Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome ...editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hard-to-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.
Abstract
In mammals, the circadian clock consists of transcriptional and translational feedback loops through DNA
cis
-elements such as E-box and RRE. The E-box-mediated core feedback loop is ...interlocked with the RRE-mediated feedback loop, but biological significance of the RRE-mediated loop has been elusive. In this study, we established mutant cells and mice deficient for rhythmic transcription of
Bmal1
gene by deleting its upstream RRE elements and hence disrupted the RRE-mediated feedback loop. We observed apparently normal circadian rhythms in the mutant cells and mice, but a combination of mathematical modeling and experiments revealed that the circadian period and amplitude of the mutants were more susceptible to disturbance of CRY1 protein rhythm. Our findings demonstrate that the RRE-mediated feedback regulation of
Bmal1
underpins the E-box-mediated rhythm in cooperation with CRY1-dependent posttranslational regulation of BMAL1 protein, thereby conferring the perturbation-resistant oscillation and chronologically-organized output of the circadian clock.
A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the ...full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.
A newly developed human immunoglobulin liquid preparation for intravenous injection was studied for efficacy, safety, and usefulness in treating severe and/or refractory infections in children ...receiving antibiotic treatment. It is suggested that C-425 is a useful intravenous preparation of human immunoglobulin for the treatment of severe and/or refractory infections in pediatrics. C-425 was administered to 87 inpatients with severe and/or refractory infections at 23 institutions nationwide. The Committee selected 61 cases for the present analysis. Physicians in charge judged clinical efficacy of C-425 to be “excellent” in 23 cases (40.4%), “good” in 24 (42.1%), “fair” in 7 (12.3%), “poor” in 3 (5.3%), and “unknown” in 4. The efficacy rate was calculated at 82.5% when the “excellent” and “good” cases were combined, and 94.7% when the “fair” cases were also included. According to the Committee's judgement, the efficacy of C-425 was “excellent” in 27 cases (44.3%), “good” in 18 (29.5%), “fair” in 7 (11.5%), and “poor” in 9 (14.8%). The efficacy rate was 73.8% when the “excellent” and “good” cases were combined. The rate increased to 85.2% when the “fair” cases were added. Organisms were identified in 31 cases, and the time course was followed in 19 instances. Organisms were eliminated in 12 cases (63.2%), decreased in number in 2 (10.5%), and persisted in 5 (26.3%). Eradication rate was 63.2%. One of the 87 patients died of fulminant hepatitis 2 days after the end of the treatment. The remaining 86 cases were analyzed for the safety of C-425. A skin rash was observed in one case. Laboratory examination revealed increase in transaminase levels in a total of 8 cases; both in GOT and GPT in 5, in GOT alone in 2, and in GPT alone in 1. These findings were not clinically important.
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