Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping

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

General and Distinct Functions of Sec1/Munc18-like Protein Families

All eukaryotes require Sec1/Munc18-like (SM) proteins for SNARE-mediated membrane fusion. SM proteins are divided into four families that function in different trafficking pathways — Sec1/Munc18, Vps33, Vps45, and Sly1. While their general function is to stimulate membrane fusion, they also have distinct, family-specific functions. Critical to the general function of SM proteins are an anti-parallel pair of helices, called the helical hairpin. Superposition of two crystal structures of SM protein Vps33 bound to its SNAREs suggests that this helical hairpin templates the R- and Qa-SNAREs in a half-assembled SNARE complex. This mechanistic model, or ‘template model,’ could explain how SM proteins stimulate membrane fusion. It is unclear, however, (1) if the template model is conserved in all four families, and (2) if the helical hairpin also mediates family-specific functions. Here, I created sequence banks and analyzed residue conservation in each SM protein family. I show that the template model appears to be a conserved mechanism for the general function of SM proteins; in all four families, the SM protein residues that would bind the SNAREs in the template model were more significantly conserved than other solvent-exposed SM protein residues. The helical hairpin also appears to facilitate family-specific functions, since several residues on the surface of the helical hairpin were conserved family-specifically. Taken together, these results suggest that the helical hairpin facilitates both general and distinct functions of SM proteins. Future investigations of the helical hairpin will need to uncouple these general and distinct functions

Seasonal variations in the nitrogen isotope composition of Okinotori coral in the tropical western Pacific : A new proxy for marine nitrate dynamics

To demonstrate the utility of coral skeletons as a recorder of nitrate dynamics in the surface ocean, we collected coral skeletons of Porites lobata and determined their nitrogen isotope composition (δ15N_[coral]) from 2002 to 2006. Skeletons were collected at Okinotori Island in southwestern Japan, far from any sources of terrestrial nitrogen. Nitrogen isotope compositions along the growth direction were determined at 800 μm intervals (∼1 month resolution) and compared against the skeletal carbon isotope composition (δ13C_[coral-carb]), barium/calcium ratio (Ba/Ca), and Chlorophyll-a concentration (Chl-a). From 2002 to 2004, ratios of the δ15N_[coral] varied between +0.8 and +8.3‰ with inverse variation to SST (r = -0.53). Ba/Ca ratios and Chl-a concentrations were also observed to be high during seasons with low SST. These results suggested that the vertical mixing that occurs during periods of low SST carries nutrients from deeper water (δ15N_[DIN]; +5∼+6‰) to the sea surface. In 2005 onward, δ15N_[coral] and Ba/Ca ratios also had positive peaks even in high SST during periods of transient upwelling caused by frequent large typhoons (maximum wind speed 30 m/s). In addition, low δ15N_[coral] (+0.8∼+2.0‰) four months after the last typhoon implied nitrogen fixation because of the lack of typhoon upwelling through the four years record of δ15N_[coral]. Variations in the δ13C_[coral-carb] and δ15N_[coral] were synchronized, suggesting that nitrate concentration could control zooxanthellae photosynthesis. Our results suggested that δ15N_[coral] holds promise as a proxy for reconstructing the transport dynamics of marine nitrate and thus also a tool for estimating nitrate origins in the tropical and subtropical oceans

Direct implantation of hair-follicle-associated pluripotent (HAP) stem cells repairs intracerebral hemorrhage and reduces neuroinflammation in mouse model.

Intracerebral hemorrhage (ICH) is a leading cause of mortality with ineffective treatment. Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into neurons, glial cells and many other types of cells. HAP stem cells have been shown to repair peripheral-nerve and spinal-cord injury in mouse models. In the present study, HAP stem cells from C57BL/6J mice were implanted into the injured brain of C57BL/6J or nude mice with induced ICH. After allo transplantation, HAP stem cells differentiated to neurons, astrocytes, oligodendrocytes, and microglia in the ICH site of nude mice. After autologous transplantation in C57BL/6J mice, HAP stem cells suppressed astrocyte and microglia infiltration in the injured brain. The mRNA expression levels of IL-10 and TGF-β1, measured by quantitative Real-Time RT-PCR, in the brain of C57BL/6J mice with ICH was increased by HAP-stem-cell implantation compared to the non-implanted mice. Quantitative sensorimotor function analysis, with modified limb-placing test and the cylinder test, demonstrated a significant functional improvement in the HAP-stem-cell-implanted C57BL/6J mice, compared to non-implanted mice. HAP stem cells have critical advantages over induced pluripotent stem cells, embryonic stem cells as they do not develop tumors, are autologous, and do not require genetic manipulation. The present study demonstrates future clinical potential of HAP-stem-cell repair of ICH, currently a recalcitrant disease

Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells

Atrial and ventricular cardiomyocytes differentiated from rat HAP stem cells had structures of mature cardiomyocytes.

(a) Double immunostaining of cTnT (red)- and MLC2v (green)-positive images, cTnT in the I-band and MLC2v in the A-band showed an alternating pattern of sarcomeres. Scale bars; 10 μm. (b) Double immunostaining of cTnT (red)- and β-catenin (green)-positive images of ventricular cardiomyocytes differentiated from HAP stem cells. Nuclear staining with DAPI (blue). Scale bars; 10 μm. (c-h) Transmission electron microscopy images of cardiomyocytes differentiated from HAP stem cells. (c, d) Non-supplemented cardiomyocytes. Microfibrils with scattered Z-bands (Z) and small-sized mitochondria (Mt). (e-h) Atrial cardiomyocyte (e, f) and ventricular cardiomyocyte (g, h) differentiated from HAP stem cells. Microfibrils with regularly Z-bands (Z). A-bands (A), I-bands (I), H-bands (H) and M-bands (M) were also organized. Large mitochondria (Mt) with complex internal structures. Ventricular cardiomyocytes had T-tubule-like structures (TT) and sarcoplasmic reticulum (SR). N: nuclear. (c, e, g) Scale bars; 2 μm. (d, f, h) Scale bars; 1 μm.</p

Beating cardiomyocytes differentiated from rat HAP stem cells.

Beating cardiomyocytes differentiated from rat HAP stem cells.</p

Rat HAP stem cells differentiated into atrial or ventricular cardiomyocytes.

(a-c) Immunofluorescence staining of the upper parts of rat vibrissa hair follicles, which were cultured for 21 days, differentiated into cTnI (a-upper; red) and cTnT (a-lower; red)-positive non-supplemented cardiomyocytes; cTnI (b-upper; red), cTnT (b-lower; red)- and MLC2a (b; green)-positive atrial cardiomyocytes; cTnI (c-upper; red), cTnT (c-lower; red)- and MLC2v (c; green)-positive ventricular cardiomyocytes. Nuclear staining with DAPI (blue). Scale bars; 100 μm. (d, e) qPCR analyses of cardiomyocytes differentiated from HAP stem cells. n = 4 per group for non-supplemented, atrial, and ventricular cardiomyocytes. Data are presented as mean ± SD. * P < 0.05, ** P < 0.005, two-sided Student’s t-test.</p