Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers.
View Article and Find Full Text PDFCRISPR prime editing () requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (), an extended version of a standard guide RNA () that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here, we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold.
View Article and Find Full Text PDFCRISPR prime editing () requires a Cas9 nickase-reverse transcriptase fusion protein (known as PE2) and a prime editing guide RNA (), an extended version of a standard guide RNA () that both specifies the intended target genomic sequence and encodes the desired genetic edit. Here we show that sequence complementarity between the 5' and the 3' regions of a pegRNA can negatively impact its ability to complex with Cas9, thereby potentially reducing PE efficiency. We demonstrate this limitation can be overcome by a simple pegRNA refolding procedure, which improved ribonucleoprotein-mediated PE efficiencies in zebrafish embryos by up to nearly 25-fold.
View Article and Find Full Text PDFThe CRISPR prime editor PE2 consists of a Streptococcus pyogenes Cas9 nickase (nSpCas9) fused at its C-terminus to a Moloney murine leukemia virus reverse transcriptase (MMLV-RT). Here we show that separated nSpCas9 and MMLV-RT proteins function as efficiently as intact PE2 in human cells. We use this Split-PE system to rapidly identify and engineer more compact prime editor architectures that also broaden the types of RTs used for prime editing.
View Article and Find Full Text PDFAlloreactive and autoreactive antibodies have been associated with the development of chronic lung allograft dysfunction (CLAD), but their pathogenic role is disputed. Orthotopic left lung transplantation was performed in the Fischer-344 to Lewis rat strain combination followed by the application of ciclosporine for 10 days. Four weeks after transplantation, lipopolysaccharide (LPS) was instilled into the trachea.
View Article and Find Full Text PDFPresenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-β (Aβ) peptide that deposits as plaques in the Alzheimer's disease (AD) brain. The M146L mutation in the PS1 gene () leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aβ42. This change is evident not only in the brain but also in peripheral cells of mutation carriers.
View Article and Find Full Text PDFHelicobacter pylori colonizes half of the global population and causes gastritis, peptic ulcer disease or gastric cancer. In this study, we were interested in human annexin (ANX), which comprises a protein family with diverse and partly unknown physiological functions, but with a potential role in microbial infections and possible involvement in gastric cancer. We demonstrate here for the first time that H.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
June 2021
CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair ("homing"). Understanding whether CRISPR-induced off-target mutations are generated in mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome.
View Article and Find Full Text PDFPrime editors have been delivered using DNA or RNA vectors. Here we demonstrate prime editing with purified ribonucleoprotein complexes. We introduced somatic mutations in zebrafish embryos with frequencies as high as 30% and demonstrate germline transmission.
View Article and Find Full Text PDFPrime editing (PE) is a versatile genome editing technology, but design of the required guide RNAs is more complex than for standard CRISPR-based nucleases or base editors. Here we describe PrimeDesign, a user-friendly, end-to-end web application and command-line tool for the design of PE experiments. PrimeDesign can be used for single and combination editing applications, as well as genome-wide and saturation mutagenesis screens.
View Article and Find Full Text PDFGene drives hold promise for use in controlling insect vectors of diseases, agricultural pests, and for conservation of ecosystems against invasive species. At the same time, this technology comes with potential risks that include unknown downstream effects on entire ecosystems as well as the accidental or nefarious spread of organisms that carry the gene drive machinery. A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration in the interest of society and humanity.
View Article and Find Full Text PDFVirtual reality (VR) is popular across many disciplines and has been increasingly used in sports as a training tool lately. However, it is not clear whether the spatial orientation of humans works equally within VR and in the real-world. In this paper, two studies are presented, in which natural body movements were allowed and demanded.
View Article and Find Full Text PDFAlthough there are many virtual reality (VR) applications in sports, only a handful of studies visualized the whole body. There is still a lack of understanding, how much of the own body must be visualized in the head-mounted display (HMD) based VR, to ensure fidelity and similar performance outcome as in the real-world. In the current study, 20 young and healthy participants completed three tasks in a real and virtual environment: balance task, grasping task, and throwing task with a ball.
View Article and Find Full Text PDFJ Pediatr Endocrinol Metab
August 2020
Objectives Based on the ellipse trial, liraglutide was recently approved for use in pediatric patients with type 2 diabetes. We report the comparative exposure of liraglutide in pediatric vs. adult patients.
View Article and Find Full Text PDFFerroptosis, a newly discovered form of cell death mediated by reactive oxygen species (ROS) and lipid peroxidation, has recently been shown to have an impact on various cancer types; however, so far there are only few studies about its role in hepatocellular carcinoma (HCC). The delicate equilibrium of ROS in cancer cells has found to be crucial for cell survival, thus increased levels may trigger ferroptosis in HCC. In our study, we investigated the effect of different ROS modulators and ferroptosis inducers on a human HCC cell line and a human hepatoblastoma cell line.
View Article and Find Full Text PDFIntroduction: The aim of the present analysis was to characterise the absorption, distribution and elimination of semaglutide by means of population pharmacokinetic (PK) models using data from nine clinical pharmacology trials conducted in both healthy subjects and those with type 2 diabetes.
Methods: Data were obtained from trials with subcutaneous and intravenous administration of semaglutide that utilised frequent PK sampling and included a total of 353 subjects with 10,573 concentration values.
Results: Semaglutide PK properties across trials, drug product strengths and populations were well characterised by a two-compartment model with first-order absorption and elimination.
Facial expressions play a crucial role in human daily life interactions, so it is possible that they contain relevant anticipatory cues. Therefore, we conducted two studies in karate kumite to analyse the influence of facial expressions on the attack recognition (study A) and on the response behaviour (study B). In study A, 10 karate athletes (age 14-26 years, national and international level) had to recognize temporally occluded karate attacks in 2 conditions (normal and blurred face of the attacker) and identify the upcoming attack (verbal reports).
View Article and Find Full Text PDFMost head and neck cancer patients are treated with combined modalities such as surgery, radiotherapy (RT), chemotherapy (ChT). Concurrent chemo-radiation has improved treatment outcomes with increased toxic effects. Reactions after RT are divided into early and late changes.
View Article and Find Full Text PDFAims: To evaluate dose levels for semaglutide, a glucagon-like peptide-1 analogue approved for the treatment of type 2 diabetes, by examining the effects of demographic factors on efficacy and safety in an exposure-response analysis.
Methods: We analysed data from 1552 adults from four randomized phase III trials of 30 to 56 weeks' duration, investigating once-weekly semaglutide doses 0.5 and 1.
Mol Ther Methods Clin Dev
March 2017
In T cells with transgenic high-avidity T cell receptors (TCRs), endogenous and transferred TCR chains compete for surface expression and may pair inappropriately, potentially causing autoimmunity. To knock out endogenous TCR expression, we assembled 12 transcription activator-like effector nucleases (TALENs) and five guide RNAs (gRNAs) from the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Using TALEN mRNA, TCR knockout was successful in up to 81% of T cells.
View Article and Find Full Text PDFObjectives: To investigate the safety, tolerability, and pharmacokinetics of liraglutide in adolescents with obesity.
Study Design: This was a randomized, double-blind, placebo-controlled trial. Twenty-one subjects, aged 12-17 years and Tanner stage 2-5, with obesity (body mass index [BMI] corresponding to both a BMI ≥95th percentile for age and sex and to a BMI of ≥30 kg/m for adults; additionally, BMI was ≤45 kg/m) were randomized (2:1) to receive 5 weeks of treatment with liraglutide (0.