence-based typing (SBT) and DNA fingerprinting PCRs (M13, DAF4 and ERIC2) were employed to understand the association of clones. PCRs were performed for the antimicrobial resistance genes ISAba1-blaOXA-51-like, ISAba1-blaOXA-23-like, blaNDM-1, blaPER-7 and blaTEM-1, and the virulence genes cpa 1, cpa2 and pkf. RESULTS: The MLST showed some degree of corroboration with the other DNA typing methods. The M13 PCR was found to give better results than the other fingerprinting methods. ST848 (CC92) was the dominant strain isolated in both June and November. All isolates were blaOXA-51-like-positive, with 16 having ISAba1 upstream of the blaOXA-51-like and blaOXA-23-like genes. Genes such as blaNDM-1 (23 %, n=4), blaPER-7 (58.8 %, n=10), pkf (82 %, n=14), blaTEM-1 (5.8 %, n=1), cpa1 (5.8 %, n=1) and cpa2 (5.8 %, n=1) were also detected. CONCLUSION: M13 PCR can be used in routine environmental surveillance for the detection of persistent antibiotic resistant clones in an ICU setting because of its reliability and simplicity. Further studies based on greater sample size, conducted at the multi-centre level, can give us a better understanding of the reliability of the molecular methods that can be used for the detection of persistent clones in the hospital setting.